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Title: Immunity in infective diseases
Author: Elie Metchnikoff
Translator: Francis G. Binnie
Release date: December 21, 2025 [eBook #77523]
Language: English
Original publication: Cambridge: University Press, 1905
Credits: Richard Tonsing and the Online Distributed Proofreading Team at https://www.pgdp.net (This file was produced from images generously made available by The Internet Archive)
*** START OF THE PROJECT GUTENBERG EBOOK IMMUNITY IN INFECTIVE DISEASES ***
IMMUNITY
IN
INFECTIVE DISEASES
CAMBRIDGE UNIVERSITY PRESS WAREHOUSE,
C. F. CLAY, MANAGER.
=London=: FETTER LANE, E.C.
=Glasgow=: 50, WELLINGTON STREET.
[Illustration: Heraldic shield quartered with lions passant in the
top-left, top-right, bottom-left, and bottom-right, and ermine patterns
in the other quarters, with an open book at the center.]
=Leipzig=: F. A. BROCKHAUS.
=New York=: THE MACMILLAN COMPANY.
=Bombay and Calcutta=: MACMILLAN AND CO., LTD.
[_All Rights reserved._]
IMMUNITY
IN
INFECTIVE DISEASES
BY
ÉLIE METCHNIKOFF
FOREIGN MEMBER OF THE ROYAL SOCIETY OF LONDON
PROFESSOR AT THE PASTEUR INSTITUTE, PARIS
TRANSLATED FROM THE FRENCH
BY
FRANCIS G. BINNIE
OF THE PATHOLOGICAL DEPARTMENT, UNIVERSITY OF CAMBRIDGE
_With 45 figures in the text_
CAMBRIDGE
AT THE UNIVERSITY PRESS
1905
=Cambridge=
PRINTED BY JOHN CLAY, M.A
AT THE UNIVERSITY PRESS.
PREFACE TO THE ENGLISH EDITION.
In preparing for the English-reading student this version of M.
Metchnikoff’s latest work, wherein he “sums up the labours of
twenty-five years,” it has been my aim to give a faithful rendering of
the ideas and argument of the original, even at the risk of an
occasional crude expression, rather than to attempt to reproduce the
brilliancy of the original by any wide verbal departure from the text.
The Table of Contents forms an admirable analytical summary of the main
subject-matters treated, but an alphabetical Index has been added to the
present edition, and, though not at all exhaustive, this may serve as a
key to the many authors cited and to the maze of detail discussed in the
work.
The marginal reference to the pages of the original work will, I hope,
commend itself to those readers who may wish to refer to the _ipsissima
verba_ of the author. It is, I believe, a novelty in scientific works,
though familiar in works in other departments of literature.
I am under deep obligations to Professor Woodhead (who has read the
whole of the proofs) and to Mr A. E. Shipley, and Mr G. H. F. Nuttall
(who have read portions) for much valuable criticism and advice.
THE TRANSLATOR.
_August, 1905._
ERRATA.
Page 93, line 2, _after_ itself _insert_ alone; line 3, _delete_ only.
Page 98, line 3 from bottom, _for_ actively _read_ vigorously.
Page 386, line 16, _for_ tortoise _read_ turtle.
Page 461, line 21, _after_ circumstances _insert reference figure_ 1.
Page 537, line 11, _for_ arise _read_ arises.
TO MESSIEURS E. DUCLAUX AND E. ROUX.
_My dear Friends_,
_Permit me to dedicate to you this work, which sums up the labours of
twenty-five years; a very great part of it has been carried out by your
side, you who have done so much to lighten my task._
_When, nearly fourteen years ago, you allowed me to share your work
alongside the venerated Master who founded the House where we have
laboured together, you were anything but partisans of my theories; they
seemed to you too vitalistic, and not sufficiently physico-chemical. In
course of time you became convinced that my ideas were not without
foundation, and since then you have given me warm encouragement to
pursue my researches in the field that I had marked out for myself._
_Working by your side and drawing largely from your vast and varied
stores of knowledge, I felt myself safe from those divagations into
which a zoologist, who had wandered into the domain of biological
chemistry and of medical science, is likely to stray. I thank you with
all my heart, and I beg you to accept the homage of this work as a
testimony of my deepest gratitude and of my warmest friendship._
ÉLIE METCHNIKOFF.
_Institut Pasteur,
3 October, 1901._
PREFACE.
When, ten years ago, I was preparing my _Lessons on the Comparative
Pathology of Inflammation_ for the press, I hoped that the other
sections of the phagocytic theory—Immunity, Atrophies, and Healing—would
soon follow this first work. This hope has not been realised, and it has
needed prolonged work ere I could publish the volume I have just
completed.
During this long period I sent out several _ballons d’essai_ under the
form of summaries of the question of Immunity, published in the _Semaine
médicale_ (1892), the _Ergebnisse_ of Lubarsch and Ostertag (1886), and
the _Handbuch der Hygiene_ by Weyl (1897). I there attempted, as far as
possible, to give a general picture of the phenomena of Immunity in the
infective diseases, and it was my desire to excite criticism and
opposition, in order to determine the fate of the theory of phagocytes
in its application to the problem of Immunity.
The most recent attempt in this direction was made at the International
Congress at Paris, in the past year (1900), when I presented my report
on Immunity before an audience which included, amongst others, my
principal opponents. It was the result of this Congress which at length
decided me to bring together my views on Immunity in a volume which I
now present to the reader.
Convinced that many of the objections raised against the phagocytic
theory of Immunity proceeded solely from an insufficient knowledge of
the theory, I thought that a work condensed into one volume might render
some service to those who are interested in the problem of Immunity. I
do not know whether I shall convert my opponents, but I am convinced
that a perusal of this book will clear away certain misunderstandings. A
very competent observer recently confessed in one of his publications
that for many years he had been unaware of the experiments of M. J.
Bordet and myself on Immunity against the cholera vibrio, experiments
which he now regards as of fundamental importance for the comprehension
of the whole question of Immunity. I hope that after the appearance of
this treatise such oversights will not be so likely to occur.
Should I not succeed in convincing my opponents of the justice of the
cause which I defend, T shall at least have informed my critics and
shall have given them an opportunity of discussing it with a thorough
knowledge of the material on which it is based. This result alone would
justify me in having undertaken this work.
At first I intended to add to my explanation of Immunity a theory of the
phenomena of healing in infective diseases, but I soon had to renounce
this project, for its execution would have increased too greatly the
bulk of the book which, without it, has already assumed considerable
proportions. It seemed to me preferable to set forth the present state
of the question without paying too much attention to the historical
sequence of the discoveries, and to reserve for a special chapter, at
the end of the work, a sketch of the history of our knowledge on
Immunity.
Before I ask the reader to glance through this work, I should mention
that I have been heartily seconded in its preparation by many of my
friends and collaborators. I offer my most sincere thanks to MM. Roux,
Nocard, Massart, and J. Bordet, who kindly undertook to read my
manuscript throughout, or such parts of it as related to their special
subjects. For example, M. Nocard rendered me a very great service by
correcting the paragraphs of Chapter xv, which treat of the vaccinations
against epizootic diseases, and M. Massart, by giving me his advice on
the subject of immunity in plants.
I owe very special thanks to M. Mesnil, who has been good enough to give
me very effective help in the dry task of correcting the manuscript and
proofs.
I beg MM. E. Rémy and L. Barnéoud to accept my thanks for the care they
have bestowed on the execution of the illustrations in this work.
ÉLIE METCHNIKOFF.
PARIS, INSTITUT PASTEUR,
_3 October, 1901_.
CONTENTS.
PAGE
PREFACE TO THE ENGLISH EDITION v
PREFACE ix
INTRODUCTION 1
Importance of the study of immunity from a general point of
view.—Part played by parasites in infective
diseases.—Intoxications by the products of
micro-organisms.—Resistance of the organism to the invasion of
micro-organisms.
Natural immunity and acquired immunity.
Immunity to micro-organisms and immunity to toxins.
CHAPTER I
IMMUNITY IN UNICELLULAR ORGANISMS 11
Infective diseases of the unicellular organisms.—Intracellular
digestion in the Protozoa.—Amoebo-diastase.—Part played by
digestion in the defence of the Protozoa against infective
parasites.—Defences of the _Paramaecia_ against
micro-organisms.—Part played by irritability in defence in the
lower organisms.
Immunity of unicellular organisms to toxins.—Acclimatisation of
bacteria to toxic substances.—Protective secretion of membranes
by bacteria.
Adaptation of the Protozoa to saline solutions—of yeasts to
poisons—of yeasts to milk-sugar.
Irritability of unicellular organisms and Weber-Fechner’s
psycho-physical law.
CHAPTER II
IMMUNITY IN MULTICELLULAR PLANTS 29
Infective diseases of plants.—Plasmodia of the Myxomycetes and
their chemiotaxis.—Adaptation of the plasmodia to
poisons.—Pathogenic action of _Sclerotinia_ upon
Phanerogams.—The cicatrisation of plants.—Defence in plants
against Bacteria.—Sensitiveness of vegetable cells to osmotic
pressure.—Adaptation of plants to modifications of osmotic
pressure.—Dependence of the chemical phenomena upon the
irritability of the vegetable cells.—The law of Weber-Fechner.
CHAPTER III
PRELIMINARY REMARKS ON IMMUNITY IN THE ANIMAL KINGDOM 40
Examples of natural immunity among the Invertebrates.—Immunity
against micro-organisms and insusceptibility to microbial
poisons are two distinct properties.—The refractory organism
does not eliminate micro-organisms by the excretory channels.—It
destroys them by a process of resorption.—The fate of foreign
bodies in the organism.—The resorption of cells.—Intracellular
digestion.—This digestion effected by the aid of soluble
ferments.—Digestion in Planarians and
Actinians.—Actinodiastase.—Transition from intracellular
digestion to digestion by secreted juices.—Digestion in the
higher animals.—Enterokynase and the part it plays in
digestion.—The psychical and nervous elements in
digestion.—Adaptation of the pancreatic secretion to the kind of
food.—Excretion of pepsin in the blood and in the urine.
CHAPTER IV
RESORPTION OF THE FORMED ELEMENTS 67
Digestion in the tissues.—Resorption of cells in the
Invertebrata.—Resorption of red corpuscles by the phagocytes of
the Vertebrata.—Phagocytes.—Various categories of these
cells.—Macrophages and microphages.—Part played by macrophages
in the resorption of the formed elements.—Digestive property of
the macrophagic organs.—Solution of the red blood corpuscles by
the blood serums.—The two substances which operate in
haemolysis.—Macrocytase and fixative.—Analogy of the latter with
enterokynase.—Escape of the macrocytase during
phagolysis.—Suppression of phagolysis.—Resorption of the
spermatozoa.—Presence of fixatives in plasmas.—Origin of
fixatives.
CHAPTER V
RESORPTION OF ALBUMINOID FLUIDS 106
Resorption of albuminoid substances.—The precipitins of blood
serum which appear as a result of the absorption of serums and
of milk.—Absorption of gelatine.—Leucocytic origin of the
ferment which digests gelatine.—Anti-enzymes.—Antirennet.—The
anticytotoxins.—Antihaemotoxic serums.—Their two constituent
parts: anticytase and antifixative.—Action of anticytase.—The
antispermotoxins.—Origin of anticytotoxins.—Ehrlich’s theory on
this question.—Origin of antihaemotoxin.—Origin of
antispermotoxin.—Production of this antibody by castrated
males.—The antispermofixative produced when the spermatozoa are
excluded.—Distribution of spermotoxin and antispermotoxin in the
organism.
CHAPTER VI
NATURAL IMMUNITY AGAINST PATHOGENIC MICRO-ORGANISMS 128
Natural immunity and the composition of the body
fluids.—Cultivation of the bacteria of influenza and
pleuro-pneumonia in the fluids of refractory animals.—Resistance
of _Daphniae_ to the Blastomycetes.—Examples of natural immunity
in Insects and Mollusca.—Immunity of Fishes against the anthrax
bacillus.—Immunity of frogs against anthrax, Ernst’s bacillus,
the bacillus of mouse septicaemia, and the cholera
vibrio.—Natural immunity in the cayman.—Immunity of the fowl and
pigeon against anthrax and human tuberculosis.—Immunity of the
dog and rat against the anthrax bacillus.—Immunity of Mammals
against anthrax vaccines.—Immunity of the guinea-pig against
spirilla, vibrios, and streptococci.—Natural immunity against
anaerobic bacilli.—Fate of Blastomycetes and _Trypanosomata_ in
the refractory organism.
CHAPTER VII
THE MECHANISM OF NATURAL IMMUNITY AGAINST MICRO-ORGANISMS 175
The destruction of micro-organisms in natural immunity is an act
of resorption.—Part played by inflammation in natural
immunity.—Importance of microphages in immunity against
micro-organisms.—Chemiotaxis of leucocytes and ingestion of
micro-organisms.—Phagocytes are capable of ingesting living and
virulent micro-organisms.—The digestion of micro-organisms in
phagocytes is most often effected in a feebly acid
medium.—Bactericidal property of serums.—Phagocytic origin of
the bactericidal substance.—Theory of the secretion of the
bactericidal substance by leucocytes.—Comparison of the
bactericidal power of serums and of blood plasmas.—The
bactericidal substance of blood serums must not be considered a
secretion-product of leucocytes; it remains within the
phagocytes, so long as they are intact.—The cytases.—Two kinds
of cytases: macrocytase and microcytase.—Cytases are
endo-enzymes, allied to trypsins.—Changes in the staining
properties and in the form of micro-organisms in the
phagocytes.—Absence or rarity of fixatives in the serums of
animals endowed with natural immunity.—The agglutination of
micro-organisms does not play any important part in the
mechanism of natural immunity.—Absence of antitoxic property of
the body fluids in natural immunity.—The phagocytes destroy the
micro-organisms without their ingestion being preceded by
neutralisation of the toxins.
CHAPTER VIII
SURVEY OF THE FACTS BEARING ON ACQUIRED IMMUNITY AGAINST
MICRO-ORGANISMS 207
The discovery of attenuated viruses and its application to
vaccination against infective diseases.—Vaccination by microbial
products.—Vaccination with serums.—The acquired immunity of the
frog against pyocyanic disease.—The acquired immunity against
vibrios.—Extracellular destruction of the cholera vibrio.—Part
played by two substances in Pfeiffer’s phenomenon.—Specificity
of fixatives.—Phagolysis and its relation to the extracellular
destruction of vibrios.—Part played by phagocytosis in the
acquired immunity against vibrios.—Fate of the spirilla of
recurrent fever in the organism of immunised
guinea-pigs.—Acquired immunity against the bacteria of typhoid
fever and pyocyanic disease.—Acquired immunity against swine
erysipelas and anthrax.—Acquired immunity against the
streptococcus.—The acquired immunity of rats against
_Trypanosoma_.
CHAPTER IX
THE MECHANISM OF ACQUIRED IMMUNITY AGAINST MICRO-ORGANISMS 250
Cytases and fixatives.—Only the latter are augmented in the
immunised organism.—Properties of the fixatives.—Difference
between them and the agglutinative substances.—The part played
by the latter in acquired immunity.—Protective property of the
fluids of the immunised organism.—Stimulant action of the body
fluids.—The protective power of serum cannot serve as a measure
of acquired immunity.—Examples of acquired immunity in which the
serums exhibit no protective power.—Phagocytosis in acquired
immunity.—Negative chemiotaxis of leucocytes.—Theory of
attenuation of micro-organisms by the fluids of immunised
animals.—Refutation of this theory.—Phagocytosis acts without
requiring any previous neutralisation of the toxins.—The origin
of the fixative and protective properties of the body
fluids.—The relation between these properties and
phagocytosis.—The side-chain theory of Ehrlich and the theory of
phagocytes.
CHAPTER X
RAPID AND TEMPORARY IMMUNITY AGAINST MICRO-ORGANISMS, CONFERRED BY
SPECIFIC AND NORMAL SERUMS, OR BY OTHER SUBSTANCES, OR BY
MICRO-ORGANISMS OTHER THAN THOSE AGAINST WHICH IT IS DESIRED TO
PROTECT AN ANIMAL 300
Immunity conferred by specific serums.—Analogy of the mechanism of
this immunity with that observed in immunity obtained with
pathogenic micro-organisms and their products.—Part played by
phagocytosis in the immunity conferred by specific
serums.—Influence of opium on the course of immunisation by
these serums.—Stimulant action of specific serums.—Protective
and stimulant action of normal serums.—Influence of fluids,
other than serums: broth, urine, physiological saline solution,
etc.
Antagonism between anthrax and certain bacteria.
CHAPTER XI
NATURAL IMMUNITY AGAINST TOXINS 325
Examples of natural immunity against toxins.—Immunity of spiders
and scorpions against tetanus toxin.—Immunity of the scorpion
against its own poison.—Antivenomous property of the blood of
the scorpion.—Immunity against tetanus toxin in the larvae of
_Oryctes_ and in the cricket.—Immunity and susceptibility of
frogs against this toxin.—Natural immunity of reptiles against
tetanus toxin.—Antitetanic property of the blood of
alligators.—Immunity of snakes against snake venom.—Immunity of
the fowl against tetanus toxin.—Immunity of the hedgehog against
poisons and venoms.—Immunity of the rat against diphtheria
toxin.
CHAPTER XII
ARTIFICIAL IMMUNITY AGAINST TOXINS 342
Adaptation to poisons.—Artificial immunity against bacterial and
vegetable toxins and against snake venom.—Principal methods of
immunisation.—Immunisation by toxins and toxoids.—Inoculation
against diphtheria toxin.—Phenomena produced in the course of
vaccination against toxins.—Rise of
temperature.—Leucocytosis.—Development of antitoxic
power.—Properties of antitoxins.—Mode of action of
antitoxins.—Action of antitoxins _in vitro_.—Their action in the
organism.—Influence of living elements on the combination of
antitoxin with toxin.—Antitoxic action of non-specific serums,
of normal serums, and of broth.—Immunity against toxins is not
in direct ratio to the amount of antitoxins in the body
fluids.—Hypersensitiveness of an animal treated with
toxin.—Diminution of the susceptibility of the organism
immunised against toxins.
Hypotheses as to the nature and origin of antitoxins.—Hypothesis
of the transformation of toxins into antitoxins.—Hypothesis of
receptors detached from cells as the source of
antitoxins.—Hypothesis of the nervous origin of tetanus
antitoxin.—Fixation of tetanus toxin by the substance of the
nerve centres.—The relations between saponin and
cholesterin.—Anti-arsenic serum.—Part played by phagocytes in
the struggle of the animal against poisons.—Probable part played
by phagocytes in the production of antitoxins.
CHAPTER XIII
IMMUNITY OF THE SKIN AND MUCOUS MEMBRANES 403
Protective function of the skin.—Exfoliation of the epidermis as a
means of ridding the animal of micro-organisms.—Localisation and
arrest of micro-organisms in the dermis.—Intervention of
phagocytes in the defence of the skin.
Elimination of micro-organisms by the conjunctiva.—Microbicidal
function of the tears.—Absorption of toxins by the
conjunctiva.—Protection of the cornea.—Elimination of
micro-organisms by the nasal mucosa.—Protection by the
respiratory channels.—Dust cells.—Absorption of poisons by the
respiratory channels.
Alleged microbicidal property of the saliva.—Part played by
microbial products in the protection of the buccal
cavity.—Antitoxic function of the saliva.
Antiseptic action of the gastric juice.—Antitoxic function of
pepsin.
Protective function of the alimentary canal.—Absence of
microbicidal power from the intestinal ferments.—Protective
function of the bile.—Antitoxic rôle of the digestive
ferments.—Favouring and retarding functions of the intestinal
micro-organisms.—Destruction of toxins by these micro-organisms.
Defensive rôle of the liver. Protective function of the lymphoid
organs of the alimentary canal.
Protective function of the mucous membrane of the genital
organs.—Autopurification of the vagina.
CHAPTER XIV
IMMUNITY ACQUIRED BY NATURAL MEANS 433
Immunity acquired after recovery from infective diseases.—Immunity
acquired in malaria.—Humoral properties of convalescents from
typhoid fever.—Preventive power of the blood of persons who have
recovered from Asiatic cholera.—Antitoxic power of the blood of
persons who have recovered from diphtheria.
Immunity acquired by heredity.—Absence of hereditary immunity
properly so called.—Immunity conferred by the maternal blood and
by the yolk.
Immunity conferred by the milk of the mother.
CHAPTER XV
PROTECTIVE VACCINATIONS 454
Vaccinations against, I. Small-pox.—II. Sheep-pox.—III.
Rabies.—IV. Rinderpest.—V. Anthrax.—VI. Symptomatic
Anthrax.—VII. Swine Erysipelas.—VIII. Pleuropneumonia in the
Bovidae.—IX. Typhoid Fever.—X. Plague.—XI. Tetanus.—XII.
Diphtheria.
CHAPTER XVI
HISTORICAL SKETCH OF OUR KNOWLEDGE OF IMMUNITY 505
Methods used by savage races for vaccination against snake venom
and against bovine pleuropneumonia.—Variolisation and
vaccination against small-pox.—Discovery of the attenuation of
viruses and of vaccinations with attenuated
micro-organisms.—Theory of the exhaustion of the medium as a
cause of acquired immunity.—Theory of substances which prevent
the multiplication of the micro-organisms in the refractory
body.—Local theory of immunity.—Theory of the adaptation of the
cells of the immunised organism.
Observations on the presence of micro-organisms in the white
corpuscles.—History of phagocytosis and of the theory of
phagocytes.—Numerous attacks upon this theory.—Theory of the
bactericidal property of the body fluids.—Theory of the
antitoxic power of the body fluids.—Extracellular destruction of
micro-organisms.—Analogy between bacteriolysis and
haemolysis.—Theory of side-chains.
Progress of the theory of phagocytes.—Attempts to reconcile it
with the humoral theory.—Present phase of the question of
immunity.
CHAPTER XVII
SUMMARY 544
Means of defence of the animal against infective
agents.—Absorption of micro-organisms.—Phagocytes, and their
function in inflammation.—The action of phagocytes in the
absorption of micro-organisms.—The cytases, phagocytic
ferments.—The cytases are closely bound up with the
phagocytes.—The fixatives and their function in acquired
immunity.—The fixatives are excreted by the phagocytes and pass
readily into the fluids of the body.—Essential mechanism of the
action of the fixatives.—Adaptation of phagocytes to destroy
micro-organisms in acquired immunity.—Difference between the
fixatives and the agglutinins.—Antitoxins and their analogy with
the fixatives.—Hypotheses as to the origin of
antitoxins.—Cellular immunity is a fact of general
import.—Susceptibility and its rôle in immunity.—Applications of
the theory of immunity to medical practice.
INDEX 571
INTRODUCTION
Importance of the study of immunity from a general point of view.—Part
played by parasites in infective diseases.—Intoxications by the
products of microorganisms.—Resistance of the organism to the
invasion of micro-organisms.
Natural immunity and acquired immunity.
Immunity to micro-organisms and immunity to toxins.
[Sidenote: [1]]
The problem of immunity in relation to infective diseases is one that
not merely concerns general pathology but has a very important bearing
on all branches of practical medicine, such as hygiene, surgery and the
veterinary art. The prevention of disease by the production of an
acquired immunity is daily assuming greater importance. With the object
of arresting the multiplication and dissemination of morbific germs, we
are seeking, by artificial means, to render individuals, who may come in
contact with them, refractory to their pathogenic action. Patients who
have just undergone a surgical operation and women in child-bed are
frequently in danger of acquiring a post-operation disease or a
puerperal affection; we are, therefore, striving to protect them by
conferring upon them an artificial immunity.
The immunisation of animals useful to man is likewise a question of such
great importance to agriculture and to industry as to have now become
the object of legislation.
[Sidenote: [2]]
This question of immunity is, however, apart from its practical aspect,
intimately connected with problems of pure theory. There can be no
question that the marked pessimism developed during the century just
closed was in a large measure prompted by the dread of disease and
premature death, scourges against which humanity is as yet powerless. It
is recognised that Byron and Leopardi, the great poets of pessimism,
both suffered from congenital anomaly and from incurable disease and
that these maladies cast a gloom over their poetry. Schopenhauer, the
founder of the pessimistic school in modern philosophy, was noted for
his exaggerated fear of disease.
During the greater part of the nineteenth century our knowledge as to
immunity has been limited to certain practical methods, often
efficacious it is true, but purely empirical, such as those employed in
immunising man against small-pox and certain domestic animals against
sheep-pox or pleuro-pneumonia.
So long as the nature of the viruses was unknown no really scientific
study of their action or of immunity from them could be made. The
revelation of the organised nature of the infective viruses opened up
the way for these researches. This discovery, the outcome of the
demonstration by Pasteur of the organised nature of the ferments, has
enabled us to establish the part played by living agents in a great
number of infective diseases, and, linked with the names of Davaine,
Obermeyer, and above all with that of Robert Koch, it has very greatly
advanced the study of susceptibility and of natural immunity in certain
infections.
A considerable forward step was made with the discovery, by Pasteur and
his collaborators Chamberland and Roux, that it was possible, in certain
infective diseases, to confer immunity by means of micro-organisms which
had had their virulence attenuated. Thanks to this discovery, science
was now in a position to take up the thorough study of acquired
immunity. The field of research was still further enlarged by the
demonstration of the immunising power of the culture products of
pathogenic micro-organisms and above all by the discovery that the blood
of immunised animals is capable of conferring immunity upon susceptible
animals.
Before taking up in detail the problem of immunity as it is revealed to
us as a consequence of these discoveries, it is essential to cast a
glance at infective and allied diseases as a whole and to indicate in
what light we look upon them in view of the present state of our
knowledge.
[Sidenote: [3]]
It has been definitely established that many infective diseases of man
and animals are due to the invasion of small parasitic organisms,
sometimes of animal nature (as in itch, trichinosis, malaria, Texas
fever, nagana, or surra and the allied condition “dourine” in horses),
sometimes belonging to the vegetable kingdom like the Moulds
(aspergillosis), the Hyphomycetes (actinomycosis, Madura foot disease,
bovine farcy) and the Yeasts (disease of the _Daphniae_, some
pseudomyxomas and septicaemias, pseudolupus). But by far the greater
number of infective diseases are due to the development in the organism
of plants of the simplest structure, Bacteria. These Bacteria produce
the gravest and most destructive infections, such as tuberculosis,
bubonic plague, diphtheria, cholera, anthrax, the pneumonias,
suppuration, erysipelas, tetanus, glanders, leprosy, &c. Among these
bacteria some are too small to be resolved individually under the
highest magnifying powers and can only be made out _en masse_. Such is
the micro-organism of the contagious pleuro-pneumonia of cattle. To this
minuteness of certain pathogenic Bacteria is very probably due the fact
that in a considerable number of infections, amongst which are
scarlatina, measles, rabies, syphilis, aphthous fever and small-pox, it
has been impossible, up to the present, to recognise any specific
micro-organisms.
It is probable that we shall succeed in discovering parasites, not only
in the diseases I have just cited, which present the characters of
infective and virulent diseases, but also in diseases of entirely
different types. In spite of the failure of various attempts to
demonstrate the parasite of malignant tumours, it may be hoped that,
with improvement in scientific methods, such a parasite will be
unequivocally demonstrated. In many other conditions which are at
present considered as not dependent on micro-organisms, an intimate
connection with such organisms will probably be established. Such are
the atrophic diseases and certain diseases of nutrition in which the
parasites, without playing a direct or immediate _rôle_, act by means of
their products, or by the changes which they set up in the affected
organism. To give an idea of this possibility it will be useful to cast
a glance at the various modes of action of the numerous etiological
agents in infective diseases. The parasites which produce them have, as
a common feature, their small dimensions; they can only be recognised
with precision by the employment of high powers of the microscope. They
are likewise distinguished by a great variability, which is not
astonishing, since among infective agents are found on the one hand
animals of high structure (such as the Acari of itch) and on the other
plants of the simplest character such as the Gonococci or the various
Cocco-bacilli.
[Sidenote: [4]]
The Acari are capable of perforating the epidermis by the mechanical
action of their feet and mouth-parts. They excavate channels in the skin
and thus provoke the irritation so characteristic of itch. The larvae of
the Trichinae in like manner produce marked lesions by the mere
mechanical act of penetration and migration in the striped fibre of
muscular tissue. In human trichinosis, however, the disease picture is
more complicated than in itch and leads us to assume that there is some
additional action of the excreta of the larvae in the production both of
the febrile state and of certain general phenomena. In the nagana
disease (transmitted by the Tsetse fly) there is equal reason to admit
the preponderating _rôle_ of the mechanical action of the flagellated
parasite (_Trypanosoma_) which obstructs the vessels of the nervous
centres.
In the diseases which are set up by Fungi, such as ringworm and
aspergillosis, the purely mechanical element still appears to play the
more important part. Even certain of the bacterial infections manifest
this same character. Thus, there is no doubt that in chronic
tuberculosis in the guinea-pig, Koch’s bacillus brings about a
substitution of tuberculous elements for the normal tissues, and this to
such a degree that, at the termination of the disease, there may remain
merely traces of the liver and of the lungs, and the animal dies for
want of these organs, whose normal action is no longer possible. In the
tuberculous guinea-pig the phenomenon of intoxication by the bacillary
poisons plays but a secondary _rôle_; yet there are examples of
tuberculosis (as in acute miliary tuberculosis in man or experimental
tuberculosis in cattle, obtained by Nocard’s method of inoculation into
the milk ducts), where the poisoning assumes much greater importance.
Among the bacterial diseases of man, leprosy may be cited as one in
which the intoxication is relegated to a subsidiary position, yielding
place to the mechanical substitution of the specific granuloma for the
normal tissues. It is only in the acute leprous exacerbations that we
perceive any signs of intoxication by the products of the leprosy
bacillus.
[Sidenote: [5]]
All the instances cited, however, constitute but a feeble minority which
is completely thrown into the shade in the presence of very numerous
infections in which the toxic element dominates the situation. Even in
carbuncular diseases an exact analysis of their morbid phenomena has
compelled us to recognise the marked influence of the poison produced by
the bacterium. The majority of the micro-organisms act as poisoners
which introduce themselves into the organism where they can secrete
toxins capable of provoking general disorders of very diverse natures.
Indeed in infective diseases a whole gamut of very remarkable variations
is produced. Thus many of the micro-organisms capable of setting up
septicaemias must multiply abundantly in the organism and be distributed
in the blood, before they can produce a general morbid condition. The
spirillum of human recurrent fever is an example of this. It multiplies
for some days and produces several generations without provoking the
least malaise; then, however, their appearance in the blood suddenly
produces intense fever and constitutional phenomena of the most
pronounced character.
On the other hand there are certain bacteria which are distinguished by
a very much feebler reproductive power, but a more marked toxic
activity. Incapable of spreading through the organism, these bacteria
remain localised at the point of entrance, where they secrete their
poisons and thus frequently set up a fatal intoxication. Some of these
bacteria, such as the bacilli of tetanus and of diphtheria, penetrate
more or less deeply into the living tissues of the affected animal.
Others can manifest their toxic action so to speak at a distance or by
simple contact with the living elements. Into this category comes the
organism of Asiatic cholera. Koch’s vibrio, once established in the
intestine, there secretes its poison; this, absorbed by the apparently
intact intestinal mucous membrane, sets up a fell disease, purely toxic
in character. It is probable that in the case of those intestinal
diseases whose etiology is still unknown, such as infantile choleras,
the poisoning by the products of micro-organisms constitutes the
essential phenomenon. The micro-organisms do not make their way into the
blood or tissues; they remain in the contents of the intestine and
thence set up their deadly intoxication.
[Sidenote: [6]]
Instances do exist in which the pathogenic micro-organism disappears
from the body, leaving there a toxin which, alone, is responsible for
death. Thus in the spirillar septicaemia of geese, the birds die at a
stage when not a single living spirillum can be found in the body. The
poisoners have been destroyed before the toxin produced by them had
completed its work. In other instances, e.g. typhoid fever of the horse,
the specific micro-organism likewise disappears before the death of the
animal; but at the period when the poison of this bacterium finishes its
fatal work, there is a secondary invasion of other micro-organisms which
have nothing to do with the typhoid fever proper of the horse.
This great variability in the action of the different pathogenic agents
is still further increased through the differing relations between the
parasites and the affected organism. Certain micro-organisms are capable
of producing a typical disease, whatever may be the mode and seat of
invasion of the organism. But these are comparatively few in number. The
bacillus of tuberculosis belongs to this minority. Whether it enters
subcutaneously, by the eye or by the respiratory, digestive or
genito-urinary passages, it invariably produces tubercular lesions more
or less grave and more or less capable of generalisation. On the other
hand, a very large number of micro-organisms only exert their pathogenic
action when they invade the organism at definite points. The anthrax
bacillus, when introduced through the slightest lesion of the skin or of
the mucous membranes, produces in man, and in a large number of mammals,
a very grave and usually fatal disease; when absorbed in the vegetative
state with food, it is almost always innocuous. With the cholera vibrio
we have an exactly opposite condition of affairs. When inoculated, even
in large numbers, below the skin in the human subject, it rapidly
disappears, producing merely insignificant disturbances; but when the
same vibrio is introduced into the digestive canal it develops and
produces Asiatic cholera, a disease so often terminating in death.
All these variations and peculiarities associated with the nature of
infective agents are of great importance from the point of view of
immunity.
[Sidenote: [7]]
Do diseases come from without or do their causes arise within the
organism? is a pressing question, long discussed by pathologists. Those
who have discovered most of the pathogenic micro-organisms have ranged
themselves on the side of the former hypothesis. For the majority of
them the essential etiological factor in the causation of infective
diseases consists in the invasion of the patient by the pathogenic
micro-organism from the outer world. This theory is in perfect harmony
with many of the admitted facts of epidemiology, according to which the
viruses of the most deadly epidemic diseases, such as Asiatic cholera,
yellow fever, and bubonic plague, must be imported into a country
previously free from the disease before an epidemic can be developed. In
anthrax and trichinosis it is recognised that the parasites must come
from without. Hence, in the study of pathogenic micro-organisms one
always follows the rule that it is essential to find the specific
micro-organism in all cases of the disease in question and to prove its
absence in healthy individuals or in those who are affected with other
diseases. Thus, Koch[1], in his classical researches on Asiatic cholera,
insisted on the fact that the cholera vibrio was always found in cases
of this disease but never in healthy persons. Almost simultaneously
Loeffler[2], in the course of his work on the etiology of diphtheria,
demonstrated the presence of a specific bacillus not only in a large
number of cases of this disease but also in the throat of a healthy
child; and this fact at first prevented him from accepting this bacillus
as the real cause of diphtheria.
This view accepted by two such eminent bacteriologists cannot however be
maintained. It is impossible to assume that each time that a pathogenic
micro-organism makes its way into a susceptible species its presence
must inevitably be followed by the production of the specific disease.
Although the discovery by Loeffler of the diphtheria bacillus in the
throat of healthy individuals has repeatedly been confirmed, it is
impossible to doubt the etiological _rôle_ of this organism in
diphtheria. Moreover, it has been established that Koch’s vibrio,
although undoubtedly the etiological factor in the production of Asiatic
cholera, has nevertheless been recognised in the digestive canal of
perfectly healthy persons.
[Sidenote: [8]]
As soon as he is born, man becomes the habitat of a very rich microbial
flora. The skin, the mucous membranes, and the gastrointestinal contents
become stocked with such a flora, but a very small number of these
micro-organisms have up to the present been recognised or described. The
buccal cavity, the stomach, the intestines and the genital organs offer
a feeding ground for Bacteria and inferior Fungi of various kinds. For
long it was thought that in healthy individuals all these
micro-organisms were inoffensive and sometimes even useful. It was
supposed that when an infective malady was set up a specific pathogenic
micro-organism was added to this benign flora. Exact bacteriological
researches have, however, clearly demonstrated that as a matter of fact
the varied vegetation in healthy persons often includes representatives
of noxious species of bacteria. Besides the diphtheria bacillus and the
cholera vibrio, which have repeatedly been found in a virulent form in
perfectly healthy individuals, it has been demonstrated that certain
pathogenic micro-organisms, e.g. the _Pneumococcus_, staphylococci,
streptococci and the _Bacillus coli_, are always, or almost constantly,
found among the microbial flora of healthy persons.
This observation has necessarily led to the conclusion that in addition
to the micro-organism there exists a secondary cause of infective
diseases—a predisposition, or absence of immunity. An individual in whom
one of the above-mentioned pathogenic species is present, manifests a
permanent or transitory refractory state as regards this specific
organism. As soon however as the cause of this immunity ceases to act,
the micro-organism gets the upper hand and sets up the specific disease.
It is thus in diabetic persons that boils make their appearance as the
result of the development of _Staphylococcus pyogenes_, a micro-organism
that is almost always found in abundance on the skin and mucous
membranes of the human subject. The diabetes is, in these cases, the
cause of the suspension of the immunity which exists in the healthy
individual.
People who carry the _Pneumococcus_ on their mucous membranes may remain
for long without being attacked by fibrinous pneumonia or any of the
other maladies due to this micro-organism. But often, in consequence of
some special circumstance, a cold for example, the refractory state
gives way to a more or less marked susceptibility.
It is unnecessary to multiply the number of such examples; they
demonstrate in the clearest fashion that, in addition to the causes of
disease which come from the outer world and which are represented by the
micro-organisms, there are yet other causes which lie within the
organism itself. When these internal factors are powerless to prevent
the development of the morbific germs, a disease is set up; when, on the
other hand, they resist the invasion of the micro-organisms properly,
the organism is in a refractory condition and exhibits immunity.
[Sidenote: [9]]
Diseases in general and infective diseases in particular were developed
on the earth at a very remote epoch. Far from being peculiar to man,
animals and the higher plants, they attack inferior forms and are widely
distributed among unicellular organisms, Infusoria and Algae. Diseases
undoubtedly play an important _rôle_ in the history of life on our
planet, and it is very probable that they have contributed in a marked
degree to the extinction of certain species. When we observe the ravages
produced by parasitic Fungi among the young fish which we are trying to
rear, or the destruction of crayfish in certain countries in consequence
of the rapid increase of epizootic germs, we are involuntarily led to
the conclusion that pathogenic micro-organisms must have brought about
the disappearance of certain animal and vegetable species.
Darwin[3], in the chapter on the extinction of species in his book _On
the Origin of Species_, states upon the authority of several observers
that insects so annoy elephants that these large mammals become
incapable of reproducing themselves in sufficient numbers. Now it is
proved that many Insects inoculate pathogenic micro-organisms and thus
transmit destructive diseases. A most formidable epizootic disease,
provoked by a flagellated Infusorium, the _Trypanosoma brucei_, is
inoculated into large mammals in South Africa by a fly, the Tsetse fly;
in certain districts this disease is so widespread and so destructive
that the rearing of domestic animals becomes impossible.
Parasites strike then with great intensity, bringing about the
destruction of numerous human beings, animals and plants. Nevertheless,
in spite of the disappearance of a large number of species, the world
continues well populated. This fact proves that, by the special means at
the disposal of the organism, without any aid of the medical art or
special human intervention, many living species have held their own
throughout the ages. Everybody has seen how dogs lick their wounds,
moistening them with a saliva full of micro-organisms. These wounds heal
well and quickly without dressings or antiseptics.
In all these examples the resistance of the organism depends on
immunity, a condition very general in nature. This immunity against
infective diseases is very complex and its thorough study could only be
undertaken after we had acquired an extended knowledge of these
diseases, and after adequate methods of research had been devised.
[Sidenote: [10]]
By immunity against infective diseases we understand the resistance of
the organism against the micro-organisms which cause these diseases. We
have here to do with an organic property of living beings and not with
the immunity which belongs to certain countries or localities. For this
reason information on the causes of the immunity in Europe and in
mountainous regions from yellow fever will not be found in this book,
nor why the majority of Europeans do not take recurrent fever. The
inhabitants of our continent do not possess organic immunity against
either the virus of yellow fever or Obermeyer’s spirillum of recurrent
fever. Indeed they are very susceptible to these diseases. It is solely
the conditions of life, in the majority of European countries, that
prevent the invasion by the specific germs and the creation of epidemic
foci. The same point of view ought also to be applied to animals. Our
small laboratory rodents, mice and guinea-pigs, are much more
susceptible to anthrax, whether inoculated beneath the skin or in any
other part of the body, than are the large domestic mammals such as the
ox and the horse. And yet these latter are very liable to epizootic
anthrax, whilst the rodents mentioned are seldom, if ever, attacked by
spontaneous anthrax. This apparent immunity in no way depends on the
existence of a true immunity of the organism, but solely on the
conditions under which mice and guinea-pigs live.
We shall therefore in this volume treat only of the phenomena of
_organic_ immunity in living beings, and the problem, even restricted
within these limits, still appears sufficiently complex. With the object
of rendering its study as easy as possible, it will be useful to
commence by giving an account of the phenomena of immunity in the lowest
organisms.
Immunity against infective diseases should be understood as the group of
phenomena in virtue of which an organism is able to resist the attack of
the micro-organisms that produce these diseases. It is impossible, at
present, to give a more precise definition, and useless to insist upon
it. Some have thought it necessary to distinguish between immunity
properly so called, that is to say a permanent refractory state, and
“resistance,” or a very transient property of opposing the invasion of
certain infective micro-organisms. It is not possible to maintain this
distinction, for in reality the limits between these two groups of
phenomena are far from being constant.
[Sidenote: [11]]
Immunity may be inborn or acquired. The former is always natural, that
is to say, independent of the direct intervention of human art. Acquired
immunity is also often natural, from the fact that it is established as
the result of the spontaneous cure of an infective disease. But in a
great number of cases acquired immunity may be the result of direct
human intervention as in the practice of vaccination.
For a long time all the phenomena of immunity against infective diseases
were collected into a single group. Later, it was recognised, as the
result of the demonstrations summarised at the beginning of this
chapter, that it is necessary to distinguish sharply between immunity
against the pathogenic micro-organisms themselves and that against
microbial poisons. Hence the idea of antimicrobial and antitoxic
immunities. In the course of this work this essential distinction must
always be borne carefully in mind.
CHAPTER I
IMMUNITY IN UNICELLULAR ORGANISMS
[Sidenote: [13]]
Infective diseases of unicellular organisms.—Intracellular digestion
in the Protozoa.—Amoebo-diastase.—Part played by digestion in the
defence of the Protozoa against infective parasites.—Defences of
the _Paramaecia_ against micro-organisms.—Part played by
irritability in defence in the lower organisms.
Immunity of unicellular organisms to toxins.—Acclimatisation of
Bacteria to toxic substances.—Protective secretion of membranes by
Bacteria.
Adaptation of the Protozoa to saline solutions—of yeasts to poisons—of
yeasts to milk-sugar.
Irritability of unicellular organisms and Weber-Fechner’s
psycho-physical law.
[Sidenote: [14]]
The immunity of unicellular organisms against infective diseases and
against toxic agents is as yet very imperfectly understood.
Nevertheless, it will be very useful for us to begin our study of the
problem of immunity on these lower organisms, because of their greater
general simplicity. It may be affirmed that if the line of comparative
pathology had been followed in our study of the etiology of diseases of
man and the higher animals, the parasitic nature of these infections
would have been established considerably earlier than was the case.
Thus, at a period when medical men and veterinary surgeons were content
to record the presence of Bacteria in the blood of their patients,
without attributing to them the slightest etiological _rôle_, botanists
and zoologists had already proved most definitely that many plants and
lower animals were subject to epidemic diseases undoubtedly set up by
the parasitism of various exceedingly simple organisms. In the same
year, 1855, that Pollender[4] published his first observations on the
bacterium found in the blood of animals affected by anthrax though he
could not trace the slightest relation between the presence of this
organism and the etiology of the disease, the celebrated botanist
Alexander Braun[5] issued his work on the genus _Chytridium_, in which
he demonstrated the fact that certain plants and flagellated Infusoria
suffer from the invasion of a small mobile parasite which, attaching
itself to their body wall, absorbs the contents and so destroys its
hosts, causing a very great mortality among them. The cycle of
development in the _Chytridia_, established by Braun, left no doubt as
to the accuracy of his view and even renders it possible for us to
interpret more accurately the earlier observations of Stein, on the
supposed evolution of certain Infusoria, by showing that the changes
observed in these organisms were in reality due to an invasion by
_Chytridia_.
Since these observations were made it has been clearly demonstrated that
among the unicellular organisms, certain Flagellata and ciliated
Infusoria are subject to infective maladies the result of parasitism of
the Chytridiaceae, a group of the lower Fungi. Small, mobile, colourless
cells attach themselves to the surface of the Protozoa, penetrate into
their interior and absorb the greater part of their living content.
Sometimes these parasites multiply in a most extraordinary fashion and
destroy enormous numbers of the Infusoria. Thus, Nowakowski,[6] who has
given a very detailed description of _Polyphagus euglenae_, the
Chytridium of the common green fresh-water _Euglena_, records the
disappearance of the _Euglenae_ from his aquaria glasses: the parasites
“were reproduced in such great abundance that ultimately they had
completely replaced the _Euglenae_.”
[Sidenote: [15]]
[Sidenote: [16]]
The Flagellata, subject to infection by _Chytridia_, are found almost
exclusively amongst those genera (_Cryptomonas_, _Chlamydomonas_,
_Haematococcus_, _Phacus_, _Volvox_, etc.) which are nourished after the
fashion of vegetables, that is by the absorption of substances dissolved
in the surrounding fluids. It is very remarkable that in the group of
ciliated Infusoria this parasitism of the _Chytridia_ is observed almost
solely in the encysted forms, that is to say, at a stage when the
animalcules, surrounded by their envelope, do not take any nourishment.
The invasion by the _Chytridia_ has been demonstrated in the case of the
cysts of the Vorticellina, Oxytrichinina, _Nassula_, etc.[7] These facts
indicate that the absence of the digestion of solid aliments, such as
occurs in almost all the ciliated Infusoria, constitutes a condition
favourable to infection by the _Chytridia_. Whilst the growth of
Volvocina, _Euglenae_ and their allies is almost always interfered with
by very destructive parasitic epidemics, the ciliated Infusoria, capable
of seizing and digesting lower organisms, may be cultivated and flourish
for a very long period. Thus Balbiani[8] has watched one of his cultures
of _Paramaecium aurelia_ multiply and thrive in splendid condition for
14 years in succession. Now these Infusoria readily adapt themselves to
ordinary water untreated to render it more hygienic. Such water swarms
with all sorts of lower organisms, among which are the _Chytridia_ and
numerous Bacteria, but the _Paramaecia_ and Infusoria in general feed
upon these organisms and contribute largely to the purification of the
water. Almost the whole body-contents in a ciliated Infusorian is made
up of a digestive protoplasm into which the captured Bacteria and other
lower organisms are conveyed; the nutrient particles becoming surrounded
by transparent vacuoles, in which the ingested organisms are killed and
digested. The food contained in the vacuoles circulates in the endoplasm
of the Infusoria by means of the streaming movements of this layer. The
digestive vacuoles become filled with a fluid having a distinctly acid
reaction. Formerly, in order to demonstrate this reaction, Infusoria
were allowed to ingest small granules of blue litmus which after a
certain time became more or less intensely red; but the use of aniline
colours has much simplified the study of digestion in microscopic
organisms. By introducing a solution of alizarin sulpho-acid into a
liquid containing Infusoria, the yellow staining (characteristic of the
acid reaction) of the digestive vacuoles can be readily made out. When
the Infusoria ingest small clumps of alkaline substances, stained violet
by this reagent, the vacuoles take on a red tint, indicating the acidity
of their contents[9]. Another aniline colour, neutral red (Neutralroth),
introduced into microscopical technique by Ehrlich[10], enables us to
demonstrate the acid reaction in the digestive vacuoles even within a
few minutes. Thus, in _Paramaecia_ treated with a dilute solution of
this reagent, the digestive vacuoles at once assume the deep rose tint,
characteristic of an acid reaction. This coloration is observed during
the life of the Infusorian, but immediately after death the vacuoles
become brownish and then completely lose their colour. This reaction,
easily demonstrated, indicates that neutralisation of the acid of the
vacuoles by the protoplasm and the surrounding water, both of which are
alkaline in reaction, has taken place.
In a medium distinctly acid the Infusoria digest their prey which, in a
very great number of cases, consists of Bacteria. These micro-organisms
are swallowed and carried into the digestive endoplasm in the living
condition; we have evidence of this in the active movements of a certain
number of the bacteria; at first they are found isolated in the interior
of the vacuoles, but later they collect into more or less compact
clumps. These masses of micro-organisms undergoing digestion, when
treated with neutral red assume a very deep rose tint, preserving their
bacillary form to the end, that is to say up to the extrusion of the
effete or waste material. There is, indeed, only very imperfect
dissolution not only of the bacilli as a whole but also of their
contents. _Paramaecia_ placed amongst cholera vibrios swallow them
greedily and in great numbers, digesting them as they would any other
micro-organism. I have never been able to see any conversion of vibrios
into granules going on within the digestive vacuoles.
All the attempts that have been made in my laboratory to extract a
digestive fluid from _Paramaecia_ have failed entirely. Very large
quantities of these Infusoria, obtained by filtration of rich cultures,
and macerated by different methods, have proved inactive even in the
case of those Bacteria which constitute their normal food.
Intracellular digestion in the Infusoria unquestionably takes place as
the result of the action of some diastase; but from the impossibility of
observing the action _in vitro_ the properties of this diastase, except
that it can act in a distinctly acid medium, cannot be determined.
[Sidenote: [17]]
Even less is known concerning the digestion of Rhizopods than concerning
that of Infusoria. It has long been recognised that, in the majority of
cases, _Amoeba_, _Actinophrys_ and Rhizopods in general, absorb a
nourishment composed of lower plants and animals, which are taken into
the protoplasmic body by means of the movements of amoeboid processes,
pseudopodia or lobopodia. Once within the Rhizopod the nutritive
particles are surrounded by a digestive fluid, in which the presence of
acid may be recognised by means of colour reactions. The addition of a
drop of Ehrlich’s neutral red to _Amoebae_ in the act of digesting
Bacteria at once gives the acid colour reaction (Fig. 1 ). Rhumbler[11]
has described very precisely and with much detail the way in which the
_Amoebae_ behave when they are incorporating filaments of _Oscillaria_
very much longer than their own bodies. He has also described the
digestion that these Algae undergo; a process most characteristic in
those cases where a portion only of the filament has been taken into the
interior of the _Amoeba_ and there subjected to the digestive action.
Whilst the free part of the _Oscillaria_ retains its normal properties
and appears of a bluish green colour, the ingested portion progressively
changes colour, assuming first a deep green tint, then becoming light
yellow, orange yellow, brown and finally reddish brown. Simultaneously
the cellulose wall of the Alga begins to soften, and the cells break up
into minute fragments which are soon extruded. The food is seldom
completely digested and there is always an abundant residual material
which is thrown out in the form of solid excreta.
[Illustration:
FIG. 1. An _Amoeba_ treated with neutral red, 1%.
]
[Sidenote: [18]]
Although it is fully recognised that, in the Rhizopods, digestion goes
on in a medium distinctly but feebly acid, and that the intervention of
some soluble ferment is essential, our ideas on this subject were very
vague until the publication of the researches of Mouton[12], carried out
with great care in the Pasteur Institute. In order that he might obtain
exact results Mouton made use of cultures of _Amoebae_ grown on agar, in
association with the _Bacillus coli_ which served them as food. The
bacilli were ingested in large numbers, became enclosed in vacuoles and
were digested by a ferment which Mouton was able to obtain _in vitro_.
To that end he collected large numbers of _Amoebae_, and, after
centrifugalising them in water, treated the deposit with glycerine. On
adding alcohol he obtained a precipitate readily soluble in water.
The fluid thus obtained exerted an undoubted digestive action upon
albuminoid substances. It readily liquefied gelatine and even attacked,
though feebly, albumen coagulated by heat; flakes of fibrin heated to
58° C. remained unaltered. There was present then, in this fluid derived
from _Amoebae_, a proteolytic diastase of feeble activity. On the other
hand, this extract contained neither sucrase, capable of inverting cane
sugar, nor lipase, capable of digesting fatty matters.
The amoebo-diastase of Mouton must be classified with the trypsins. It
is very active in a distinctly alkaline medium and continues the
diastatic action even when the medium becomes weakly acid (a feature
that corresponds to the reaction observed in _Amoebae_ treated with
appropriate staining agents). The amoebo-diastase is affected at as low
a temperature as 54° C. and at 60° C. is rendered completely inactive.
A question of especial importance is that concerning the action of the
amoebo-diastase upon Bacteria. The numerous experiments of Mouton
directed to the solution of this point, and made with living _Bacillus
coli communis_, gave negative results. If, however, these bacilli were
previously killed by heat or by chloroform, they were at once attacked
by the soluble amoebo-ferment. Opalescent emulsions of these dead
bacilli, incapable of undergoing self-digestion of any kind, became
transparent after remaining for some time in contact with the extract of
_Amoebae_. The amoebo-diastase, then, undoubtedly digests dead bacilli
_in vitro_, whereas in the body of the _Amoebae_ the ingested bacteria
are attacked whilst still living. As a result of these observations it
must be concluded that only a fractional part of the diastase is
extracted in the solution prepared by Mouton.
[Sidenote: [19]]
This intracellular digestion in the Protozoa serves not merely for the
nutrition of these organisms, but also as a protection against infective
parasites. The protoplasm of the Infusoria, with its vacuolar
secretions, has a general digestive action on everything that comes
within its reach. If the internal structures, such as the nuclei and the
pulsatile vacuoles, resist this process, it is undoubtedly because they
possess a power of defending themselves against the attack of the
digestive secretions. Thus, as brought out in the beautiful researches
of Maupas[13], the macronucleus of the _Paramaecia_ is, at a certain
stage in the life of the Infusorian, completely digested by the
protoplasm just as is any other nutrient substance introduced from
outside. It must be admitted that in this case the nucleus has ceased to
produce the protective substance which, under ordinary conditions,
interferes with its being digested.
A struggle similar to that observed between the nucleus and the
digestive content of the Protozoa goes on between these latter organisms
and infective microbes. All organisms which, in any way whatever,
penetrate into the body of an Infusorian or Rhizopod, are brought into
contact with the digestive endoplasm of these Protozoa. If the intruders
are killed and partially digested by the digestive secretions, or are
expelled as excrementitious matter, the Protozoon remains uninjured and
continues its normal and routine existence. Here, then, we have an
example of natural immunity, due to intracellular digestion. On the
other hand, when the foreign parasitic organism resists this digestive
action, it instals itself permanently in the body of the Protozoon, and
should it reproduce itself in small numbers merely, excrete no poison
and, in general, exercise no injurious influence upon its host, the
parasite may readily become a commensal. Thus, it is not rare to find in
the contents of Infusoria and Radiolaria small vegetable organisms of
the genera _Zoochlorella_ or _Zooxanthella_ which not only set up no
disease but, owing to their assimilation of carbonic acid, may even be
useful to their hosts. There are cases, however, where the parasites act
in a manner more or less injurious to the Protozoa containing them; in
such cases a true and sometimes fatal infection results.
[Sidenote: [20]]
Among the infective diseases of the Protozoa the one that has been most
thoroughly studied is that set up by several representatives of a
particular genus of micro-organisms discovered by Johannes Müller in
1856 and made the subject of an investigation carried out in my
laboratory by Hafkine[14]. I have already discussed these researches in
my work on the comparative pathology of inflammation[15] and need here
recapitulate only very briefly. _Paramaecia_ are sometimes affected by
needle-shaped or spirillar parasites which penetrate, sometimes into the
macronucleus, sometimes into the micronucleus, reproducing prolifically,
giving rise to a marked hypertrophy of the affected organs. The
Infusorian, in spite of this invasion, may continue to exist and carry
on its reproductive processes; it is, thus, enabled in many cases to
recover from the disease. On the other hand the _Paramaecium_ into whose
body the spores of the parasite are introduced treats them as it would
any other ingested foreign body. Not being able to digest them, owing to
the resistance offered by the membrane of the spore, the _Paramaecium_
expels them just as it would any other excrementitious matter. The
Infusorian behaves in the same way in regard to bacterial endospores.
Hay bacilli, which occur so commonly in the infusions in which the
_Paramaecia_ live, are digested in the endoplasmic vacuoles of the
latter, but the spores of these bacilli, after a more or less prolonged
sojourn in the vacuoles, are expelled with the excrement.
As by far the greater part of the body of a Protozoon is made up of
digestive protoplasm, it is natural that infective epidemics should be
very rare among these animalcules. The Infusoria and Rhizopods,
organisms specially well adapted to live upon the lower Algae and
Bacteria, are, practically, never subject to bacterial diseases. The
infections observed in the Protozoa are due in most cases to the
invasion of the lower Fungi, such as the _Chytridia_, the Microspheres,
the _Saprolegniae_ or the special organisms mentioned as occurring in
the nuclei of _Paramaecia_. Further, these infections are met with most
frequently in Protozoa which are incapable of carrying on true
intracellular digestion or which are in the encysted stage, at which
period the Infusoria, leading a passive existence, neither absorb nor
digest nutriment. As an exception to the above general statement I ought
to mention the epidemic in _Amoebae_ caused by the _Microsphaera_[16]
and the disease in _Actinophrys_ observed by K. Brandt[17] and
attributed to Fungi allied to the genus _Pythium_. In these two
instances we have to do with parasites which live and develop in the
interior of the active protoplasm of these Protozoa. Certainly a
proportion of the parasites are expelled with the excrementa; but there
remain others which instal themselves in the protoplasm, multiply there
and cause the death of their hosts. In these cases the digestive action
of the protoplasm must be neutralised or paralysed by the secretions of
the parasite. This aspect of the question, however, has so far not been
considered.
[Sidenote: [21]]
In addition to intracellular digestion and the expulsion of parasites by
the excretory function, the resistance offered by Protozoa to infective
diseases should, in part, be ascribed to their great irritability.
Anyone who will watch the manœuvres of _Amoebae_ or of certain Infusoria
in the midst of a rich microscopic flora and fauna, will at once be
struck by the preferences which these Protozoa exhibit in the choice of
their food. _Amoebae_ are often seen making search for Diatoms only,
disdaining all other Algae, or again they may single out one species of
Palmellaceae from a very varied flora. The Infusoria also have their
likes and dislikes in the matter of food. Many of the ciliated Infusoria
choose Bacteria to the exclusion of almost everything else; others, as
_Nassula_, have a special partiality for the _Oscillariae_. A most
striking example of this is afforded in _Amphileptus claparedei_, a
voracious Ciliate, which chooses _Vorticellae_ to the exclusion of all
other animalcules; these it devours, and then becomes transformed into a
cyst upon the peduncle of the _Vorticellae_ it has devoured. This
irritability clearly must control and guide the Protozoa in their
relations with other organisms and enable them to escape the invasion of
parasites.
In this connection I must mention a very interesting observation made by
Salomonsen[18] and communicated to the Paris International Medical
Congress in 1900. He was able to demonstrate the fact that almost all
the ciliated Infusoria, on becoming aware of the proximity of dead
bodies of kindred organisms, rapidly draw away, thus manifesting a very
marked negative chemiotaxis. This property must, it is evident, protect
them from any contamination by the parasites contained in the bodies of
Infusoria that have succumbed to infective diseases.
We have, then, quite a number of facts which throw light on the natural
immunity of the Protozoa against the action of pathogenic
micro-organisms. Up to the present, however, we know nothing concerning
the existence or the possibility of an acquired immunity among the lower
animalculae against infective diseases. We are better informed as to the
resistance of unicellular organisms to the action of soluble poisons,
which is, in general, much more easily studied than is immunity against
the micro-organisms themselves.
[Sidenote: [22]]
As a very large number of the higher animals are sensitive to the toxic
action of poisons of bacterial origin, the question has been put, “May
not the Infusoria also be poisoned by these micro-organismal products?”
With the object of answering this question Gengou[19] has studied the
influence of the toxins of tetanus and diphtheria on the ciliated
Infusoria. He was unable, however, to bring forward proof that these
substances exert any special toxic action on the _Paramaecia_. These
Infusoria withstand, perfectly well, doses of cultures of the diphtheria
and the tetanus bacillus grown in broth and deprived of the bacilli by
filtration as large as those of ordinary broth alone in which no bacilli
have been cultivated. Gengou argues from this that the _Paramaecia_
possess a natural and absolute immunity against these two toxins. When
we take into consideration the fact that these poisons act but feebly at
ordinary temperatures and are often innocuous to “cold-blooded” animals
we may perhaps be tempted to attribute the immunity of the Infusoria to
the temperature that was maintained in the incubator whilst Gengou’s
experiments were being carried on. Led by this train of thought Mme
Metchnikoff tried the action of the blood-serum of eels, which is very
toxic, not only for warm-blooded Vertebrates but also for cold-blooded
Vertebrates and the Invertebrates, on the _Paramaecia_, and this at a
low or medium temperature. This eel’s serum, however, exerted no greater
toxic action than did the blood-serum of other animals.
The microbial toxins are innocuous not only to the ciliated Infusoria
but also to many other unicellular organisms. It is now well recognised
that these toxins, exposed to the air, are soon inhabited by quite a
rich flora of micro-organisms, amongst which Bacteria and Yeasts
predominate. I have been able to prove[20] that these organisms are not
only unaffected in their normal life by the presence of the toxins of
diphtheria or tetanus but that they rapidly bring about the more or less
complete destruction of these poisons. Gengou, also, observed that
yeasts thrive luxuriantly in these bacterial toxins. The rapid increase
of micro-organisms and the destruction of these poisons take place at
temperatures varying from 15° to 37° C.
[Sidenote: [23]]
Whilst the lower organisms are refractory to bacterial toxins which in
quite small doses are capable of killing man and the higher animals,
many micro-organisms manifest a special sensitiveness to certain fluids
of animal origin. In a succeeding chapter we shall treat at greater
length of this microbicidal property of the humours. Here it is merely
necessary to indicate certain facts concerning this property, regarding
them solely from the point of view of the immunity of the lower
organisms. The most striking example of the bactericidal power of an
animal fluid is certainly that afforded in the action of the blood-serum
of the rat on the anthrax bacillus. This fact, discovered in 1888 by von
Behring[21], led to the conclusion that the blood of the rat contains an
organic base capable of killing and dissolving a considerable number of
anthrax bacilli. Several observers have confirmed von Behring’s
observation and have supplemented it by the fact that the bacillus can
be readily accustomed to the toxic action of this serum. Thus
Sawtchenko[22], in an investigation carried out in my laboratory, was
able, by successive cultures, to accustom the anthrax bacillus to an
existence in the pure serum of the rat. In this case, therefore, there
has been produced a real acquired immunity of a lower plant against a
toxic substance of animal origin. More recently Danysz has demonstrated
the same thing and has added several other facts which seem to throw
light upon the means by which the bacterium becomes adapted to the
poison. He has shown, in a work carried out in the Pasteur
Institute[23], that the anthrax bacillus protects itself against the
toxic action of the serum by surrounding itself with a thick sheath
composed of a kind of mucus which fixes the toxin of the rat’s blood and
renders it harmless. This same mucus, but in smaller quantity, is
likewise produced in a culture of the bacillus grown in ordinary broth.
When such a culture is freed from the contained bacilli by filtration
through porcelain and a little of this fluid is added to the rat’s
serum, this latter becomes less bactericidal than is a mixture of the
same serum with ordinary broth. Danysz suggests that this is to be
explained by the presence in the filtrate of a certain quantity of the
mucous substance produced by the bacillus, which fixes and neutralises a
portion of the “rat toxin.” If, in place of sowing the ordinary
bacillus, sensitive to this toxin, we inoculate the broth with an
anthrax bacillus which has previously been accustomed to the rat’s
serum, we find that the liquid of this culture when filtered neutralises
a larger proportion of the toxin. Danysz concludes from this that the
acclimatised bacillus has acquired the property of producing more mucus
than does the ordinary bacillus and that, for this reason, a greater
quantity of this protective substance passes into the fluid of the
culture.
[Sidenote: [24]]
The formation of a transparent sheath has several times been observed in
the anthrax bacillus, notably in cases where this organism happens to be
in “a state of defence” against various noxious influences. For example,
this sheath is well developed in the anthrax bacillus which invades the
blood of lizards, animals which are in general very resistant to
anthrax[24]. Under analogous conditions the streptococci which, as a
rule, do not produce a mucous sheath, will develop one of exceptional
size. The guinea-pig is in general very resistant to the streptococcus
against which it exhibits a very effective reaction. Sometimes, however,
this immunity gives way; in such instances, as demonstrated by J.
Bordet[25], the streptococcus, in order to overcome the natural
resistance of the guinea-pig, is found to have surrounded itself with a
sheath of a thickness such as is seldom to be met with in the world of
bacteria (Fig. 2).
[Illustration:
FIG. 2. Streptococcus surrounded by a protective envelope.
]
[Illustration:
FIG. 3. Tubercle bacillus surrounded by a transparent envelope and
enclosed in the giant cell of a gerbil.
]
[Sidenote: [25]]
Analogous facts are also observed in cases where the micro-organism is
defending itself against the action of substances enclosed in animal
cells. I may cite as an example the tubercle bacillus in the interior of
the giant cells of a gerbil (_Meriones shawii_), where, under the
influence of noxious substances contained in these cells, the tubercle
bacillus (Fig. 3) envelops itself in a transparent sheath similar to
that of the bacillus or of the streptococcus. As the action of the giant
cell still does not cease, the tubercle bacillus secretes a second
sheath (Fig. 4) and continues to surround itself with quite a series of
such envelopes (Fig. 5), thus coming to resemble a palmellaceous Alga
surrounded by successive layers of membranes or certain other vegetable
cells whose principal means of defence against all kinds of injurious
influences consists in the production of these protective membranes.
[Illustration:
FIG. 4. Another tubercle bacillus surrounded by two membranes.
]
[Illustration:
FIG. 5. Tubercle bacillus surrounded by a series of concentric layers.
]
[Sidenote: [26]]
Quite recently Trommsdorf[26], in Buchner’s laboratory in Munich, has
carried out a series of experiments on the adaptation of the cholera
vibrio and of the typhoid bacillus to the bactericidal substance found
in the blood of the rabbit. He has been able to confirm the results of
his predecessors and by various experiments has convinced himself that
these two micro-organisms are capable of adapting themselves to
existence in the defibrinated blood and in the blood-serum of the
rabbit.
The immunity, or acclimatisation of injurious organisms to different
toxins, presents an undoubted analogy to the phenomena of adaptation
shown by these organisms to mineral or organic poisons. It has long been
known that the same species of Protozoa are met with in both fresh and
salt water and that it is possible to gradually accustom Infusoria and
_Amoebae_ to tolerate an amount of sea salt which at first is absolutely
fatal to them. This toleration is not acquired unless care be taken to
increase the amount of salt very gradually: too abrupt a rise inevitably
causing death. By this means Cohn[27] accustomed the fresh-water
_Euplotes_ to a life in artificial sea water containing 4% of sodium
chloride. In Balbiani’s experiments[28] the fresh-water Monads
(_Menoidium incurvum_ and _Chilomonas paramaecium_) died very quickly on
the addition of ½% of this salt; but when it was added in small
successive doses (0·05 per day), they readily became accustomed to a
concentration of 1%. In the encysted state the Protozoa are even more
resistant than in the active state to the different salts that may be
added to their normal culture medium. It is probable that the wall of
the cyst interferes with the penetration of these substances into the
endoplasm. If a small quantity of an aniline dye be added to a fluid
containing encysted Infusoria, it is seen that the cyst-membrane becomes
very intensely coloured but the body of the Infusorian remains
unstained. The membrane absorbs a large amount of colouring matter,
after which, being saturated, it ceases to take it up; but it does not
allow the dye to penetrate into the endoplasm.
[Sidenote: [27]]
Balbiani (_loc. cit._ p. 580), having compared the action of the salts
of sodium with that of the salts of potassium and lithium on Infusoria,
comes to the conclusion that the injurious influence of these substances
can only be partially explained by osmotic phenomena. In addition to
these a purely chemical action must be invoked. He bases his opinion on
the fact that the isotonic solutions of the three salts acting on
Infusoria of the same species and same origin exert a different
influence. The salts of potassium and of lithium act in a much more
energetic fashion than do the sodium salts. Consequently, the Protozoa
are able to adapt themselves progressively not only to noxious
influences of a physiological character but also to those of a chemical
nature. Thus Infusoria and Rhizopods can be accustomed to the action of
high temperatures, to an intense light, etc. On the other hand they can
also be habituated to the toxic actions of true poisons. Davenport and
Neal[29] have established the fact that Stentors kept for two days in a
weak solution of corrosive sublimate (0·00005%) acquire a tolerance to a
dose of this poison four times as great as the lethal dose for
individuals previously kept in pure water. The same thing has been
observed in connection with the toxic action of quinine. This immunity
cannot be attributed to the selection and persistence of those Infusoria
which possess a natural resistance to the sublimate. It is really
acquired as the result of a direct and gradual chemical influence on the
protoplasm of the Stentors which, once adapted, all survive doses which
are lethal for the unacclimatised control organisms.
[Sidenote: [28]]
The vegetable micro-organisms, which are much more easily cultivated
than are the Protozoa, frequently manifest most characteristic phenomena
of acclimatisation. The first systematic researches in this direction
were carried out by Kossiakoff[30] in the laboratory of Duclaux. He
studied the antiseptic action of borax, of boracic acid, and of
corrosive sublimate on the anthrax microbe and several other bacilli
(_Bacillus subtilis_, _Thyrotrix scaber_ and _T. tenuis_). He found that
all these micro-organisms can be gradually accustomed to doses which are
absolutely bactericidal to the same species when not so acclimatised.
The acclimatised _Thyrotrix tenuis_ withstands almost double the amount
of bichloride of mercury that the non-acclimatised bacillus will resist.
The ordinary anthrax bacillus will not develop at all if the culture
medium contains more than 0·005 of boracic acid whilst the same
organism, when accustomed by passage through successive cultures in
which this substance is present in gradually increasing proportions,
grows well in spite of the presence of 0·007 of the same antiseptic.
Since these observations were made similar facts have been demonstrated
by several other observers, and the ready acclimatisation of Bacteria to
poisons is now generally admitted. Danysz (_loc. cit._), with the object
of elucidating the mechanism of this adaptation, has studied the action
of arsenic acid on the _Bacillus anthracis_. He has demonstrated that
this bacillus will gradually accustom itself to grow in broth containing
a quantity of arsenic acid which at first inhibited all development.
During this phenomenon of adaptation, which is acquired after a series
of passages through media more and more highly arsenicated, the bacillus
secretes a coating of mucous substance which protects the sensitive
parts of the microbial cell. Here, therefore, is formed something
exactly corresponding to what the same observer has demonstrated in
anthrax bacilli that have acquired a tolerance for rat’s serum. This
analogy extends even to the throwing out of the protective substance
into the culture fluid. When one sows an ordinary unadapted bacillus in
arsenicated broth to which has been added some of the fluid from a
culture of the adapted bacillus, development takes place in a marked
fashion. On the contrary when the same material is “seeded” into
arsenicated broth of the same composition but to which has been added
the filtrate from an unadapted culture, the bacillus does not develop
nearly so well. The difference is explained by the presence, in the
fluid in which the adapted bacillus had grown, of a certain quantity of
the mucous substance which fixes the arsenic and prevents it from acting
on the protoplasm of the micro-organisms.
[Sidenote: [29]]
The Yeasts, also, adapt themselves very readily to antiseptics. This
property has even had a practical application. We know that small doses
of hydrofluoric acid are capable of preventing the proliferation of the
yeast of beer, and Effront[31] has accustomed this plant to live in
media containing an amount of hydrofluoric acid which is absolutely
inhibitory to the unadapted yeast. Under these conditions the adapted
cells undergo a stimulation which causes the production of a greater
quantity of alcohol. The yeast, in adapting itself to antiseptic doses
(300 mm. of hydrofluoric acid per 100 c.c. of beer wort), acquires a
kind of immunity which it did not possess in the first instance.
Moreover this acquired property can be hereditarily transmitted to new
generations developed in ordinary beer wort to which hydrofluoric acid
has not been added. The stimulating action of this substance on the
fermentative property does not depend upon the acid reaction of the
hydrofluoric acid, for other acids which are non-antiseptic, such as
tartaric acid, are incapable of inducing it.
The acquired immunity against hydrofluoric acid is strictly specific,
the yeasts that have been adapted to this substance becoming even more
susceptible to the action of other poisons.
Duclaux[32] has already insisted on the relations which exist between
antiseptics and foods. Formic aldehyde which has a very powerful
coagulative and therefore strongly antiseptic action on protoplasm may
actually serve as a food for micro-organisms. The _Thyrotrix tenuis_,
studied in this connection by Péré[33], adapts itself to the presence of
this aldehyde and utilises it for its nutrition. Here is produced
something that recalls the case of the Protozoa that digest parasitic
organisms.
It is now a current idea in microbiology that Bacteria and Yeasts which
primarily do not make use of certain substances, adapt themselves to use
them as nutrient substances. Dienert[34] has published a detailed work
on the adaptation of the yeasts to milk-sugar. This sugar is usually
disdained by the yeasts that set up the fermentation of glucose; it is
not difficult, however, to adapt them to galactose which they then
attack and transform into alcohol and carbonic acid.
The Protozoa can be progressively accustomed not only to poisons but
also to altered physical conditions. Thus, Dallinger[35] succeeded in
raising the temperature of the water in which flagellated Infusoria were
growing from 15°·5 to 23° C. without causing their death. By prolonging
the experiment over several months, he was even able to habituate them
to an existence at a temperature of 70° C. In the opinion of
Davenport[36], a view which is shared by many other observers, this
resistance to high temperatures was dependent on the abstraction of
water from the protoplasm. Dallinger has also observed that in Infusoria
that are accustomed to life in hot water, the vacuoles become smaller
and smaller and may even actually disappear.
[Sidenote: [30]]
This adaptation, then, is a property that is very general and widespread
in the microcosm of the unicellular organisms. It is connected with the
intracellular digestion of solid food and with the absorption and
transformation of soluble substances. These phenomena, chemical in
character, are intimately linked with the irritability of microscopic
organisms, which represents one of the fundamental properties of living
organisms.
A Protozoon, which is refractory to a parasite, may protect itself by
flight or it may devour and digest the parasite; another, which acquires
a tolerance in regard to a toxin or to a mineral poison, absorbs, fixes
and transforms this substance. Consequently, in all these instances of
immunity there is a reaction of the living elements of the organism,
this being a direct consequence of the irritability of the protoplasm.
Before an Infusorian retreats from the dead body of an allied organism,
before a Protozoon secretes a digestive fluid around the prey it has
ingested, before a Bacterium secretes a glairy layer for its defence,
etc., these unicellular organisms must receive sensations which provoke
the above-mentioned reactions. It is to a celebrated botanist, Pfeffer,
that we owe the most important researches on this irritability of
unicellular organisms, researches which may be summed up in the general
statement that this property is subject to the psycho-physical law of
Weber-Fechner. Pfeffer, by the observation of the movements of Bacteria
under the influence of increasing stimulations, has established the fact
that, conformably to this law, when the stimulus increases in
geometrical ratio, the irritability increases in arithmetical ratio,
that is to say, the reaction is proportional to the logarithm of the
stimulation. In order that a motile bacterium (_Bacterium termo_), grown
in a peptonised solution, may perceive a difference of medium, it is
necessary to place it in a peptone solution of five times the original
concentration; weaker solutions, in which the concentration is but three
or four times greater than the original fluid, do not attract the
bacteria at all; consequently these differences are below their
chemiotactic sensibility.
The different reactions that are exhibited in the immunity of
unicellular organisms, reactions which are dependent on the irritability
of their protoplasm, therefore, come undeniably under the category of
purely cellular phenomena.
CHAPTER II
IMMUNITY IN MULTICELLULAR PLANTS
Infective diseases of plants.—Plasmodia of the Myxomycetes and their
chemiotaxis.—Adaptation of the plasmodia to poisons.—Pathogenic
action of _Sclerotinia_ upon Phanerogams.—The cicatrisation of
plants.—Defence in plants against Bacteria.—Sensitiveness of
vegetable cells to osmotic pressure.—Adaptation of plants to
modifications of osmotic pressure.—Dependence of the chemical
phenomena upon the irritability of the vegetable cells.—The law of
Weber-Fechner.
[Sidenote: [31]]
For several reasons this immunity in the vegetable kingdom cannot be
treated in a satisfactory fashion. Much attention has been devoted to
the pathology of plants and the etiology of a number of vegetable
diseases was well established at a period when we were still groping in
the dark for the causes of infective diseases in man and the higher
animals. In spite of this, the botanist has relegated the study of the
phenomena of immunity to a secondary position, and up to the present no
work specially devoted to this subject has appeared. It is only
incidentally that the question of the resistance of certain plants to
morbific factors capable of infecting or intoxicating them has been
touched upon. We should require, therefore, to carry out special
researches in this direction and to make a very complete study of
botanical literature, before we should be able to present to our readers
a _résumé_ of the question of immunity in the vegetable kingdom. Such a
programme being impossible we must content ourselves with borrowing from
the botanists certain facts which throw light on some aspects of the
general problem in which we are interested.
[Sidenote: [32]]
Many of the higher plants are subject to infective diseases set up by
the lower plants, of which the most important are the Fungi. Whereas in
the animal kingdom the majority of the infections are due to Bacteria,
these micro-organisms rarely occur in plants; moreover when they are
present the part they play is nearly always a secondary one. This
difference is due mainly to the chemical composition of the “humours” in
the two kingdoms, the cell-juice of plants being generally acid; under
this condition the Fungi develop much better than do the Bacteria.
The various modes of defence against infective diseases that have been
met with in unicellular organisms are also found in the multicellular
plants. Whereas in almost all plants the cells are rigid, owing to the
presence of a well-developed membrane, some of the lower plants have
preserved a condition in which the protoplasm is completely naked and
capable of movement. Myxomycetes are specially distinguished by an
amoeboid stage of existence and by the formation of large plasmodia
which protrude protoplasmic processes and exhibit a kind of locomotion
similar to that met with in the Rhizopods and the Sporozoa.
[Sidenote: [33]]
Infective diseases among the Myxomycetes must be very rare since, up to
the present, they have not been noted by a single observer. It is very
probable that the plasmodia get rid of the infective germs, as do the
Protozoa, both by expulsion of the parasites and by means of
intracellular digestion. This latter takes place in a medium which is
distinctly acid and by means of a soluble ferment described by
Krukenberg[37] as a kind of pepsin. I need not here enter into further
detail as I have already treated this subject in my _Lectures on the
comparative pathology of inflammation_. The fact that the Myxomycetes
can ingest living organisms has been demonstrated by Celakovsky,
jun.[38], who has observed that the spores of the various Fungi can
germinate in the interior of the plasmodium. Whilst our conceptions
concerning the resistance of the plasmodia in regard to micro-organisms
are merely based upon analogies and hypotheses, our ideas as to their
immunity against soluble substances rest on well-established
experimental facts. We owe to Stahl[39] our first information as to the
mode by which the plasmodia resist poisons. When they are placed in
contact with solutions of salts, of acids or of sugar in a sufficiently
concentrated form to bring about an injurious action, the plasmodia make
use of their amoeboid power of motion to escape from these fluids. Hence
they exhibit a _negative chemiotaxis_, exactly parallel to that so often
observed in the case of the unicellular organisms. Consequently there is
in the Myxomycetes a natural immunity due to the activity of their
movements. Further, a kind of acquired immunity in these plants has also
been demonstrated by Stahl. The following is the passage in his paper
referring to this subject, a passage very important from a general point
of view[40]: “If we replace the water in a vessel by a 1 or 2% solution
of glucose, we observe either the death of the plasmodia, if the action
is too rapid, or merely their retreat from the glucose solution. Even
solutions of ½ or ¼% are at first avoided by the plasmodia and, should
the action be too rapid, may cause their death. Usually the plasmodia
emigrate into those portions of the substratum remote from the solution,
to return after some time, often only after several days, and immerse
themselves in the solution of glucose as they do in an infusion of tan,
although with more hesitancy. _Consequently the Myxomycetes accommodate
themselves slowly_[41] to a more concentrated solution, probably by
giving up a certain proportion of their water. I was able to observe the
same phenomena with even much more concentrated solutions (2%). A
plasmodium which at the end of several days had adapted itself to a 2%
solution of glucose and had sent out numerous processes into it, found
itself injuriously affected when the sugar solution was suddenly
replaced by pure water. Those that remained alive had retired to a great
distance from the upper layer of the fluid and did not descend again
until the end of the second day. After a fresh change of fluid we were
able to observe first the repulsion and later the attraction of the
plasmodia, but a certain time always elapses before the plasmodia become
accustomed to the change in concentration. We obtain the same result
when we replace a 2% solution, not by pure water, but by a ½ or a 1%
solution” (p. 166).
[Sidenote: [34]]
De Bary[42] had already interpreted these facts as being due to an
immunity acquired by the plasmodia, the result of an adaptation of these
organisms to solutions which they had, at first, carefully avoided. He
threw out the suggestion that a similar adaptation might take place in
regard to solid substances ingested by the Myxomycetes.
As these phenomena of acquired immunity, in organisms so primitive and
of so simple a structure, are of immense importance from the point of
view of Immunity in general I felt bound to submit them to a personal
investigation. I found it an easy matter to accustom the plasmodia of
_Physarum_ to solutions of arsenious acid which at first repelled them
in a very striking manner. This adaptation manifests itself by movements
of the plasmodia and by the change from negative chemiotaxis (repulsion)
to positive chemiotaxis (attraction).
It is impossible in the present state of our knowledge to state
precisely the modifications that the plasmodia undergo during this
process of adaptation. Stahl supposes that they depend “on some special
properties of the plasmodia (probably in a greater or less richness in
water)”; and that it is a case “not of simple phenomena, easy of
explanation, but of extremely complicated phenomena of irritability.”
It is evident that, in this case of acquired immunity, we have not to do
with a question of physical or chemical modification of the solutions
employed but solely with reactive phenomena on the part of the living
plasmodia.
After a phase of active life, during which the Myxomycetes move, feed,
digest and expel waste products as do the lower animals, there comes a
stage when they become immobile and transform themselves into a number
of sporangia filled with rounded spores. Before leaving their animal
aspect for that of true plants, the plasmodia exhibit entirely new
attributes. They reject all nourishment and no longer ingest foreign
bodies; they avoid the moisture which previously attracted them and
cease to shrink from the light.
[Sidenote: [35]]
Having come to maturity, the Myxomycetes declare themselves true plants
and lead a passive life until the new generation comes forth. Most
plants are restricted to this passive phase of the Myxomycetes. In these
latter it persists only for a short period, whereas in almost all plants
it is the permanent condition. It is at this stage that these organisms
are liable to the attack of parasites against which it is necessary for
them to oppose all their means of defence. Our knowledge of these means
of defence is as yet, as I have already stated, very imperfect, and the
example of _Sclerotinia libertiana_ (or _Peziza sclerotiorum_) which has
been the subject of the researches of de Bary[43] remains up to the
present the one that has been most thoroughly studied.
This Fungus, belonging to the group of the Discomycetes, invades many
species of plants and often produces great ravages amongst the
cultivated plants of our fields and gardens, such as colza, hemp
petunias, dahlias, etc. The mycelium of this _Sclerotinia_ develops in
the stems of herbaceous plants and produces sclerotia inside them, forms
of resistance, which in this instance are black and resemble small
particles of mouse excrement.
The spores of the _Sclerotinia_ germinate and form mycelial threads on
the surface of the plants. In order that they may penetrate into the
tissues these threads must attack the cell-membrane and for this purpose
they secrete a fluid, which contains both a digestive ferment and oxalic
acid, the latter being essential for the action of the ferment.
The presence of this “toxin” has been demonstrated by de Bary by
macerating the mycelium of the _Sclerotinia_. The resultant extract has
a well-marked action on the tissues of many plants (carrot, Jerusalem
artichoke, chicory, etc.). Under its influence the protoplasm of the
cells contracts, a genuine plasmolysis is set up, the cell-membrane
swells and its layers between the cells are dissolved. As the result of
this digestive action, the cells become separated and the tissue
softens. This extract, when heated to 52° C., loses its digestive action
on the cellulose membrane, but still retains its power of setting up
plasmolysis. This reaction to temperature confirms the view that the
juice of the Fungus contains a soluble ferment. The results of de Bary’s
researches have been confirmed and in part supplemented by the
experiments of Laurent[44].
[Sidenote: [36]]
It is a fact of common observation that the _Sclerotinia libertiana_
invades for the most part young plants. It may therefore be asserted
that the disease produced by this Fungus is, like scarlatina or measles
in the human subject, an “infantile” disease. De Bary suggested that the
immunity of adult plants must depend on the greater resistance which
their cell-membranes offer to the fluid secreted by the mycelial
filaments. Direct experiments have shown the accuracy of his suggestion.
Whilst the fluid extracted from the _Sclerotinia_ readily digests the
tissue of young plants it leaves intact that of adult plants of the same
species.
In the course of this disease we have a struggle going on between two
plants. The parasite brings into play toxic and digestive secretions
with which it seeks to impregnate its host. The attacked plant defends
itself by the secretion of membranes capable of resisting the action of
the secretions of the Fungus. This struggle by means of chemical
substances is, however, directed by the activity of the living cells of
the two belligerent plants, an activity dependent upon the irritability
of their protoplasm.
The example we have just studied may serve as a type for our examination
of the phenomena of immunity in the vegetable kingdom. The crux is above
all to prevent the access of the parasites to the vital parts of the
plant by opposing to them membranes as resistant as possible.
Consequently the majority of plants, directly the smallest lesion is
produced, react by an abundant cell-proliferation and by the
suberisation of the outer layers. The cell-membranes of the latter
thicken, the cellulose is transformed into suberin; a layer of cork not
very permeable to fluids and gases being thus formed. By suberisation
the plant reacts against grosser lesions, incisions or burns, as well as
against the decay set up by micro-organisms.
Massart[45], in an extremely interesting memoir, has brought together
the known data concerning cicatrisation in plants and has demonstrated
the fact that it is a very variable process. In many leaves after being
damaged there is no attempt to react by forming cicatricial tissue. Many
aquatic and marsh plants react but feebly. Their tissues die and turn
brown, the plants failing to defend themselves by cicatrices, probably
owing to the ease with which the lost parts can be replaced. When,
however, in the same plants, there is produced a lesion of parts which
are of great importance for the preservation of the integrity of the
individual or a lesion of the organs which enable the plant to continue
its existence through the winter, cicatrisation of the wounds takes
place rapidly.
[Sidenote: [37]]
The old or adult parts in most cases react differently from the young
parts. Thus, the young leaves of _Clisia_ (the example selected by
Massart) react to traumatism very promptly and form a genuine callus
which makes good the injury, but the adult leaves merely produce a layer
of cork in the immediate neighbourhood of the lesion.
The essential mechanism of cicatrisation has not yet been satisfactorily
analysed, but it is evident, when all is said and done, that it is
directed by the irritability of the living protoplasm of the vegetable
cells.
Many plants protect their wounds with a kind of dressing, using for that
purpose juices which harden on exposure to the air. Sometimes these
juices, _e.g._ latex, are preformed in the plant and are as it were
always ready for use; at other times they may be formed only as the
result of an injury. In this latter case the resins and gums which serve
to close the wound and to protect the living parts receive the name of
“cicatricial secretions” (Wundsecrete). According to the view first
formulated by de Vries, those juices which harden under the action of
air prove of great service both as natural “dressings” and as safeguards
against the attacks of plants and animals. Indeed many of these
secretions contain essences whose antiseptic and toxic action is now
generally recognised[46].
[Sidenote: [38]]
The suberisation, the formation of a callus, and the secretion of juices
which close the wounds, are all means readily utilised and very potent
in ensuring the resistance of plants against all sorts of injurious
influences which may be set up by a morbid condition. But these
processes are not the only means which plants have at their disposal.
The living elements of plants usually secrete a cell-juice of acid
reaction which plays a very important part in the defence of plants
against pathogenic agents. Laurent[47] has studied this phase of the
immunity of plants against bacterial decay. A variety of the _Bacillus
coli communis_, according to this observer, attacks the potato by means
of its secretions in a fashion analogous to that already described when
discussing _Sclerotinia_. This bacillus produces a soluble ferment which
has the power of digesting the cellulose membrane in the tuber of the
potato, and at the same time secretes an alkaline juice without which
this digestion cannot go on. Heating to 62° C. destroys the soluble
ferment and the fluid thus heated is no longer able to digest the layers
of the cell-membrane between the cells. In spite of exposure to this
temperature, however, it still retains intact one or even several
substances which may continue to cause contraction of the protoplasm and
ultimately kill it.
When Laurent placed cut halves of tubers coming from races of potato
which were most resistant to bacterial decay in the fluid produced by
the _Bacillus coli_ and afterwards inoculated them with the bacillus
itself, he invariably found that the vegetable cells were profoundly
affected.
The alkaline secretions of the bacillus studied by Laurent may be
neutralised by the acid juice of the potato, and when certain races of
tubers prove immune from decay, it is, according to this observer,
because of the production of sufficiently acid cell-juices. Moreover he
actually succeeded in communicating an artificial immunity to varieties
of the potato which were most susceptible to decay by immersing them for
several hours in solutions of certain organic acids. On the other hand,
when he treated varieties endowed with a well-marked natural immunity
with alkaline solutions, the tubers became very susceptible to the decay
set up by the bacillus.
The struggle between the potato and the _Bacillus coli_ reduces itself,
then, to the chemical reaction between the alkaline cell-secretions of
the micro-organism and the acid secretions of the potato. This general
fact, according to Laurent, explains the part played by certain manures
in determining the susceptibility or the resistance manifested by the
potato and many other plants against infective diseases.
We know that the addition of phosphates to the soil increases the
immunity of certain cultivated plants. These substances are greedily
absorbed by the roots and produce acid salts which are dissolved in the
cell-juice. The nitrogenous manures, on the other hand, both potassic
and lime, diminish the resistance of the same plants, probably from the
fact that they bring about a diminution of the acidity of the
cell-juice.
But these manures can act differently on different plants. Thus the same
phosphates which confer immunity on the potato against bacterial decay
render the Jerusalem artichoke more susceptible to the attacks of the
_Sclerotinia_.
[Sidenote: [39]]
Laurent explains this fact as due to the difference in the reaction of
the medium, which favours the action of one or the other of the soluble
ferments of the two parasites. The ferment of the bacillus digests the
cell-membrane in an alkaline or feebly acid medium, whereas the
hyperacidity which results from the absorption of the phosphates
prevents this digestion and consequently aids the plant in its struggle.
On the other hand, the ferment of _Sclerotinia_, as is seen from the
researches of de Bary, will digest cellulose even in a distinctly acid
medium. The hyperacidity, induced by the phosphated manure, in this case
favours the parasite and enables it to gain the upper hand in the
struggle with the tissues of the artichoke.
In addition to neutralising the microbial products the acids of the
cell-juice also act injuriously on most bacteria, which will only
develop in neutral or alkaline media; it is for this reason that
bacterial diseases are so much rarer in plants than in animals.
The secretion of cell-juices is consequently a very important element in
the defence of plants; it will be useful, therefore, to ascertain as
definitely as possible the essential mode of its action. Vegetable cells
are as a rule very sensitive to the influences to which they are
exposed; they distinguish with great precision the changes which take
place in their surroundings. They are, indeed, capable of discerning not
only the physical properties but also the chemical composition of the
medium in which they live.
Vegetable cells estimate very accurately the osmotic pressure of the
fluid which bathes them, and they react towards this solution by
increasing or diminishing their own internal pressure. Van
Rysselberghe[48], in an investigation very carefully carried out,
demonstrated that when vegetable cells (especially the epidermic cells
of certain species of _Tradescantia_) are placed in a solution of
greater density than that to which the cells are accustomed, the
intracellular pressure increases; in a solution of less density the
pressure diminishes. These changes in osmotic pressure are due to
variations in density of the cell-juice, whilst these variations are in
turn determined by chemical transformations. Thus, if the cell be
treated with a too concentrated solution it produces oxalic acid, which
dissolving in the cell-juice, is, owing to the smallness of its
molecule, very osmotic.
[Sidenote: [40]]
With the purpose of confirming this by exact facts van Rysselberghe has
studied the acids of the cell-juice of _Tradescantia_. In the normal
juice he found that malic acid was constantly present and, in rare cases
only, traces of oxalic acid. He then determined the acids present in the
leaves of the same plant after they had been several days in contact
with fairly concentrated solutions of cane sugar. In each analysis he
found oxalic acid in quite appreciable quantity. There is then, in the
plant which adapts itself to more concentrated solutions of the medium,
a production of oxalic acid which serves the purpose of increasing the
pressure of the cell-juice.
The origin of this oxalic acid could not be accurately demonstrated, but
van Rysselberghe considers that it is probably formed at the expense of
the glucose.
According to the researches of Giessler oxalic acid is localised
specially in the epidermis and generally in the peripheral tissues of
plants; it is very probable, therefore, that it fulfils a protective
_rôle_ against all kinds of injurious influences. Botanists hold indeed
that oxalic acid keeps herbivorous animals, especially slugs and plant
lice, from attacking plants that are rich in this substance. It is of
use, also, in retaining the moisture in the superficial cells. It is
very probable that it also plays an important part as a factor in the
maintenance in plants of immunity against bacterial diseases.
The vegetable protoplasm, which is capable of increasing the production
of acids in order to raise the osmotic pressure, can also, in case of
need, cause a diminution.
When the cells of _Tradescantia_ are transferred from a concentrated
solution into one much more dilute there may often be observed a
precipitation, in the cell-juice, of crystals of oxalate of lime; this
brings about a diminution in the osmotic pressure. When the density of
the medium is altered, and the vegetable tissue is again transferred to
a stronger solution, the oxalate crystals are seen to dissolve, as a
result of a new production of acid.
These chemical processes, so important to the life of plants in general
and for ensuring to them immunity against infective agents in
particular, are dependent upon the irritability of the protoplasm.
Imprisoned in its resistant and more or less thick membrane, the living
part of the vegetable cell estimates with nice discrimination every
change that takes place around it.
[Sidenote: [41]]
Massart[49] has shown that the stimulation produced by traumatism is
often propagated a considerable distance and may excite a reaction in
very remote cells. If the mid-rib of a leaf of _Impatiens sultani_ be
cut near the base of the limb the wound does not cicatrise but, a few
days later, the leaf becomes detached from the stem.
Irritability is a fundamental property of all living beings. The plant
may react by rapid movements, as in the case of the _Mimosa pudica_, or
more slowly—by chemical reactions—as in the case of adaptation to
density of medium. These reactions are produced as the result of various
irritabilities which exhibit a specific character.
It is this specificity that determines whether the reaction that is
manifested by the movements shall be produced in this direction or in
that. The stem, owing to the specific irritability of its living parts,
turns to the light; whilst the root, guided by a different irritability,
grows down into the soil.
The irritability of plants, like that of unicellular organisms, is
subject to the psycho-physical law of Weber-Fechner. Pfeffer[50] first
demonstrated this for the motile spermatozoids of the Cryptogams.
Massart[51], by a series of ingenious experiments on the irritability of
a Mould (_Phycomyces nitens_) to light, has shown that the same law
regulates the movements of this plant towards the source of light. This
irritability of the Fungus to light is much more delicate than is the
chemiotaxis of the spermatozoids of the Mosses and the Ferns and than
that of the Bacteria.
Errera concluded from a consideration of the experiments of van
Rysselberghe that the osmotic reaction of plants must also come under
this psycho-physical law. His pupil at his request made systematic
researches on the subject and the results have entirely confirmed his
prevision. According to the data obtained by van Rysselberghe[52], the
cellular osmotic reaction increases in arithmetical progression as the
osmotic stimulation increases in geometrical progression. The osmotic
reaction is therefore proportional to the logarithm of the stimulation.
[Sidenote: [42]]
To sum up, the phenomena of adaptation and of immunity in plants are, as
in the unicellular organisms, very widely distributed. Plants defend
themselves by means of their resistant membranes and by secretions whose
physical and chemical properties they are able to modify. These
phenomena are dependent on the living parts of the cell which regulate
them according to their greatly developed irritabilities. Thanks to this
power, plants can gradually adapt themselves to concentration of the
medium and to the presence of poisons which, at first, set up serious
disturbances. Plants therefore, alongside a natural immunity, possess an
acquired immunity against many pathogenic agents.
CHAPTER III
PRELIMINARY REMARKS ON IMMUNITY IN THE ANIMAL KINGDOM
Examples of natural immunity among the Invertebrates.—Immunity against
micro-organisms and insusceptibility to microbial poisons are two
distinct properties.—The refractory organism does not eliminate
micro-organisms by the excretory channels.—It destroys them by a
process of resorption.—The fate of foreign bodies in the
organism.—The resorption of cells.—Intracellular digestion.—This
digestion effected by the aid of soluble ferments.—Digestion in
Planarians and Actinians.—Actinodiastase.—Transition from
intracellular digestion to digestion by secreted juices.—Digestion
in the higher animals.—Enterokynase and the part it plays
in digestion.—The psychical and nervous elements in
digestion.—Adaptation of the pancreatic secretion to the kind of
food.—Excretion of pepsin in the blood and in the urine.
[Sidenote: [43]]
As shown in the two preceding chapters unicellular organisms and plants
afford evidence of numerous phenomena of immunity. Alongside natural
immunity we find in them undoubted evidence of an adaptation to the
presence of morbific agents, evidence which warrants us in inferring
that cases of acquired immunity are frequent. This being the case it is
quite natural that the animal kingdom should be no exception to the
general rule. Indeed, immunity against pathogenic agents is widely
distributed in animals, and we continually see manifestations of natural
immunity not only against parasites and their toxins, but against
poisons in general. Just as frequently we find cases of acquired
immunity against these morbific agents.
[Sidenote: [44]]
As yet we know but little concerning the phenomena of immunity in the
lower animals belonging to the great group of the Invertebrata. But it
may be affirmed with certainty that these also are often endowed with a
natural immunity against micro-organisms and bacterial toxins. As an
example I may cite the case of the large white larvae of the Rhinoceros
beetle (_Oryctes nasicornis_) frequently met with in tanner’s bark. Very
susceptible to the cholera vibrio—¹⁄₈₀₀₀ of a culture[53] of this
organism being sufficient to set up a fatal septicaemia—these larvae
exhibit a very remarkable natural immunity against the bacilli of
anthrax and diphtheria. A large dose of bacteria of the second anthrax
vaccine, fatal to rabbits, guinea-pigs and mice, is borne without any
inconvenience by the larvae of the Rhinoceros beetle. They are equally
refractory to large doses of the diphtheria bacillus. And yet, there are
not wanting species of insects which are susceptible to these same
micro-organisms. Thus, according to A. Kovalevsky[54], crickets contract
anthrax very readily even at moderate temperatures (22°–23° C.). On the
other hand they are, according to the same author, refractory to the
bacillus of avian tuberculosis. Many of the Invertebrata, studied from
this point of view, present analogous facts, with which, however, we
need not at present occupy ourselves.
In the Vertebrata in general and in Man in particular, natural immunity
against many infective diseases and soluble poisons is so widespread
that we are at no loss to find examples for citation. We have a whole
series of human infections whose study is rendered particularly
difficult simply because of the natural immunity of all other species of
animals from these infections. Such are syphilis, scarlatina, leprosy,
exanthematous typhus, etc. On the other hand, a large number of
diseases, very infective for domestic animals, are quite innocuous to
man. In this group we have cattle plague, strangles, contagious
pleuro-pneumonia, fowl cholera, pneumo-enteritis of pigs, and a number
of other diseases.
As in a very large majority of instances pathogenic organisms act
through the agency of their toxic products, one might believe—and this
has been assumed repeatedly—that natural immunity against infective
diseases is dependent on the insusceptibility of the refractory organism
to the specific poisons.
[Sidenote: [45]]
Such a supposition cannot survive criticism. We have undoubted instances
of a species of animal being resistant both to a micro-organism and to
its toxin. Such instances, however, are rare and usually an organism
that is refractory or only slightly susceptible to the micro-organism
itself is very susceptible to its toxic products. Even those
micro-organisms which come almost constantly in contact with the human
organism without becoming pathogenic, may produce toxins capable of
gravely affecting health. Let us take as an example the bacillus of blue
pus. This organism is most widely diffused in human surroundings.
According to Schimmelbusch[55] it is met with on the skin of the
arm-pits and of the inguinal region of one-half of mankind. From the
skin it very often passes into the dressings of wounds which then assume
the characteristic and so long recognised blue colour. The same bacillus
is also found in the intestines of both sick and healthy persons.
Jakowski[56] has met with it in the faeces coming from intestinal
fistulae in two women who had undergone operations. Now, in spite of
these specially favourable conditions for the production of infection,
the _Bacillus pyocyaneus_ has remained harmless. It is only in children,
and even then rarely, that it can be convicted of exciting disease. Man,
then, usually enjoys a true natural immunity against the _Bacillus
pyocyaneus_. And yet it is not to his insusceptibility to the pyocyanic
toxin that he is indebted for this immunity. Schaffer[57], having
injected himself in the shoulder with half a c.c. of a sterilised
culture of _B. pyocyaneus_, developed fever and an erysipelatous
swelling. Bouchard and Charrin[58] injected pyocyanic toxin into
patients who reacted with more or less fever and by other toxic
symptoms.
[Sidenote: [46]]
Another extremely common saprophyte, the _Micrococcus prodigiosus_, is
incapable of setting up an infective disease, but this does not prevent
its products from exercising a toxic action, often very grave, in man.
The frog, which is refractory to the cholera vibrio, undergoes a fatal
intoxication when cholera toxin is injected. One of the most striking
examples is furnished in the case of the human tubercle bacillus and
tuberculin. Man is much more resistant than is the guinea-pig to the
pathogenic action of this organism, yet he is incomparably more
susceptible to its toxin (tuberculin). According to the researches of
Behring and Kitashima[59], the sheep, of all species of mammals, is most
susceptible to the tubercular poison; the Bovidae and the guinea-pig
occupy an inferior rank in the scale of susceptibility. On the other
hand, the guinea-pig is very susceptible to the tubercle bacillus; the
Bovidae are less so and the sheep is still more resistant to
tuberculosis. It is unnecessary to multiply instances. Immunity against
microbial infection and against intoxication are two distinct
properties, so that it is impossible to reduce the former to an
insusceptibility to toxins. We must therefore consider these two kinds
of immunity separately and we will first consider the resistance of the
animal organism against living infective micro-organisms.
Refractory human beings and animals may be inoculated with a large
number of micro-organisms without being affected. Thus Opitz[60]
injected 10,000,000 organisms into the blood of a dog. Twenty minutes
later he could find no more than 9000. It is then quite natural to ask,
What becomes of these micro-organisms after they have made their way
into the interior of the refractory organism? It has been suggested that
the animal gets rid of the pathogenic germs much as it does of all kinds
of soluble poisons. Certain of these poisons, such as iodine and
alcohol, are in great part eliminated by the kidneys; others, such as
iron, by the alimentary canal. Why, it is asked, should not
micro-organisms also be eliminated by the same channels? Flügge has
adopted this view and has expounded it in his work on ferments and
micro-organisms[61]. Moreover he suggested to Wyssokowitch[62] that he
should carry out a large series of experiments with the object of
verifying this theory. But numerous very careful researches have given a
result quite at variance with the forecast made by Flügge.
Micro-organisms of various species, injected into the blood vessels of
rabbits and dogs, were, in those cases where these animals are
refractory, never eliminated, either by the kidneys or by any other of
the excretory channels which were studied. When bacteria pass into the
secretions, lesions of the tissues, more or less grave, are invariably
present.
[Sidenote: [47]]
This result has been repeatedly confirmed and has been accepted as a
general experience. The elimination of micro-organisms by the urine
indicates not merely the absence of immunity, but implies, also, a
susceptibility of the organism. In many septicaemias, such as those
produced by the anthrax bacillus, the streptococcus and other bacteria,
or in less generalised diseases, such as typhoid fever, bacteria are
found in the urine, often in large numbers. In these cases it is a
question of anything but a refractory condition even of the slightest
degree.
In recent years, however, several works have been published the aim of
which was to demonstrate the inaccuracy of this apparently
well-established thesis. Biedl and Kraus[63] in Vienna took the
initiative and announced in a detailed work that micro-organisms can
readily pass intact into the kidney and that this organ in virtue of its
physiological function eliminates them. The organisms were said to leave
the blood capillaries by the normal process of diapedesis and were then
eliminated with the urine. The liver in a physiological condition,
according to the researches of these authors, is equally capable of
allowing of the passage of micro-organisms; indeed it aids in
discharging them from the system. On the other hand, the pancreas and
the salivary glands were incapable of fulfilling this function. Von
Klecki[64] obtained similar results. He also holds that the kidney is
the principal organ of elimination for micro-organisms which have
penetrated into a refractory organism.
With these contradictions before him, Opitz[65] set himself to study
this question in Flügge’s laboratory at Breslau. Having critically
reviewed the technical methods of his predecessors and carried out a
series of new experiments, he declared categorically “that a
physiological excretion, by the kidneys, of the micro-organisms which
circulate in the blood, does not exist.” For Opitz “the frequent
appearance of micro-organisms in the urine of animals into whose blood,
a short time previously, living bacteria have been injected, is due to
mechanical and chemical lesions of the vessel wall and of the renal
epithelia.”
[Sidenote: [48]]
This question might be looked upon as definitely settled in favour of
the first results obtained by Wyssokowitch were it not that other voices
had been raised in favour of a physiological excretion of the
micro-organisms by the renal channels. Pawlowsky[66] has recently
published a long work on this subject in which he attempts to
demonstrate that certain micro-organisms, even when introduced into the
subcutaneous tissue of animals, pass very rapidly (at the end of a
quarter of an hour) into the uropoietic organs and are eliminated with
the urine.
It was necessary to put an end to these controversies and Métin[67]
undertook a series of researches at the Pasteur Institute with the
object of clearing up this question. He guarded himself against the
objections justly made against his predecessors and conducted his
experiments under unexceptionable conditions. He injected several
species of micro-organisms into the veins of rabbits and into the
subcutaneous tissue of guinea-pigs. At various intervals he performed
laparotomy on these animals, pulled out the bladder and drew off the
urine in such a fashion that no trace of blood could get into it. The
results were most conclusive. Never, when the experiment was conducted
under the rigorous conditions just mentioned, did the micro-organisms
traverse the kidneys of resistant animals nor were they ever met with in
their urine.
Métin’s researches on the passage of micro-organisms through the liver
in refractory animals gave the same results. In no case was he able to
find in the bile any of the organisms that had been injected into the
blood or under the skin. At the end of his memoir Métin sums up his
results as follows: “(1) The kidneys and the liver are impermeable to
bacteria introduced into the organism, subcutaneously or intravenously;
(2) when the culture tubes contain colonies of the injected
micro-organism, it is because there has been a certain amount of blood
in the fluid inoculated, this being an indication of a vascular or
epithelial lesion, either mechanical or chemical.” We were present at M.
Métin’s experiments and can bear witness to their exactitude.
[Sidenote: [49]]
There can no longer be any doubt then on this point. The elimination of
the micro-organisms from the refractory animal takes place, as indicated
in Wyssokowitch’s first investigation, neither by the kidneys nor by the
liver. Some observers have asserted that this elimination may take place
by the sudoriparous glands. Thus, Brunner[68] made experiments with
young pigs and cats into which he had previously injected
micro-organisms, for the most part pathogenic. Then producing a
transpiration by means of pilocarpin, he “cultivated” the sweat and
noted the development of the same bacteria as he had introduced into the
blood. In a single experiment with a saprophyte (_Coccobacillus
prodigiosus_) he obtained a positive result, from which he concludes
that the refractory animal gets rid of bacteria which circulate in its
blood by way of the sudoriparous glands. It is scarcely allowable to
draw any conclusion from this experiment from the fact that the snout of
the pig, the seat of the transpiration, is very liable to small vascular
lesions which might furnish the bacteria that developed on Brunner’s
plates. Nevertheless, even in the case of pathogenic organisms, which
swarm in the blood, the sweat is usually free from them. This has been
shown by Krikliwy[69] in the case of cats inoculated with anthrax whose
sweat, in spite of the passage of numerous bacteria into the
circulation, contained none.
[Sidenote: [50]]
Micro-organisms, then, after their entrance into the refractory animal,
are not eliminated by any of the excretory channels which serve for the
elimination of many of the soluble poisons. It was necessary therefore
to seek some other process capable of affording an explanation of the
disappearance of the micro-organisms which so often and by such varied
means make their way into the interior of a resistant organism. For it
is a well-established fact that in these cases the micro-organisms do
disappear completely. This has been observed so often that it is
unnecessary to offer any demonstration of the fact. Perhaps in the
refractory organism the micro-organisms undergo the fate of the foreign
bodies which penetrate, or which are introduced, into the circulation.
It has long been known, thanks especially to the work of Hoffmann and
Recklinghausen[70], and of Ponfick[71], that particles of carmine or
vermilion when injected into the blood are deposited in several organs.
They are found in the spleen, the lymphatic glands and the bone-marrow.
A certain number of these foreign particles may even be fixed in the
liver and kidneys, but, instead of passing into the bile and the urine,
they remain lodged in the interstitial tissue of the organs. The
observers just cited noted that the coloured granules do not remain long
in either the blood or the lymph but will be found in the interior of
the cellular elements. These granules persist for weeks without any
appreciable modification, differing in this from the micro-organisms
which, as a rule, after several days or even after a few hours,
disappear from the refractory organism. This disappearance might be more
justly compared to the resorption of corpuscular elements which results
in a more or less complete atrophy. The facts concerning the resorption
of pus, of extravasated blood, of the mucosa of the uterus in pregnancy,
etc., have long been known, and it is among these that one should seek
analogies with the disappearance of the micro-organisms. When bacteria
of various species are injected into refractory or not very susceptible
animals, we always observe a local reaction in the form of inflammation,
accompanied by the appearance of white corpuscles. Gradually the
organisms disappear from the point at which they are introduced; the
exudation becomes sterile and ultimately is completely absorbed.
Numerous researches, which will be set forth in the succeeding chapters,
have, indeed, demonstrated the remarkable analogy that exists between
the disappearance of the micro-organisms from the refractory animal and
the resorption of corpuscular elements or of animal cells.
The analysis of the phenomena of this resorption will help us
considerably in our study of immunity against micro-organisms. When in
any part of the animal organism a collection of pus, an effusion of
blood, or any other organic lesion is produced, these lesions are
usually repaired after the lapse of a longer or shorter interval. In
those cases where the cells retain their integrity, they are taken into
the lymphatic vessels and then pass into the circulating blood. In the
course of his researches on the transfusion of blood, Hayem[72] observed
“that blood injected into the peritoneum is absorbed unaltered and
passes with its anatomical elements into the general circulation.” He
was able to demonstrate “that the lymphatic channels play an important
part in this absorption.” Lesage of Alfort[73] confirmed this result. He
found that in the dog “one hour after an abundant haemorrhage into the
peritoneum, induced experimentally, the red corpuscles commenced to pass
freely, without alteration and in very large numbers, into the thoracic
duct.” I have observed a similar resorption of the red blood corpuscles
of the guinea-pig when injected into the peritoneal cavity of other
individuals of the same species. The white corpuscles can also be taken
up by the lymphatic vessels without being modified in any way. At the
end of an inflammatory reaction of feeble intensity, set up in
cold-blooded animals, especially in the tadpole, the direct passage of
leucocytes from the exudation into the lymphatic system may be observed.
[Sidenote: [51]]
The examples I have just cited are, however, quite exceptional. In the
great majority of cases the cellular elements that are undergoing
resorption are seized by the amoeboid cells and are taken into their
substance. Even in the resorption of the red corpuscles, lying free in
the peritoneal cavity of the same species of animal, a certain number of
the globules do not pass directly into the circulation but are first
ingested by the amoeboid elements. This fact is insisted upon by Lesage.
In inflammatory exudations the leucocytes also become the prey of their
fellows. The ingested white corpuscles may be recognised for some time
lying in the interior of other leucocytes; they are soon broken up,
however, and finally disappear completely. When, instead of isolated
cells such as leucocytes, we introduce fragments of tissues or of organs
into any part of the organism, the same mode of resorption may always be
observed. The introduced fragments are first surrounded and infiltrated
by amoeboid cells and are then taken up into their interior.
[Sidenote: [52]]
The mode of absorption just described is very general. It applies to all
kinds of cells and is observed in the absolutely normal organism, as
well as in a large number of pathological conditions. For more than
fifty years, the existence of cells which contain red blood corpuscles
(“blutkörperchenhaltige Zellen” of German writers) has been recognised;
they were met with in the spleen, the lymphatic glands and in many
pathological products. For long we could not explain how the red
corpuscles come to be inside other cells. Virchow[74] thought that they
got there as the result of a mechanical pressure. Later histologists
succeeded in determining the true nature of cells containing red blood
corpuscles and in recognising that the leucocytes had really ingested
the corpuscles. There has been much discussion, also, on the presence of
leucocytes in the interior of large cells in exudations. It was thought
that these were mother-cells which contained a new generation of small
cells. Writers even described a fusion between the large cell and those
found inside it; but Bizzozero[75] first recognised that the former was
an amoeboid cell which had ingested pus corpuscles. Since this
observation was made numerous cases have been described in which
different cell elements have been found in the large cells. There could
no longer be any hesitation in interpreting these cases as instances of
ingestion by leucocytes or similar cells.
The changes that the ingested elements undergo within amoeboid cells may
be compared with those that take place in intracellular digestion. If
the modifications of the particles ingested by the _Amoebae_ be studied
side by side with those which take place in ingested cells in the
process of resorption, a striking analogy may be observed. To establish
this satisfactorily it is essential to begin with a study of
intracellular digestion properly so called, especially as in this
phenomenon we have the fundamental basis of the whole of the theory
developed in this work.
In our first two chapters we have already cited examples of this
intracellular digestion in the Protozoa (_Amoebae_, Infusoria, etc.) and
in the plasmodium stage of the Myxomycetes. In all these cases it goes
on in the organism, in a distinctly acid medium, by the aid of ferments
which could be demonstrated in the _Amoebae_ and Myxomycetes, and which
are analogous sometimes with trypsin, sometimes with pepsin.
In the lower Invertebrata we find the principal source of our knowledge
of intracellular digestion in the digestive organs. This form of
digestion is met with in Sponges, in the whole of the Coelenterates
(Medusae, Siphonophora, Ctenophora, etc.), in the great majority of the
Turbellaria (Planarians, Rhabdocoela), and in certain of the Mollusca
(the lower Gasteropods). In the Invertebrata higher in the animal scale,
intracellular digestion in the digestive organs becomes more and more
rare, and sometimes it manifests itself only in the larval condition
(_Phoronis_); ultimately it gives place permanently to digestion by
juices secreted into the gastro-intestinal canal.
[Sidenote: [53]]
In his sketch of the comparative physiology of digestion, Krukenberg[76]
sought to establish two types: protoplasmic or cellular digestion and
secretory digestion. The former is effected, according to this observer,
by a vital action independently of any production of soluble ferments.
Secretory digestion alone, characteristic of the Vertebrates and of
almost all the higher Invertebrates, is effected by means of these
ferments (diastases or enzymes). Many observers, adopting this view,
maintain that intracellular digestion presents a purely vital phenomenon
essentially different from that of chemical digestion due to juices
containing soluble ferments secreted in the gastro-intestinal canal.
That this theory is absolutely erroneous the succeeding pages of this
work will furnish ample proof.
[Sidenote: [54]]
The Protozoa, from their small size, are unsuitable for researches on
the essential phenomena of intracellular digestion. Amongst animals
higher in the scale the Planarians lend themselves most readily to the
observation of this process. These flat worms are very common in both
fresh and sea water and are easily fed in captivity. They are very
voracious animals and, among other things, devour the blood of man or
animals with avidity. One has merely to allow them to fast for a few
days, and then to give them a drop of blood in order to see their
digestive canal fill itself with this fluid (fig. 6). The white
Planarian, _Dendrocoelum lacteum_, is well adapted for these researches.
In a worm that has sucked blood from a Vertebrate, owing to its great
transparency, the whole length of its intestine with its numerous
ramifications may be seen. For some time this organ remains of a bright
red colour, but gradually the tinge becomes brownish or faintly violet.
These changes of colour recall those observed in effusions of blood in
or under the human skin resulting from contusions. A microscopical
examination of Planarians that have been fed with blood shows that the
coloration of their digestive canal is due to red blood corpuscles in
different stages of digestion. Immediately after the taking in of the
blood by the Planarian all the red blood corpuscles are ingested by the
epithelial cells of the intestine. Connected with the wall by slender
stalks, these elements appear as large amoeboid cells whose free end
projecting into the lumen of the intestine sends out protoplasmic
processes which seize the red blood corpuscles and convey them into the
interior of the cell. This goes on very rapidly, and in a very short
time all the red corpuscles are found within the epithelial cells. As a
result of the increase in volume of these cellular elements the
intestinal cavity is completely occluded.
[Illustration:
FIG. 6. Young Planarian some time after having sucked goose’s blood.
]
[Sidenote: [55]]
[Illustration:
FIG. 7. Intestinal cell of a Planarian, filled with red blood
corpuscles, undergoing digestion, of the goose.
]
Once inside the cells of the intestine the red blood corpuscles exhibit
changes which are readily followed under the microscope. It is better
still to feed the Planarians with the blood of those lower Vertebrates
whose red corpuscles are nucleated. In my researches I have used the
blood of the goose. The red blood corpuscles of this bird, when ingested
by the epithelial cells of the intestine of Planarians, are usually
collected into compact groups (fig. 7), only a few remaining isolated.
The majority of these red corpuscles soon lose their normal appearance
and contour; they become rounded and fused together, but the nucleus and
the haemoglobin enable us to recognise them without any difficulty.
Later the red colouring matter begins to diffuse into the digestive
vacuoles which form around the corpuscles. These corpuscles empty
themselves, retaining their nuclei and capsules, which shrivel more and
more. The nucleus also undergoes almost complete digestion, its
membranous layer alone persisting (fig. 8). Even several days after the
digestion of the blood has begun one can still find _debris_ of
perfectly recognisable red corpuscles, but the red colour has been
replaced by a more or less pronounced brown tint. In the last stage of
the digestive process, as the red corpuscles disappear, the protoplasm
of the intestinal cells becomes filled with round vacuoles, containing
brown irregular concretions—excreta—which are expelled into the
intestinal cavity.
[Illustration:
FIG. 8. Digestion of red blood corpuscles of the goose within an
intestinal cell of a Planarian.
]
This slow digestion of a substance usually so easily assimilable as
blood takes place entirely within the epithelial cells of the intestine.
Continuous microscopical observation demonstrates most clearly the
complete absence of any extracellular digestion of the blood corpuscles
in the intestinal content.
[Sidenote: [56]]
When goose’s blood mixed with blue litmus powder is given to Planarians,
the coloured grains may be found some hours afterwards inside the
epithelial cells of the intestine, but only a few of the blue litmus
granules change colour, taking on a light violet tinge; the great
majority retain their blue coloration. It might be concluded from this
that in Planarians intracellular digestion is effected in a neutral or
nearly neutral medium. If, however, the preparations of intestinal cells
gorged with goose’s blood are treated with a 1% solution of neutral red,
we at once notice that the red corpuscles and the vacuoles which contain
them are stained bright red, assuming a tint similar to that given with
picrocarmine staining (fig. 9). This colour reaction indicates,
according to our researches on neutral red, an acid reaction, more
feeble, however, than that met with in _Paramaecium_ and many other
Protozoa.
[Illustration:
FIG. 9. Portion of an intestinal cell of a Planarian, treated with 1%
neutral red.
]
Macerations of Planarians in normal saline solution to which has been
added a small quantity of the red corpuscles of the goose’s blood
exhibit _in vitro_ a very distinct solvent action on these corpuscles,
which become rounded and lose their haemoglobin, this latter diffusing
into the surrounding fluid, and at the close of the experiment there
remain simply the membranes and the nuclei of the corpuscles.
[Sidenote: [57]]
The study of these Planarians shows us, then, that the food of these
animals undergoes exclusively intracellular digestion in a feebly acid
medium and by means of a soluble ferment, and it furnishes us with proof
that typical intracellular digestion is essentially a chemical process
due to the intervention of enzymes. Now there can be no question, here,
of a protoplasmic action proper, but the branched digestive canal, so
intimately associated with the parenchyma, cannot be completely isolated
from the rest of the Planarian, and it is impossible to study _in vitro_
its digestive action apart from other tissues. To attain this end we
must turn to animals of larger size and those in which the digestive
organs can be isolated more easily. In the Coelenterata intracellular
digestion is general. Many of them are so transparent that they can be
examined _in vivo_. It is easy to observe that the particles of food are
seized by amoeboid processes of the entodermic cells and that they pass
into the substance of these elements there to be digested. For the
systematic study of the digestive phenomena, however, it is not
sufficient merely to examine all that takes place in the living animal.
Experiment _in vitro_ is also necessary. For this purpose the Actinians
or sea-anemones offer us really excellent material. As these animals are
very common in all our seas and are easily kept alive for long periods
in aquaria, they have been used for various researches, among others for
the study of the process of digestion.
[Sidenote: [58]]
The Actinians are easily fed in captivity; they devour morsels of flesh,
of shrimps, of mollusca and other marine animals with avidity. The
ingenious English observers Couch and G. H. Lewes[77] long ago
demonstrated that morsels of food when introduced enclosed in perforated
quills or wrapped in test paper or gutta percha silk and swallowed by
the anemones were afterwards ejected surrounded by mucus but with no
trace of digestion. Having failed in their search for digestive juices
in the large gastric or coelenteric cavity of the Actinians, Lewes
concluded that digestion in these animals is effected in a purely
mechanical fashion. The greatly developed muscles of the Actinians were
supposed to squeeze the food and extract its fluid which is then
absorbed by the walls of the general cavity. It was not until very much
later that the problem of digestion in the Actinians could be resolved
in any accurate and definitive fashion. More than twenty years ago I
demonstrated[78] that the digestion in these polyps is intracellular. In
order that a clear conception of this phenomenon may be obtained it may
be useful to recall in a few words the fundamental features of the
organisation of Actinians. They are cylindrical bodies, sometimes as
large as the fist, attached by their base to stones, shells, or other
submarine objects, and furnished at their free extremity with one or
more series of tentacles. In the middle of this extremity is an
elongated opening, the mouth, which leads into a spacious sac, often
spoken of as the stomach. It is, however, only a kind of oesophagus,
through which the food passes into the large coelenteric cavity which is
divided by septa into numerous compartments lined by the entodermic
epithelium. These septa give origin to many very long and tortuous
filaments, spoken of as mesenterial filaments from their resemblance, a
purely superficial one, to the mesentery of higher animals (fig. 10).
When the Actinian is hungry it protrudes its tentacles in order to seize
marine animals, which it conducts to its mouth. The lips and the
oesophagus are used to estimate the quality of the capture, and if it is
found unsuitable the anemone rejects it, first surrounding it with a
layer of mucus. If however the food is found to be suitable, the
Actinian retains it in its large cavity and throws around it a multitude
of its mesenterial filaments. These penetrate it in all directions, and
as their epithelial cells are capable of sending out amoeboid processes
they seize and ingest the particles, which immediately enter the
protoplasmic content. This work is done with such precision and nicety
that the sea-anemone is able to extract the contents of a shrimp from
the carapace, which latter alone it rejects.
[Illustration:
FIG. 10. Longitudinal section of an Actinian (after Hollard).
]
[Illustration:
FIG. 11. An Actinian in which carmine after absorption has passed into
the mesenterial filaments.
]
[Sidenote: [59]]
The epithelium of the mesenterial filaments is therefore the organ of
digestion in the Actinians. The nutritive parts of their prey pass into
the amoeboid epithelial cells and there undergo a purely intracellular
digestion. If we add to the shrimp-muscle or other food a little carmine
or blue litmus powder, the mesenterial filaments ingest it also and
become pigmented. After eating carmine they assume a very brilliant rose
colour (fig. 11); blue litmus colours them rose violet. This change of
colour in the interior of the cells of the filaments indicates a
decidedly acid reaction of their contents[79]. When one adds to the
mesenterial filaments which are carrying on the process of digestion a
drop of a 1% solution of neutral red they assume various shades of red
(fig. 12).
[Illustration:
FIG. 12. Portion of mesenterial filament of an Actinian, stained with
1% neutral red.
]
This intracellular digestion in the Actinians has been confirmed by
several observers, amongst whom may be cited Chapeaux[80] and
Bjelooussoff[81]. It has often been asserted, however, that, along with
a digestion in the interior of the cells of the mesenterial filaments,
there is, in the Actinians, a secretion in the coelenteric cavity of
their body of fluids which digest nutritive matter by means of a soluble
ferment. A ferment similar to trypsin has been extracted from Actinians
by Léon Frédéricq and Krukenberg. But, in presence of contradictory
assertions, it remained undecided whether, in the enzymatic digestion,
this ferment does its work in the fluid of the coelenteric cavity or
whether it represents the active factor in intracellular digestion.
With the object of definitely elucidating a problem of such general
importance, Mesnil, the superintendent of my laboratory, has been good
enough to carry out a fresh series of experiments on the digestion of
the Actinians and has studied this process not only in animals kept in
captivity in aquaria but also in Actinians living under natural
conditions in the sea[82].
[Sidenote: [60]]
As intracellular digestion is of interest to us specially in connection
with the resorption of formed elements in the tissues and cavities of
animals, Mesnil directed his attention to the digestion of the red
corpuscles of the blood. He made use of the red corpuscles of several
species of Vertebrata, but he made a special study of the digestion of
nucleated red blood corpuscles. These corpuscles are very delicate, and
may even undergo a certain degree of maceration in ordinary sea water.
In spite of this these red corpuscles are not digested in the
coelenteric cavity of the Actinians but, once ingested by the entodermic
cells of the mesenterial filaments, they are completely dissolved by the
intracellular digestion. Mesnil also observed that fibrin is not
digested except in the cells of the filaments. The facts cited by
Chapeaux in favour of an extracellular digestion in the fluid of the
coelenteric cavity in no way support his hypothesis, and reduce
themselves, according to Mesnil, to a digestion by the diastase of blood
itself fixed by the fibrin, after the bleeding, at the moment of the
formation of the clot.
For a certain period the red corpuscles may be met with inside the cells
of the mesenterial filaments. They are ingested in their normal
state—oval red corpuscles with a nucleus. As several hours are required
for the ingestion, it is evident that the fluid of the coelenteric
cavity has been incapable of attacking the red corpuscles. In the
protoplasm of the entodermic cells the red corpuscles become rounded,
their walls become permeable, and the haemoglobin begins to diffuse from
them. It passes first into the vacuoles of the digestive cells and is
then, in part, ejected into the general body cavity. The haemoglobin is
transformed into a green substance which reminds one of biliary pigment.
The membranes and nuclei of the red corpuscles are also digested and
ultimately disappear completely.
The digestive cells of the entoderm ingest not only blood corpuscles or
fibrin, but also fragments of muscular fibre and particles of carmine
and litmus. These latter, as already stated, indicate a marked acid
reaction.
[Sidenote: [61]]
In the Actinians, then, the mesenterial filaments, or rather their
entodermic portion, represent the real organ of intracellular digestion.
There are indeed other regions of the entoderm which also carry on this
function, but in an insignificant degree as compared with the
mesenterial filaments which are capable, however, not only of ingesting
and digesting solid substances, but also of absorbing solutions. Mesnil
has demonstrated this by injecting soluble colouring matters, such as
eosin, carminate of ammonia, etc., into Actinians. These solutions,
although in great part absorbed by the digestive cells of the
mesenterial filaments, can, however, also be retained by other elements,
amongst others, the cells of the ectoderm.
As the digestion of the food-particles goes on within the entodermic
cells of the mesenterial filaments and as these organs can easily be
isolated from the rest of the Actinian, Mesnil was able to study with
great precision and care the phenomena of digestion outside the
organism. With this object he prepared extracts of the filaments in sea
water and studied their action on various nutritive substances. He
confirmed the discovery of a soluble ferment made by Léon Frédéricq and
demonstrated that it is capable of digesting albuminoid substances
(fibrin, coagulated albumen) in media which are neutral, slightly
alkaline or weakly acid. In this respect the _actino-diastase_ (the name
given by Mesnil to the soluble ferment of the Actinians) approaches most
nearly to papain. On the other hand, it is distinguished by its greater
sensitiveness to an excess of acid and also by its more powerful action
on coagulated albumen.
The actino-diastase acts vigorously at any temperature between 15° and
20° C., but the optimum temperature for its digestive action is between
36° and 45° C. Higher temperatures weaken the diastatic power, and
heating to 55–60° C. inhibits it completely. Among the products of the
digestion of albuminoids by actino-diastase, Mesnil, like his
predecessors, found not only a notable quantity of peptone but also
products of the disintegration of the albuminoid molecule, such as
tyrosin and proteino-chromogen. Consequently actino-diastase resembles
Mouton’s amoebo-diastase in certain respects.
[Sidenote: [62]]
The nucleated red blood corpuscles of the lower Vertebrata are very
convenient objects on which to observe the process of intracellular
digestion within the cells of the mesenterial filaments. Mesnil has also
studied them _in vitro_ under the influence of actino-diastase. Under
these conditions the phenomena of digestion recall very clearly those
that have been observed within the digestive cells. The oval red
corpuscles of the fowl and goose become spherical as a result of the
solvent action on their membrane, and the haemoglobin diffuses into the
fluid. The membranes and the nuclei of the corpuscles are, however,
little altered and may be recognised under the microscope. The
difference between this and digestion within the cells reduces itself to
a more feeble digestive action of the aqueous extract. It is evident
that the preparation of this extract is only capable of bringing into
prominence a certain proportion of the actino-diastase contained in the
entodermic cells of the filaments.
Mesnil has fed the same Actinians with repeated doses of blood with a
view to make out whether the cells, under these conditions, acquire any
special aptitude for the production of the actino-diastase.
Notwithstanding numerous attempts, he could never assure himself that
this takes place; the rapidity with which the red corpuscles were
dissolved by the extract of the mesenterial filaments was the same
whether this was prepared from Actinians that had been several times fed
on blood or from those that had received none at all.
From what I have just described no doubt can exist that intracellular
digestion is not a “protoplasmic” process essentially different from
that which is brought about by the digestive juices secreted in the
intestinal canal. In both cases we have a diastatic action, due to
soluble ferments, produced by living elements. In intracellular
digestion, however, the diastases carry on digestion in the interior of
the cells, principally in the vacuoles, whilst in extracellular
digestion this process goes on outside the cells, in the lumen of the
gastro-intestinal canal.
It cannot be doubted that, in the animal scale, intracellular digestion
represents an earlier and primitive condition for the solution of the
food substances. This follows from the fact that it is widely
distributed amongst the lowest animals, such as the Protozoa, Sponges,
Coelenterata and Turbellaria. Intracellular digestion only gives way
step by step to digestion by secreted juices. The higher Invertebrata
furnish us with conclusive testimony on this point. Thus, among the
gasteropod Mollusca, there are some which exhibit the two modes of
digestion in the same animal. In _Phyllirhoë_, a beautiful mollusk,
without a shell and quite transparent, which floats on the surface of
the sea, the food can be seen passing into the cavity of the digestive
canal, where it undergoes a preliminary digestion by secreted juices;
the result is a magma of small solid particles which are at once seized
by the amoeboid epithelium of the coecal appendages, two on each side of
the body. Intracellular digestion then completes the process and ends by
dissolving the nutritive substances and reducing them to their final
stage previous to absorption. On adding to the food some particles of
carmine these may be found along with the digestible particles in the
interior of the epithelial cells of the coeca.
[Sidenote: [63]]
This example furnishes us with a real link between primitive
intracellular digestion and the perfected and derivative extracellular
digestion. In the same group of Gasteropods may be followed out several
stages of this evolution so that in the higher representatives of the
group, such as the slugs and the snails, we meet with digestion carried
on only by secreted juices in the gastro-intestinal contents. In these
Mollusca a voluminous glandular organ, the liver, which is certainly
derived from coecal appendices similar to those of _Phyllirhoë_, is now
met with. Regarded from this point of view the liver is, as Claude
Bernard has stated, an organ of second digestion. I think that a
detailed study of the liver of the Mollusca, guided by this idea, will
give results of considerable importance.
In the Vertebrata intracellular digestion in the gastro-intestinal canal
almost disappears and is replaced by digestion carried on by means of
ferments contained in secreted juices. We cannot, of course, offer to
the reader anything like a complete account of this extracellular
digestion in the higher animals. It is necessary, however, to draw
attention to several aspects of this function which have been
established, thanks to the progress made during recent years, in
obtaining digestive juices and in the study of their action.
[Sidenote: [64]]
For the study of intracellular digestion the sea-anemone is the most
suitable animal for our purpose; for that of extracellular digestion the
dog. In this latter animal, an omnivorous flesh-eater, the food
substances are treated by digestive juices of great activity which
contain a whole series of soluble ferments. The stomach secretes two of
these: rennet and pepsin. The pancreas elaborates three: trypsin,
amylase and saponase, which act on the three main groups of food
substances. To these the small intestine adds a special ferment,
described by Pawloff[83] under the name of enterokynase. Every one
recognises the proteolytic function of pepsin and trypsin and the
analogies and differences between these two diastases. Nor need I dwell
on amylase or on the ferment which saponifies fats. But enterokynase
merits special attention in connection with the study of immunity.
Pawloff entrusted to his pupil Chépowalnikoff the study of the digestive
_rôle_ of the intestinal juice concerning which, up to this, very little
was known. It was known indeed that this juice contained weak
saccharifying and inverting ferments, but it was generally regarded as a
secretion of little importance. Chépowalnikoff[84] has demonstrated that
this view is absolutely erroneous. The intestinal juice fulfils the very
important function of accelerating the action of the three pancreatic
ferments. The duodenal juice of the dog, especially, contains
enterokynase. When this juice is mixed with a pancreatic juice that by
itself actively digests fibrin and albumen, digestion takes place still
more rapidly, the action being from three to four times as great. The
part played by the intestinal juice becomes even more evident when it is
mixed with a pancreatic juice that has little or almost no activity, as
is the case of that from dogs that have recently been operated upon.
Thus pancreatic juice, which has no action upon albumen, digests it
promptly when a certain quantity of duodenal juice is added. When
Chépowalnikoff took 500 c.c. of inactive pancreatic juice diluted with
500 c.c. of water or soda solution and added to it but a single drop of
intestinal juice, the mixture exerted a manifest digestive action on
coagulated albumen.
If, in place of pancreatic juice, we take the aqueous or glycerinated
extract of the pancreas, which by itself exerts a very insignificant
digestive action on albumen, and add to it intestinal juice, digestion
takes place immediately. If it be admitted, as several physiologists
maintain, that the inactivity of the pancreas is due to the fact that we
have zymogen present in place of trypsin, one might conclude with
Chépowalnikoff that “the intestinal juice possesses the power of
transforming the zymogen into trypsin, and that this transformation
takes place in a much more marked degree than in the presence of acids
or the oxygen of the air” (p. 137).
The intestinal juice, from whatever region of the small intestine it be
derived, exercises an undoubtedly favourable influence on the digestion
of starch by the pancreatic juice, but this action is much more feeble
than that on trypsin digestion. The action of the intestinal juice on
the saponification of fats is even less marked. But here it is to the
bile that the more important _rôle_ is transferred. This fluid also
augments the activity of the pancreatic juice, but in a manner different
from the intestinal juice, for it acts especially by accelerating the
digestion of fatty substances.
[Sidenote: [65]]
The action on the pancreatic digestion is not in any way interfered with
when the bile is heated to boiling point. On the other hand the
intestinal juice, under these conditions, completely loses its
accelerating _rôle_. It follows from this, as has been formulated by
Pawloff, that, in the intestinal juice, the existence of a soluble
ferment which is destroyed by heat must be admitted; to this ferment he
proposes to give the name of enterokynase. Without exercising a
digestive power on any of the alimentary substances, it may act as a
ferment of the pancreatic ferments.
Delezenne, at the Pasteur Institute, has repeated Chépowalnikoff’s
experiments. He has confirmed the accuracy of his results and has added
new data of great importance, not only as regards the physiology of
digestion but also in relation to the study of immunity. Enterokynase
appears from Delezenne’s experiments to be a true ferment; carried down
by the same precipitants (collodion, phosphate of lime, alcohol) which
enable us to obtain the greater number of the known ferments; it is
sensitive to high temperatures, and even that of 65° C. is sufficient to
do away with the greater part of its activity. Yet another property of
enterokynase, which it possesses in common with the soluble ferments and
which has for us a very special interest, is the facility with which it
attaches itself to fibrin. By means of flakes of this substance we can
at any time remove from a fluid the whole of the enterokynase contained
therein. This fixative property is very important in connection with the
part which enterokynase plays in digestion. The fibrin to which it has
become attached absorbs trypsin with great avidity. If we introduce
flakes of fibrin impregnated with enterokynase along with other flakes
which have not been in contact with this ferment into a solution of
trypsin, the former are digested with great rapidity, whilst the latter
do not undergo any change. The fibrin that has fixed enterokynase is
capable of clearing a fluid of its trypsin. On the other hand, that
which has not been acted upon by the intestinal juice leaves it there
almost unaltered.
[Sidenote: [66]]
It is of the utmost importance that we should inform ourselves as to the
origin of the enterokynase of the intestinal fluid. This fluid, when
obtained from a fistulous opening, for example, contains mucus and a
considerable amount of _débris_ of various kinds of cells. What are the
elements which furnish such a remarkable ferment? Delezenne has obtained
a very precise answer to this question. The enterokynase is not
contained in the mucus and is not secreted by the intestinal glands; it
comes from the lymphoid organs.
If the small intestine of a fasting dog be washed carefully with water
all the pre-existing enterokynase is removed from it. The Peyer’s
patches are then removed and treated with chloroform water. The other
parts of the small intestine are similarly treated. This fluid dissolves
the enterokynase, as it does the other soluble ferments. We find that
the Peyer’s patches furnish enterokynase, but that the rest of the
intestine, including Lieberkühn’s glands, give none.
We know that the Peyer’s patches are lymphoid organs in which are a
large number of amoeboid mononucleated cells, and that these elements
are even capable of ingesting foreign bodies and of submitting them to
intracellular digestion. It is therefore not at all astonishing that
Delezenne should have succeeded in finding enterokynase in the
mesenteric glands of several Mammals (dog, pig, rabbit). These glands,
when treated by the method just mentioned, yield a substance which
assists the action of trypsin just as does the intestinal juice. Having
reached this point, Delezenne asked himself whether the mononucleated
white corpuscles, so closely allied to the mononucleated cells of the
lymphoid organs, may not also contain enterokynase. With the object of
settling this point he collected exudates that were rich in
mononucleated leucocytes; in these also he found this same soluble
ferment. Moreover, the leucocytic layer of the blood showed itself
equally capable of increasing, very energetically, the action of
trypsin.
The results of the old experiments carried out by Schiff and by Herzen
on the adjuvant _rôle_ of the extract of the spleen in pancreatic
digestion, must without doubt be ranged alongside those we have just
indicated. In fact the mononucleated cells of the spleen, like those of
Peyer’s patches and of the mesenteric glands, contain a substance which
acts like enterokynase. Delezenne has given us a definite demonstration
of its presence and action.
[Sidenote: [67]]
In intracellular digestion it is the chemical side which has been most
difficult of demonstration. The purely physiological functioning, the
sensitiveness of the digestive cells and the amoeboid movements of their
protoplasmic processes are, on the other hand, so manifest that it has
even been suggested that intracellular digestion should be looked upon
as a protoplasmic phenomenon purely vital in character.
In extracellular digestion through the agency of secreted juices we have
a very different condition. Here the chemical side is the striking
feature, the physiological factor being veiled more or less completely.
Nevertheless, thanks to recent advances and above all to the labours of
Pawloff’s disciples in St Petersburg, this problem has been elucidated
in a very remarkable fashion.
The secretion of digestive fluids follows definite laws, the most potent
factor being the reflex action of the nervous system. To use the
expression of Pawloff, the study of the process of salivary secretion
has revealed a real psychology of these organs. You may fill the mouth
of a dog with small polished pebbles or with snow; you may pour into it
very cold water—the saliva will not flow. But merely allow the animal to
see sand in the distance—the glands at once begin to secrete fluid
saliva. Tempt the dog with flesh—and immediately a thick saliva appears;
show him dry bread—saliva is secreted in abundance, even if the dog has
no great desire to eat.
The same phenomena may be observed in the stomach. Mechanical
stimulation by inert bodies, such as stones, provokes no secretion; but
the suggestion of a meal or the sight of food is sufficient to call
forth a large quantity of gastric juice. The quantity and quality of the
gastric juice are regulated by the quantity and quality of the food.
Bread given to a dog provokes the secretion of a gastric juice endowed
with the greatest digestive power. That which flows after the ingestion
of milk contains only one-fourth as much pepsin.
[Sidenote: [68]]
In spite of these differences in the gastric secretion in relation to
food, Pawloff and his pupils have never been able to assure themselves
that there was any prolonged and chronic adaptation of the gastric
function. They were struck by the uniformity of the digestive power of a
great number of their dogs. Samoïloff[85] had under observation three
dogs placed on different diets. In spite of the very long periods during
which these diets were given, the gastric juice, in all the dogs,
presented the same properties and manifested no appreciable difference.
This result harmonises with that indicated above as obtained in the
Actinians fed with blood by Mesnil. In spite of repeated feedings on
blood from the same species of animal, the extract from the mesenterial
filaments was in no way different from that of the fasting Actinians
used for control.
The pancreatic secretion is, in many respects, a more perfect type. We
have here to do with the principal agent in the digestive function,
without which the organism could not continue to exist. The advances
made in surgery have enabled us to remove the stomach, first in the dog
and then in man, and there are already several persons[86] from whom the
stomach has been removed and who, in spite of this operation, have
continued to live. A portion of the small intestine may also be removed,
but, in order that life may not be endangered, a considerable portion of
it must be left intact. It is evident then that the pancreatic digestion
is an admirably organised function both in animals and in man. One of
the main regulators of this process of digestion consists in the great
sensitiveness of the intestinal mucous membrane. Just as the organs of
the buccal cavity possess in the specific sense of taste an excellent
means of discrimination in the choice of foods, so the mucous membrane
of the small intestine is endowed with a special sensitiveness,
comparable to the chemiotaxis of unicellular organisms and of the cells
of more highly developed organisms. Hirsch and Mehring have satisfied
themselves that the passage of the contents of the stomach through the
pyloric orifice depends on a reflex mechanism which proceeds from the
upper reaches of the small intestine. To the researches of the school of
Pawloff, however, we owe what light has been thrown on this question.
The duodenal mucous membrane is endowed with a well-developed
chemiotaxis for acid substances. The passage of the acid content of the
stomach into the duodenum determines this chemiotaxis and brings about a
secretion of alkaline juice which neutralises the acid. This contest
between acid and alkali forcibly calls to our mind the analogous
phenomena in those plants that defend themselves against the alkaline
secretions of parasites by the production of an acid (see Chapter II).
As in these lower organisms, this battle of the chemical secretions is
regulated by the action of living and sensitive parts.
When the acidity of the mass which passes through the pylorus is too
marked, the reflex contraction starting from the duodenal mucosa arrests
its passage. Then takes place a neutralisation of the acid, thanks to
the alkaline secretion, and the pylorus is again allowed to open. This
mechanism thus regulates the passage of the contents of the stomach into
the duodenum, the passage taking place in instalments.
[Sidenote: [69]]
The sensitive intestinal mucous membrane can estimate not only the
degree of acidity, but also the other chemical characters of the
aliments which pass into the duodenum. This chemiotaxis is, as it
were, the starting-point of the reflex action which excites the
pancreatic secretion with its contained three ferments. The passage of
bread through the pylorus excites the secretion of a juice very rich
in amylase and very poor in saponase. The passage of milk into the
duodenum brings forth, on the other hand, a juice very much richer in
saponase but poorer in amylase and in trypsin. Flesh-meat provokes the
secretion of a pancreatic juice which is less rich in amylase than the
juice poured on bread, but richer in saponase. Fat causes the
secretion of a juice still richer in saponase than is the juice poured
out in the presence of bread or milk. These facts now carefully
established—especially by Walter[87]—demonstrate that the pancreatic
function is carefully regulated as regards its adaptation to the
characters of the food substances on which it is to act. Such
adaptation may even become permanent.
Whilst, as already stated, the stomach, under the influence of a fixed
diet, is incapable of effecting any lasting modification in the
composition of its secreted juice, the pancreas may reach this degree of
perfection. When a dog is fed for several weeks on bread or on milk and
is then placed on flesh diet its pancreatic juice is found to become
progressively richer in trypsin. Whilst this augmentation of the
proteolytic power is being brought about, the juice becomes poorer and
poorer in amylase. Wassilieff[88] has carried out a large number of
experiments on this point and has demonstrated a very remarkable
adaptation of the pancreatic juice to the wants of nutrition, an
adaptation that may become permanent. A dog which has been accustomed to
digest bread and milk adapts itself to this nourishment: its pancreatic
juice contains less and less trypsin, but, on the other hand, becomes
richer in amylase. Pawloff observed that in dogs great variations in the
composition of the pancreatic juice are often present; this he
attributes to the diet to which these animals had been previously
subjected.
[Sidenote: [70]]
Not only does the quality of the digestive juices accommodate itself to
the wants of digestion; their quantity also undergoes variations
according to the part that these juices have to play. Thus, Pawloff has
observed that his dogs secreted a saliva which was very fluid and very
abundant when he gave them acids, bitter substances or other substances
they did not like. On the other hand, the presence of food in the mouth,
or even the sight of it, excited the secretion of a thick saliva
containing a large quantity of mucin. In the first case the part played
by the saliva was that of diluting the injurious substances as much as
possible, in the second that of facilitating the deglutition of the
food.
In general the organism manifests a tendency to produce more digestive
ferments than it actually needs for digestion. It is for this reason
probably that they are often found outside the digestive canal. Among
these ferments pepsin and amylase, especially, have been definitely
proved to be present in the urine of man and of some mammals, notably
the dog. The data as to rennet and trypsin are not so well established.
But, as several of these ferments, such as amylase and trypsin, may be
derived from several sources in the organism, their elimination by the
urine is less important for the thesis I have just formulated than is
that of pepsin.
Pepsin was found in the urine by Brücke exactly forty years ago. It is
more frequently found in the morning urine, but is absent from that
passed immediately after the principal meal. Leo and Senator[89] found
only traces of pepsin during the prolonged fast of the Italian Cetti;
but the day he broke his fast they were able to demonstrate the presence
of a considerable quantity of this ferment in his urine.
[Sidenote: [71]]
Delezenne and Froin, with the object of seeking the source of the
urinary pepsin, extirpated the stomach of a dog. After the animal had
recovered, they fed it well and examined its urine at different periods
of the day. By the methods which had shown the presence of pepsin in all
the normal dogs taken as controls they could never discover the faintest
trace of this diastase in the urine of the dog that had been operated
upon. On the other hand, the urine of a dog whose stomach had simply
been isolated, contained very much the same quantity of pepsin as that
of normal dogs. This experiment proved among other things that the
pepsin, before it could be eliminated by the kidneys, must have been
re-absorbed by the wall of the stomach. From these data, combined, it
must therefore be admitted that the pepsin found in the blood and which
passes thence into the urine can only be of gastric origin. As it serves
no useful purpose in the organism we must conclude that a portion of the
pepsin, secreted by the stomach and not used for digestion, has been
rejected as superfluous.
The study of the digestive function of animals gives us information on a
large number of points of the highest importance for the comprehension
of immunity. Intracellular digestion, a function so widely distributed
in the lower animals, is very intimately connected with the phenomena
which are observed when micro-organisms are destroyed in the animal
organism. Extracellular digestion furnishes us with information
concerning many of the features of progressive adaptation, similar to
those which are observed in connection with acquired immunity.
When we examine the phenomena of intracellular digestion and those of
secretory digestion as a whole, we see that, in both, the chemical
processes are subjected to the influence of the living parts of the
organism. In the lower animals, it is the protoplasm of the amoeboid
cells which regulates the chemical processes in digestion; in the higher
animals, this _rôle_ is taken by a very complicated apparatus, in which
the nervous system plays a predominant part.
CHAPTER IV
RESORPTION OF THE FORMED ELEMENTS
Digestion in the tissues.—Resorption of cells in the
Invertebrata.—Resorption of red corpuscles by the phagocytes
of the Vertebrata.—Phagocytes.—Various categories of these
cells.—Macrophages and microphages.—Part played by macrophages
in the resorption of the formed elements.—Digestive property
of the macrophagic organs.—Solution of the red blood
corpuscles by the blood serums.—The two substances which
operate in haemolysis.—Macrocytase and fixative.—Analogy of
the latter with enterokynase.—Escape of the macrocytase during
phagolysis.—Suppression of phagolysis.—Resorption of the
spermatozoa.—Presence of fixatives in plasmas.—Origin of
fixatives.
[Sidenote: [72]]
It is usually understood that nutritive substances must necessarily be
subjected to the influence of the digestive juices in the
gastrointestinal canal before they can be utilised for the nutrition of
the organism. This is a very old idea. It was based on a well-known
experiment by Schiff who injected several animals intravenously with
solutions of cane sugar and egg albumen and others with the same
substances after they had been artificially digested. In the first case
the food substances passed into the urine, in the second they only
appeared there when injected in large quantities.
[Sidenote: [73]]
At the recent International Congress of Medicine held in Paris in 1900,
the question of extra-buccal nutrition was much discussed[90]. It has
been accepted that fats, when injected into the subcutaneous tissues,
are, at least in part, absorbed by the organism, but that carbo-hydrates
and albuminoids are never absorbed. This is perhaps true from the point
of view of clinical medicine. But, in principle, it must be admitted
that food substances of very diverse natures, when introduced into the
organism by channels other than the gastrointestinal canal, still
undergo profound changes.
When we inject milk, blood serum, or white of egg, that is to say,
materials very rich in albuminoid substances, under the skin or into the
peritoneal cavity of laboratory animals, we find that after a time they
disappear. At the same time they give rise to modifications of the
organism which indicate that these injected substances have there
undergone profound changes.
After injecting eel’s serum into rabbits, Th. Tchistovitch[91] found a
substance in the blood of the injected animals which gave a precipitate
with eel’s serum. Shortly afterwards Bordet[92] observed that the blood
of animals into which he had injected cow’s milk acquired a new
property: it gave a precipitate with this milk, a condition never
observed in the serum of untreated animals.
The injection of white of egg into rabbits, carried out by Myers[93] and
Uhlenhuth[94], brought about the same changes in the blood serum. The
researches of the latter of these two observers have for our present
purpose a special interest. He demonstrated first that the injection of
white of egg into the peritoneal cavity of rabbits was followed by the
appearance in the blood serum of these animals of a substance which
precipitates egg albumen _in vitro_. Uhlenhuth then obtained this same
acquired property of the blood in rabbits which had been made to swallow
a considerable quantity of the white of hens’ eggs. Twenty-four days
after the commencement of this regimen the serum of the rabbits
precipitated white of egg in the test-tube. This example affords a
marked analogy between the results of digestion in the alimentary canal
and those of resorption into the tissues. Uhlenhuth points out, indeed,
that his rabbits which received the injections of white of egg into the
peritoneal cavity flourished under this treatment.
[Sidenote: [74]]
A certain number of similar examples are now recognised. They all
indicate that various nutritive substances, when introduced into the
peritoneal cavity or under the skin of animals, are retained there for a
longer or shorter time and are subjected to certain modifying influences
on the part of the organism. The proof that these substances are not
eliminated intact by the kidneys has been furnished by a large number of
experiments. Recently Lindemann[95] and Néfédieff[96], working in my
laboratory, have established the fact that normal blood serum, when
injected under the skin of animals, does not provoke albuminuria at all,
or at least produces it in a very insignificant and transitory degree.
The mechanism by which the organism modifies these nutritive substances,
introduced by a channel other than the digestive canal, is not as yet
sufficiently known; and is therefore not easy to define. But we know,
very definitely, that each injection of serum, whether of white of egg,
milk or fatty matter, is followed by a rather considerable aseptic
inflammation at the point at which these substances are introduced. We
might conclude from this that the organism digests the food substances
outside the gastro-intestinal canal, by means of an inflammatory
reaction. In order to determine more exactly the phenomena that appear
under these conditions, it may be useful to consider first, not the
fluid substances but the solid elements that are introduced into the
tissues and cavities.
Let us begin with the lower animals in which the anatomical organisation
and all the functions are of a much more simple character than they are
in the Vertebrata. In my _Comparative Pathology of Inflammation_
(Lecture IV) I have directed some attention to the digestion of the
Sponges.
The nutritive substances—small organisms—whether they may have entered
by the small openings, so numerous on the surface of Sponges, or have
been introduced through a rent in the body wall, undergo the same fate.
They are seized by vibratile or amoeboid cells which ingest the food and
digest it by an intracellular digestion. These two kinds of cells, which
come under the category of _Phagocytes_, have a great resemblance to one
another, and we may say that digestion and resorption are two very
closely related phenomena.
[Sidenote: [75]]
When we examine somewhat higher Invertebrata, such as the Medusae or
certain other Coelenterates, we can still trace a close analogy between
the true digestion of the food that goes on within the epithelial cells
of the entoderm and the resorption of certain foreign bodies which make
their way by an extra-buccal channel into the intermediary tissue. Here
these bodies are surrounded by amoeboid cells which fulfil their
function as phagocytes by ingesting and digesting the substances that
have come from outside.
It is, here, unnecessary to go over the whole gamut of the perfecting of
the organisation of the Invertebrata, in its relation to the resorption
of foreign bodies, especially as it has already been treated in my
Lectures on Inflammation. Let us choose merely some of the more common
and better-known representatives of the Invertebrata and dwell for a few
moments on the phenomena manifested in their organism, into the midst of
which have been introduced a few nucleated red blood corpuscles[97].
If a small drop of defibrinated blood from a goose be injected beneath
the skin of a snail and another under the skin of a cockchafer larva,
the red corpuscles are disseminated in the blood fluid which, of itself,
is incapable of modifying them, but at the end of a few hours the
leucocytes of the two invertebrates that we have chosen for the
experiment will have ingested a certain number of the injected red blood
corpuscles. The next day red blood corpuscles are still to be found
intact in the blood plasma, but the great majority have been devoured by
the leucocytes (Fig. 13). Inside these cells the red corpuscles undergo
constant and marked changes. In the snail they become round and their
walls permeable. In the vacuoles that are produced around the ingested
red corpuscles dissolved haemoglobin is found (Fig. 14); a portion of
this colouring matter passes into the nucleus of the red corpuscles, so
that it also has undergone a profound change (Fig. 14). Many of the
nuclei become emptied, only the peripheral layer remaining. This layer
and the membrane of the red corpuscle are the parts that resist the
action of the leucocytes longest and they are found for some time after
their ingestion. The white corpuscles of the snail, having devoured one
or more red corpuscles, may themselves become the prey of their fellows.
[Sidenote: [76]]
[Sidenote: [77]]
In the “ver blanc” (French popular name for the larva of the cockchafer)
the phenomena of resorption of the red corpuscles of the goose resemble
those just described. The blood plasma leaves intact the red corpuscles
which undergo no change until they have been ingested by the leucocytes.
The haemoglobin diffuses into the leucocyte, whilst the nucleus and the
membrane persist for a very considerable period (Fig. 15), though they
lose their normal aspect, shrivel, and become transformed into an
irregular mass of brown pigment which may remain in the substance of the
leucocyte (Fig. 15, _p_) for weeks.
[Illustration:
FIG. 13. Leucocytes of a cockchafer larva containing red blood
corpuscles of a goose.
]
[Illustration:
FIG. 14. Red blood corpuscles of a goose, free, and ingested by
leucocytes of a snail (_Helix pomatia_), 24 hours after their
injection.
]
[Illustration:
FIG. 15. Leucocyte of a cockchafer larva, 7 days after last injection
of goose’s blood.
]
[Illustration:
FIG. 16. Leucocyte from peritoneal cavity of a gold-fish after
ingesting red blood corpuscles of a guinea-pig.
]
Having once injected goose’s blood into snails and “vers blancs,” if we
repeat the injection several times, the phenomena observed are
invariably the same. The red corpuscles are unacted upon by the plasma
and undergo the same changes within the leucocytes. These changes are in
fact comparable to those described in the preceding chapter in
discussing the intracellular digestion of the red corpuscles by the
intestinal cells of the Planarians. In both cases the red corpuscles are
seized by amoeboid cells and subjected to the influence of their
contents. In the intestinal phagocytes of the Planarian, as in the
phagocytes of the blood (leucocytes) of the snail and “ver blanc,” the
haemoglobin diffuses through the wall of the red corpuscle, whose most
resistant parts are the nucleus and the membrane. These resistant
residual fragments, impregnated with haemoglobin, become brown in the
Planarian, in the “ver blanc,” and also, but in a less degree, in the
snail. The most appreciable difference consists in the formation of
excretory vacuoles, containing concretions, in the Planarian, and the
absence of these vacuoles in the blood phagocytes of the other
Invertebrata. We have, however, less right to attribute a fundamental
importance to this difference, in that the phenomena in the Actinians,
which ingest the red blood corpuscles by the amoeboid cells of their
entoderm, are in all respects (with the exception of the presence of
these special excretory vacuoles) comparable to the phenomena observed
in the Planarians. From the fact that in these two examples we have to
do with a true intracellular digestion, it must be admitted that the
modifications of the red blood corpuscles within the phagocytes of the
blood in the snail and in the larva of the cockchafer, must also be
placed in the same category of phenomena.
[Sidenote: [78]]
In order to make a more thorough study of this intracellular digestion
in the phagocytes of the blood, we must direct our attention to larger
and more highly organised animals than the snail and the “ver blanc.”
Let us take, first, an example among the inferior cold-blooded
Vertebrata. The red blood corpuscles of a few drops (0·25 c.c.) of the
blood of a guinea-pig injected into the peritoneal cavity of a gold-fish
(_Cyprinus auratus_) are not appreciably changed by the peritoneal fluid
itself; but the numerous leucocytes that are found in the peritoneal
fluid seize them and ingest them, just as do the phagocytes of the blood
of Invertebrata, or the intestinal phagocytes in the Planarians and
Actinians in the case of the red blood corpuscles of the goose. Each
leucocyte of the _Cyprinus_ ingests several red blood corpuscles and
subjects them to intracellular digestion. The stroma of the red
corpuscles becomes permeable; the haemoglobin diffuses into the
nutritive vacuoles and at the end of a shorter or longer period the
whole is dissolved and decolorised (Fig. 16). Here no brown pigment is
produced and the red corpuscles are completely digested, leaving no
“remains”; in this respect differing from the process in the
Invertebrata mentioned.
This result depends, probably, partly upon the more feeble resistance
offered by the non-nucleated red corpuscles of Mammals, and partly upon
the more active digestive power of the leucocytes of Fishes.
As the result of several injections of guinea-pig’s blood into the
peritoneal cavity of _Cyprinus_, the peritoneal fluid acquires new
properties[98]. If, a fortnight after the first injection, a little of
the peritoneal exudation in the gold-fish be withdrawn, it is found that
a drop of the serum which floats on the surface produces, almost
immediately, well-marked agglutination of the red corpuscles of the
guinea-pig, this being soon followed by the rapid solution of these red
blood corpuscles in the fluid. This new property, which does not exist
in the untreated fish, also makes its appearance in the blood serum of
_Cyprini_ treated with guinea-pig’s blood. The experiment is very
successful at a temperature of 18°–19° C.
As the solution or lysis of the red blood corpuscles in the serum is
exactly like that which takes place within the leucocytes of _Cyprinus_,
we are justified in assuming that, in both cases, it is produced by the
same substance. And, since the solvent or haemolytic power of the serum
is only acquired as the result of the intracellular digestion of the red
blood corpuscles by the leucocytes, it is probable that the solvent
substance represents the intracellular ferment derived from the
leucocytes.
[Sidenote: [79]]
The subject we have just broached is of fundamental importance in
connection with the study of resorption and of the phenomena of immunity
dependent upon it. It is necessary, therefore, that we should go more
fully into its analysis. With this object we must first review the
processes that go on during resorption in the higher animals and
continue our examination of the changes that injected or extravasated
blood undergoes in various positions of the organism.
This study is rendered comparatively easy for us by the numerous
researches that have been carried out by pathological anatomists for the
purpose of ascertaining the fate of effusions or extravasations of blood
so frequently met with in disease. It has long been known that in
subcutaneous, cerebral and other haemorrhages, or in hepatised lungs,
there are found in the escaped blood a great number of cells containing
red corpuscles. As was mentioned in the preceding chapter, these cells
were evidently amoeboid cells that had ingested red blood corpuscles. To
Langhans[99] especially we owe a detailed study of the phenomena that
follow extravasation of blood produced artificially in the subcutaneous
tissue of the pigeon, rabbit and guinea-pig. In all these animals the
haemorrhage is early followed by exudative inflammation, during which
the leucocytes come up in great numbers and ingest the red blood
corpuscles which are modified in the interior of the leucocytes. There
is a formation or deposition of pigment and finally all traces of the
red corpuscles disappear. In Mammals the pigment is brown or brownish,
just as it is in the Planarians and in the “ver blanc”; in the pigeon it
is green and resembles that found in the Actinians. In short there is a
great analogy between the resorption of red corpuscles and the true
intracellular digestion of the red blood corpuscles that goes on in the
intestinal cells of the Invertebrata.
But what is the nature of these amoeboid elements that intervene in the
resorption of the extravasated blood? At the period when Langhans
carried out his investigation, we were unable to differentiate the cells
at all satisfactorily. It is only since the publication of Ehrlich’s
classic researches on the white corpuscles that we have been able to
bring more order into this question. Thanks to the use of various
aniline stains, Ehrlich was able to arrange the leucocytes found in the
Vertebrata into several definite groups.
The question has already been touched upon in our eighth lecture on
inflammation; it is therefore unnecessary to treat it here at length. We
must, however, before entering on the analysis of the essential
phenomena in the resorption of cells, as we now understand them, give a
rapid survey of the different varieties of amoeboid cells that are found
in the Vertebrata.
[Sidenote: [80]]
Beside mobile amoeboid cells, represented by several forms of white
corpuscles, we must distinguish fixed amoeboid cells. These are
permanently fixed in certain situations in the body; this, however, in
no way prevents them from throwing out amoeboid processes in various
directions and seizing foreign bodies or certain elements of the same
organism. The nerve cells, the large cells of the splenic pulp and of
the lymphatic glands, certain endothelial cells, the cells of the
neuroglia, and perhaps some connective tissue cells, belong to the
category of fixed amoeboid cells. All these elements, under certain
conditions, are able to ingest solid bodies; consequently, they act as
phagocytes. With the exception of the cells of the nerve centres, all
these fixed phagocytes are of mesoblastic origin. It has been much
discussed whether certain processes of the nerve cells may not really
serve to seize foreign bodies and carry them into the cell contents. It
appears to us that sometimes they undoubtedly do fulfil this function.
For example, it is only by means of such amoeboid movements that leprosy
bacilli can be introduced into the interior of ganglion cells and cells
of the spinal cord[100]. We must not dwell on this question, as the
phagocytic property of the nerve elements plays no part in the
resorption of cells. On the other hand, the neuroglia cells contribute
largely to this process and their phagocytic function is now admitted by
many observers[101].
For long the large “dust” cells of the respiratory channels were looked
upon as being epithelial cells which were capable of ingesting carbon
particles, micro-organisms and other foreign bodies. The researches of
N. Tchistovitch, carried out in my laboratory more than twelve years
ago, made it evident that these elements are nothing more than white
corpuscles that have immigrated into the alveoli and bronchi.
[Sidenote: [81]]
It is probable that the same is the case as regards the stellate cells
of the liver, known as Kupffer’s cells. First described by Kupffer as
cells of a nervous type, having long processes, they were later
recognised by several observers as belonging to the endothelial tissue
of the blood vessels of the liver. Kupffer[102] himself has accepted
this view and in his recently published monograph on these stellate
cells, he describes them as endothelial cells that have retained their
independence. Some researches on the resorption of blood, of which I
shall speak shortly, have led me to think that these cells are nothing
but white corpuscles that have been arrested in the hepatic capillaries.
I have asked Mesnil, head of my laboratory, to study this question for
me. His investigation is not yet concluded, but the demonstration
already made that the livers of guinea-pig embryos and new-born rabbits
do not possess any Kupffer’s cells is an argument in favour of my
hypothesis.
Certain white corpuscles have undoubtedly been often mistaken for
epithelial or connective tissue cells. We must not conclude from this,
however, that these elements are never capable of sending out amoeboid
processes and of ingesting foreign bodies. It would, however, be useful
to collect new and incontestable proofs of the accuracy of this thesis.
In spite of this uncertainty, it may be accepted as fully demonstrated,
that certain fixed amoeboid cells, such as the large elements of the
splenic pulp, of the lymphatic glands, and of the omentum, play an
important part in the resorption of cells. It is there that elements
filled with red corpuscles and white corpuscles in process of being
destroyed are so often found.
[Sidenote: [82]]
Just as certain fixed cells do not function as true phagocytes, so also
in some leucocytes this function is undoubtedly absent. The suggestion
has been made several times that any cell element, provided it be young,
is capable of ingesting foreign bodies. The examination of white
corpuscles proves exactly the contrary. The smaller white corpuscles
found in fairly large numbers in the blood and the lymph, and which are
commonly known as _lymphocytes_ or _small lymphocytes_, are simply
leucocytes with very little protoplasm which in this state never fulfil
phagocytic functions. It is only when it becomes older, when its
nucleus, single and rich in chromatin, becomes surrounded by an ample
layer of protoplasm, that the lymphocyte becomes capable of ingesting
and resorbing foreign bodies. Several authors, with Ehrlich at their
head, still assign to these larger cells the same name—lymphocytes.
Others, however, give them the name of large mononuclear cells.
Confusion is thus possible, especially as Ehrlich includes under the
same term the large mononucleated leucocyte, a very rare form of cell in
human blood, which is distinguished by the greater staining capacity of
its nucleus. To avoid this inconvenience I propose to designate the
large lymphocytes by the name of blood macrophages and lymph macrophages
(_haemomacrophages_, _lymphomacrophages_). This term is preferable to
that of mononuclear leucocytes, especially as in exudations we
frequently meet with macrophages with two and even several sharply
separated nuclei. Giant cells, moreover, are nothing but polynucleated
macrophages. On the other hand, the leucocytes so often designated by
the name of polynuclear in reality contain but a single nucleus. Even
Ehrlich, who introduced this term, acknowledged its imperfection but he
retained it for some time because it was already very extensively used
and could, he thought, give rise to no misunderstanding. In his
excellent work on anaemia, published jointly with Lazarus[103], he now
agrees that the name of “cells with polymorphous nuclei” would be more
exact.
These polymorpho-nuclear leucocytes are very numerous in the blood and
in many exudations and are distinguished by the greater selective
affinity of their nucleus for basic aniline dyes and by a certain
tendency of the protoplasm to become stained by acid aniline colours,
such as eosin. The true macrophages are without granulations, but the
“polymorpho-nuclear” leucocytes contain many. These granulations are
sometimes “eosinophile,” “pseudo-eosinophile” (or “amphophile”) or even
“neutrophile” (as in man and the horse).
[Sidenote: [83]]
These two main groups of leucocytes are generally distributed in the
Vertebrata; and we already meet with them in one of the lowest
vertebrate forms—the _Ammocoetes_ (the larva of the lamprey). The
macrophages of this fish present all the principal characters of the
group to which they belong (protoplasm without granules, easily stained
with methylene blue, large nucleus rich in nuclear juice). In the
“polynuclear” forms in this lower vertebrate the protoplasm does not
stain with methylene blue, but assumes a faint rosy tint with eosin; the
single nucleus is divided into several lobes. In Vertebrates which are
much higher in the scale these characters change. Thus in the cayman
(_Alligator mississipiensis_), according to the researches of Madame
Podwyssotsky, carried out in my laboratory, the two great varieties of
leucocytes are readily found in the blood, lymph and exudations. The
macrophages, however, especially in the exudations, are very often
furnished with two or several nuclei, whilst the small leucocytes
possess only a single nucleus, which is not divided into lobes. In spite
of this peculiarity the two groups are readily distinguished. The
staining reactions of the macrophages are identical with those of the
corresponding corpuscles in all the other Vertebrata; whilst the small
leucocytes, in spite of the absence of a polymorphous nucleus, are
easily recognised by their eosinophile granulations and by the special
affinity of the nucleus for basic aniline dyes. Under these
circumstances it would be quite inappropriate to designate those
leucocytes, which are really polynuclear, that is to say, possessing two
or several nuclei, by the name of “mononuclear,” and to reserve the name
of “polynuclear” for the small corpuscles which possess only a single
nucleus undivided into lobes. For this reason it is much more rational
to retain for these so-called polynuclear cells my proposed name of
_microphages_. Moreover, the microphages are true phagocytes. It was
formerly thought that the eosinophile leucocytes, such as the “‘overfed’
cells (Mastzellen)” of Ehrlich, which are identical with the
_clasmatocytes_ of Ranvier, never ingested foreign bodies. But,
(especially after the researches of Mesnil[104]), we have been compelled
to change our opinion on this point. The true eosinophile cells are able
to devour foreign bodies, especially micro-organisms, and must therefore
be regarded as phagocytes belonging to the group of microphages.
[Sidenote: [84]]
It is the peculiar merit of Ehrlich and of his school that they have
thoroughly established the fact that, in Mammals at any rate, the two
principal groups of white cells are distinguished, amongst other
characters, by the diversity of their origin. The lymphocytes and the
mononuclear cells are developed in the spleen and lymphatic glands,
whilst the “polynuclear” cells arise from the granular mononucleated
myelocytes of the bone marrow. This is now generally accepted as
applicable in the great majority of cases. In _Ammocoetes_, however, the
two chief varieties of leucocytes arise from one and the same organ,
regarded by several observers as a kind of primitive spleen, which runs
along and in part surrounds the intestine. Mesnil has been good enough
to make sections of this primitive organ in which it may be demonstrated
that the macrophages and the microphages in the larva of the lamprey
have the same seat of origin. Frog tadpoles and Cartilaginous Fishes
also possess microphages which do not arise from the bone marrow, since
in them this tissue is completely absent. But even in Mammals, at least
in certain pathological conditions, Dominici[105], in a research
executed with much care and a perfect technique, has demonstrated the
myelogenous transformation going on in the spleen. Thus in the adult
rabbit affected with septicaemia by the typhoid bacillus, he found in
the spleen developmental centres of amoeboid elements which, normally,
appear to develop in the bone marrow only, _i.e._ the megacaryocytes, or
large cells with budding nuclei, the neutrophile myelocytes
(amphophiles), basophiles and eosinophiles.
The mesoblastic phagocytes of the Vertebrata are divided, then, into
fixed phagocytes—the macrophages of the spleen, endothelia, connective
tissue, neuroglia, and muscle fibres—and free phagocytes. These latter
are sometimes haemo- or lympho-macrophages, sometimes microphages. The
fixed macrophages and the free macrophages resemble one another so
greatly that it is very often extremely difficult, if not impossible, to
differentiate them. For this reason it is often very useful, when the
exact origin of a large phagocyte is not known, simply to name it
“macrophage.”
[Sidenote: [85]]
The two principal groups of phagocytes—(1) fixed and free macrophages,
(2) microphages—are distinguished not only by their morphological
characters; they also give evidence of very marked physiological
differences. All phagocytes are endowed with amoeboid movement which
allows them either to move about freely or merely to put out
protoplasmic processes. These movements are regulated by a very great
sensitiveness, often different in the two groups. Besides a tactile
sense, the phagocytes possess a kind of sense of taste or chemiotaxis
which enables them to distinguish the chemical composition of the
substances with which they come in contact. The existence of this
chemiotaxis could be anticipated from the moment that an important part
in the life of the organism began to be ascribed to the amoeboid cells.
Leber[106], Massart and Charles Bordet[107] have, however, demonstrated
it by rigorous experiment. Following the method used by Pfeffer to
demonstrate the chemiotaxis of the vegetable spermatozoids and of
Bacteria, these investigators introduced into the bodies of higher
(rabbits and guinea-pigs) and lower (frogs) Vertebrates small glass
tubes filled with different solutions (peptone, broth, salts, bacterial
products, etc.). The leucocytes, guided by their positive chemiotaxis,
made their way into the tubes and there formed plugs which were often
very voluminous; when, on the other hand, the chemical composition of
the solutions excited their negative chemiotaxis, the leucocytes avoided
the tubes.
[Sidenote: [86]]
Having acquired information as to the chief characters of the
leucocytes, we may ask, To which group do those amoeboid cells, which,
according to the observations of Langhans and many other investigators,
bring about the resorption of the red corpuscles of the blood, belong?
This resorption goes on more rapidly and is observed much better if,
instead of introducing blood of the same species into any part, we
inject defibrinated blood, or red blood corpuscles from which the serum
has been removed by washing, from another species of Vertebrate. It will
be found best to inject the nucleated red corpuscles of lower
Vertebrates into Mammals, or (as already described above) to introduce
the non-nucleated red blood corpuscles of Mammals into lower
Vertebrates. In all these cases the injection of such blood or
corpuscles sets up an aseptic inflammation which attracts a large number
of free phagocytes to the seat of injection. In subcutaneous, peritoneal
or intraocular exudations produced under these conditions, we find, in
addition to a number of microphages, many macrophages. Whilst the former
ingest the injected red corpuscles merely in isolated cases, the
positive chemiotaxis of the macrophages manifests itself much more
actively. In the resorption of the red blood corpuscles the more
important part is played by the macrophage. To get a clear idea of the
phenomena that accompany this resorption, let us take a concrete
example. Inject defibrinated goose’s blood into the peritoneal cavity of
guinea-pigs[108]. During the first few hours after injection the oval
nucleated red corpuscles are found intact in the fluid of the peritoneal
lymph. The plasma, by itself, exercises no destructive or solvent action
on the red corpuscles of the goose.
Immediately after the injection the lymph of the peritoneal cavity
begins to show important changes. The white corpuscles which, in the
normal condition, are fairly abundant, disappear almost completely; some
small lymphocytes presenting their ordinary aspect may indeed be found,
but the few macrophages and the microphages that remain show signs of
very grave lesions. They lose their mobility, run together into clumps
and become incapable of ingesting foreign bodies. At this moment the
phagocytes undergo a critical change which we have designated by the
name of _phagolysis_. This condition lasts for about an hour, sometimes
it continues longer, according to case and circumstance, but after this
the peritoneal fluid becomes filled with leucocytes that have newly come
on to the scene. These cells make their way, by diapedesis, through the
walls of the congested vessels of the peritoneum. A true aseptic
inflammation is produced which induces an exudation of a large number of
white corpuscles, amongst which are found microphages and still more
numerous macrophages. The latter show a very pronounced positive
chemiotaxis towards the injected red corpuscles of the goose. Soon after
their appearance, that is to say two or three hours after the injection
of the blood, the macrophages send out very small protoplasmic processes
and affix them to the surface of the red corpuscles. There follows an
aggregation of the macrophages of the guinea-pig with the red corpuscles
of the goose and characteristic masses, in which can be recognised both
kinds of cells, are produced. This union with the very small pseudopodia
is the first stage in the ingestion of the red corpuscles by the
macrophages (Fig. 17). The red corpuscle, seized by amoeboid processes,
passes into the interior of the macrophage. This macrophage seldom rests
contented with ingesting a single red corpuscle. Usually it devours a
large number and sometimes enormous macrophages may be seen filled with
a score of red corpuscles.
[Sidenote: [87]]
If the quantity of goose’s blood injected into a guinea-pig is large
(5–7 c.c.), the ingestion of red corpuscles by the macrophages continues
for a considerable period—often for three to four days. During the whole
of this time a certain number of the red corpuscles remain free in the
peritoneal plasma, but, in spite of this prolonged stay, none of them
undergo extracellular solution.
[Illustration:
FIG. 17. Macrophage of guinea-pig in process of devouring and
digesting red blood corpuscles of goose.
]
[Illustration:
FIG. 18. Macrophage of guinea-pig in the act of ingesting and
digesting red corpuscles of goose. _Intra vitam_ staining with
neutral red.
]
[Sidenote: [88]]
The red blood corpuscles, anchored by the amoeboid processes of the
macrophages, at first present a normal appearance. Later their membrane
begins to wrinkle, but as soon as they have passed within the phagocytes
the wrinkles disappear and the corpuscles regain their normal aspect. If
a little neutral red solution be added to a drop of peritoneal exudation
(Fig. 18) we observe that the nucleus of the ingested red corpuscle and
even its contents are stained red, whilst the red corpuscles adherent to
the surface of the phagocytes retain their normal yellow colour. This
reaction enables us to see that the red corpuscles are seized by the
macrophages whilst still in their normal condition, but that they
undergo a change immediately after they have been ingested. Little by
little the devoured corpuscles are digested within the phagocytes. The
haemoglobin diffuses into the contents of the macrophage through the
stroma, which has become permeable; the nucleus of the ingested red
corpuscle also becomes stained by the haemoglobin. Part of this
colouring matter is excreted by the phagocyte. The body of the red
corpuscle is pretty soon digested, but the nucleus, impregnated with
haemoglobin, persists for a much longer period. It divides into several
fragments, recognisable by their yellow colour, and in certain cases
these remnants of red corpuscles may be met with for weeks in the
interior of the macrophages. These macrophages do not remain permanently
in the peritoneal fluid. Some (3–4) days after injection the lymph of
the peritoneum contains only leucocytes that have newly come up and
which contain neither red corpuscles nor their remains. We must open the
guinea-pig to find any macrophages that have devoured red corpuscles.
They are to be met with in large numbers in the glandular portion of the
omentum, in the mesenteric glands, in the liver and in the spleen. They
are fairly easily recognised by the characteristic aspect of the
_débris_ of the red blood corpuscles. Having devoured the red corpuscles
the macrophages leave the peritoneal fluid and the digestion is
completed in the positions just mentioned. In the liver they are seen as
large mononuclear cells often with highly developed processes. In this
condition they remind one of Kupffer’s stellate cells—a fact that
suggested to me the idea that these elements are nothing but white
corpuscles which have immigrated into the vessels of the liver.
Following up the fate of the macrophages that have resorbed the red
blood corpuscles, we find them in the large hepatic vessels, in the vena
cava and even in the blood of the heart. But in these latter situations
they contain merely a few scarcely recognisable traces of their prey.
These phagocytes, which left the blood during the inflammation that
followed the injection of red corpuscles of the goose, re-enter it,
having fulfilled their function, during the final period of the
resorption. This resorption must undoubtedly be regarded as an
intracellular digestion. When we compare the essential phenomena taking
place inside the macrophages containing red blood corpuscles with those
we have described in the intestinal phagocytes of the Planarians or
Actinians after a meal, the analogy between the two becomes very
apparent. In both cases the red blood corpuscles undergo a marked change
which results in a diffusion of the haemoglobin. The membrane and
nucleus of the red blood corpuscles persist longer but they also are
ultimately digested. The excretion of haemoglobin from the phagocytes,
just mentioned in the case of the macrophages of the guinea-pig, is also
observed in the Actinians, whose coelenteric cavity is tinted by a
rose-coloured solution.
[Sidenote: [89]]
We have seen that in the Actinians intracellular digestion takes place
in a distinctly acid medium, whilst in the intestinal cells of the
Planarians it takes place in one that is only weakly acid. The
macrophages of the guinea-pig, during the resorption of red blood
corpuscles of the goose, carry on the digestive process in a medium
which shows a still weaker acidity. When made to ingest granules of blue
litmus there is no change of colour. Nor does alizarin sulpho-acid give
any reaction, probably owing to the fact that it exerts a toxic action
on the protoplasm of the macrophages. If, however, we add to a drop of
the peritoneal exudation of a guinea-pig, containing macrophages filled
with red blood corpuscles of the goose, a little of Ehrlich’s 1%
solution of neutral red, the red brick tint at once makes its appearance
in the content of these phagocytes. This coloration is identical with
that described in the _Amoebae_ which digest Bacteria or in the
intestinal phagocytes of the Planarians. It may, then, be regarded as an
indication of weak acidity. This coloration is maintained for some
hours, after which it gives place to complete decoloration, a phenomenon
that must be attributed, as in many other cases, to the neutralisation
of the acid by the alkaline protoplasm that has been macerated in the
fluid after the death of the macrophages.
The example we have chosen—the destruction of red blood corpuscles of
the goose by the macrophages of the guinea-pig—may serve as a prototype
of the resorption of formed elements in general. If, instead of red
blood corpuscles of the goose, we inject into the guinea-pig’s
peritoneal cavity pigeon’s or fowl’s blood, the essential phenomena will
be the same. The red blood corpuscles will always induce positive
chemiotaxis, especially of the macrophages, which in turn will ingest
the nucleated red corpuscles. It may be that in certain cases, when
fowl’s blood containing red corpuscles that are not very resistant is
injected, a certain number of the corpuscles immediately undergo a
partial solution in the peritoneal fluid[109]. Here also the stromas and
the nuclei of all the red blood corpuscles, as well as many of the
corpuscles unacted upon by the plasma of the phagolysed exudation,
undergo digestion inside the macrophages.
[Sidenote: [90]]
When, instead of blood, we inject white corpuscles from the bone marrow,
spleen or lymphatic glands of animals into the peritoneal cavity, we may
still observe their final disappearance in the macrophages. The
spermatozoa of man or of various mammals (bull, rabbit, guinea-pig,
etc.), when injected into the peritoneal cavity of the guinea-pig or
rabbit, are well adapted for this line of investigation. Here again the
immediate result of injection is the very marked phagolysis of the
leucocytes. This phenomenon gives place to an exudative inflammation
which brings into the peritoneal cavity a number of phagocytes. These,
especially the macrophages and in a much smaller degree the microphages,
devour the spermatozoa which in no case are dissolved, even partially,
in the plasma of the exudation. The macrophage seizes the spermatozoa
which sometimes, by the active movements of their flagella, exhibit
great vitality. At the end of several hours all the spermatozoa are
found inside phagocytes where they are completely destroyed. The
flagellum is digested first, but the head and medial portion soon suffer
the same fate. Neutral red reveals the feebly acid reaction, perhaps
with even more distinctness than in the case of the red blood
corpuscles.
The _résumé_ of Langhans’ investigation given in this chapter would lead
us to expect that resorption in the subcutaneous tissue will follow the
same rules as that going on in the peritoneal cavity. As a matter of
fact, blood injected at this position sets up a diapedesis of phagocytes
which ingest the red blood corpuscles. In some cases only is there a
partial solution of these corpuscles in the fluid of the subcutaneous
exudation. It is for this reason that goose’s blood, injected under the
skin of a guinea-pig, gives rise to a fluid exudation coloured a bright
rose red by the dissolved haemoglobin. This haemoglobin is derived from
red blood corpuscles which are damaged by the goose’s blood serum that
was added to the plasma of the exudation. The stroma and nuclei of the
red blood corpuscles cannot, however, be dissolved in this fluid. They
undergo the same fate as the red corpuscles that have remained intact,
that is to say they are ingested by the macrophages which immigrate into
the subcutaneous tissue and which finally digest all these elements. The
cells, less fragile than certain red corpuscles, are, in the
subcutaneous tissue, as in the peritoneal cavity, destroyed solely in
the interior of the phagocytes.
[Sidenote: [91]]
The analogy between the modifications undergone by the red blood
corpuscles and other cells inside the macrophages and the changes that
take place in the intestinal cells of Planarians and Actinians, suggests
that the resorption of formed elements must undoubtedly be regarded as a
true intracellular digestion. It would, however, be a very important
matter to be able to support this conclusion by even more convincing
proofs. The study of the artificial digestion that is observed _in
vitro_ in the case of the macerated mesenterial filaments of Actinians
has furnished a very valuable argument in favour of the enzymatic nature
of intracellular digestion. Animal exudations are not well adapted for
this special line of study. We can only obtain them as the result of the
injection of different substances, solid or fluid, which are greedily
absorbed by phagocytes. If we collect the exudations at a moment when
the number of these cells is still considerable we must withdraw along
with them many digestive substances which interfere with our
observation. We may therefore with advantage turn our attention to
masses of phagocytes collected in organs. As it is mainly the
macrophages which effect the resorption of cells, it is evident that we
must choose the centres where they are formed in order to investigate
the digestive ferments. Let us take, then, the lymphatic glands of the
mesentery, the glandular portion of the omentum and the spleen, the
three pre-eminently macrophagic organs, and let us see if, with an
extract of them, prepared with physiological salt solution (0·75% of
sodium chloride), any digestive effect is to be obtained.
Macerate the three organs mentioned of a guinea-pig and mix the extracts
thus obtained with red blood corpuscles of the goose, corpuscles that
have already given us information in connection with the phenomena of
resorption in the living organism. In almost all the guinea-pigs a
solution of the red blood corpuscles of the goose by the extract of the
glandular portion of the omentum may be observed. The mesenteric glands
likewise give an extract which in most cases has a solvent action. The
extract from the spleen is only active in a limited number of cases. In
all these examples the extracts from macrophagic organs bring about the
solution of the haemoglobin, but leave intact the membrane and nucleus
of the corpuscles. In this respect there exists, then, a certain
difference between this and the digestion of red corpuscles in the
macrophages of exudations, where the membrane and even the nucleus are
in the end completely dissolved. This difference may be explained by the
fact that in the preparation of the extract in physiological salt
solution, one part only of the soluble digestive ferment may be set at
liberty.
[Sidenote: [92]]
The solvent action of extracts of macrophagic organs must in fact be
attributed to the presence of a soluble ferment in the cells of which
these organs are made up. As the diastases are distinguished, in
general, by their great sensitiveness to heat, we tried the action of
our extracts after a preliminary heating, when it was found that a
temperature of 56° C., applied for three quarters of an hour, completely
abolished the solvent action of the extracts upon the red blood
corpuscles of the goose. The soluble ferment of macrophagic organs, to
which we propose to give the name of _macrocytase_[110] or macrophage
ferment, is in many respects analogous to the actino-diastase of Mesnil,
described in the preceding chapter.
With a view to obtain more complete information on the cytases I
suggested to Tarassewitch that he should make a detailed study of them;
this he has carried out in my laboratory. He has demonstrated that the
macrophagic organs of other mammals than the guinea-pig, especially
those of the rabbit and dog, exert the same solvent action on the red
blood corpuscles. He has also established the fact that this action
applies not only to the red corpuscles of the goose but extends also to
those of several other birds and mammals. Tarassewitch succeeded in
confirming the injurious action of heat on macrocytase. Extracts of
macrophagic organs which contain much _debris_ in suspension, when
heated for an hour at 55°·5 C. in certain cases lose their solvent
property for red blood corpuscles; sometimes this temperature brings
about merely a weakening of the macrocytase. In order to destroy it
surely and completely, the suspensions must be heated at 58°·5–62° C.
for an hour. If, however, instead of heating the entire suspension, we
first pass it through filter paper, the clear fluid filtrate is deprived
of its diastatic action even after it has been heated at 55°·5 C. for
three quarters of an hour.
[Sidenote: [93]]
Of all the other organs of which extracts have been kept in prolonged
contact with the red blood corpuscles of birds, the pancreas alone has
shown a very well-marked digestive action. Extracts of the salivary
glands exerted a feeble solvent action on a certain quantity of the red
corpuscles. The other organs, such as the liver, kidneys, brain, spinal
cord, ovary, testicles, suprarenal capsules and placenta, exercised no
such action. Even bone marrow, in agreement with my results published
some years ago, showed itself quite inactive.
The blood serum of guinea-pigs which I employed in my researches, as
well as that of the animals examined by Tarassewitch, has not shown
itself capable of dissolving the red blood corpuscles of the goose,
although the macrophagic organs dissolve them easily. It has long been
known, however, that the serum of the blood of many animals will destroy
the red corpuscles of a different species. This demonstration was
afforded during the period when attempts were being made to transfuse
the defibrinated blood of mammals, especially of the sheep, into man.
This practice had to be abandoned, in consequence of the difficulties
resulting from the solution of the human red corpuscles. Later,
Daremberg[111] and Buchner[112] set themselves to study this haemolytic
action of serums systematically. They found that it was due to a
particular substance to which Buchner gave the name of _alexine_ or
protective substance. Of indeterminate chemical composition, this
substance is allied to albuminoid substances. It is destroyed when
heated to 55°–56° C. and only acts in the presence of certain salts.
When these salts are removed from the serum by dialysis, it loses its
haemolytic power; but as soon as the salts are replaced in proper
proportion this power reappears. Later, Buchner[113] compared the action
of alexine to that of soluble ferments and referred it to the category
of the digestive diastases. According to him the same alexine is capable
of dissolving the red blood corpuscles of several species of
Vertebrates. Bordet[114], in a series of researches made in the Pasteur
Institute, confirmed this view. He came to the conclusion that the
alexines of the various species of animals differ from one another.
Thus, the alexine of the blood serum of the rabbit is not the same as
that found in the serum of the guinea-pig or dog. Nevertheless each of
these alexines is capable of exerting a solvent action on the red blood
corpuscles of several species.
[Sidenote: [94]]
[Sidenote: [95]]
Ehrlich and Morgenroth[115], in a series of memoirs on the solution of
red blood corpuscles, have combated the idea that there is only a single
alexine in one and the same serum. Moreover, they state that alexine
always requires for its action the aid of another substance, and that
matters are much more complicated than at first sight appears. They
maintain that in each normal serum a number of different substances are
found, each one of which only attacks a single species of red blood
corpuscle. They point out that the solution of the red corpuscles by the
normal serum takes place through the combined action of two different
substances and cite several cases where a normal serum, after being
heated to 55° C. and so deprived of its haemolytic power, again becomes
capable of dissolving the red corpuscles when some normal serum from
another species, which of itself is destitute of the solvent property,
is added to it. Let us quote an example from Ehrlich and Morgenroth. The
normal serum of the goat readily dissolves the red blood corpuscles of
the rabbit and guinea-pig, but if heated for half-an-hour at 55° C., it
loses this power. On the other hand, the normal serum of many horses
shows itself powerless to dissolve the red corpuscles of these rodents.
Here, then, are two serums, equally incapable of effecting the solution
of the red corpuscles of the rabbit and guinea-pig. Yet, when they are
mixed together and to them a few drops of blood from one of the rodents
cited is added, haemolysis takes place readily. The heated goat’s serum
then, has, retained in it something that resists a temperature of 55°
C., a substance which, by itself, leaves the red blood corpuscles
intact; but which, when combined with a second substance present in the
horse’s serum, causes their solution. Ehrlich gives to the first
substance, that is to say that found in the heated goat’s serum, the
name of _intermediary body_ (“Zwischenkörper”). The second substance,
present in the unheated horse’s serum, is designated by him the
_complement_. In order that a normal serum may dissolve the red
corpuscles, it is not sufficient that it should possess a single
substance, the alexine of Buchner. It must, to exert this action,
contain two distinct substances which are very often found together in
the same normal serum. Unheated goat’s serum was only capable of
dissolving the red blood corpuscles of the rabbit because a particular
complement and intermediary substance were both present. Deprived of its
complement at 55° C., the serum is solvent only when we add to it
another substance that is contained in the normal serum of a different
species (horse). Continuing their researches in this direction, Ehrlich
and Morgenroth have come to the conclusion that the normal serum of a
single species may contain several intermediary substances, each one
acting on a single species of red blood corpuscles. Further, that normal
serum must contain several or even many different complements.
[Sidenote: [96]]
Ehrlich and Morgenroth carried on researches on the intermediary
substances in normal serums and found several in addition to that
already mentioned. The serum of the normal dog readily dissolves the red
blood corpuscles of the guinea-pig. When heated to 57° C. it loses this
property; but with the addition of normal guinea-pig’s serum the
property is regained. In the serum of the normal dog there exists, then,
besides the complement, at least one intermediary substance. The same
result can be obtained with several combinations of serums of normal
mammals, heated or unaltered[116]. Yet it often happens, as Ehrlich and
Morgenroth themselves point out, that the demonstration of the presence
of the intermediary substance in normal serums is accompanied with
marked difficulties. Bordet, also, who has studied this question very
thoroughly, often failed completely in his attempts to make normal
serums, that were incapable of producing haemolysis, active by the
addition of heated serums of other species of animals. Thus he observed
that normal fowl’s serum readily dissolves the red corpuscles of the
rabbit. When heated to 55°–56° C. this serum loses its haemolytic power
which cannot be restored by the addition of any normal serum. He thinks
therefore that, in this example, haemolysis is produced solely by the
alexine, without the assistance of any intermediary substance in the
serum of the normal fowl. P. Müller[117], whilst confirming Bordet’s
experimental results, considers that, in this case also, there is the
intervention of an intermediary substance. When he mixed heated fowl’s
serum with a small quantity of unaltered fowl’s serum the solution of
the red corpuscles of the rabbit is not brought about. When, however,
instead of adding a little unheated normal fowl’s serum, he added the
same quantity of serum from a fowl previously treated with physiological
salt solution, the red corpuscles of the rabbit were dissolved without
any difficulty. Müller explains this difference as due to the fact that
the serum of the treated fowl contains more complementary substance than
does that of the normal fowl.
We see, then, from this example that the analysis of the phenomena
taking place in the solution of the red corpuscles by normal serums is
beset with very great difficulties. For this reason it is much more
profitable to make researches in this direction, using more active
serums, where the demonstration of the two substances can be made simply
and exactly. This desideratum has been supplied by J. Bordet, when
_preparateur_ in our laboratory; he described an easy method of
increasing the haemolytic power of serums.
As stated above, guinea-pigs that have received an intraperitoneal
injection of goose’s blood digest the corpuscles, although the
peritoneal fluid exerts no haemolytic action. _In vitro_, the extract of
their macrophagic organs certainly dissolves the red corpuscles, whilst
the blood serum usually fails to do so. Now, if a second or a third
injection of goose’s blood be made into the peritoneal cavities of the
same guinea-pigs, partial solution of the corpuscles takes place in the
peritoneal plasma and the serum of the blood acquires new properties: it
becomes capable of clumping the red corpuscles, that is to say of
agglutinating them; afterwards it dissolves them _in vitro_.
[Sidenote: [97]]
J. Bordet[118] has shown that the injection of the blood of one species
of Vertebrate (mammal or bird) into the peritoneal cavity or under the
skin of an animal of a different species, always produces in the blood
serum of the latter the haemolysing substance. This haemolysing
substance is specific or nearly so, that is to say it dissolves the red
corpuscles of the species which has furnished the injected blood and
also, but more feebly, the red corpuscles of allied species.
Consequently, with guinea-pig’s serum, treated with goose’s blood, we
obtain the greatest solvent action on the red corpuscles of the goose,
though there is a certain haemolysis of the red corpuscles of some other
birds. This rule, thoroughly established by Bordet, has been the
starting-point for a large number of researches on haemolysis and
amongst others of those which bear on the intermediary substance of
normal bloods.
Bordet demonstrated very definitely a fact of fundamental
importance—that in the blood serums of animals treated with blood from a
different species, there exist two distinct substances which only
dissolve the red blood corpuscles when they are combined. Here the
duality of the haemolytic agent cannot be doubted, as it may in certain
examples of normal serums. Each time that we deprive the serum of a
treated animal of its solvent action by heating at 55°–56° C., this
property can be restored to it with certainty by the addition of a
little normal serum which, by itself, is incapable of bringing about
haemolysis. The heated serum of these injected animals loses the power
of dissolving the corresponding red corpuscles, but it retains its other
acquired property—the agglutination of the corpuscles. The red
corpuscles, aggregated into voluminous masses quite visible to the naked
eye, remain intact indefinitely, if left in the prepared and heated
serum. But as soon as we add to them a trace of normal blood (taken from
one of a number of species of Vertebrates), the solution of the red
corpuscles is not long in taking place. Under these conditions an action
of two substances is set up; one of these substances is found in the
heated serum of the injected animal, and the other in unheated normal
serum. The first of these substances which not only resists a
temperature of 55°–56° C., but stands, without alteration, heating to
60°–65° C., corresponds to the intermediary substance of Ehrlich. By
Bordet it has been termed “substance sensibilisatrice[119].” The second
substance, a common one, found in normal serums and destroyed at 55°–56°
C., is the alexine of Buchner and of Bordet, or the complement of
Ehrlich.
[Sidenote: [98]]
The ease with which one can demonstrate the co-operation of two
substances in the haemolysis by the serums of animals treated with the
blood of a different species, is due to the fact, that during the course
of this treatment the animal organism produces a quantity of an
intermediary or sensibilising substance. In fresh animals which have not
been treated, it is often very difficult to demonstrate the presence of
this substance. Bordet has established the fact that the serum of
animals which have been injected several times with the blood of a
different species, contains almost the same amount of alexine as does
untreated serum. On the other hand, the sensibilising substance makes
its appearance in large quantity as the result of these injections. Von
Dungern[120] has confirmed this result and contributes the interesting
fact that the sensibilising substance is found even in great excess in
the serum of treated animals. When he adds to this serum blood that has
not been heated, he produces a haemolysis that is more than thirty times
as active as when the serum of the prepared animal alone is used. From
the quantitative point of view, then, there is no relation between the
amount of the two substances in the serum of prepared animals.
It may be suggested that the sensibilising or intermediary substance is
the same as that which produces the agglutination of the red corpuscles.
But careful researches have thoroughly demonstrated the difference
between the two substances that have this character in common, both
resist heating to 55°–60° C. and even beyond this point.
[Sidenote: [99]]
Having established this co-operation of two substances in haemolysis the
intimate mechanism of their action was next studied. Here I must give
pride of place to the discovery by Ehrlich and Morgenroth that the
intermediary (or sensibilising) substance links itself to its
corresponding red corpuscles. A serum, capable of dissolving the red
corpuscles of a different species, is heated to 56° C. which causes it
to lose this solvent property. When a certain number of these corpuscles
are added to it, such corpuscles remain intact although they are
agglutinated. It is sufficient, after some hours of contact, to
centrifugalise the mixture in order to separate a limpid serum from the
mass of red corpuscles, the former being now entirely deprived of its
intermediary substance, that is to say it has become incapable of
dissolving the red corpuscles even with the addition of a large quantity
of the “complement” (normal serum, unheated). On the other hand, the red
corpuscles, having fixed (linked) all the intermediary substance,
dissolve very rapidly when placed in contact with normal serum which
contains the necessary quantity of the complement (or alexine). This
fundamental experiment has been confirmed and repeated by many observers
and has now become classic. The idea that the intermediary (or
sensibilising) substance links itself to the red corpuscle, without
dissolving it, is generally accepted and may be regarded as permanently
settled. We should do well, then, instead of designating by all sorts of
synonyms the substance in serums which resists the action of a
temperature of 55°–65° C., to apply to it, once for all, the name of
_fixative substance_ or simply that of _fixative_. This name is short,
expresses the essential character of the substance and gives rise to no
misunderstanding, as do the other names proposed up to the present
(amongst them that of _philocytase_ employed by myself in some of my
earlier publications).
Another of Ehrlich and Morgenroth’s experiments has furnished the proof
that the complement does not fix itself to the red corpuscles only. A
normal serum, unheated, which, by itself, is quite as incapable of
dissolving the red corpuscles as the fixative alone, is mixed with some
defibrinated blood. After the centrifugalisation of this mixture, it is
easy to demonstrate that the supernatant fluid has lost none of its
complement (alexine), whilst the red corpuscles have fixed none.
If, instead of an inactive serum, we take a serum which is capable of
dissolving the red corpuscles and which consequently contains the two
haemolysing substances, and if we place it in contact with the
corresponding red corpuscles, at a temperature between 0° and 3° C., the
solution will not take place (Ehrlich and Morgenroth). Under these
conditions the fixative certainly attaches itself to the red corpuscles,
but the alexine remains in solution, unused. It is only necessary,
however, to heat the mixture up to 30° C. to bring about rapid
haemolysis.
[Sidenote: [100]]
From their very ingenious experiments, as a whole, Ehrlich and
Morgenroth conclude that the fixative possesses two different
affinities: one for the red corpuscle and another for the complement. Of
these two affinities the stronger is that which links it to the red
corpuscle, for this is manifested at a very low temperature. In order
that the fixative may combine with the complement a much higher
temperature is requisite. Ehrlich comes to the conclusion that the
molecule of the fixative possesses two haptophore groups, or groups
capable of chemical combination. The first of these links it to a
corresponding molecule of the red corpuscle to which he gives the name
of receptor; the second combines the fixative with the molecule of the
complement and in this way introduces the latter into the red corpuscle.
These investigators give a diagram which greatly facilitates the
understanding of their hypothesis (Fig. 19). They seek to prove that the
combinations of the fixative with the red blood corpuscle and with the
complement follow the law of definite multiples and that these phenomena
must, in consequence, be looked upon as being of a purely chemical
character.
[Illustration:
FIG. 19. Schema of Ehrlich’s theory.
_c_, complement (alexine, cytase)—_am_, amboceptor (fixative)—_r_,
receptor of the red corpuscle.
(After Levaditi in the _Presse médicale_.)
]
The hypothesis advanced by J. Bordet does not accord very well with the
theory we have just set forth. He could never convince himself that the
fixative combines with the complement. He was of opinion rather that the
fixative, retained by the corpuscle, exercises upon it a mordant action
which enables it to absorb the alexine. The alexine is supposed to
attach itself to the sensibilised red blood corpuscle as a dye attaches
itself to a mordanted element. Bordet rests his interpretation mainly on
the fact that the absorption of alexine by the sensibilised corpuscles
does not follow the elementary laws of chemical combination, especially
those of definite multiples.
Nolf[121] has sought to define more accurately the part played by these
two substances in the solution of the red blood corpuscles. He agrees
with Bordet, that in this phenomenon the fixative plays the same part
that the mordants do in dyeing. Linked to the red corpuscle the fixative
renders it more greedy for alexine, exactly as the mordant facilitates
the fixation of the dye on the fibre of the textile fabric. Under these
conditions the alexine (complement), finding itself in large quantity
inside the red corpuscle, exercises upon it its hydrating action, thus
bringing about the diffusion of the haemoglobin and often even the
solution of the corpuscular stroma.
Nolf compares the solvent action of alexine upon the red corpuscle to
that of certain mineral salts, such as ammonium chloride. He passes in
review the various properties of alexines and finds them very similar to
the solvent action of certain salts. Even the peculiarity of alexine, of
remaining inactive at a temperature of 0°–3° C., is shared by ammonium
chloride which, alone of all the salts studied by Nolf, exercises no
solvent action under these conditions. But Nolf found it impossible to
push these analogies further, and especially to sensibilise, by the
fixative, the red corpuscles to the action of quantities, which were of
themselves inactive, either of ammonium chloride or of any other salt.
[Sidenote: [101]]
London[122] hoped by fresh experiments to solve the problem of the mode
of action of the two substances which act in haemolysis. He pronounced
in favour of the theory that they entered into chemical combination with
the red corpuscles. But the facts accumulated up to the present do not
enable us to make a positive statement as to the exact nature of the
reaction which is set up during the solution of the red blood
corpuscles; this is not astonishing in view of the fact that it is
impossible to isolate the haemolysing substances in a pure state.
It may, however, be admitted that the action of alexine (complement)
comes under the category of phenomena that are produced by soluble
ferments. Buchner[123] maintains that there is an analogy between this
substance and the diastases (or enzymes); Bordet[124], from the
appearance of his first publications on haemolysis, has expressed
himself in favour of this view. Ehrlich and Morgenroth[125], in their
two first memoirs, very distinctly put forward the same idea. “We shall
not deceive ourselves”—they say—“if we attribute to the addiment (syn.
complement, or alexine) the character of a digestive ferment.” In one of
their last memoirs[126] they no longer express themselves in so decided
a fashion. Nevertheless we are still quite justified in maintaining this
proposition. The substance which dissolves the red blood corpuscles of
Mammals or a portion only of those of Birds, undoubtedly presents very
great analogies to the digestive ferments. As has been mentioned
repeatedly, it is very sensitive to the action of heat and is completely
destroyed by heating for one hour at 55° C. In this respect it closely
resembles the macrocytase of macrophagic organs which also dissolves red
corpuscles. As it is the macrophages which ingest and digest the red
blood corpuscles in the organism, it is evident that alexine is nothing
but the macrocytase which has escaped from the phagocytes during the
preparation of the serums.
[Sidenote: [102]]
We know that the leucocytes contain quite a series of soluble ferments
of which some are set at liberty after the blood has been drawn from the
vessels. It is thus that plasmase, or fibrin-ferment, is set free from
the leucocytes to combine with fibrinogen to produce the clot. This is
not the only soluble ferment of leucocytic origin. It has been known for
some time that in addition to this coagulating ferment the leucocytes
contain ferments which are especially digestive or decoagulating. Thus
Rossbach[127] has demonstrated the presence of amylase in the leucocytes
of different organs, especially the tonsils. Arthus has confirmed this
discovery and Zabolotny[128] has completed it by his observations on the
phenomena which appear in the peritoneal cavity of animals into which
wheat flour or starch were injected. He observed that the small granules
are quickly ingested by isolated leucocytes, whilst the large granules
are surrounded by quite a layer of phagocytes. He agrees with several
other writers, that the amylase found in defibrinated blood has its
origin in leucocytes.
[Sidenote: [103]]
Leber[129], in the course of his researches on inflammation, made the
observation that the pus of a hypopyon that was absolutely aseptic
digests coagulated fibrin at a temperature of 25° C. and liquefies
gelatine very readily. Achalme[130] has confirmed this and has added
several other interesting data. He investigated the soluble ferments of
pus and directed his attention amongst others to experimental pus, set
up by the injection of spirit of turpentine. In addition to amylase and
a ferment which liquefies gelatine, Achalme has discovered in pus,
saponase (lipase), casease, and a ferment closely allied to trypsin.
This last readily digests fibrin and also attacks coagulated white of
egg; in the products of this digestion Achalme found peptone but could
not always obtain leucin and tyrosin. He never succeeded in
demonstrating the presence of sucrase, inulase, emulsin or lactase in
pus. On the other hand he found large quantities of oxydase, thus
confirming the discovery of Portier[131] who was the first to
demonstrate that these ferments met with in the blood are, in the living
animal, found inside leucocytes. By a large number of experiments,
carried out on most diverse representatives of the animal kingdom,
Portier was able to establish the important fact that the oxydases which
are found in many organs or in the fluid of blood withdrawn from the
organism really originate in leucocytes as they deteriorate and break
up. In this respect, then, they resemble fibrin-ferment very closely.
To complete the list, already considerable, of leucocytic ferments, I
must further cite the anticoagulating soluble ferment whose existence in
Mammals has been so well demonstrated by Delezenne.
All this evidence encourages us, then, to support the thesis that
alexine is one of the numerous intraleucocytic soluble ferments and that
it only passes into the fluids as the result of rupture or of damage to
the phagocytes. Nolf (_l.c._) has recently pronounced against this view;
we must therefore examine his arguments closely. In the first place he
takes his stand on the analogies between the solution of the red blood
corpuscles by the serums and by certain salts. It must not be forgotten,
in connection with his theory, that haemolysis is but one example, out
of many, of the action of alexines. Of all the formed elements the red
corpuscles are the most delicate; they are readily broken up by all
sorts of agents (moderate heat, water, salts, etc.). Further, there are
numerous other cells (white corpuscles, spermatozoa, and inferior
organisms) which resist the action of salts much better, which,
nevertheless, are very injuriously affected by the action of the
alexines.
Nolf lays special stress on the experiments in which, after keeping red
blood corpuscles in prolonged contact with active serums, he has looked
in vain for the peptone reaction. He prepared his mixtures in sealed
tubes or flasks, and kept them in an incubator at 37° C. for 24–48
hours, or even for weeks. Under these conditions the haemoglobin is
transformed into metahaemoglobin, but peptones never appear. Nolf
concludes therefrom “with confidence, that the alexines do not exert the
slightest peptonising effect on the albuminoids of the corpuscle”
(_l.c._ p. 672).
[Sidenote: [104]]
To this conclusion it must be objected that peptone is not the only
product of the digestion of albuminoids by soluble ferments. Under
certain conditions the disintegration is carried much further, in others
it is arrested at an earlier stage. Thus human urine which contains
pepsin, never gives the peptone reaction with fibrin; the digestion of
the latter only goes on up to the stage of protalbumose. When, however,
the urinary pepsin is fixed on flakes of heated fibrin which are
submitted to digestion in acidulated water the digestion proceeds
further and gives as final products deuteroalbumose and peptone[132].
Now, under the conditions in Nolf’s experiments the digestion would be
very quickly stopped, because, at the temperature of 37° C., alexine
very soon loses its strength. Investigators who have experimented with
haemolytic serums know well that, even when kept at a low temperature,
alexine may lose its activity within 24 hours.
It has been mentioned above that Nolf sought in vain for a parallel
between haemolysis by salts and that by serums, in what relates to the
action of the fixative. He was unable to find anything comparable to
this action amongst salts, although digestion by soluble ferments offers
undoubted analogies. I need only recall further the discovery of
enterokynase, the soluble ferment of the digestive juice of the dog,
which actively stimulates the action of pancreatic ferments, and
especially that of trypsin. The recent researches of Delezenne
(communicated to the International Congress of Physiology held at Turin
in September 1901) support this conclusion in a very important fashion.
As already pointed out in Chapter III the enterokynase of the intestinal
juice exerts an action comparable with that of the fixatives of
haemolytic serums. Alone, it does not act as a solvent ferment, but when
it attaches itself to the fibrin it aids the action of the trypsin in a
marked degree. In pancreatic digestion enterokynase plays the part of
the fixatives in the solution of red corpuscles.
[Sidenote: [105]]
The analogy between the resorption of formed elements and intestinal
digestion extends even beyond this. When we inject, into the peritoneal
cavity or under the skin of various animals, blood from a different
species, the blood serum of the former becomes haemolytic for the red
corpuscles of the latter. The solution of these red corpuscles is
effected by the alexine of the serum, whose activity is rendered very
great owing to the presence of a quantity of specific fixative. This
same fixative appears also in the fluids of animals to whom, instead of
injecting blood, we simply give it by the mouth. This fact has been
established by Metalnikoff[133].
Another fact in favour of the close relationship between the fixatives
and enterokynase consists in the presence of both in the lymphatic
(lymphopoietic) organs. The fixatives which aid the solution of red
corpuscles are found specially in the mesenteric glands. Enterokynase,
as demonstrated by Delezenne, is found not only in the intestinal juice,
but also in Peyer’s patches, the solitary glands, the mesenteric glands,
and the leucocytes of exudations and of the blood.
Supported by these various facts we are quite justified in regarding the
haemolysing substance of serum as containing two soluble ferments, of
which one, alexine, corresponds to trypsin, the other, the fixative,
resembling enterokynase. The alexine, whose nature is gradually
disclosing itself with more precision, should bear the name of _cytase_
or cell-ferment. The cytase of the macrophagic organs, or _macrocytase_,
comes under this category. According to the researches of Tarassewitch
it also acts more actively when there is added to it some of the
fixative found in the serum (heated to 56° C.) of prepared animals.
[Sidenote: [106]]
We have said that in the living animal the macrocytase is localised in
the phagocytes of the organs and of the blood. Thus, when goose’s blood
is injected into the peritoneal cavity of the guinea-pig the red blood
corpuscles are digested within the macrophage and not in the fluid of
the peritoneal exudation. When, however, the same kind of blood is
injected a second or a third time, it is found that a certain number of
the red corpuscles become permeable and lose their haemoglobin, which
they give up to the fluid of the exudation, and only the membrane and
the nucleus remain. These are at once ingested by the macrophages which
under these conditions manifest a real excess of activity. Instead of
sending out small processes, as they do after the first injection of
blood, these phagocytes move about like true Amoebae, sending out broad
pseudopodia, and ingest not only the remains of the red corpuscles but
also those still intact[134] (Fig. 20). Under these conditions
macrocytase must undoubtedly be found in the peritoneal plasma. It is,
however, easily demonstrable that this ferment was not preformed in the
fluid but has escaped from the leucocytes that have undergone
phagolysis. After the rapid injection of alien blood the phagocytes of
the peritoneal lymph gather into clumps, become immobile, and for a time
lose their phagocytic power. It is only after the lapse of a longer or
shorter period that the leucocytes recover from the phagolysis, arrive
in great numbers in the peritoneal cavity and display their phagocytic
energy.
[Illustration:
FIG. 20.—Rapid ingestion of red corpuscles of the goose by
macrophages.
]
If the damage to the phagocytes—the phagolysis—is the actual cause of
the setting free of the intraleucocytic ferment, we have only to prevent
this phagolysis in order to inhibit the solution of red blood corpuscles
in the fluid of the exudation. For this purpose it is sufficient to
prepare guinea-pigs (which have already received several injections of
goose’s blood) by means of an injection of fresh broth, of physiological
salt solution, or of carbonic acid into the peritoneal cavity on the eve
of the decisive experiment. Such injection at once provokes phagolysis,
which is then followed by an abundant exudation of leucocytes. When,
next day, a dose of red blood corpuscles of the goose (deprived of serum
by centrifugalising) is introduced into the peritoneal cavity thus
prepared phagolysis is no longer produced, or very feebly, and is of
very short duration. Under these conditions the solution of the red
corpuscles by the peritoneal fluid is reduced to a minimum, and in its
place an extremely rapid and considerable ingestion of red corpuscles by
the macrophages may be observed. In order that the experiment may be
completely successful it is advisable to use goose’s blood heated to 37°
C. or thereabouts for the injection.
[Sidenote: [107]]
Even when the red corpuscles of the goose are introduced, not into the
peritoneal cavity but into the subcutaneous tissue of guinea-pigs that
have received several injections of goose’s blood, we can easily prevent
the extracellular solution of the red corpuscles which takes place, as
already indicated, in the normal guinea-pig. As in this case the goose’s
serum which is mixed with the corpuscles contributes to the haemolysis,
it must be suppressed by centrifugalising the defibrinated goose’s blood
and by washing the corpuscles with normal saline solution.
Collectively, the facts I have just described clearly indicate that the
phagocytes must be regarded as the source of the haemolytic ferment. The
macrocytase remains in the body of these cells so long as they are in a
normal condition; but immediately they are injured, in consequence of
the sudden introduction of foreign substances into the peritoneal
cavity, a portion of the macrocytase escapes and acts on the red
corpuscles as if it had been employed _in vitro_.
As the conclusion I have just formulated is of fundamental importance in
the study of resorption and immunity it is necessary to support it by as
many arguments as possible. For this reason, therefore, I feel obliged
to draw the attention of the reader to another example of the resorption
of formed elements.
We have already spoken of the resorption of spermatozoa in the
peritoneal cavity, and of the part played by the macrophages in this
phenomenon. As a result of this resorption, just as after that of red
blood corpuscles, the organism acquires new properties of the same
character. Landsteiner[135] and the writer[136] have shown that the
blood serum and the peritoneal fluid of animals that have been injected
with the spermatic fluid of bull, rabbit, or man, become spermotoxic,
that is to say, they render the corresponding spermatozoa motionless and
kill them. These fluids, however, never acquire the power of dissolving,
even partially, these elements. The disappearance and final solution of
the spermatozoa is only effected within phagocytes, and almost
exclusively in the macrophages.
[Sidenote: [108]]
Moxter[137] has demonstrated that the spermotoxin which appears in the
serum of prepared animals consists of two substances, corresponding to
those present in the haemolytic serums. These are the macrocytase
(alexine, complement) and the fixative (intermediary or sensibilising
substance). For him they are identical with those which dissolve the red
corpuscles. Without dwelling on the subject we may say that the
macrocytase which dissolves the red corpuscles and that which arrests
the motion of the spermatozoa are really identical in the same species
of animal, as is accepted and developed by Bordet. On the other hand, it
is impossible to accept Moxter’s theory of the identity of the two
fixatives. They must be regarded as different; this we have attempted to
prove in one of our memoirs[138] and is in accordance with the law of
the specificity of fixatives in general.
The question which interests us more especially at this moment is where
are these two constituent substances of the spermotoxin to be found and
how do they behave in the living organism? This question has been very
thoroughly studied by Metalnikoff[139] in my laboratory. His experiments
have been closely followed by me, and in presenting their principal
results I can bear witness to their correctness.
The spermotoxin obtained by Metalnikoff is distinguished from the
haemotoxins we have discussed up to the present in that they develop,
not as a result of the injection of cell elements from a different
species, but as a result of the introduction into the organism of
spermatozoa from the same species, the guinea-pig. We have here, then,
to deal with what has been termed autospermotoxin.
The serum of the normal guinea-pig acts but feebly on the spermatozoa of
this species, which, under its influence, remain motile for several
hours. When, however, guinea-pigs have received one or several
injections of the spermatozoa of their own species, their serum and
peritoneal lymph become distinctly toxic and render the spermatozoa
motionless in a few minutes. In male guinea-pigs so prepared the serum
acquires this toxic property not only for the spermatozoa of other male
guinea-pigs, but likewise for those of the individual itself which
furnishes the serum. This latter, then, becomes distinctly
autospermotoxic.
[Sidenote: [109]]
If the spermotoxin were diffused in the plasma and other fluids of the
guinea-pig which furnishes it, it ought to render motionless the
spermatozoa contained in the genital organs. Experiment demonstrates,
however, that this is not the case. If the male organs be removed from a
guinea-pig whose serum is very autospermotoxic _in vitro_, we find,
especially in the epididymis, a mass of very virile spermatozoa which
for a long time retain their motility in physiological salt solution.
The macrocytase, then, has not reached the spermatozoa in the living
animal; this is because it is not found in the plasmas. Let us inject
into a guinea-pig, whose serum is strongly autospermotoxic, one portion
of sperm into the subcutaneous tissue and another portion into the
peritoneal cavity. In the first site a soft oedema, filled with
transuded fluid, in which the very active spermatozoa retain their
motility for a couple of hours, is produced. In the peritoneal fluid the
same spermatozoa become motionless in a few minutes. This great
difference is explained by the fact that, under the skin, there are no,
or almost no pre-existing leucocytes, whilst in the peritoneal fluid
they are abundant. The phagocytes injured by the introduction of sperm
into the peritoneal cavity, abandon a portion of their macrocytase,
sufficient to render the spermatozoa motionless. But when Metalnikoff
injected physiological salt solution into the peritoneal cavity of his
autospermotoxic guinea-pigs, and then, on the following day, a quantity
of sperm, the spermatozoa continued very active for more than an hour.
In this case phagolysis is very transitory and insignificant; it is soon
followed by a great afflux of leucocytes which bring about a rapid
ingestion of the spermatozoa. Many of these elements are devoured in a
living state; for even when their body is enclosed in the macrophage,
their tail, left outside, continues to move very actively.
[Sidenote: [110]]
All these experiments demonstrate that in the normal state the
macrocytase remains within the phagocytes and only escapes during
phagolysis, or at the moment when the blood, after it has been withdrawn
from the organism, coagulates. Is it the same for the fixative? It is
easy to prove that this soluble ferment circulates in the plasmas of the
living organism. We have already said that the spermatozoa of a
guinea-pig whose serum is very autospermotoxic, remain alive for some
time in the physiological salt solution. But if we introduce them, _in
vitro_, into the serum of a normal guinea-pig they remain motile but a
short time (some 10–20 minutes), whilst the spermatozoa of a normal
guinea-pig will live in the same serum for several hours. This
difference is explained by the fact that the spermatozoa of the
autospermotoxic guinea-pig, although very active, have absorbed the
fixative during the life of the animal. This fixative is, as we have
stated, found in the body fluids and has been able to penetrate to the
male organs. Here the spermatozoa become charged with the fixative and,
once transported into the serum of the normal guinea-pig, rich in
macrocytase, they lose their movements very quickly. At the same time
the spermatozoa used for control, not having absorbed any fixative, are
able to live for a long time in the same serum.
As the macrocytase remains fixed to the phagocytes there can be no doubt
as to its origin; it is elaborated by these cells. Whence however comes
the fixative which is free in the body fluids and which is precisely the
substance that is developed in so large a quantity in the treated
animals? The exact solution of this question is not easy; nevertheless
there are many facts which indicate that this fixative is also of
phagocytic origin. We know already that the serums of normal animals
contain only small quantities or sometimes, perhaps, none of the
fixative. This fixative only appears abundantly as the result of the
resorption of the corresponding elements, red corpuscles or spermatozoa.
This resorption, as we have said, is almost exclusively the work of the
macrophages. It is just in those cases where the red corpuscles,
injected into the peritoneal cavity of an animal of the same species,
pass directly into the lymph, without being injured or, save
exceptionally, ingested by the phagocytes, that the fixative is not
formed. When the red blood corpuscles of the goose, introduced with
defibrinated blood below the skin of a guinea-pig, undergo there a
partial solution in the fluid of the exudation, and where the
phagocytosis is more limited than in the peritoneal cavity, the
production of fixative is small. When the injection of the same goose’s
blood is made into the peritoneal cavity of a guinea-pig and is followed
by complete phagocytosis, the fixative is produced in greater abundance.
There exists, then, in all these cases a constant relation between the
degree of phagocytosis and the amount of the fixative produced. As this
fixative facilitates the access of the cytase to the cells and as the
resorption of these elements takes place specially in the macrophages,
we are bound to come to the conclusion that the fixative is a second
phagocytic ferment which is produced in abundance during the process of
intracellular digestion. Only, instead of remaining in the substance of
the phagocytes, this fixative is in part thrown out from these elements.
It passes into the plasma of the blood and into the other fluids and
ends by disappearing from the organism, probably being eliminated by the
excretory channels.
[Sidenote: [111]]
In the Invertebrata, where, as we have seen, the alien red blood
corpuscles are also digested within the phagocytes, we have never been
able to demonstrate any haemolytic property of the blood fluid, even
after repeated injections of blood. We must conclude from this that in
these animals the quantity of fixative is merely sufficient to bring
about the solution of the red corpuscles which are within the
phagocytes. In the case of fishes and higher animals (we may recall the
example of the red corpuscles of the guinea-pig when resorbed into the
organism of the gold-fish) the production of the fixative is much more
abundant, and this ferment can be easily demonstrated by its action _in
vitro_.
This over-production of a ferment which acts in the phagocytic
resorption, finds its analogue in the passage of certain digestive
ferments, such as amylase and pepsin in man and the dog, into the blood
and urine, as mentioned in the preceding chapter.
One of the best arguments in favour of the thesis here developed, has
been furnished to us by the analysis of the phenomena observed in
connection with the autospermotoxic serums of the guinea-pig. This idea
of autotoxins was originally put forward by Ehrlich in his memoirs,
published in conjunction with Morgenroth and already repeatedly cited.
Ehrlich asked himself whether the organism which resorbs, not red
corpuscles of an alien species, but red corpuscles of its own species,
would also be capable of developing haemolytic substances. With this
object he injected blood obtained from goats into these same goats or
into other individuals of the same species. He and Morgenroth[140] were,
under these conditions, able to obtain isotoxic serums, that is to say
serums which dissolve the red corpuscles of the goat, coming from other
individuals than those which had been treated by the blood and which
furnished the serum. In order to obtain this result, however, they had
to inject, not unaltered blood but blood mixed with water. The red
corpuscles of the unaltered blood pass readily into the circulation of
the animal of the same species, without being attacked by the
phagocytes. Now, we know from the experiments of Bordet that the stromas
of the red corpuscles suffice for the production of the fixative, whilst
the haemoglobin does not incite to the development of this ferment by
the organism. As the stromas, injected with a mixture of blood and
water, must be devoured by the macrophages, we can readily understand
that these phagocytes may serve for the elaboration of the fixative.
[Sidenote: [112]]
The resorption of the red corpuscles and that of spermatozoa which we
have presented as examples, may serve as types for the resorption
phenomena of formed elements in general. When other species of cells are
introduced into the organism, the resulting process always reveals the
same character: inflammatory reaction with preponderant intervention of
the macrophages; intraphagocytic digestion of the introduced elements;
excessive production and excretion of the fixatives. Whilst the
macrocytase is always the same in the same species of animal, the
fixatives are different and specific. In addition to the haemofixatives
and spermofixatives already described, we may obtain, as the result of
the injection of the corresponding cells, leucofixatives,
nephrofixatives, hepatofixatives, trichofixatives, etc. It does not
enter into our programme to treat the subject here[141]. We wish simply
to insist on those aspects of the resorption of cells which are closely
connected with the problem of Immunity. In the next chapter we must,
however, recur to certain features of the phenomena of resorption.
CHAPTER V
RESORPTION OF ALBUMINOID FLUIDS.
Resorption of albuminoid substances.—The precipitins of blood serum
which appear as a result of the absorption of serums and of
milk.—Absorption of gelatine.—Leucocytic origin of the ferment
which digests gelatine.—Anti-enzymes.—Antirennet.—The
anticytotoxins.—Antihaemotoxic serums.—Their two constituent
parts: anticytase and antifixative.—Action of anticytase.—The
antispermotoxins.—Origin of anticytotoxins.—Ehrlich’s theory on
this question.—Origin of antihaemotoxin.—Origin of
antispermotoxin.—Production of this antibody by castrated
males.—The antispermofixative produced when the spermatozoa are
excluded.—Distribution of spermotoxin and antispermotoxin in the
organism.
[Sidenote: [113]]
We stated at the beginning of the last chapter that various fluid
substances of very complicated chemical composition may be absorbed by
the tissues and utilised by the organism without requiring to be
modified by the digestive juices of the intestinal canal. We must now
describe, exactly, the phenomena observed in these cases and endeavour
to establish the mechanism of the absorption of fluids in the living
organism.
[Sidenote: [114]]
We have already cited the examples of blood serum, milk, and white of
egg, all of which are readily utilised by the organism which receives
them directly into the peritoneal cavity or below the skin. The proof
that these substances are modified—digested by the tissues, is furnished
by the observation that their injection necessarily brings about
appreciable changes in the properties of the blood. Th.
Tchistovitch[142], in a research carried out in the Pasteur Institute,
was the first to demonstrate that the resorption of the blood serums of
the eel and horse by the organism of the rabbit, excites in the blood of
the latter animal the production of specific precipitates. The blood
serum of rabbits that have been vaccinated against the toxic eel’s serum
gives a precipitate with eel’s serum; the serum of rabbits treated with
horse’s blood gives a similar precipitate with horse’s serum, etc. This
property has since been confirmed and studied by several observers, who
have made use of it for the recognition of human blood in medico-legal
investigations[143].
Bordet[144] has made the discovery that intraperitoneal injections of
the milk of cows into rabbits provokes in the blood serum of the latter
the property of giving a specific precipitate with cow’s milk only. This
precipitation bears a great resemblance to the coagulation of casein;
which, however, does not justify us in identifying the precipitating
substance with rennet. This fact has been confirmed for several other
species of milk, and Schütze[145], in an investigation carried on in the
Berlin Institute, essayed to apply it to the differentiation of the
various kinds of milk. In the same order of ideas, researches have been
made on the artificial precipitins that develop in the blood as the
result of injection of white of egg and other albuminoids[146].
Leclainche and Vallée[147] have prepared animals in such a fashion that
their serum produces a precipitate with urinary albumen. The biological
precipitin reactions are more sensitive than any of the chemical
reagents properly so called. These specific substances in the serums
must be looked upon as belonging to the group of soluble ferments,
approximating to the fixatives rather than to the cytases, since they
are unaltered by being heated to 56° C. Their action gradually declines
after passing 60° C. but is only destroyed at a temperature beyond 70°
C.
[Sidenote: [115]]
An analogous soluble ferment has been discovered in the blood serum of
animals treated with injections of gelatine. We owe to Delezenne, who
has studied this question in his laboratory at the Pasteur Institute,
the most important and most complete data on the resorption of gelatine.
The blood serum of normal animals possesses only a very feeble power,
sometimes even none, of liquefying gelatine. When however this substance
is injected several times, the serum, as is the rule for the formed
elements and quite a series of fluid substances, acquires a much more
pronounced activity. The gelatine, without giving any precipitate, is
simply dissolved and will no longer solidify when it is cooled. The
ferment of the serum that produces this effect resembles the precipitins
in that it withstands the action of a temperature of 56° C. and is only
destroyed beyond 60° C. Like the trypsins it acts in a weakly alkaline,
neutral, or weakly acid medium; but digestion takes place best in a
slightly alkaline medium.
The question of especial interest to us is that of the origin of this
ferment which digests gelatine. If several c.c. of a 10% solution of
this substance be injected into the peritoneal cavity of a laboratory
animal, there is provoked with certainty, within a few hours, a marked
leucocytosis of the peritoneal fluid. A considerable afflux of
leucocytes, amongst which the microphages are even more numerous than
the macrophages, takes place. When to a hanging drop of such an
exudation a trace of Ehrlich’s neutral red solution is added, there
appears almost at once an intense coloration of the numerous droplets
inside the two kinds of leucocytes. It is, therefore, manifest that the
gelatine excites a powerful positive chemiotaxis of the mobile
phagocytes and that it is absorbed by these cells. This experiment
demonstrates that the phagocytes can not only ingest solid bodies, such
as the various formed elements, coloured granules, etc., but that they
are also capable of absorbing fluid substances introduced into the
tissues or cavities of the organism.
The data brought forward by Delezenne demonstrate very clearly the part
played by the mobile phagocytes in the digestion of gelatine. He
obtained his best results in the dog. We know that it is easy in this
animal to provoke an aseptic exudation, very rich in leucocytes. This
exudation when deprived of its serum and washed with physiological salt
solution gives a solution which exerts a feeble digestive action on
gelatine. If the exudation be produced in a dog that has previously
received several injections of this substance, we obtain leucocytes
whose extract, obtained by the same method, will digest gelatine much
more actively. The digestive power of the leucocytes of the treated dog
is sometimes five times greater than that of the leucocytes of the
normal dog. Here, then, we undoubtedly have an acquired digestive power
which reveals a great reinforcement of the phagocytic activity.
[Sidenote: [116]]
In the prepared dogs the leucocytes have a much greater digestive action
on gelatine than has the blood serum of the same animals, a fact which
indicates that the source of the soluble ferment must be sought for in
the phagocytes themselves. The results of these researches are of great
service to us in the study of immunity properly so called.
For some time past attempts have been made to show that the soluble
ferments, diastases, or enzymes, are closely allied to albuminoid
substances. Nencki and Mme Sieber[148] support this view by their recent
researches on the chemical composition of pepsin. In all the above cases
there is this in common between the two categories of substances, their
absorption by the organism is followed by the appearance in the blood of
antagonistic ferments. Just as after the injection of milk, white of
egg, serums, etc. into the cavities or tissues, specific precipitins are
produced, so the injection of certain enzymes provokes the formation in
the organism of antienzymes or antidiastases.
[Sidenote: [117]]
It has been known for some time that the blood serum of many animals
prevents the action of certain enzymes. Thus Röden has shown that normal
horse’s serum retards or even completely prevents the coagulation of
milk by rennet. It has often been observed, too, that normal serums
hinder, more or less, the digestion of albuminoids by trypsin. It is
only quite recently, however, that we have begun to prepare antienzymes
by the injection into animals of corresponding enzymes. Thus,
Hildebrand[149] has succeeded in obtaining an antiemulsin in the serum
of rabbits, into which he had injected several separate doses of
emulsin. Fermi and Pernossi[150] have prepared an antitrypsin, and von
Dungern[151] has obtained an antidiastase against the proteolytic
enzymes of some bacteria. But of all the antienzymes, the one that has
been best studied up to the present is indisputably antirennet, obtained
independently by Morgenroth[152] and Briot[153]. The former of these
investigators treated goats with increasing quantities of rennet and was
able to assure himself, by comparative detailed researches, of the
appearance and increase in quantity of antirennet in the blood serum.
The goat which gave the best result ceasing to develop antirennet it was
impossible to make the antirennet potency go beyond a certain point.
Briot also obtained antirennet in rabbits into which he had injected
fluid rennet on several occasions. He was able to convince himself that
the antirennet of horse’s serum is a non-dialysable substance which is
precipitated by alcohol and certain salts. Like the precipitins and the
diastase which digests gelatine, antirennet resists a temperature of
55°–56° C.; even heating to 58° C. has no effect on the antirennet
serum. At 60° C. however, the heat begins to exert an injurious effect,
and after three hours at 62° C. the serum has lost all power to prevent
the coagulation of the casein by antirennet. Morgenroth and Briot both
state that the antirennet neutralises the rennet by a direct action.
The cell poisons, or cytotoxins, of animal origin which were treated in
the preceding chapter, likewise set up the production of special
antibodies, or anticytotoxins. The consideration of these latter has a
very special interest for those who study the question of immunity from
a general point of view. The first discovery of these anticytotoxins was
made in connection with the study of the toxic power of the blood serum
of eels. Camus and Gley[154] and, independently of them, H. Kossel[155]
demonstrated that animals when treated with increasing doses of eel’s
serum acquire an antitoxic property which protects their corpuscles
against the haemolytic action of ichthyotoxin, or the toxic substance of
the blood of eels. Th. Tchistovitch[156] has not only confirmed this
discovery, but has added to it new and interesting data.
[Sidenote: [118]]
When antitoxic serum is mixed _in vitro_ with red blood corpuscles of
the species which furnished the serum and there is added to it some
haemolytic eel’s serum, it will be found that the red corpuscles remain
quite unaltered. In the control tubes, however, in which the antitoxic
serum is replaced by normal serum of the same species, the red
corpuscles are very readily dissolved under the toxic influence of the
eel’s serum. In animals (rabbits) that are treated with this latter
fluid, there is established not only an antitoxic power of the blood,
but the red corpuscles acquire a resisting power more or less pronounced
against the ichthyotoxin of eel’s serum. When the red corpuscles are
separated from the serum of rabbits (treated with eel’s serum) and some
ichthyotoxin is added to them, solution very often does not take place
at all. According to the experiments of Tchistovitch there is no direct
relation between this acquired resistance and the antitoxic power of the
blood. Sometimes even a kind of antagonism is observed between the two
properties; that is to say, the red corpuscles of a rabbit whose serum
is very antitoxic may be extremely sensitive to the poison of the eel
whilst the converse may also hold good [cf. _infra_ p. 120].
The toxic action of the eel’s serum upon the red corpuscles of a great
number of Vertebrates is a natural property which demands no previous
treatment of the eel. It is the antitoxic power, directed against the
ichthyotoxin, which is developed only as a result of the preparation of
the animals by the administration of increasing doses of eel’s serum.
Nevertheless we also find natural antitoxins present in the blood of man
or animals that have not been treated and which act against the cell
poisons, cytotoxins, so widely distributed in the blood of a large
number of species of animals.
Besredka[157] has demonstrated that the blood serum of Man and many
Vertebrates contains a substance which prevents the solution of red
corpuscles under the influence of blood serums of a different species.
To reveal the presence of these antitoxins it is useful to heat the
serums to 56° C. and then to add to them red corpuscles of the same
species and some haemolytic serum of a different species. Under these
conditions the solution of the red corpuscles does not take place,
whilst their mixture with haemolytic serum alone, inevitably provokes
haemolysis.
[Sidenote: [119]]
Along with these natural antihaemolysins there exist a number of
artificial antihaemolysins or antihaemotoxins. Jules Bordet[158] was the
first to draw attention to this important subject. He first obtained
these antihaemolysins by injecting blood serum of the fowl, which
possesses a very great haemolytic power on the red corpuscles of the
rabbit, into individuals of this latter species. After some injections,
the serum of these treated rabbits was found to be antihaemotoxic
against the fowl’s serum. Later[159], Bordet obtained a serum against an
artificial haemotoxin. The serum of the guinea-pig is innocuous to the
red corpuscles of the rabbit. But when rabbit’s blood was injected
several times into guinea-pigs the serum of the latter became very
solvent for the red corpuscles of the rabbit. To prevent this action it
is sufficient to inject the haemotoxin of treated guinea-pigs several
times into rabbits. The serum of these rabbits becomes antihaemotoxic
and protects the red corpuscles of the rabbit against the solvent action
of guinea-pig’s serum.
In the normal haemolytic serums, such as the serums of the eel and fowl,
the presence of two substances which act by combining could not be
demonstrated. On the other hand, in the serums that were obtained as a
result of the treatment of animals by the injection of blood from a
different species, it was easy to demonstrate, as we have shown in the
preceding chapter, the presence of two constituent substances which are:
the macrocytase (alexine, complement) and the fixative (amboceptor of
Ehrlich, sensibilising substance of Bordet). For this reason the study
of the antihaemotoxins obtained against artificial haemotoxins is
endowed with special interest. As the solution of the red corpuscles, in
this case, can be prevented either by an antitoxic action directed
against the cytase, or by a neutralisation of the fixative (for the
concurrence of these two substances is indispensable in order that the
solution may take place), Bordet asked whether the antitoxic serum,
obtained by him in rabbits, is anticytatic or antifixative, or whether
it contains both properties. Before resolving this problem it was
necessary to establish some of the essential characters of artificial
antihaemotoxic serums. The principal one amongst them is the resistance
of these antihaemotoxins to a temperature of 55–60° C.; even when heated
to 70° C. the antihaemotoxins retain, at least in part, their
fundamental property. In this respect these substances differ from the
cytases and approach the precipitins, fixatives and agglutinins.
[Sidenote: [120]]
[Sidenote: [121]]
The very exact experiments carried out by Bordet have demonstrated that
in the serum of rabbits, treated with the specific haemotoxic serum of
guinea-pigs, two substances, an anticytase and an antifixative, are
found in combination. The former of these antitoxins is found in
abundance, but the amount of antifixative is very small. Bordet was led
to this result in the following way. To prevent the solution of the red
corpuscles of the rabbit in the haemotoxic serum of the guinea-pig, it
was necessary for him to add a considerable dose (10 to 20 times) of the
antitoxic serum. When, however, he heated the latter to 55° C. the
quantity of this serum necessary to prevent haemolysis could be reduced
very considerably. In place of its being necessary to add to the
haemotoxic serum 10 or 20 volumes of antitoxic serum, it was sufficient
to add three or sometimes only two volumes of this heated serum. As we
know already, heating to 55° C. destroys the macrocytase which should be
found in the antitoxic blood of the rabbit. This cytase by itself is
incapable of dissolving the red corpuscles of the same species; but when
it is added to the fixative of the haemotoxic serum of the guinea-pig
the macrocytase of the rabbit’s serum dissolves them very readily. Hence
the conclusion that in the haemotoxic serum of the guinea-pig there must
be present a quantity of fixative sufficient to allow of the solution of
the red corpuscles by the macrocytase of the rabbit’s serum. This
antitoxic serum, therefore, which only prevents the haemolysis on the
condition of being added in comparatively large quantity, contains very
little antifixative. When, by heating this serum to 55° C. we destroy
the rabbit’s macrocytase, the mixture of antitoxic serum of the rabbit
and haemotoxic serum of the guinea-pig, which ordinarily dissolves the
red corpuscles of the rabbit, now leaves them intact. The reason is that
the free fixative contained in this mixture does not find any available
macrocytase: that of the rabbit being destroyed by the heating, and that
of the guinea-pig neutralised by the antitoxic serum. The experiment I
have just described proves that this antitoxic serum contains specific
anticytase. This anticytase is capable of neutralising the guinea-pig’s
macrocytase, but is altogether powerless against that of the rabbit.
This last circumstance allows us to investigate whether the antitoxic
serum of the rabbit contains, in addition to anticytase, a specific
antifixative. Bordet prepared a mixture of antitoxic serum of the
rabbit, heated to 55° C., with haemotoxic serum of the guinea-pig, also
heated to 55° C. In this mixture the two macrocytases (that of rabbit
and that of guinea-pig) have been destroyed by heat, but the antitoxins
of the rabbit’s serum and the fixative of the haemotoxic serum have
remained intact. This mixture owing to its want of macrocytases was
incapable of dissolving the red corpuscles of the rabbit. By adding to
it some fresh unheated serum from a normal rabbit the rabbit’s
macrocytase was introduced. As the latter could not be neutralised by
the anticytase of the antitoxic serum and was incapable, by itself, of
dissolving the red corpuscles of the rabbit, it was unable to produce
haemolysis except on the condition that there is in the mixture a
sufficient quantity of unneutralised free specific fixative. As a matter
of fact, the red corpuscles of the rabbit are not dissolved in the
mixture described; this proves that the fixative had become inactive in
consequence of the presence of an antifixative in the antitoxic serum of
the rabbit. I need not enter into further details of Bordet’s
experiments, which have fully demonstrated the fact that in the
antitoxic serum of his rabbits there were really two antitoxins; an
anticytase abundant in quantity, and an antifixative present in much
smaller amount.
Ehrlich and Morgenroth[160] quite independently of Bordet have shown
that an antihaemotoxic serum is very rich in anticytase. After making a
number of injections of normal horse’s serum (very rich in cytase) into
a goat, they obtained in the blood serum of the latter an anticytase
very active against the cytase of the horse. This antitoxic serum of the
goat, as might be anticipated, contains no antifixative, the horse’s
serum that served for the injections coming from normal horses which
contained no, or very little, fixative. Even in another case, where
these investigators[161] injected a dog with sheep’s serum very rich in
fixative specific for the red corpuscles of the dog, they did not
succeed in obtaining any antifixative. These observations in no way
diminish the value of the discovery of the antifixative by Bordet,
though they demonstrate that this antitoxin cannot, in certain cases, be
found in the serum. Ehrlich and Morgenroth themselves throw out, in this
connection, the suggestion that in these cases the antifixative remains
linked to the cell which produces it, without being thrown off into the
blood.
[Sidenote: [122]]
The very precise data that we have just summarised do not seem to agree
with the statements of certain other investigators. Thus Schütze[162],
from his researches on the antihaemotoxic serum of guinea-pigs, directed
against the rabbit’s haemotoxin, has arrived at the conclusion that in
the former an antifixative only is produced. As he merely injected into
his guinea-pigs haemotoxic rabbit’s serum that had been heated to 60° C.
and consequently deprived of the macrocytase, he concluded that in this
serum there remained only the specific fixative capable of provoking the
formation of an antitoxin. This must consequently be an antifixative.
Paul Müller[163] came to a similar conclusion, after injecting rabbits
with the heated haemotoxic serum of fowls. These injections caused the
formation in the rabbit’s serum of an antitoxin that Müller regarded as
an antifixative.
Ehrlich and Morgenroth[164] objected to this interpretation, taking
their stand on experiments made with the serums of normal animals. They
were able to show that these serums, when injected in the fresh state or
after being heated to 60° C., caused the production of a corresponding
antihaemotoxin which is nothing but an anticytase. When Schütze and Paul
Müller concluded that by heating the serums they had entirely deprived
them of cytase elements they did not take into account the possibility
of the cytases being transformed, under the influence of heat, into
other bodies unable to produce haemolysis, but quite capable of
provoking the formation of anticytases. Ehrlich and Morgenroth give to
these new bodies, derived from cytases under the influence of
temperatures between 55°–60° C., the name of _complementoids_; and these
complementoids appear in the experiments of Schütze and Müller to have
caused the production of antitoxins—anticytases.
In all the investigations just summarised the anticytases have been
obtained by the injection into animals of various blood serums, fresh or
heated. Wassermann[165] has discovered another method of arriving at the
same result. He injected into guinea-pigs the leucocytes of rabbits,
carefully deprived of all traces of serum. After some time the blood
serum of guinea-pigs thus treated became weakly but distinctly
anticytatic. From this experiment Wassermann draws the conclusion that,
as has been often affirmed by several observers, the leucocytes really
contain cytases.
[Sidenote: [123]]
How do the anticytases act upon the cytases? On this point all observers
who have studied this question have but one answer, the action of the
anticytases is direct. Bordet thinks that the two substances combine so
intimately that they cannot be again separated by heat. We know that the
cytases are very sensitive to heat and that their haemolytic property is
destroyed at 55° C. The anticytases, on the other hand, as already
noted, are much more resistant to the action of heat. Bordet has
prepared mixtures of haemolytic cytase serum and of antihaemolytic
serum, neutral mixtures, that is to say, inactive for red corpuscles or
with a very feeble action upon red corpuscles that have been
sensibilised by the specific fixative. These mixtures no longer exhibit
antihaemotoxic properties or they exercise this power in a very feeble
degree. If in these mixtures the cytases remain uncombined alongside the
anticytases, it is to be expected that heating them to 55° C. will
restore the antihaemotoxic function of the anticytases; the cytases
being destroyed at 55° C. there will remain in the mixtures only active
anticytase. The experiments made on this point have demonstrated that
the heating of these mixtures does not restore the antihaemotoxic
action, that is to say, the anticytase is definitely combined with the
cytase.
Ehrlich and Morgenroth have satisfied themselves that their
antihaemotoxin exerts no influence, either upon the red corpuscles or
upon the fixative, and is only capable of preventing the action of the
cytase. They introduced red corpuscles of the rabbit into a mixture of
goat’s serum, heated to 56° C. and thus only retaining its fixative, and
anticytase serum. The fluid bathing the red corpuscles was then removed
by centrifugalisation and the corpuscles were mixed with normal
haemolytic horse’s serum. Solution of the red corpuscles took place at
once as the anticytase had been completely removed during
centrifugalisation, being combined with neither the red corpuscles nor
the fixative.
These investigators have obtained various anticytases by injecting serum
of various species of animals into other mammals. They observed,
however, that injections of the serum of an allied species did not bring
about the formation of anticytases. Thus the injection of goat’s serum
into sheep, or of that of sheep into goats, never produced anticytase
serum.
[Sidenote: [124]]
In addition to antihaemotoxic serums several other analogous
anticytotoxic serums have now been obtained. Thus Delezenne has prepared
serums which prevent the action of neurotoxin and of the cell poison
which destroys the liver cells. We[166] have been able to obtain a
rabbit’s serum which prevents the spermatozoa of this rodent being
rendered motionless by the specific spermotoxin of the guinea-pig. More
recently Metalnikoff[167], working in my laboratory, has prepared
another antispermotoxic serum which prevents the specific spermotoxin of
the rabbit from arresting the movement of the guinea-pig’s spermatozoa.
[Sidenote: [125]]
As the history of these antispermotoxins presents certain interesting
general features we may with advantage, perhaps, dwell on some of their
characters. The two antispermotoxins mentioned above are distinguished
by certain peculiarities. When Metalnikoff set to work to inject
rabbit’s spermotoxin into guinea-pigs, he thought that he had an easy
task before him and that after a few injections the guinea-pig’s serum
would become antispermotoxic. This, however, was not the case. The serum
from these animals when mixed with spermotoxic serum was powerless to
prevent the immobilisation of the spermatozoa of the guinea-pig. It was
only when he heated the serum of his treated guinea-pigs to 56° C. that
the antispermotoxic power appeared with the greatest distinctness. The
inefficacy of the unheated serum must therefore depend on the toxic
action of the guinea-pig’s macrocytase, because it is this substance
alone that can have been destroyed by the heating process. Now, in order
that this macrocytase may act, the presence of the fixative is
necessary, which leads us to the conclusion that the serum of the
guinea-pigs injected by Metalnikoff contained no antifixative. This
hypothesis was fully confirmed by experiment. Metalnikoff introduced a
drop of guinea-pig’s serum into a mixture of antispermotoxic serum,
heated to 56° C., with spermotoxic serum. The spermatozoa continued
their movements in normal fashion. But when afterwards he added a few
drops of unheated serum from a normal guinea-pig the motions of the
spermatozoa were arrested almost instantaneously. Consequently there was
present in the mixture rabbit’s macrocytase which had been neutralised
by the anticytase of the prepared guinea-pig’s serum and for that reason
the spermatozoa remained motile. But in the same mixture we had also the
specific fixative, coming from the rabbit’s spermotoxic serum, which
remained free and not neutralised. The motile spermatozoa had become
impregnated with this fixative and a little guinea-pig’s macrocytase
(against which the anticytase was powerless) was sufficient to make them
suddenly cease their movements.
There is no doubt, then, that the serum of guinea-pigs that have been
treated with spermotoxin contains anticytase only and no, or almost no,
antifixative. Such is not the case with the antispermotoxin obtained by
us in rabbits that were treated with spermotoxic toxin of guinea-pigs.
Several consecutive injections were sufficient to render the serum of
the rabbits so treated capable of preventing the action of the
spermotoxic serum of the guinea-pig on the motility of the rabbit’s
spermatozoa. In the mixture of antispermotoxic serum and spermotoxic
serum these spermatozoa continue to move for a considerable time, whilst
in the control mixture prepared with normal rabbit’s serum and
spermotoxic serum they become motionless at the end of a few minutes. To
obtain this marked effect it was not necessary to heat the
antispermotoxic serum as in Metalnikoff’s case. Indeed I have performed
almost all my experiments with fresh serums, unheated. As the rabbit’s
serum contains macrocytase capable of rendering the spermatozoa,
sensibilised by the fixative, motionless and as this macrocytase cannot
be neutralised by the anticytase that is active against the guinea-pig’s
macrocytase, the fact I have just pointed out indicates that the
antispermotoxic serum of my rabbits contains antifixative. The
difference between the antispermotoxic serum obtained by Metalnikoff and
that prepared by me is similar to that observed between the
antihaemotoxic serums. Some contain only anticytase but others
undoubtedly contain antifixative also.
[Sidenote: [126]]
As this result appeared to me to be of far-reaching importance I felt
bound to verify it by another method. I injected certain rabbits with
spermotoxic serum of the guinea-pig and others with normal guinea-pig’s
serum. The amount of cytases being about the same in both, the strength
of the serums obtained as the result of injections of normal serum and
of specific serum should be the same if the antispermotoxic serums
contain anticytase only. Experiment demonstrates just the contrary. The
antispermotoxic serum of rabbits treated with normal guinea-pig’s serum
was on every occasion much less active than the serum of rabbits
injected with the spermotoxic serum of prepared guinea-pigs. The former
contains anticytase only, whilst the latter contains in addition
antifixative. Weichhardt’s[168] experiments carried out in my laboratory
corroborated the conclusion I have just formulated.
Having made ourselves acquainted with the constitution of the
anticytotoxins we may now pass to the question of the origin of these
bodies and of analogous ferments which act in the resorption of
albuminoid substances in the blood and in the tissues.
We have already mentioned that the leucocytes are charged with a soluble
ferment which digests gelatine, and that in animals treated with
injections of gelatine these cells elaborate a much larger amount of the
ferment. Here we have evidence of a kind of education of the leucocytes
to produce a greater amount of digestive ferment, in a manner quite
analogous to that which has been described in Chapter III in connection
with the augmentation of the pancreatic ferments in intestinal
digestion. It is, then, quite permissible to look upon leucocytes, and
probably phagocytes in general, as the source of the soluble ferment
that digests gelatine.
[Sidenote: [127]]
Is this the case with the other substances which take an active part in
the resorption of albuminoid substances in the fluids and tissues of the
organism? Up to the present the origin of precipitins and antiferments,
such as antirennet, has not been studied. The problem being very complex
and difficult, it appears to be impossible at present to solve it. It is
known indeed that the introduction of these substances into the organism
provokes a reaction similar to the one we have described in the case of
the injection of gelatine into the peritoneal cavity of guinea-pigs.
Thus Morgenroth[169] observed that in his goats the subcutaneous
injection of sterile rennet caused the formation of extensive
infiltration at the seat of inoculation, this being accompanied by
fever; we are justified in concluding from this that rennet provokes a
marked leucocytic reaction. Hildebrandt[170] has demonstrated by direct
experiment that rennet, when enclosed in capillary glass tubes and
introduced below the skin of rabbits, induces a marked positive
chemiotaxis. This led to the formation of a leucocytic plug several
millimetres long. Now we know from Briot that the rabbit is capable of
producing antirennet. Hildebrandt has further shown that several other
diastases, or hydrolytic ferments, such as sucrase and emulsin, give
rise to a similar chemiotactic phenomenon. The leucocytic reaction is
consequently a general phenomenon following the introduction into the
tissues of substances of complex chemical composition capable of
provoking the formation of antibodies. We are tempted from this fact to
accept it as a law that the leucocytes are capable of producing these
latter substances. Although this hypothesis may be very probable, the
number of facts at our disposal is not yet sufficient to justify the
statement that its truth is demonstrated.
Since it is the red corpuscles which are affected by the haemotoxins it
might be asked whether it may not be that these elements defend
themselves by the production of antihaemotoxins the overplus of which is
thrown into the blood and fluids in general? The researches that have
been made on this point relate especially to the antihaemotoxin of the
blood serum of rabbits in relation to the ichthyotoxin of eel’s serum.
We must therefore examine the collected evidence bearing on
anticytotoxins and analogous bodies and endeavour to form some idea as
to their probable origin. A large accumulation of exact data bearing on
the antihaemotoxins does not afford us sufficient information as to the
source of these substances.
Let us first examine the question, is it possible to attribute to the
red corpuscles the function of producing the antihaemotoxins? If these
elements are really the source of the antihaemotoxins it is probable
that the red corpuscles of animals whose serum is antihaemotoxic will
exhibit marked resistance to the toxins; thus we know that the white
corpuscles which produce gelatinase digest gelatine much better than
does the serum of the same animals. From the experiments of Tchistovitch
(_l. c. supra_ p. 110) on rabbits that have been immunised against eel’s
ichthyotoxin, it must be accepted that the red corpuscles of these
animals are often very sensitive to the action of the poison at a period
when the blood serum of the same rabbits exhibits a marked
antihaemotoxic power. It is not until later in the process of
immunisation, when the serum loses a great part of this power, that the
red corpuscles become resistant to the ichthyotoxin.
[Sidenote: [128]]
But before we abandon the hypothesis of the production of
antihaemotoxins by the red corpuscles we must see if it cannot be
reconciled with the facts, by the application of Ehrlich’s side-chain
theory[171]. This theory was evolved with the object of explaining the
production of antitoxins and their action on bacterial and vegetable
toxins. Later, Ehrlich has extended it to the cytotoxins, anticytotoxins
and bactericidal substances.
[Sidenote: [129]]
According to Ehrlich the complex molecule of albuminoid substances
contains, besides the central stable nucleus, a number of side-chains,
or “receptors,” which fulfil various accessory functions and serve
especially for the nutrition of the cell. These receptors have a great
affinity for the various substances necessary for the maintenance of the
life of the cell. Under normal conditions these receptors seize
nutritive molecules, as a leaf of _Dionaea_ seizes the fly that serves
it as food. Under special conditions these receptors lay hold of complex
molecules of albuminoid substances, such as the various toxins. In this
case the receptor, instead of combining with a molecule which supports
life, fixes a molecule which poisons the cell. According to Ehrlich’s
theory on the constitution of toxins their molecules contain an atomic
group which poisons—the _toxophore_, and another group which combines
with the receptor—the _haptophore_. The toxic group of a complex poison,
such as ichthyotoxin, cannot penetrate into a red corpuscle except by
the help of the haptophore group and of the corresponding receptor. When
a red corpuscle has absorbed a large number of molecules of
ichthyotoxin, the united action of the toxophore groups renders life
impossible and the corpuscle is dissolved. But when a red corpuscle has
been touched by only a few toxic molecules, too few to compromise life,
there is merely immobilisation of the receptors which are combined with
the haptophore groups of the ichthyotoxin. As these receptors fulfil an
important function in the nutrition of the red corpuscles, the latter
reproduce them in larger numbers than were originally present. We know
that in the phenomena of repair an over-production of the new-formed
parts often takes place and, according to Ehrlich, to this
over-production the presence of antitoxins in the fluids of the body is
due. The receptors, developed in excess by the red corpuscles, fill
these cells, and no longer finding room therein are extruded from them
and overflow into the blood and other fluids of the organism. When a
fresh injection of toxin makes its way to the blood it there meets with
a number of free receptors, endowed with an affinity for the haptophore
group of the molecule of the toxic substance. The chemical combination
between the two substances takes place at once in the plasmas, a fact
which prevents the haptophore group of the toxin from uniting with the
receptor of the red corpuscles and so injuring these cells by
introducing the toxophore group into them. According to this theory the
same receptors which, in the free state in the fluids, fulfil the
_antitoxic_ function become in the interior of the red corpuscles the
vehicles of intoxication and consequently fulfil a _philotoxic_
function. This opposite _rôle_ of the receptors has often been compared
to a lightning-conductor; so long as the receptors are attached to the
molecule of the living protoplasm they attract the toxin just as a
lightning-conductor attracts the lightning when it is badly insulated.
[Sidenote: [130]]
So interpreted, it is easy to conceive that the red corpuscles of
animals whose fluids are antihaemotoxic may be sensitive to the toxic
action of eel’s serum, as has been observed by Tchistovitch. As soon as
the protective fluids have been removed from the red corpuscles of the
immunised organism, the corpuscles when placed in contact with
ichthyotoxin (eel’s serum) attract the haptophore groups of the poison
by means of their numerous receptors. These haptophores in their turn
introduce the toxophore groups which dissolve the red corpuscles without
the slightest difficulty. This theory does not explain the cases, which
are numerous, in which the red corpuscles of rabbits that are vaccinated
against eel’s poison resist this poison. Camus, Gley, and Kossel,
working independently, have arrived at the result that the red
corpuscles of immunised rabbits, from which the serum has been carefully
removed, are not dissolved when submitted to the action of ichthyotoxin,
whilst the red corpuscles of untreated rabbits placed under the same
conditions, undergo a rapid solution. Tchistovitch confirming this fact
has added to it the observation that the resistance of the red
corpuscles of the rabbit is most often found when the serum loses its
antitoxic power. If the receptors of the red corpuscles of immunised
rabbits, owing to their great affinity for the haptophore group of the
ichthyotoxin molecule, only attract the toxophore group of this poison,
as the lightning-conductor when badly insulated attracts the lightning,
the red corpuscles should never manifest resistance. To explain this
contradiction we must not suppose that the red blood corpuscles which
have become resistant have got rid of their receptors. In fact, if these
receptors are so necessary to the nutrition of the cell that their
absence has set up this extraordinary over-production which has
inundated the fluids, it is evident that one cannot admit the existence
of red corpuscles entirely deprived of corresponding receptors.
When examined from different points of view the hypothesis of the
production of antihaemotoxin by the red corpuscles is surrounded with
very great difficulties. It appears to be probable, therefore, that the
source of this antitoxin must be sought for in other cell elements, and
we may be allowed to recall to mind those cells which manifest a general
and local reaction of the most constant kind after each injection of
ichthyotoxin. Tchistovitch has observed that eel’s serum when introduced
into rabbits in non-fatal but immunising doses excites a marked
hyperleucocytosis.
[Sidenote: [131]]
The question of the origin of anticytotoxins being so complicated, it
has been necessary for its elucidation to seek an experimental method of
excluding the organ in which the antibody is supposed to have its
origin. As we cannot think of eliminating the red or white corpuscles,
nor the greater part of the tissues and organs, there remains only one
way of bringing about this result. It is the suppression of the male
genital organs. We know already that the injection of semen readily
excites the production of a spermotoxin, and that this spermotoxin gives
rise to the development of a corresponding antispermotoxin. If it is the
spermatozoa, that is to say the elements having a particular affinity
for the spermotoxin, which elaborate the antitoxin we must conclude that
castrated males would be incapable of producing it. With this in view we
have carried out a great number of experiments which have amply proved
to us that male rabbits when deprived of their sexual organs are fully
as capable of developing antispermotoxin in their fluids as are control
rabbits in which the male genital apparatus remains intact. Doe-rabbits,
and young, sexually immature rabbits of both sexes, also react to
injections of spermotoxin by producing the corresponding
antispermotoxin. The specific elements which are sensitive to the action
of a cytotoxin undoubtedly are not indispensable for the development of
the corresponding anticytotoxin. This result is in complete harmony with
the hypothesis above put forward, that the red corpuscles cannot be
regarded as the source of the antihaemotoxin. In the case of
antispermotoxin this fact can be rigorously established by experiment.
Here arises the following question. We have seen that the anticytotoxins
are composed of two different substances: an anticytase and an
antifixative. The former is an antitoxin capable of neutralising
macrocytase, the soluble ferment which will attack indifferently all
kinds of cell elements. It is not to be wondered at, then, that the
exclusion of the spermatozoa in no way prevents the production of
anticytase by an organism which receives injections of cytotoxins. These
latter, as we have already said, contain cytase along with the specific
fixative; the macrocytase can attack any kind of animal cell provided
that it can find some fixative or any other means to penetrate into the
interior of these formed elements. We have seen that the
antispermotoxin, obtained by Metalnikoff in guinea-pigs, does not
contain any anticytase. Amongst his animals treated with spermotoxin was
a castrated male guinea-pig which also produced anticytase. There is
nothing astonishing in this fact, the injected cytase must have linked
itself to many other cells which were able to develop anticytase.
[Sidenote: [132]]
But the example of the antispermotoxin of the rabbits in my own
experiments is very different. In order that it might manifest its
action the serum of these rabbits did not need to be heated to 56° C.;
it was not necessary to rid it of its own macrocytase which could have
acted under the influence of the fixative, if this latter for want of
antifixative had remained free in the added spermotoxin. This
antifixative, then, is undoubtedly found in the serum of castrated males
which have shown themselves capable of producing not only anticytase,
but also antifixative. This result has been further verified by
comparative experiments on castrated male rabbits, some of which
received spermotoxic guinea-pig’s serum whilst the others received only
normal guinea-pig’s serum. It has been demonstrated that the amount of
cytases remains almost constant in both normal and vaccinated
animals[172]. If, then, the antispermotoxins contain only anticytase,
the injection of specific guinea-pig’s serum and that of normal
guinea-pig’s serum should produce the same result, that is to say the
serums of castrated rabbits, when treated by these two kinds of
guinea-pig’s serum, should exhibit the same antispermotoxic power.
Experiments have, however, proved that this is not the case. The serum
of castrated rabbits that have been injected several times with normal
guinea-pig’s serum becomes distinctly antispermotoxic, but its power to
protect the spermatozoa of the rabbit against being deprived of motility
by the guinea-pig’s spermotoxin is greatly inferior to that which is
developed in the serum of other castrated rabbits that I injected with
spermotoxic guinea-pig’s serum. Of course all the other conditions of
the experiment were the same for the two groups of rabbits.
[Sidenote: [133]]
Several series of facts, then, focus to this fundamental point, that the
organism of an animal that has been deprived of its male sexual organs
is in a condition to produce antispermofixative. Against the argument
that we have drawn from the fact that the antispermotoxic serum of
castrated rabbits that have been treated with spermotoxic serum acts
without being heated, might be cited certain experiments made by Ehrlich
and Morgenroth. The antispermotoxic action in this case, as already
stated, demonstrates that the serum of prepared rabbits contains
antifixative. Otherwise, had the fixative not been neutralised, it would
have allowed the macrocytase of the rabbit’s serum to arrest the
movements of the spermatozoa. Now the two above-named observers have
demonstrated[173] that the injection of different serums into animals is
capable of exciting in their blood the development of anticytases. The
macrocytase of castrated rabbits which, before treatment with the
spermotoxin, was capable of arresting the movements of rabbits’
spermatozoa acted upon by a fixative, might become inert after the
injections of spermotoxic serum of guinea-pigs. To clear up this point I
asked M. Weichardt[174], who has carried out work on this subject in my
laboratory, to try by means of unheated serums of normal animals, to
restore the activity of spermotoxin that had been mixed with
antispermotoxic serum. Spermatozoa of rabbits were put into a definite
mixture of spermotoxic guinea-pig’s serum, heated to 56° C., and
antispermotoxic serum, also heated to 56° C., obtained from castrated
rabbits that had been treated with spermotoxin. The spermatozoa remained
very active in this mixture which contains specific fixative (in the
spermotoxic guinea-pig’s serum) and antispermotoxin. To this mixture is
added a little normal rabbit’s or horse’s serum, unheated. These serums
contain cytases and would be quite capable of arresting the movements of
the spermatozoa if there was found in the mixture any free fixative that
would enable the macrocytase to be linked to the spermatozoa. Under
these conditions the spermatozoa remain motile for a long time. The
fixative, then, was no longer active; it was neutralised by the
antifixative of the antispermotoxic serum of castrated rabbits. A
control experiment was made with the same substances; but the castrated
rabbits’ serum that had been treated with spermotoxic serum was replaced
by the serum of other castrated rabbits treated with normal guinea-pig’s
serum. In these latter mixtures the spermatozoa became motionless at the
end of a very short time; the fixative, not being neutralised, readily
allowed the rabbit’s and horse’s cytases to affect the spermatozoa.
It follows from all this that the antispermotoxic serum of castrated
male rabbits, when treated with normal guinea-pig’s serum, contains
anticytase only; whilst the serum of castrated male rabbits, treated
with specific and spermotoxic guinea-pig’s serum, contains anticytase
and antifixative. The latter, then, has been produced independently of
the sensitive elements,—the spermatozoa.
[Sidenote: [134]]
Having established the fact that antispermotoxin does not come from the
male organs, it was necessary to try to ascertain its true source. With
this object in view we injected spermotoxic serum into young rabbits
(quite capable of producing antispermotoxin) and tried to follow the
fate of the spermotoxin in the organism. When spermotoxic guinea-pig’s
serum is injected into the peritoneal cavity of the rabbit a notable
amount of spermotoxin is found in the thickened portion of the omentum
made up of lymphoid tissue. But the greater portion of the poison passes
into the circulation whence it goes to fix itself in various organs,
especially the spleen. At the moment when the spermotoxin is found in
the blood a certain quantity of this fluid was drawn off into tubes
containing some drops of extract of leeches’ heads. After the blood thus
treated had been centrifugalised the plasma was decanted and its power
of arresting the movements of spermatozoa was compared with that of
serum of the same blood prepared in the usual way. From these researches
it results that the plasma is always richer in spermotoxin than is the
corresponding serum. Sometimes the difference in favour of the plasma is
very great.
A part of the spermotoxin passes into the kidneys and the suprarenal
capsules. It is probable that, as is the case with so many soluble
poisons, a certain proportion of the spermotoxin may be eliminated by
the uropoietic organs. A small quantity of this poison is found also in
the male and female sexual glands of young non-castrated rabbits.
The search for some main centre of origin for the production of
antispermotoxin has as yet led to no positive result. The power of
arresting the movements of spermatozoa first appears in the blood
plasma, and it is this same fluid which, later, is more antispermotoxic
than is any organ. Amongst the tissues which fix spermotoxin the genital
organs play not the slightest part in the production of antispermotoxin.
The experiments with castrated rabbits afford sufficient proof of this.
On the other hand it becomes more and more probable that the phagocytic
system, disseminated in many organs, and especially the leucocytes,
furnish the antispermotoxic substance. The fixation of the spermotoxin
by the leucocytes of the blood, such as the cells of the omentum and of
the spleen, already offers us a valuable indication. The absence of any
particular organ that might have the monopoly of fixing the spermotoxin
and which should later be found charged with a predominant amount of
antispermotoxin also speaks in favour of the phagocytic origin of this
antitoxin.
[Sidenote: [135]]
After a single intraperitoneal injection of spermotoxic guinea-pig’s
serum into young rabbits, the blood of the latter is distinctly
spermotoxic for several days; later it becomes indifferent, but eight or
ten days after the commencement of the experiment the blood begins to
exhibit an antispermotoxic power. In these cases the plasma shows itself
more active than the serum. When the rabbits are killed at this stage of
commencing antitoxic production, it is found that an extract of the
organs is not antispermotoxic or only feebly so. In all cases this
power, when it exists, is more feeble than that of the blood fluid. The
results obtained with extracts of organs are not constant. Sometimes the
spleen possesses more antitoxic activity, whilst the liver, thymus,
omentum, lymphatic glands and genital glands exhibit none of this
property. In other cases the survival of the spermatozoa that are
influenced by the spermotoxin has been longest in the extract of the
suprarenal capsules. Sometimes the extract of the omentum exhibits the
greatest antispermotoxic power. This great variability in the
development of the property of protecting the spermatozoa accords well
with the idea that the elements which produce antispermotoxin are
wandering cells which, under diverse influences, may be localised in
very diverse points of the organism.
We must not deceive ourselves. The facts which have been collected up to
the present do not allow us as yet to form a final opinion on the origin
of anticytotoxins, but we are quite justified in regarding as very
probable the hypothesis that the phagocytes play a most important part
in the process. It is in all cases beyond doubt that the amoeboid cells
which resorb the formed elements play a very important part in the
resorption of fluids of very complex molecular composition.
CHAPTER VI
NATURAL IMMUNITY AGAINST PATHOGENIC MICRO-ORGANISMS
Natural immunity and the composition of the body fluids.—Cultivation
of the bacteria of influenza and pleuro-pneumonia in the fluids of
refractory animals.—Resistance of _Daphniae_ to the
Blastomycetes.—Examples of natural immunity in Insects
and Mollusca.—Immunity of Fishes against the anthrax
bacillus.—Immunity of frogs against anthrax, Ernst’s bacillus, the
bacillus of mouse septicaemia and the cholera vibrio.—Natural
immunity in the cayman.—Immunity of the fowl and pigeon against
anthrax and human tuberculosis.—Immunity of the dog and rat
against the anthrax bacillus.—Immunity of Mammals against anthrax
vaccines.—Immunity of the guinea-pig against spirilla, vibrios,
and streptococci.—Natural immunity against anaerobic bacilli.—Fate
of Blastomycetes and _Trypanosomae_ in the refractory organism.
[Sidenote: [136]]
In the third chapter reference has been made to the frequency of cases
of natural immunity against infective diseases. Examples of this
immunity occur in the lower animals—the Invertebrata—and are widely met
with among the Vertebrata. We have already mentioned that this natural
immunity can be attributed neither to insusceptibility to microbial
toxins nor to the elimination of the micro-organisms by the excretory
channels. Nevertheless the pathogenic agents which have penetrated into
the tissues of the refractory organism disappear, without being
eliminated. To facilitate the study of their disappearance it has been
necessary to pass in review the phenomena that follow the introduction
of foreign bodies into the organism and to present a brief analysis of
the process of resorption of cell elements in its relations to
digestion. We have tried to demonstrate that resorption is nothing more
than a process of digestion which, instead of going on in the intestinal
canal, takes place in the tissues; that it is, indeed, an intracellular
digestion exactly comparable to that which serves for the nutrition of
certain of the lower animals.
[Sidenote: [137]]
A knowledge of all these facts is necessary before we can deal with the
subject to which the present chapter must be devoted—the innate natural
immunity of animals and man against pathogenic micro-organisms. As,
under natural conditions, it is the micro-organism and not its toxic
products which invades the organism, it is clear that we must give the
first place to the study of immunity against the micro-organism. The
more so because this form of immunity is much more frequently met with
than is an insusceptibility to toxins.
Since the animal organism has a very variable composition it might be
concluded that the micro-organisms find in the refractory species simply
a chemical medium in which they cannot live. We cannot go far in the
discussion of this supposition without seeing that it may be rejected.
Among the pathogenic micro-organisms some are distinguished by a great
fastidiousness and sensitiveness as regards the medium in which they are
placed. Such, for example, are the parasites of malaria and their
allies. They live inside the red blood corpuscles of Vertebrata and
appear to be extremely discriminating in regard to their requirements.
All animals, even monkeys, are refractory to human malarial fevers. It
might be concluded from this that here at least the immunity may be due
to the fact that the chemical composition of the contents of the red
corpuscles in the immune animals is different from that of the red
corpuscles of man. But when we see, as was first demonstrated by
Ross[175], that the malaria parasite of Laveran, having made its way
into the digestive canal of certain mosquitos (_Anopheles_), there
develops abundantly, it is difficult to maintain this thesis.
Among other micro-organisms of animal origin we have the _Trypanosoma_,
the parasite of the terrible disease propagated by the Tsetse fly which
commits such ravages amongst mammals. Man alone escapes it, exhibiting a
natural immunity that nothing apparently can overcome. Are we to affirm
that it is the difference in the chemical composition of the human body
which assures to man his immunity against a parasite that attacks
indifferently an herbivorous animal, such as the ox or rabbit, or a
carnivorous animal, such as the dog? In these examples I have chosen
merely those micro-organisms which it has never been possible to
cultivate on any artificial nutrient medium and which are kept alive
with very great difficulty outside the living organism.
[Sidenote: [138]]
What is to be said then of the vegetable micro-organisms which, in this
respect, are much less exacting? The most important of these and the
most numerous of all pathogenic micro-organisms, the Bacteria, can as a
rule be cultivated without difficulty not only in the blood and fluids
of animals that are susceptible or refractory to their morbific action,
but also on all kinds of vegetables and artificial media: broths, fluids
composed of mineral salts and of certain organic substances. It is
really not possible to attribute the natural immunity of the dog and the
fowl against the anthrax bacillus—so fatal to a great number of mammals,
man included,—to its incapacity to feed on the fluids of these animals,
when we see that this same bacillus is capable of killing lower animals,
such as the cricket, and can thrive on carrots, potatoes and other
vegetables.
Even when, among the bacteria, we take those that are most exacting in
the choice of their food, we still find it impossible to explain natural
immunity as being due to the want of power on the part of these
organisms to obtain their nutriment from the juices of refractory
species. The bacillus discovered by R. Pfeiffer[176] in influenza does
not develop on media that are ordinarily employed in bacteriology in the
cultivation of a great number of micro-organisms. It needs a special
food, which is prepared for it by spreading a drop of fresh blood on the
surface of agar. Pfeiffer has established the fact—confirmed by many
observers—that the best species of blood to use for this purpose is that
of the pigeon. We should have to believe, then, did the immunity really
depend on the composition of the fluids, that the pigeon is the least
refractory of all animals. Experiment has demonstrated the erroneousness
of such a supposition: the pigeon is quite as refractory to Pfeiffer’s
bacillus as are most other species of animals.
[Sidenote: [139]]
As a second example the bacterium of bovine pleuro-pneumonia may be
cited. It is the smallest of all known bacteria. The difficulties
surrounding the discovery and identification of this organism were very
great, and the ingenuity of Nocard and Roux[177] was required for the
demonstration of its existence. Very exacting in its choice of nutritive
material, it was first cultivated in the fluids of the rabbit, a species
endowed with an absolute immunity against bovine pleuropneumonia. It is
unnecessary to multiply examples to obtain a general proof that natural
immunity against micro-organisms cannot be explained by the incapacity
of these pathogenic agents to live in the fluids of the refractory
organism.
We must, however, ascertain what takes place in resistant animals
inoculated with micro-organisms. Here, again, it is preferable to begin
with the lower animals of simple organisation. We have already seen that
examples of natural immunity are not rare in the Invertebrata. When
engaged in the study of the disease found in _Daphniae_, small crustacea
so common in fresh water, I was able to show that the special
Blastomycetes which cause it meet with a vigorous resistance on the part
of the organism. As the _Daphniae_ are small, transparent, and
consequently easily observed under the microscope, I was able without
difficulty to establish the main phenomena observable in these
organisms. I can be the more brief in describing these phenomena of
resistance as, in addition to devoting a special memoir to the _Daphnia_
disease[178], I have, in my _Lectures on Inflammation_ (pp.
97–103)[179], described at some length the reaction of their organism to
the _Monospora_. It is nevertheless necessary that I should recall, very
briefly, the mechanism by which these small crustaceans secure immunity.
The spores of the parasite—very delicate and rigid needles—are swallowed
with the food. By means of their sharp points they perforate the
intestine and penetrate into the body cavity, full of blood, where they
find themselves exposed to the attacks of leucocytes. These leucocytes,
guided by their tactile sense, gather around the foreign body, ingest it
completely and destroy it. It is remarkable that the spore, which is
furnished with a very resistant membrane, once in the interior of the
mass of leucocytes, undergoes modifications which afford evidence of the
presence in these cells of an extraordinary digestive power. The surface
of the spore, from being smooth and regular, becomes pitted and sinuous,
the spore breaks up into fragments and is reduced to a mass of _débris_
which, in the form of brown granules, remains indefinitely in the
contents of the leucocytes. From this it is evident that these
phagocytes must produce a ferment which is capable of digesting the
cellulose or analogous substance which forms the membrane of the spore.
Unfortunately, the small size of the _Daphniae_, so useful for the
direct observation of the phenomena of immunity, presents an
insurmountable obstacle to the study of its leucocyte ferments,
especially _in vitro_.
[Sidenote: [140]]
The destruction of the spores of the parasite by the leucocytes secures
to the _Daphnia_ a real immunity. Of a hundred _Daphniae_ taken in my
aquarium and carefully examined under the microscope, fourteen only were
found to be infected by the budding conidia of the parasite, whilst
fifty-nine of the others contained the remains of spores that had been
destroyed by the phagocytes. When transferred to pure water containing
no new source of contagion, these _Daphniae_ flourished and lived a
normal life, giving birth to a numerous progeny.
The immunity of the _Daphnia_, due to the intervention of phagocytes, is
an example of natural, individual immunity. It is not the specific or
racial possession of these crustacea, for when the leucocytes do not
seize the spore, at once, on its penetration into the body cavity, it
commences to germinate and gives rise to a whole generation of budding
cells. These cells, then, secrete a poison which not only repels the
leucocytes, but kills and completely dissolves them. Under these
conditions the _Daphnia_ is disarmed; the parasites grow in the
organism, deprived of its arm of defence, as in a culture tube, and the
animal rapidly succumbs.
Since I first observed this struggle between the _Daphnia_ and its
parasite, some eighteen years ago, no other example has been found that
is so easily observed and so demonstrative of the protective action of
phagocytes in an animal that can be kept under observation, alive, under
the microscope. Cases, however, are not wanting in the Invertebrata in
which the different phases of this struggle may be observed with
sufficient accuracy to warrant the conclusion that in these cases also
the phenomena are analogous to those observed in the case of the
_Daphniae_.
[Sidenote: [141]]
It has already been stated in Chapter III. that the larvae of the
rhinoceros beetle (_Oryctes nasicornis_), although very sensitive to the
cholera vibrio, are very refractory to anthrax and diphtheria. In order
that we may obtain some idea of the mechanism of this immunity let us
inject into the body cavity of these large white grubs a trace of
anthrax culture. In the blood, drawn off the following morning, the
injected bacilli are found, not in the plasma, but inside many of the
leucocytes. Here there has occurred, as in the _Daphnia_, an ingestion
of the parasites which have then been destroyed by the intracellular
digestion of phagocytes. The process is the same, then, as that by which
the resorption of the red corpuscles of the goose takes place when they
are injected into the blood of cockchafer larvae. In both cases the
foreign bodies are ingested and destroyed by the leucocytes of the
blood; this act of resorption, however, taking a very long time.
Although the leucocytes of the larvae of the rhinoceros beetle exhibit a
positive chemiotaxis for the bacillus, these same cells behave in a very
different fashion in presence of the cholera vibrio. Very small
quantities of this vibrio, when injected into the blood of the larvae,
give them a fatal disease: the vibrios excite in the leucocytes a
negative chemiotaxis and flourish without hindrance in the blood plasma.
The larva is soon transformed into a culture vessel and the numerous
vibrios that develop in it cause the death of the animal.
The difference in action of the two bacteria cannot be explained by any
corresponding difference in their mode of life in the blood. Removed
from the organism the blood plasma of the white larvae of the rhinoceros
beetle is a culture medium just as favourable to the growth of the
anthrax bacillus as to that of the cholera vibrio. Moreover, the former
of these micro-organisms is quite capable of setting up a fatal disease
in other representatives of the class of Insects. Kovalevsky[180] has
discovered in the house cricket four phagocytic organs, with a great
appetite for all kinds of foreign particles that may penetrate into its
body. The blood of mammals, when injected below the skin of the cricket,
is rapidly absorbed by the cells of the four “spleens” (for so
Kovalevsky designates these phagocytic organs). The resorption of the
red blood corpuscles goes on within these phagocytes owing to their
power of intracellular digestion. When Kovalevsky kept crickets at a
temperature of 22°–23° C. and injected them with anthrax bacilli he
noted that these bacilli also were ingested by the cells of the spleens.
There was, thus, no manifestation of negative chemiotaxis of these
elements towards the bacillus. The ingestion of the bacilli by the
phagocytes was not sufficient, however, to protect the animal. The
bacilli reproduced themselves rapidly in the blood fluid; the
intracellular lacunae of the spleens were full of them and the crickets
quickly succumbed to the infection.
[Sidenote: [142]]
Nevertheless these crickets are quite capable of resisting certain other
bacteria. Balbiani[181] has shown that they are refractory to a great
number of bacilli belonging to the group of _Bacillus subtilis_. He
observed that when injected into the body of the cricket these bacilli
are devoured and destroyed by the leucocytes of the blood and by the
large cells of the pericardial tissue corresponding to the elements of
the spleens of Kovalevsky. Whilst the crickets and other Orthoptera,
which are rich in phagocytes, exhibit a real immunity against these
bacilli, insects which have very few leucocytes such as butterflies,
flies and Hymenoptera are found to be much more susceptible to infection
by the same bacilli. In this case the direct relation between immunity
and phagocytosis is very marked.
[Sidenote: [143]]
The Mollusca also furnish some interesting examples of natural immunity.
Karlinsky[182] has observed that anthrax bacilli, when injected into the
blood of slugs and snails, soon disappear from their bodies; these
pulmonate Gasteropods are absolutely unaffected by this bacillus so
formidable for many species of animals. From the rapidity of this
disappearance of the bacilli it has even been concluded that it was
impossible for this bacillus to live in the fluids of Mollusca.
Kovalevsky (_l.c._ p. 443) has studied this question with the
carefulness that characterises all his work. He confirms the fact that
snails (_Helix pomatia_) resist the introduction of a large quantity of
anthrax bacilli into their bodies; he notes also that these bacteria
disappear from the blood. But he finds them again in the tissues of the
foot, and especially in the cells which surround the pulmonary vessels.
“The greater number of the bacteria are found in the cells of that part
of the pulmonary region in _Helix_ which adjoins the heart and kidney.
All the bacteria were ingested by the cells and I easily succeeded in
demonstrating this not only in sections but also in bulk” (p. 444). The
snails remained in good health in spite of the presence in their
phagocytes of numerous bacteria which maintained themselves there for
some time. At the end of ten or twelve days and more these bacteria
still presented their usual aspect; this accords well with the slowness
with which intracellular digestion goes on in the majority of the
Invertebrata. These bacteria were, however, no longer living, although
still undigested. Morsels of the pulmonary tissue of the snails that
were injected with anthrax bacilli still gave cultures 48 hours after
injection and contained bacilli capable of giving fatal anthrax to mice.
Later, media seeded with similar particles remained sterile, and mice
inoculated therewith continued to live. From these experiments it may be
accepted that bacteria, living in the blood plasma, become the prey of
phagocytes which render them inoffensive and kill them. This example
demonstrates once again that the organism gets rid of bacteria by the
same mechanism as that which serves for the resorption of any of the
formed elements. The snail reacts to bacteria as it does to the red
corpuscles of the goose.
It is unnecessary to insist further on the natural immunity of the
Invertebrata, and it is useless to multiply examples which always point
in the same direction: to the importance of phagocytic reaction and of
intracellular digestion in resorption and immunity. We must pass on to
the examination of the reaction phenomena of the vertebrate organism
towards pathogenic micro-organisms, following, as hitherto, the
comparative method. We will commence with the study of the natural
immunity of fishes as lower representatives of the great group of the
Vertebrata.
[Sidenote: [144]]
It is well known that fishes are liable to infective diseases and
pisciculture has often to deplore considerable losses brought about
sometimes by certain of the lower Fungi (_e.g. Saprolegniae_), sometimes
by Bacteria. The pathogenic microbes which produce epidemics in fishes
are still little understood; but among the bacteria which kill many of
the higher animals are some which cause fatal maladies in certain
fishes. Thus the anthrax bacillus so virulent for many mammals is
capable also, as we have seen, of producing an infection in the cricket,
and may cause the death of small marine osseous fishes, the
_Hippocampi_. Sabrazès and Colombot[183], who have studied this
question, have demonstrated that the anthrax bacillus, which is virulent
for the rabbit, when inoculated into these fishes first produces
swellings at the seat of inoculation and ultimately becomes generalised
throughout the body, producing a fatal septicaemia. As these experiments
have given this result at a temperature of 14°–16° C., it is quite
evident that the bacillus, in order to manifest its pathogenic effect,
in no way needs the high temperature of the mammalian body for its
action.
Now among fishes there are not wanting species which resist the anthrax
bacillus. Mesnil[184] has, in our laboratory, thoroughly studied the
mechanism of this immunity. He has shown that several fresh-water
fishes, _e.g._ the perch (_Perca fluviatilis_), the gudgeon (_Gobio
fluviatilis_), and the gold-fish (_Carassius auratus_), will resist an
injection of a considerable number of bacilli into the abdomen. When
kept at temperatures of 15°–20° C. or even 23° C., a temperature at
which the bacilli are able to develop very abundantly, these fishes
destroy a large number of the bacteria in their bodies. Soon after the
introduction of the bacilli into the peritoneal cavity, the numerous
leucocytes accumulate around them and ingest them by the same mechanism
that is observed in the Invertebrata or in the same fishes when
absorbing the red blood corpuscles of alien species. In the gudgeon, at
as early as six and a half hours, a very marked, nay, an almost complete
phagocytosis is set up.
It is impossible to doubt the fundamental fact that the bacilli, at the
moment of their ingestion, are in a perfect condition of vitality and
virulence. The fluid of the peritoneal exudation, when withdrawn from
the animal, is of itself incapable of preventing the development of the
anthrax bacilli. The peritoneal lymph of the above-mentioned fishes is,
_in vitro_, even a good culture medium for these bacilli.
[Sidenote: [145]]
When, long after the completion of the phagocytosis by the leucocytes of
the peritoneal exudation, a drop of the exudation is withdrawn and kept
outside the organism under suitable conditions of temperature and
moisture, a number of the ingested bacilli begin to multiply and give an
abundant culture. This experiment proves, indisputably, that the bacilli
are devoured in the living state. If a little of the peritoneal
exudation, withdrawn several (up to nine) days after the injection of
the bacilli, be injected below the skin of guinea-pigs these animals die
from generalised anthrax, a fact which demonstrates that the bacilli,
which have been ingested alive, have retained their virulence a long
time after they have been devoured by the leucocytes. But, if the
peritoneal exudations that have been withdrawn at still longer periods
after injection be examined, it is found that they no longer contain
bacilli capable of developing in culture media or of setting up the
disease in the most susceptible animal. Hence it follows that in the
organism of the refractory fish, the bacteria are not destroyed by the
fluids but by the phagocytes, which take a long time to bring about the
complete intracellular digestion of ingested micro-organisms.
The phagocytes which assure immunity to the osseous fishes that were
studied by Mesnil belong principally to the group of haemomacrophages.
These are leucocytes with abundant protoplasm which stain readily by
basic aniline dyes, mononuclear cells whose nucleus, however, is
sometimes divided into lobes. It is to be noted that in the perch these
are the sole representatives of the motile phagocytes, and that in this
fish not only the eosinophile but every other variety of granular
leucocyte is completely wanting. In the gudgeon, in addition to
haemomacrophages, some microphages whose protoplasm stains faintly with
acid aniline colours are met with. These facts will be useful to us when
we come to study the part played by phagocytes in immunity from a
general point of view.
Another class of cold-blooded animal, the Amphibia, has been much more
frequently studied from the point of view of infection and immunity. The
frog, an animal so convenient for many physiological and pathological
researches, has been much employed for the study of immunity against
pathogenic micro-organisms. Quite a literature, which has been
excellently summarised in the memoir of Mesnil already cited, and to
which we shall have occasion to return more than once, has been
accumulated on the subject.
[Sidenote: [146]]
The immunity of frogs against the anthrax bacillus was early
demonstrated and studied in Robert Koch’s celebrated memoir[185] on
anthrax. This observer, after injecting an emulsion of anthrax spleen
into the lymph sac of the frog, recovered the bacilli from the interior
of round cells which burst readily when transported into water. Koch,
accepting the view then generally held, thought that the bacilli found a
favourable culture medium in the contents of certain cells, but that, in
spite of this, the frog was capable of manifesting a real immunity
against anthrax. Gibier[186] made the interesting discovery that frogs
when subjected to the influence of high temperature (about 37° C.) lose
their natural immunity and readily contract fatal anthrax.
Since that time the mechanism by which the organism of the frog secures
immunity against the anthrax bacillus has repeatedly been studied. In a
memoir which appeared in 1884[187] I insisted that the principal part
played in this immunity belonged to the phagocytes which devour the
injected bacteria and subject them to intracellular digestion. The round
cells described by Koch are nothing but the leucocytes of the lymph sac
which have seized upon the anthrax bacilli. These bacilli instead of
thriving in the cell contents find there a very unfavourable medium, and
perish at the end of a longer or shorter period. When the activity of
the phagocytes is impeded by unfavourable influences, _e.g._ high
temperature, they exhibit a very feeble reaction, incapable of assuring
to the frog that immunity which, under normal conditions, it possesses.
The conclusions I have just summarised have raised very lively
opposition from a large number of observers. Baumgarten[188], with his
pupils Petruschky[189] and Fahrenholtz[190], have endeavoured to
demonstrate that phagocytosis plays no part in immunity and that the
frogs resist anthrax simply because the bacilli are incapable of
maintaining themselves alive in the fluids of this Batrachian.
Nuttall[191], of Flügge’s school, also maintained that frogs resist
anthrax owing to the bactericidal power of their fluids. This view has
been defended by several other observers and appeared for some time to
become quite dominant.
[Sidenote: [147]]
Nevertheless, it is possible to demonstrate that the plasmas of the frog
not only are not inimical to the life of the bacillus, but serve as a
good culture medium for it[192]. All that is necessary for the
demonstration of this fact is to introduce below the skin of frogs
anthrax spores enclosed in a sac of reed pith, or simply enveloped in a
small piece of filter paper. The plasma of the lymph sac at once
permeates the spores and allows them to germinate and produce quite a
generation of bacilli. But, as soon as the leucocytes pass through the
paper, they seize upon the young bacilli, digest them in their substance
and prevent their pathogenic action. The germination of the spores may
take place even where they have been introduced below the frog’s skin
without being protected in any way whatever. But, under these
conditions, only a certain number of the spores germinate, the majority
not having time to do so before the arrival of the leucocytes. The
small, very short bacilli which proceed from the germinated spores, are,
along with the spores that have not germinated, soon ingested by the
phagocytes. But, whilst the rods are in the end digested within these
cells, the ingested spores remain intact for a very long time: they do
not germinate, but they are not destroyed and retain their vitality
indefinitely, in spite of the influence of the phagocytes. It is
sufficient to withdraw from a frog, that has been inoculated with
anthrax spores some time before and kept at a moderate temperature
(15°–25° C.), a little lymph and sow it in any nutrient medium (of those
employed in the culture of bacteria), in order to see the spores
germinate and produce a whole generation of absolutely normal
filamentous bacilli. All these phenomena have been carefully studied by
Trapeznikoff[193] in a work executed in my laboratory. It is obvious
from his experiments that the phagocytes of the frog are quite capable
of protecting the organism against the anthrax bacillus by ingesting and
digesting the bacilli in the vegetative state and by preventing the
germination of the ingested spores. This phagocytic action is very
important in presence of the fact that the plasmas of the frog allow the
spores to germinate and the bacilli to develop and produce abundant
cultures.
[Sidenote: [148]]
The immunity of frogs against the anthrax bacillus that we have just
described and which is guaranteed by the activity of the phagocytes, is
constant under the conditions of temperature above mentioned (15°–25°
C.), conditions which are sufficient, however, to ensure the death of
susceptible cold-blooded animals, such as the cricket or _Hippocampus_,
from anthrax. The edible frog, a species that readily accommodates
itself to a temperature of 35° C., resists, even under these conditions,
infection by the bacillus, as pointed out by Mesnil in a work already
cited when treating of the immunity of fishes. The green frog (_Rana
esculenta_) when kept for a long time at this high temperature, so
suitable for the development of the anthrax bacillus, reacts by the same
phagocytic mechanism. The leucocytes of the lymph and blood, the cells
of the splenic pulp and Kupffer’s stellate cells of the liver, seize the
introduced bacilli and digest them as in any other case of phagocytosis.
The brown frog (_Rana temporaria_) adapts itself but slightly and with
great difficulty to the high temperature and dies whether it has been
inoculated with anthrax or not. Under these conditions the bacteria
develop in the body of the dead or dying frogs, but Mesnil insists on
the fact that a true anthrax infection is not produced, as has been
maintained by Gibier as the outcome of his researches.
[Sidenote: [149]]
Dieudonné[194], however, has found a method of removing the natural
immunity of the frog against the anthrax bacillus, by inoculating it
with an artificial bacterial race which he had adapted to develop fairly
luxuriantly at the low temperature of 12° C. Under these conditions all
the inoculated frogs, even those which had resisted the inoculation with
ordinary bacteria (grown at 37°·5 C.), died within a period of 48 to 56
hours, containing many bacilli in the blood and organs. Dieudonné has
not studied the essential mechanism that accompanies this loss of
immunity; but it is very probable that, for one thing, we have here to
do with a reinforcement, special for the frog, of the bacillus that has
become accustomed to develop at a low temperature. This bacillus must
multiply, in frogs that have been maintained at a low temperature, much
more rapidly and profusely than would the ordinary bacillus. On the
other hand, the susceptibility of Dieudonné’s frogs must depend on a
less resistance of the organism under the conditions of his experiments.
Unfortunately, we cannot find in his memoir sufficient data on these
points; he does not even state the temperature at which the frogs that
had been inoculated with bacteria adapted to cold lived. Dieudonné
invokes the analogy of his results with those obtained in the case of
the immunity and susceptibility of frogs as regards a septicaemic
bacillus.
This bacillus (_Bacillus ranicida_) has been made the subject of an
interesting study by Ernst[195]. It is a small, very slender bacillus,
which, in frogs, produces a fatal malady epidemic in spring, but ceasing
completely during summer. Taking this fact as a basis, Ernst has
succeeded in conferring immunity upon frogs in autumn by placing them in
an incubator at 25° C. In spite of the injection of a considerable dose
of the small bacillus, the frogs living at this temperature remained in
good health, whilst control animals exposed to a low temperature died of
septicaemia. The counter-test was made in summer. Inoculated frogs that
were kept in the laboratory were unaffected, whilst those that had been
kept in a refrigerating apparatus at 6°–10° C. invariably died. It may
be asked, Is this evident influence of temperature on immunity and
receptivity exercised on the organism of the frog or upon the pathogenic
bacillus? In the case where a bacillus can only develop at low
temperatures its harmlessness at the higher temperature may be readily
understood. The experiments of Ernst have demonstrated, however, that
this small bacillus develops much better at 22° C., and even at 30° C.,
than at lower temperatures. It must be concluded, therefore, that the
high temperature which confers immunity acts not by weakening the
bacillus, but rather by reinforcing the resisting power of the organism.
The low temperatures (6°–10° C.) that are favourable to a fatal
infection have a different action; that is to say, they weaken the
reaction of the inoculated frogs.
[Sidenote: [150]]
Although Ernst has not studied the mechanism of this resistance fully,
it is evident, from the data he has supplied, that it consists in a
phagocytic reaction. He was able to demonstrate the ingestion of the
bacilli by the phagocytes in the susceptible refrigerated frogs, as well
as in the refractory frogs, kept at a higher temperature; but in the
former case the phagocytosis was so feeble that 24 hours after
inoculation a considerable number of free bacilli were still found in
the lymph of the dorsal sac, whilst in the refractory frogs the much
more active phagocytosis brought about the disappearance of the free
bacilli during the first day. If, as is very probable, the analogy of
this septicaemia with anthrax in frogs, upon which Ernst insists, really
exists, it must be concluded that the susceptibility of these
Batrachians to the modified race of the bacillus depends on their weak
phagocytic resistance.
Since, in these two examples of natural immunity in the frog, we have
seen that the phagocytic activity exhibits itself in an active form
against bacteria which readily develop in the fluids of the same animal,
we might conclude that the reaction of the phagocytes constitutes a
general mode of defence in cold-blooded animals. But Lubarsch[196], a
very cautious observer, has expressed an opposite view, based on his
studies on the bacillus of mouse septicaemia. He convinced himself that
frogs will resist injections of even considerable quantities of this
bacillus, without any co-operation on the part of the phagocytes. As we
have, here, to do with a matter of fact, Mesnil (_l.c._) set himself to
verify these observations, with the object of establishing whether it
was a case of a real exception or of a simple misunderstanding. He was
able to demonstrate, by irrefutable observations and experiments, that
the bacilli of mouse septicaemia when inoculated into frogs, set up a
very pronounced positive chemiotaxis on the part of the phagocytes,
which seized and digested the bacilli just as they do the anthrax
bacillus. This apparent exception, therefore, becomes transformed into
an additional argument in favour of phagocytic reaction being a general
factor in immunity. In support of this hypothesis I may adduce a further
example, already mentioned in a preceding chapter when discussing
another question. The frog is very refractory against the cholera
vibrio. When these vibrios are inoculated into the dorsal lymphatic sac
or into any other part of the body the animal retains its health
unimpaired. An examination of the exudation at the point of inoculation
demonstrates that the vibrios meet with a vigorous opposition on the
part of the phagocytes, which ingest and completely digest them. This is
of special interest from the fact that the frog is very sensitive to the
toxin of the cholera vibrio. When injected in a weak dose it kills the
frog very quickly. Two small frogs died in less than an hour from the
effect of 0·5 c.c. of cholera toxin.
[Sidenote: [151]]
The natural immunity of the frog against the cholera vibrio affords,
then, an example in which the organism, destroying the vibrio by
phagocytosis, prevents the production of the poison, which, otherwise,
would infallibly kill it.
Having demonstrated that phagocytic reaction manifests itself in the
frog in all cases of natural immunity that have been sufficiently
studied, we must dwell for an instant on the question of the condition
of the bacteria at the moment of their ingestion by the phagocytes. It
is very evident that this phagocytic defence is only efficient on
condition that it is exercised against bacteria which, in its absence,
might injure the organism by their multiplication and their virulence.
For this reason the question as to whether the micro-organisms, before
being ingested, were living and capable of producing their pathogenic
action has been widely discussed. It has even been suggested that the
phagocytes are only capable of ingesting the dead bodies of
micro-organisms that have been killed by other agents. Frogs are very
suitable for a study of this question. When a drop of the exudation is
removed some time after inoculation with a motile organism, such as the
_Bacillus pyocyaneus_ or the cholera vibrio, the organism was often
found moving rapidly within the vacuoles inside leucocytes. The
experiment will succeed even more completely if a drop of frog’s lymph
be mixed, on a slide, with a trace of a culture of these motile
micro-organisms, the latter being soon found in the clear vacuoles
included in leucocytes and executing extremely rapid movements.
Besides this direct proof we can assure ourselves of the living
condition of the micro-organisms in another way. Withdraw a drop of the
exudation at an advanced stage of the process when there are no longer
any free micro-organisms; inside the phagocytes a few scattered
bacteria, more or less well preserved, can still be seen. It is
sufficient to keep a hanging drop of such an exudation at a temperature
of about 30° C., care being taken to keep it from drying, but without
adding to it any nutrient medium. Under these conditions the leucocytes
die more or less rapidly, but the bacteria regain vigour: they begin to
multiply, and at the end of a short time produce a generation of
bacteria within the dead leucocyte. The multiplication of the bacteria
goes on progressively and the hanging drop is transformed into a real
pure culture. Mesnil was able to confirm these data with the exudations
of frogs that had been inoculated with either the bacilli of anthrax or
of mouse septicaemia.
[Sidenote: [152]]
The bacteria, ingested in the living state by phagocytes, retain their
original virulence. Some authors think, and I was formerly of this
opinion, that at the end of a more or less prolonged sojourn within the
leucocytes, anthrax bacilli undergo an attenuation in their virulence.
Later, numerous researches have, however, demonstrated that this opinion
is incorrect, and that the virulence is maintained in the bacteria
included in the phagocytes of frogs the whole time that these bacteria
remain alive. Dieudonné has insisted on this fact as regards the anthrax
bacillus. Mesnil has confirmed it for this same species and for the
bacillus of mouse septicaemia. It is impossible, therefore, to doubt
this general result, that frogs which are refractory against certain
bacteria resist because of the phagocytosis which is exercised against
living and virulent micro-organisms.
We have insisted sufficiently on the analysis of the natural immunity of
the frog, and need not tarry over the facts relating to other amphibia
which, moreover, have been much less studied. The reptiles, those higher
representatives of the Vertebrata called cold-blooded, often present
examples of really remarkable immunity. Thus alligators will resist
enormous doses of various bacteria, such as the anthrax bacillus, that
of human tuberculosis or the cocco-bacillus of typhoid fever. When, some
time after an injection is made, the exudation at the point of
inoculation is withdrawn there is found a large number of leucocytes,
amongst which may be recognised many eosinophile microphages, though the
majority are macrophages with one, two or more nuclei. Really giant
cells are found in the exudation. It is the macrophages which specially
manifest phagocytosis and they are often found crammed with the injected
bacteria, as I was able to assure myself after injections of typhoid
cocco-bacilli. The natural immunity of alligators (_Alligator
mississipiensis_) persists not only at the temperature of the incubator
(37° C.), but also at room temperature (20°–22° C.).
[Sidenote: [153]]
Passing in review the animal kingdom we must pause for a moment to
consider the natural immunity of birds or lower warm-blooded
Vertebrates. The classic example of this immunity is that of the fowl
against anthrax. It has long been known that birds resist inoculation
with anthrax or only exhibit a feeble receptivity; though smaller birds
are for the most part susceptible to anthrax, the pigeon is much less so
and the fowl presents a case of the most pronounced immunity. It was
believed to be absolutely refractory until the experiments of Pasteur
and Joubert[197], who found a sure method of suppressing this immunity.
Fowls that had been inoculated with the bacillus were immersed up to the
thighs in cold water in order to bring down their temperature. It was
found that, under these conditions, the anthrax bacillus developed at
the seat of inoculation and later became generalised in the blood, and
invariably caused death. It was concluded from this that the natural
immunity of the fowl was dependent on its very high normal temperature
(41°–42°) which interfered with the pathogenic functions of the anthrax
bacillus.
Hess[198] studied the mechanism of this immunity of the fowl and pointed
out the important part that phagocytosis plays in the destruction of the
inoculated bacteria.
These researches were resumed in my laboratory by Wagner[199]. Having
established that the anthrax bacillus develops readily in the blood and
the blood serum of fowls, outside the organism, at high temperatures
(42°–43° C.), he came to the conclusion that the lowering of the
temperature of the body of the fowls by immersing them in water
produced, not a reinforcement of the bacillus, but a weakening of the
resisting power of the animal. He was able to convince himself that this
resistance exhibits itself in the activity of the phagocytes which
ingest and destroy the anthrax bacillus in its vegetative state. In the
normal fowl the phagocytosis is rapid and very pronounced, whilst in a
fowl that has been refrigerated this reaction is very slight or absent.
To corroborate this general conclusion, Wagner, instead of lowering the
temperature by means of cold water, made use of antipyrin and chloral.
The application of this treatment likewise caused enfeeblement of the
natural defence of the organism and suppressed the immunity of the fowl
against anthrax.
[Sidenote: [154]]
Trapeznikoff[200] has studied carefully the fate of anthrax spores when
injected into fowls. He observed that most of them are devoured by the
leucocytes. Some of the spores were first transformed into small rods,
sometimes growing into real bacilli, but finally they all became the
prey of phagocytes and perished in their interior. Those in the
vegetative condition are soon digested, the spores, however, persist for
some time inside the phagocytes, but ultimately disappear. The
phagocytosis in fowls inoculated with spores is very marked, and
preparations, stained by Ziehl’s method, demonstrate most clearly the
reality of this reaction phenomenon. These preparations have for long
been used in the course in bacteriology at the Pasteur Institute for the
demonstration of phagocytosis.
[Sidenote: [155]]
In the face of these facts, well established and confirmed many times,
it is impossible to accept Thiltges’[201] denial of the ingestion of
these bacteria by the phagocytes of the fowl. Some fault of technique,
which I am not at the moment in a position to indicate exactly, has
evidently slipped into this author’s work. The positive data, however,
on phagocytosis in the fowl, obtained by Hess, Wagner, and Trapeznikoff,
data confirmed by myself, render unnecessary any fresh researches for
the purpose of explaining the negative results obtained by Thiltges. As
regards his experiments on the bactericidal action of defibrinated blood
and of blood serum of fowls against the bacillus and its spores,
experiments whose results are opposed by those of Wagner, the
contradiction may be explained pretty easily, at least in part. Thiltges
mentions several times that the bacilli, when sown in the blood serum of
the fowl, were aggregated in clumps. Nevertheless, he has failed to
guard against this source of error and has attributed the diminution in
number of the colonies on plates to the destruction and not to the
agglutination of the bacilli. Thiltges gives so few particulars of the
conditions under which his experiments were performed that we do not
even know at what temperature he kept his tubes containing blood and
serum sown with bacilli. As Wagner kept his at 42°–43° C., a temperature
which corresponds to that of the body of the fowl, I asked M. Gengou to
make a series of experiments on the bactericidal power of the plasma and
blood serum of fowls on the anthrax bacillus, keeping his tubes at 37°
C. The result of his experiments was in complete accord with those of
Wagner. Under the conditions that I have just stated the fluids of the
fowl are no more bactericidal than they are under the conditions
maintained in Wagner’s experiments.
In summing up these data on the natural immunity of fowls against
anthrax, we are certainly justified in concluding that it is due to the
phagocytosis and not to any bactericidal property of the “humours.”
The pigeon is more susceptible than the fowl to the action of the
anthrax bacillus, still it manifests a certain degree of resistance
against the microbe. After what we have said on the subject of the fowl
we need make but few remarks on the pigeon, in spite of the very
animated discussions that have taken place on the mechanism of its
immunity. When Baumgarten was offering a systematic opposition to the
part played by phagocytic reaction in immunity, he set his pupil
Czaplewski[202] to investigate the resistance of pigeons against
anthrax. The results of this investigation were absolutely negative as
regards phagocytosis. The latter was said to have no importance in the
defence of the organism, which resisted simply because it was impossible
for the bacillus to live in the body of the pigeon. I then set myself to
study this question[203], and I was able to demonstrate that the anthrax
bacillus is quite capable of keeping alive in the pigeon, that it can
develop in its fluids, but that it is unable to defend itself against
the aggression of the phagocytes which ingest it and completely digest
it. By isolating the phagocytes that had ingested the bacilli injected
into the body of the pigeon, I was able to prove that a number of these
bacilli were still alive. The enfeeblement and death of the phagocytes
when outside the body allowed the anthrax bacilli again to get the upper
hand in this struggle, to develop and to give virulent cultures. The
part played by phagocytes in this example of natural immunity was thus
placed beyond doubt.
[Sidenote: [156]]
Later, Czaplewski[204] himself became convinced that his previous
negative results would not stand criticism, and Thiltges, in his work
already mentioned, when discussing the fowl, was able to confirm the
importance of phagocytosis in the defence of the organism of the pigeon
against anthrax. He was struck by the difference between these two
species of birds. In the pigeon it was easy for him to prove that in the
individuals that succumb to anthrax the phagocytic reaction is very
feeble, whilst in those which ultimately resist the bacillus it is very
pronounced. Thiltges likewise observed that the blood and blood serum of
pigeons when sown _in vitro_ with the anthrax bacillus, manifest only an
insignificant bactericidal power, a fact that further warrants him in
attributing great importance to phagocytosis in the maintenance of the
natural immunity of the pigeon. It is remarkable that, in presence of
these facts, it did not occur to the author to ask whether this
fundamental difference in the mechanism of the resistance, which he
thought possible in two birds so closely allied as are the pigeon and
the fowl, really did exist in nature. I infer that his experiments on
the fowl were made before those on the pigeon, and that the difference
in his results depended specially on the fact that he had acquired
greater skill in executing his later experiments.
Having observed that frogs die readily when inoculated with an anthrax
bacillus that was adapted to develop at a low temperature, Dieudonné
(_l.c._) endeavoured to suppress the immunity of the pigeon by using
bacilli adapted to a high temperature. But the inoculation of a second
generation of the anthrax bacillus, cultivated at 42° C., was borne by
five pigeons without inconvenience. Even bacilli that were rendered
capable, by cultivation through sixteen generations, of developing at
this temperature were not in a condition to kill more than five pigeons
out of thirteen inoculated. These attempts to explain immunity as due to
the properties of the bacilli rather than to those of the organism of
the pigeon, have therefore led to a result very different from that
anticipated by Dieudonné.
[Sidenote: [157]]
The pigeon is further of interest to us because of its natural immunity
against the bacillus of human tuberculosis. It resists considerable
doses of this bacillus, so virulent for man and for the majority of
mammals, and even for some birds (canaries and parrots). Dembinski[205],
studying the mechanism of this immunity, was able to prove that the
bacilli of human tuberculosis encounter in the organism of the pigeon a
very great resistance from the phagocytes, especially from the
macrophages. These cells fuse together around masses of bacilli and
imprison them within real giant cells or polynucleated macrophages (Fig.
21). The microphages in this struggle play only a secondary part, but
the resistance offered by the macrophages is a most effective one.
Incapable of completely destroying the bacilli, these phagocytes
exercise over them an unfavourable influence and prevent them from
multiplying and exhibiting their noxious action. The importance of the
defence by the macrophages comes out still more clearly when compared
with what takes place if, instead of the bacillus of human tuberculosis,
we inoculate into pigeons the bacillus of avian tuberculosis. In the
latter case the microphages certainly promptly seize the bacilli, but
being powerless against them they perish, whilst the macrophages only
intervene later on and in small numbers. The result is that in the
pigeon the avian bacillus becomes generalised in the organism and sets
up a fatal tuberculosis.
[Illustration:
FIG. 21. Reaction of the phagocytes of the pigeon against the bacilli
of human tuberculosis.
]
It must be admitted, then, that the immunity of the pigeon against the
bacillus of human tuberculosis is due to the defence by the macrophages.
This conclusion is corroborated by the fact that in the fowl—equally
refractory against the same bacillus—there is also observed a very
strong macrophagic reaction.
[Sidenote: [158]]
Nocard[206], who for several years has been carrying on studies on the
relations between the bacilli of human and avian tuberculosis, conceived
the idea of adapting the former to the organism of the fowl. With this
object he enclosed a culture of the bacillus of human tuberculosis in a
sac of collodion which he then introduced into the peritoneal cavity of
fowls. Under these conditions the bacillus, protected against the
aggression of phagocytes, continued to live inside the sac through whose
walls the fluid part of the peritoneal lymph could diffuse. After
several passages from sac to sac the human bacillus becomes acclimatised
to the body of the fowl and is transformed into a variety quite
comparable to the bacillus of avian tuberculosis. This experiment has
definitely settled the question so long under discussion of the specific
difference between the two tubercle bacilli. It has resolved it in the
sense of affirming their unity; the avian bacillus is only a modified
race of the same bacillus which sets up tuberculosis in man and other
mammals.
In spite of the great difference between the anthrax bacillus and that
of human tuberculosis, the immunity against these two bacteria, which is
shown in birds, depends in every case upon the reaction of the
phagocytic system.
Having rapidly glanced at natural immunity as we ascend the scale of the
animal series we now come to it as it presents itself in the highest
class, Mammals, a question on which it is necessary to dwell at greater
length because of its great importance, and also because of the fuller
study that has been given to it.
[Sidenote: [159]]
As the immunity of the Invertebrata and of the lower Vertebrata against
the anthrax bacillus has furnished us with several important indications
we will first endeavour to throw light on the mechanism of the
resistance offered to anthrax by certain mammals. The representatives of
this class being, however, for the most part extremely susceptible to
this disease, examples of true natural immunity are very rare. The first
place among resistant mammals is occupied by the dog. Although young
dogs, as demonstrated by Strauss[207], readily take fatal anthrax, the
canine species may nevertheless be regarded as possessing a real
immunity, as adult dogs withstand, without inconvenience, the
inoculation of large quantities of bacilli. When introduced beneath the
skin these bacilli excite a local inflammation, accompanied by a very
marked diapedesis of white corpuscles which at once begin to devour the
bacilli. This phagocytosis has already been observed by Hess[208],
Malm[209], myself, and several other investigators, so that its
existence cannot be doubted. Recently, Martel[210] has demonstrated a
very distinct phagocytic reaction in all those cases where he has had to
deal with dogs that were refractory or not very susceptible. This
reaction is shown by the ingestion of the bacteria and by the large
accumulation of leucocytes at the seat of inoculation. His researches
are of special interest by reason of the counter-test that he was able
to make upon dogs that were susceptible to anthrax. It was demonstrated
some years ago that the natural immunity of the dog against the
bacillus, although very real, is, nevertheless, relative and limited.
Thus Bardach[211] established the fact that dogs from whom the spleen,
an organ full of phagocytes, had been removed, became susceptible to
anthrax. Even dogs into whose veins he injected fine wood-charcoal
powder suspended in water, with the purpose of “diverting” the
phagocytosis, readily succumbed to anthrax.
Martel endeavoured to suspend the natural immunity of dogs by injecting
into them phloridzin or pyrogallic acid. But he obtained much more
constant results by inoculating the bacillus into rabid dogs. The
organism, weakened by this terrible disease, became very susceptible to
anthrax, and the rabid animal succumbed to anthrax before the rabies had
completed its evolution. By its passage through the rabid dog the
anthrax virus is so augmented in virulence that it becomes fatal for
normal dogs. Martel succeeded also in reinforcing the bacillus isolated
from a cow affected with anthrax. In all these cases where the
reinforced bacilli set up a severe and rapidly fatal infection, Martel
could demonstrate only a feeble phagocytic reaction.
Researches on the phagocytosis of dogs, inoculated with the anthrax
bacillus, have always demonstrated a regular and constant relation
between this reaction and the resistance of the organism. On the other
hand, experiments undertaken for the purpose of establishing the part
played by the body fluids in this immunity, have always given negative
results.
[Sidenote: [160]]
As the dog, of all mammals, exhibits the greatest natural immunity from
anthrax, it is very natural that in the bactericidal property of its
blood the key to the enigma has been sought. Thus Nuttall[212] concludes
from his experiments that the anthrax bacillus is readily destroyed by
defibrinated dog’s blood. But, as this result was not in accord with my
observations[213] that the bacillus is easily cultivated in dog’s blood,
and as several observers, especially Lubarsch[214], had arrived at
conclusions opposed to those of Nuttall, systematic researches were made
for the purpose of solving this complicated problem. Denys and
Kaisin[215] sought to remove the objections formulated against the
explanation of the immunity of the dog as due to the bactericidal
property of its blood by affirming that this power, which is absent in
the inoculated dog, develops whilst the animal is under the influence of
the bacillus. Immunity is reduced, then, in this case to the
establishment of a new property in the fluids during the course of the
struggle of the organism against the inoculated bacillus. None of the
observers, however, who have repeated these experiments, _e.g._
Lubarsch[216] and Bail[217] were able to confirm the results of the
Belgian observers. Denys himself, indeed, having resumed this study with
Havet[218], had to reject the conclusions of his former work executed in
collaboration with Kaisin. He is persuaded that their error was due to
the fact that in their experiments _in vitro_, the living leucocytes
ingested the bacilli and prevented their development. As the result of
these new researches Denys and Havet have come to the conclusion “that
the main, the predominating part of the bactericidal power of the dog’s
blood must be ascribed to the leucocytes acting as phagocytic elements”
(_loc. cit._ p. 15).
[Sidenote: [161]]
As a result of the investigations I have summarised the conclusion is
forced upon us that the natural immunity of the dog from anthrax is a
function of the phagocytes. In presence of this uniformity of the
experimental results it becomes very important to make a more profound
study of the phenomena that manifest themselves during the destruction
of the bacilli by the phagocytes of the dog. What are the phagocytic
elements which play the principal part in this struggle, and by what
means do they attain this result? Gengou[219] undertook a detailed
investigation in my laboratory to answer these questions. He was able to
convince himself, in agreement with the statements of his predecessors,
that not only was the serum of dog’s blood not bactericidal for the
anthrax bacillus, but that the plasma of the blood is no more so. The
fluid of the aseptic pleural exudation obtained after injection of
gluten-casein, was likewise incapable of killing the anthrax bacillus.
When Gengou, by means of centrifugalisation, isolated the leucocytes
from these exudations, washed them in physiological salt solution, froze
them, and then macerated them in broth, he obtained suspensions of white
corpuscles, to which he added bacilli. He was able to demonstrate that
when the exudations contained macrophages principally, as is observed in
exudations taken at the end of two or three days, the bactericidal power
of the suspensions was _nil_ or insignificant. When, on the other hand,
the leucocytes came from exudations only twenty-four hours old and were
composed almost exclusively of microphages, the destructive action on
the bacilli of the extract of the microphages in broth was most marked.
Now it is fully demonstrated that in the exudation set up in the
refractory dog by the injection of anthrax bacilli, it is especially the
microphages which exhibit the phagocytic reaction against this bacillus.
This is how the question of the immunity of the dog from anthrax stands
at present. The natural immunity of this species, which although not
unlimited, is very real, depends on the activity of phagocytes. These
elements, under the stimulus of the bacillus and its products, exhibit a
positive chemiotaxis of the most marked character, they approach the
bacilli, ingest them by a physiological act, and destroy them by means
of a substance which is not found in either the plasma or the blood
serum, but which can be demonstrated in an extract of the microphages.
[Sidenote: [162]]
In spite of the uniformity and precision of these data, it is impossible
to rest satisfied with describing, as an example of natural immunity
from anthrax, the single case of the dog. If the resistance of the rat
against this disease was merely of historical interest because of the
large number of works devoted to this question, we might relegate it to
the chapter reserved for the history of our knowledge on immunity. But
it is not so. The anthrax of rats is a subject full of very valuable
instruction, and von Behring was quite justified in saying that whoever
wished to get a true conception of natural immunity from a virus should
pay special attention to this example.
As a matter of fact, it may be stated that the grey rat (_Mus
decumanus_), the black rat (_Mus rattus_), and white rats are far from
enjoying a true immunity from anthrax. They, nevertheless, exhibit a
more or less marked resistance against this disease and are always less
susceptible than are the other laboratory rodents: mice, guinea-pigs and
rabbits. Rats resist attenuated bacilli (anthrax vaccines) better than
do these three species, and in order to induce in them fatal anthrax it
is necessary to inoculate a much larger number of virulent bacilli. On
the other hand, rats are distinguished by a great irregularity in the
resistance they offer to the bacillus. At times they resist very
virulent bacilli; at others they contract a fatal disease after an
injection of very attenuated bacilli (Pasteur’s first vaccine).
[Sidenote: [163]]
In my first memoir on anthrax[220] I noted the fact that in rats the
phagocytosis against the bacillus when injected subcutaneously was more
marked than after the same inoculation into the rabbit and guinea-pig.
Later, this fact was disputed by several observers, who refused to
accept the extent and importance of the phagocytic reaction in the rat.
This opposition was strengthened by a very interesting discovery made by
von Behring[221], namely, that the blood serum of the rat possessed a
remarkably destructive power for the anthrax bacillus. When this
observer added a certain quantity of anthrax bacilli to some blood serum
of the rat, instead of elongating into filaments and dividing they
underwent a change, lost their normal refraction and took on staining
reagents very imperfectly. The membranes alone remained of the bacilli
thus treated. Von Behring considered that this bactericidal action of
the serum depends on the presence of an organic base dissolved in the
blood fluid. He had merely to neutralise the serum by means of an acid,
and there was at once a very abundant development of the bacillus. From
these researches von Behring came to the conclusion that the natural
immunity of the rat from anthrax can be reduced to terms of the chemical
action of the blood on the bacillus.
In one of his most recent publications this author[222] returns to the
question of anthrax in rats and sums up his present point of view as
follows. He regards the immunity of these rodents as being relative, not
absolute. “The anthrax bacilli”—he says—“die in rat’s serum _in vitro_;
and in the cases where the inoculation of these animals with the anthrax
virus is not fatal, it is at least reasonable to assume that the blood
fluid likewise produces this protection in the organism of the living
rat. Now, an immunity that manifests itself without the aid of any
activity of the cell must undoubtedly be regarded as being of a humoral
character” (_loc. cit._ p. 202).
[Sidenote: [164]]
Let us begin by analysing the facts as presented in rats into whose
subcutaneous tissue we have injected anthrax virus. A certain number of
them resist, without exhibiting any lesion other than a certain
exudative inflammation at the seat of inoculation. The exudation is, in
this case, very rich in leucocytes which quickly exert their phagocytic
function and destroy the ingested bacilli. In this reaction it is the
microphages that play the chief part, the macrophages intervening later
and in a much less pronounced fashion. Usually, however, the inoculated
rats exhibit a more serious illness: the bacilli multiply at the point
of inoculation and excite the formation of an extensive oedema, rich in
serous fluid, transparent, and very poor in leucocytes. It is only later
that these cells intervene in any considerable number. The exudation
becomes thicker and turbid, the numerous white corpuscles devour the
bacilli and cause their disappearance. Under the influence of this
marked reaction the animals in most cases recover, as has already been
established by Frank[223]. But even in those individuals which succumb
to anthrax death occurs more or less tardily, an examination of the
internal organs then revealing a considerable phagocytic reaction. The
spleen, often of enormous size, contains numerous macrophages which are
filled with normal or more or less altered bacilli. In the liver these
macrophages, which have devoured several microphages and some bacteria,
are also found (Figs. 22 and 23).
When instead of bacteria in the condition of rods, anthrax spores are
inoculated subcutaneously or into the anterior chamber of the eye, we
can observe their germination. There is developed a whole generation of
bacilli which behave like those we have already described, that is to
say, they excite an exudation and are ultimately digested within the
phagocytes (Figs. 24 and 25). All these phenomena of phagocytosis I
described in detail more than ten years ago in my memoir on the anthrax
of rats[224]. Since then not a single fact has been brought forward to
invalidate the results there set forth.
[Illustration:
FIG. 22. Macrophage from the liver of a rat affected with anthrax.
]
[Illustration:
FIG. 23. Macrophage containing bacilli, from the liver of a rat
affected with anthrax.
]
[Illustration:
FIG. 24. Microphage of rat filled with bacilli.
]
[Illustration:
FIG. 25. Two microphages of rat that have ingested bacilli.
]
[Sidenote: [165]]
How is this paradoxical fact to be explained, that anthrax which grows
in the body of the rat, there setting up a disease more or less grave
and sometimes fatal, is so readily destroyed by the serum and blood when
removed from the organism? From numerous experiments, carried out by
Hankin[225] and by Roux and myself[226], it has been demonstrated that
the bactericidal power of the fluids of the rat cannot be invoked as the
cause of the animal’s resistance to anthrax. Those rats which show
themselves very susceptible to this disease and die from anthrax
infection, furnish, nevertheless, a serum that will prevent anthrax in
other rats, and which will protect even mice into which the bacilli have
been injected. Rats into which we inoculate on one side of the body a
little anthrax culture, and on the other side the same quantity of
bacilli mixed with blood serum from the same animal, manifest oedema at
the former place only. It is from this latter point that the general
infection takes place, the side where the anthrax bacilli mixed with
serum was introduced remaining unaffected. Sawtchenko[227], who has
investigated the immunity of the rat in my laboratory, has to the facts
just mentioned added the observation that when the injection of bacilli
causes haemorrhage the rat survives. When, on the contrary, the
injection is made with a fine needle and without effusion of blood, the
rat contracts a fatal anthrax.
[Sidenote: [166]]
[Sidenote: [167]]
It follows from these facts that the blood, immediately it has escaped
from the vessels, undergoes a change in its composition and becomes
bactericidal for the anthrax bacillus, whilst, when it is circulating in
the organism, it exhibits no such power. Sawtchenko has studied the
substance in the serum which kills the bacilli and has demonstrated that
it will resist heating to 56° C.; even when heated to 61° C. the serum
still exercises a certain amount of bactericidal power for very
attenuated bacilli (Pasteur’s first vaccine). Researches on the
distribution of this bactericidal power in the living rat have convinced
Sawtchenko that none of it passes into the fluid of the passive oedema
set up by the slowing of the circulation, nor into that of the active
oedema developed as the result of the inoculation of anthrax bacilli. He
observed that even the bacillus of Pasteur’s first vaccine grows
abundantly in the oedematous fluid produced by the injection of virulent
bacilli. The peritoneal lymph, however, exerts a very marked
bactericidal action on the bacilli. Having demonstrated this fact
Sawtchenko put to himself the question: May not the great difference
between the action of these fluids depend on the fact that the lymph is
rich in leucocytes, whilst in the fluid of the oedema they are almost
absent? Pursuing this question, Sawtchenko made a comparative study of
the bactericidal power of the serum, prepared outside the body, and of
the blood plasma obtained by means of an extract of the heads of
leeches, and he concluded from his researches that the bactericidal
substance circulates in the plasma of the living rat and that it is not
derived from the microphages, but must be looked upon rather as a
secretion of the macrophages in the blood and of endothelial cells. This
result was not confirmed by Gengou[228], who also took up the study of
this important question in my laboratory. Instead of preparing the
plasma by means of the addition of an extract of leeches he made use of
a method much more perfect and free from sources of error. He introduced
no foreign substance capable of affecting the results of his
experiments. Collecting the rat’s blood in paraffined tubes, and
centrifugalising it in similar tubes, he obtained a fluid which
approaches much more closely the plasma of circulating blood than does
serum. This fluid, however, will coagulate at the end of a fairly long
interval, which proves that it cannot be looked upon as blood plasma.
Gengou examined the bactericidal power of the fluid portion of the
“plasma,” obtained by the process just described, on the anthrax
bacillus, and also that of serum prepared in tubes in the ordinary way.
The difference between the two fluids is very marked; whilst the serum
destroys the bacilli sown in it very rapidly and dissolves their
contents, the fluid of the “plasma” has no similar action. These
results, confirmed several times, demonstrate very definitely that the
plasma of the circulating blood does not contain any bactericidal
substance. This, during the life of the animal, is found inside
leucocytes and only escapes from them when the cells burst or undergo
profound lesions, this taking place when the clot is formed and when the
serum is prepared outside the organism, or in the effused and coagulated
blood, or again in the peritoneal lymph during phagolysis. This
phagolysis is inevitably produced as a result of rapid injection of
foreign fluids into the peritoneal cavity, _e.g._ of broth or of
physiological salt solution, containing bacteria in suspension.
[Sidenote: [168]]
The facts we have brought together on the subject of anthrax in rats
form a whole whose several parts are in complete harmony. The phagocytes
of this species of rodent contain a bactericidal ferment, a kind of
cytase, which resists temperatures approaching 60° C. This cytase is
very active against the bacilli, but in the living animal it can only
act within the phagocytes, or, in a transitory and incomplete fashion,
outside these cells, when phagolysis is taking place in the peritoneal
cavity. The resistance offered by the rat to anthrax depends, then, on
this phagocytic activity. For its manifestation it is necessary, first
of all, that the phagocytes should manifest a positive chemiotaxis for
the bacilli, and then that they should seize and ingest these organisms.
These are the vital acts that decide the result of the struggle. When
the phagocytes show themselves inactive the bacilli multiply in the
oedematous fluid which contains no bactericidal cytase, and pass into
the plasmas of the lymph and of the blood, which also are incapable of
killing these bacteria. The animal may, then, die of anthrax, in spite
of the presence in its body of a large quantity of bactericidal cytase
which is to be found in situations to which the bacilli have not
penetrated. In those cases, on the other hand, where the phagocytes
accomplish their function, where they rush up to the menaced point and
devour the inoculated bacteria, these bacilli are placed in contact with
the intracellular cytase and undergo complete digestion. The organism in
this case gets rid of its enemies and victoriously resists infection.
[Sidenote: [169]]
Anthrax in rats, then, presents one of the most instructive examples of
natural immunity. But the detailed analysis of the mechanism of this
resistance demonstrates very clearly the great part played by the
phagocytes in this process. In this respect the organism of the rat
presents, in a general fashion, a great analogy to the natural immunity
of the dog, of birds, and of other representatives of the animal kingdom
that we have examined. Under these conditions it is useless to insist at
any length on other examples of resistance against anthrax which,
moreover, have relation much more often to a natural immunity against
attenuated bacilli than to one against true anthrax virus. Rabbits and
guinea-pigs, so sensitive to this virus, often resist the inoculation of
Pasteur’s vaccines. The rabbit is, in general, refractory to the first
anthrax vaccine; it may even resist the second vaccine. The guinea-pig,
a more sensitive animal, does not exhibit any natural immunity except
against the first vaccine. In all these cases the mechanism is similar
to that which the rat and the dog oppose to virulent anthrax. The
bacilli, into whatever part of the body they are injected, set up an
exudative inflammation which brings up a large number of leucocytes to
the point menaced. These cells readily exert their phagocytic function
and rid the organism of the introduced bacteria. In order to obtain a
complete grasp of the part played by this reaction it will be found
useful to inject beneath the skin of one ear of a rabbit a little
anthrax vaccine and beneath the skin of the other the same quantity of
virulent bacilli. The difference between the reaction in the two cases
is very striking. The ear inoculated with vaccine soon becomes the seat
of a circumscribed inflammation with a purulent exudation, all the
bacilli in which have been devoured by the leucocytes. The other ear, on
the contrary, presents, around the injected virus, only a serous or
blood-tinged exudation containing no, or few, leucocytes; the bacilli
are found free in the liquid and multiply without let or hindrance.
Meeting with no opposition the virus becomes generalised throughout the
organism and brings on death by anthrax septicaemia. Rabbits, into which
anthrax vaccines only are introduced, oppose to the invasion of the
bacilli a leucocytic barrier which arrests their extension. The natural
immunity of the sheep, rabbit and guinea-pig is also a phagocytic
immunity, but it is only capable of being exercised against bacilli
previously attenuated in virulence. The researches of Mme
Metchnikoff[229] on the reaction of the phagocytes of these animals to
the bacilli of Pasteur’s two anthrax vaccines have demonstrated the
importance of the destruction of these bacilli by the leucocytes. All
the other examples of natural immunity against anthrax are also merely
relative. The fowl that resists an anthrax virus strong enough to kill
an ox or a horse, succumbs to a special variety of anthrax cultivated by
Levin[230]. The dog, as we have seen, in spite of its pronounced natural
immunity against anthrax, is killed by the special anthrax bacillus
prepared by Martel.
In this immunity against anthrax we have to deal with a bacillus capable
of living and reproducing itself in extremely varied media. Hence the
reason, it may be said, that the bactericidal influence of the fluids is
so little pronounced in this case. To bring it into relief we must,
therefore, choose a bacterium less capable of adapting itself to the
chemical composition of various culture media. In this matter we cannot
do better than select pathogenic spirilla of extremely delicate nature
and analyse the mechanism of the natural immunity of certain species of
animals with respect to them. It must not be forgotten, however, that
here we are making use of representatives of an infinitely small
minority of pathogenic bacteria, the majority resembling the anthrax
bacillus in the facility with which they can be cultivated in all sorts
of nutritive media.
[Sidenote: [170]]
[Sidenote: [171]]
[Illustration:
FIG. 26.—Leucocyte of guinea-pig in the act of ingesting two spirilla.
]
[Illustration:
FIG. 27.—The same leucocyte, half-an-hour later.
]
[Illustration:
FIG. 28.—The same leucocyte, ten minutes later than Fig. 27.
]
[Illustration:
FIG. 29.—Leucocyte of guinea-pig in the act of ingesting a very active
spirillum.
]
[Illustration:
FIG. 30.—The same leucocyte, forty minutes later.
]
[Illustration:
FIG. 31.—The same leucocyte, half an hour later than Fig. 30.
]
The spirillum of recurrent fever of man (_Spirochaete obermeyeri_) was
the first pathogenic microbe found in an infective disease distinctly
human. Discovered a third of a century ago, it has passed through the
hands of the most skilful bacteriologists, who have tried all possible
methods of cultivating it outside the body. Koch himself tried to solve
the problem, but, in spite of his incomparable skill, did not succeed.
Later, Sakharoff[231], at Tiflis, discovered a spirillum very similar in
appearance which produced a fatal septicaemia in the goose. He, also,
tried to cultivate it, but in vain. His successors have not been more
fortunate in this respect. Here, then, are two micro-organisms, against
which natural immunity should be easily obtainable and in a fashion
quite other than that against anthrax. Nothing, indeed, is more frequent
than examples of very stable natural immunity against the spirilla of
Obermeyer and of Sakharoff. As I wished to obtain a clear idea of the
mechanism by which the guinea-pig resists injections of the spirillum of
goose septicaemia (_Spirochaete anserina_) I made injections of goose’s
blood, containing a quantity of these organisms, into the peritoneal
cavity of guinea-pigs. This injection, as usual, causes the
disappearance of most of the leucocytes, as the result of a very marked
phagolysis. We know that, under these conditions, the damaged leucocytes
allow a certain quantity of the bactericidal cytase to escape. In spite
of this the spirilla remain intact and exhibit very active movements in
the peritoneal exudation. This exudation, after a period of phagolysis,
which lasts for two or three hours, begins to be stocked again with
leucocytes which come up in increasing numbers, a fact that does not
prevent the spirilla moving about with great rapidity. Even seven hours
after the injection of goose’s blood we still find many extremely active
spirilla among a large number of recently migrated leucocytes, some of
which even at this stage contain red corpuscles of the blood of the
goose. It is not until later that the ingestion of these spirilla by the
leucocytes commences, the leucocytes at last damaging and completely
destroying them. This act of phagocytosis may be readily observed in
hanging drops of the peritoneal exudation of inoculated guinea-pigs. The
attention of the observer is drawn to certain macrophage leucocytes
which throw out one or two conical-looking processes (Figs. 26–28).
These pseudopodia attach themselves to spirilla which exhibit very
violent movements as though wishing to extricate themselves from the
grasp of the leucocyte. Sometimes the spirillum succeeds in escaping,
but usually it becomes surrounded by the protoplasm and sinks more and
more deeply into the substance of the leucocyte. Even when almost
surrounded the free part of the spirillum still continues to move (Figs.
29–31). These movements cease only after the complete ingestion of the
spirillum. Once inside the phagocyte the spirillum is digested and soon
becomes unrecognisable.
[Illustration:
FIG. 32.—Macrophage of guinea-pig filled with spirilla of recurrent
fever (after Sawtchenko).
]
[Illustration:
FIG. 33.—Macrophage of guinea-pig containing three _Spirochaete
obermeyeri_ (after Sawtchenko).
]
[Sidenote: [172]]
Recently, Sawtchenko[232] took advantage of an epidemic of recurrent
fever at Kazan to make similar investigations on the natural immunity of
the guinea-pig against Obermeyer’s spirillum. He observed that these
organisms, when injected into the peritoneal cavity, remained there,
alive, for 24 and even 30 hours, whilst these same spirilla, when kept
at 37° C. outside the organism in their natural medium, died at the end
of some (4–7) hours. The injection of human serum containing spirilla
into the peritoneal cavity of guinea-pigs set up a phagolysis succeeded
by a considerable afflux of leucocytes. In spite, however, of the
arrival of quite an army of these cells, the spirilla continued to move
rapidly; for a long time they evaded the phagocytes which, however, in
the end always ingested them. But it is only the macrophages which
fulfil their phagocytic function (Figs. 32 and 33); the microphages
obstinately exhibit an absolutely negative chemiotaxis. Now, as the
macrophages do not make their way into the peritoneal cavity until after
the microphages have appeared, it is easy to understand that
phagocytosis can only take place at a late period. Sawtchenko came to
the conclusion that “in the peritoneal cavity of animals naturally
refractory, the spirochaetes perish as the result of a slow phagocytosis
and not from the action of the bactericidal substances of the fluids.”
In conformity with this result this observer has often noted the
ingestion of living spirilla by the macrophages, in hanging drops of the
peritoneal exudation of inoculated guinea-pigs. The phenomenon
corresponds exactly to that described in connection with the spirillum
of the goose.
In spite of the great difference between the spirillum and the anthrax
bacillus from the point of view of their adaptation to surrounding
media, the general result is the same with both these microbes: animals
endowed with natural immunity get rid of them through the agency of
their phagocytes.
[Sidenote: [173]]
[Sidenote: [174]]
It would be impossible and even useless here to pass in review all the
cases of natural immunity against infective micro-organisms. We must
consequently limit ourselves to several examples which may have an
interesting bearing on the study of the problem as a whole. The
spirilla, whose history we have just recorded, remain in the peritoneal
fluid, without change of form, up to the moment when they are captured
by the macrophages. Let us see by what mechanism the natural immunity
against micro-organisms, characterised by a very special sensitiveness
to external influences and by a considerable change of shape, is
produced. The cholera vibrio and its allies best satisfy this postulate.
When they find themselves placed under unfavourable conditions, these
vibrios immediately become transformed into small spherical bodies which
are much more like cocci than vibrios. The cholera vibrio is pathogenic
for the laboratory rodents, especially for the guinea-pig, when a fairly
large quantity of a culture is injected into the peritoneal cavity.
Against smaller doses, however, the natural immunity is a most marked
one. If we take a race of the cholera vibrio of medium virulence, and
inject into the peritoneal cavity of guinea-pigs a sublethal dose of a
culture, the following phenomena may be observed[233]. The inoculated
vibrios move actively in the peritoneal fluid, from which almost all the
leucocytes have disappeared. There remain only a few lymphocytes which
appear to be indifferent to the influences that set up a real
phagolysis. But, little by little, fresh leucocytes come into the
exudation and engage in a struggle with the vibrios which, so long as
they are free, retain their curved form and complete motility. The
microphages, especially, swarm into the peritoneal cavity. Some of them
begin to ingest vibrios, but this phagocytosis is at first slight. Later
it becomes much more active. The microphages and macrophages seize
vibrios that are evidently living and uninjured, which, sometimes, may
be observed inside the vacuoles of the leucocytic contents exhibiting
very lively movements. Once ingested, however, many of the vibrios
become transformed into round granules. This change of shape is constant
when inside microphages, but is completely absent when inside
macrophages (Figs. 34 and 35). Finally, the phagocytosis becomes
complete, and the organism gets rid of the vibrios solely by means of
this reaction. Even seven hours after injection of the vibrios, when the
peritoneal fluid, crammed with leucocytes, has become thick and turbid,
there still remain a few scattered vibrios which always retain their
shape and their normal activity. A drop of this exudation, maintained at
38° C. outside the organism, gives, in a few hours, an abundant culture
of very active vibrios. It must, therefore, be concluded that the fluid
part of the exudation was powerless to destroy the vibrios or even to
render them motionless, whilst the living leucocytes have shown
themselves capable of ingesting and digesting them. The peritoneal
exudation, withdrawn at a period when it no longer contains any free
vibrios, still gives cultures of the organism for some time. Soon,
however, there comes a period when the inoculated exudation remains
sterile, this proving that the vibrios, ingested in a living state by
the phagocytes, have at length been killed by the microphages and
macrophages.
[Illustration:
FIG. 34.—Microphage of guinea-pig filled with cholera vibrios, the
majority of which are transformed into granules.
]
[Illustration:
FIG. 35.—Macrophage of guinea-pig filled with cholera vibrios not
transformed into granules.
]
[Sidenote: [175]]
When, instead of cholera vibrios of medium virulence, we take a variety
completely deprived of pathogenic activity, it is sometimes observed
that certain of these organisms, when injected into the peritoneal
cavity of the normal guinea-pig, become transformed into spherical
granules in the fluid of the exudation without any direct co-operation
of the phagocytes. This transformation into granules was first studied
by R. Pfeiffer[234] and hence has been termed Pfeiffer’s phenomenon. It
is of limited occurrence in natural immunity and is produced, as I have
been able to demonstrate, only under certain well defined conditions.
Pfeiffer’s phenomenon is observed in the peritoneal fluid. It commences
soon after the injection of the vibrios and takes place during the
period of phagolysis. In other parts of the body of the guinea-pig,
notably in the subcutaneous tissue and in the anterior chamber of the
eye, Pfeiffer’s phenomenon does not manifest itself; the animal, none
the less, resists the inoculation of the vibrios. Even in the peritoneal
cavity, moreover, it is easy to check the granular transformation of the
vibrios by means which prevent the production of phagolysis. When we
inject into the peritoneal cavity of a guinea-pig a foreign fluid,
capable of exciting the phagocytic action, e.g. veal broth,
physiological salt solution, urine, etc., we first excite a transitory
phagolysis. To this stage succeeds another in which the leucocytes
become very numerous and much more resistant than before. If we take
advantage of this period of leucocytic stimulation to inject vibrios
which have been attenuated as much as possible, we shall observe that
they soon become the prey of the peritoneal phagocytes, without
manifesting any sign whatever of Pfeiffer’s phenomenon.
It is evident, then, that this extracellular destruction of the vibrios,
sometimes observed in the peritoneal cavity, is really the work of the
microcytase that has escaped from the phagocytes during their period of
transient injury.
[Sidenote: [176]]
[Sidenote: [177]]
[Illustration:
FIG. 36.—Peritoneal exudation from guinea-pig showing free
streptococci and microphages that have ingested _Proteus_ bacilli.
]
Having analysed the mechanism of natural immunity against certain
bacilli, spirilla and vibrios, it will be interesting to determine
whether the same rules are to be applied in the case of the cocci.
Choice is not difficult since we may equally well fix upon the
staphylococci, the pneumococci, streptococci or gonococci. Should we
decide upon the streptococcus it is solely because the natural immunity
against this micro-organism has attracted the special attention of
several observers. A second advantage of the streptococcus, however, is
the high degree of natural immunity manifested against it by a
laboratory animal so convenient as the guinea-pig. Dr Jules Bordet[235]
studied this subject in my laboratory. He observed that the injection of
streptococci into the peritoneal cavity sets up a marked leucocytosis
which ends in a complete destruction of the micro-organisms. The
leucocytes rapidly ingest the great majority of the streptococci and
destroy them; there remain only a few isolated and free individuals
which are protected by a clear zone (aureola) which develops around
them, but in the end they also become the victims of the voracity of the
phagocytes. When we increase the dose of streptococci injected,
phagocytosis still goes on, but some of the streptococci succeed in
escaping, and we see a new generation produced which is distinguished by
the thickness of the protective aureola. In spite of the afflux of a
large number of leucocytes, they no longer ingest the streptococci and
generalisation of the infection results, followed by the death of the
animal. Natural immunity, then, can be suppressed under certain definite
conditions. Dr Jules Bordet[236] wished to satisfy himself whether the
leucocytes failed to fulfil their phagocytic function because of the
paralysis of their movements, or as the result of some other weakness.
With this object he injected into the peritoneal cavity of guinea-pigs,
at the moment when the streptococci begin to get the upper hand of the
leucocytes, a definite quantity of a culture of _Proteus vulgaris_.
These small bacilli in a short time become the prey of phagocytes which,
however, still refuse to ingest streptococci (fig. 36). There is thus in
the peritoneal cavity a kind of selective process as regards the
ingestion of these microbes. The _Proteus_ disappears as the result of
phagocytosis, whilst the streptococci thrive in the fluid of the
exudation and continue to multiply. This experiment, which readily
succeeds, demonstrates very clearly the difference between the positive
susceptibility of the leucocytes (with respect to the _Proteus_) and the
negative (with respect to the streptococcus). Bordet, in accordance with
the view now generally accepted, regards this sensitiveness as a
chemiotaxis, that is to say a perception of the chemical composition of
the surrounding medium. It must be admitted that the substance which
excites the chemiotaxis of the leucocytes does not readily diffuse and
may not, therefore, be found in a state of solution in the plasma of the
peritoneal exudation. Otherwise the leucocytes would refuse to ingest,
not only the streptococci, but also the small _Proteus_ bacilli, bathed
in the same repellent fluid. It is more probable that the substance
which excites the negative chemiotaxis is contained in the aureola that
surrounds the streptococci, from which it only escapes with difficulty
and for a short distance.
[Sidenote: [178]]
Marchand[237] continued the investigation of the same subject in Denys’
laboratory at Louvain. He studied the natural resistance of the
guinea-pig, rabbit and dog against the streptococcus. He, also, came to
the conclusion that phagocytosis constitutes the principal means of
defence of these mammals in their struggle against one of the most
formidable of the pathogenic micro-organisms. Starting from a single
colony, Marchand obtained two distinct races, one very virulent for the
rabbit, the other encountering a most effective natural resistance. This
resistance is due to the activity of the phagocytes which destroy the
streptococci in the ordinary fashion. He states as the general result of
his investigation that “an attenuated streptococcus is a streptococcus
readily devoured by phagocytes” whilst “a very virulent streptococcus is
a microbe that is not attacked by the leucocytes,” and he adds that “a
streptococcus is virulent because it is not devoured by phagocytes”
(_l.c._ p. 270). Up to this point the views of Marchand are in accord
with those of Bordet; but here they diverge, in fact as soon as it
becomes a question of the explanation of the origin of the difference in
the behaviour of the leucocytes. Marchand refuses to apply the theory of
chemiotaxis and asserts “that the phagocytosis depends on some physical
property of the streptococcus and is consequently dependent on the
tactile functions of the leucocytes” (p. 292). The experiments upon
which he founds his conclusion cannot, however, be regarded as
absolutely demonstrative. Thus, Marchand observed that the attenuated
streptococci, when conveyed in the culture-fluid of the virulent
variety, are as readily devoured by the phagocytes as when they were
injected alone. According to him, therefore, there was in the
culture-fluid of the virulent streptococcus no soluble substance capable
of exciting the negative chemiotaxis of the leucocytes. But is it quite
proved that this substance must necessarily pass into the filtrate of a
virulent culture? If it adheres closely to the glairy aureola, as we
have suggested, may it not remain behind with the bodies of the
streptococci, without passing through the filter in any appreciable
amount? The question cannot be regarded as definitely settled, but
probability appears to be on the side of the theory of chemiotaxis.
Marchand also investigated whether the immunity against the attenuated
streptococcus might not be explained by the bactericidal activity of the
fluids of refractory animals. His results were unvarying and definite.
The blood serum of his animals never exhibited any bactericidal power
against the streptococcus, and the attenuated race, like the virulent
one, grew well in the serums of the rabbit, dog and guinea-pig.
More recently, Wallgren[238] has taken up the study of the immunity and
susceptibility of rabbits with respect to the streptococcus. His
conclusions are, on the whole, in accord with those of his predecessors.
He found that if the injected streptococci were not very virulent
phagocytosis began immediately after the injection into the peritoneal
cavity and continued as long as there were any streptococci to be
attacked. In those cases, on the other hand, where the streptococcus was
endowed with a greater virulence, a transitory phagocytosis took place
at the beginning of the infection; but the streptococci soon succeeded
in adapting themselves to the struggle with the leucocytes and kept them
at a distance. The multiplication of the streptococci could then go on
without restraint and the animal soon succumbed to a generalised
infection. Wallgren considers that, in the defence of the organism
against the streptococcus, the products of the destroyed leucocytes may,
sometimes, play a part.
[Sidenote: [179]]
As the mechanism of natural immunity against the groups of
bacteria—bacilli, spirilla (and vibrios) and cocci—presents a very great
analogy in all three, it might be considered superfluous to continue our
analysis of this phenomenon. Our review, however, would be incomplete if
we omitted to take note of the natural immunity of the animal organism
against micro-organisms which are distinguished by an exceptional
toxicity. The first place in this group must undoubtedly be assigned to
the bacillus of tetanus.
[Illustration:
FIG. 37.—Leucocytes of rabbits filled with tetanus spores.
]
[Sidenote: [180]]
It may appear very inconsequent to be told that animals very susceptible
to tetanus, such as the guinea-pig and rabbit, are endowed with a
natural immunity against the tetanus bacillus. And yet this fact,
paradoxical as it may seem, has been demonstrated beyond doubt by
Vaillard and his collaborators Vincent and Rouget[239]. When a small
quantity of a culture of the tetanus bacillus was injected into one of
the animals just mentioned, tetanus was not long in declaring itself.
After a period of incubation, certain muscles became stiff and a
tetanus, local at first, soon became general and had a fatal issue. Now,
when much larger quantities of bacilli are inoculated, but care is taken
to rid them of the tetanus poison elaborated in the culture-fluid, the
animals resist without exhibiting any trace of tetanus. This experiment,
repeated many times, always with the same result, demonstrates that the
tetanus bacillus, when deprived of the co-operation of the toxin,
encounters, in these animals so susceptible to the latter, a most
effective opposition. Why is this? It was supposed that, in diseases
like tetanus so markedly toxic in character, the resistance was in no
way dependent on the phagocytic function. Thus Vaillard and Vincent were
quite prepared to attribute no share to the phagocytes in the example of
natural immunity which they had discovered. A detailed analysis of the
facts convinced them, however, that in this they were in error.
Guinea-pigs and rabbits do not contract tetanus, after the inoculation
of a quantity of spores and bacilli of tetanus deprived of their toxin,
solely because of the occurrence of very pronounced phagocytosis. Such
an injection is soon followed by a very marked invasion of leucocytes
which cram themselves with spores and bacilli without being in any way
inconvenienced thereby (Fig. 37). Once the phagocytes have devoured all
these organisms, the latter become incapable of producing their morbific
effect. The spores cannot germinate within the phagocytes, but there
undergo a marked degeneration and finally, after a longer or shorter
interval, disappear.
When, on the other hand, the tetanus bacilli or their spores are
accompanied by the pre-formed toxin, the latter, according to Vaillard,
excites a negative chemiotaxis of the leucocytes which keep away from
the organisms and which are thus allowed to multiply and to secrete
fresh quantities of toxin. The natural immunity of the animal’s organism
against the tetanus bacillus can be suppressed whenever the phagocytic
defence is hampered in any way. Under natural conditions it is usually
the adjuvant micro-organisms that aid the tetanus infection by hindering
the phagocytes from seizing the spores with sufficient rapidity to
prevent their germination. This fundamental result, established by
Vaillard and Vincent, has often been gainsaid on the evidence of
insufficient experiments (Sanchez-Toledo, Klipstein, Roncali), but,
ultimately, its accuracy has been completely confirmed. Cases have been
cited in which the tetanus spores, deprived of their toxin, still set up
a fatal tetanus. When a small fragment of an agar culture of tetanus,
previously heated to 85° C. for the purpose of destroying the toxin, is
inoculated, we produce tetanus. Vaillard and Rouget have demonstrated
that, under these conditions, the leucocytes penetrate merely into the
superficial layer of the agar, the spores germinating and the bacilli
multiplying in the deeper part. We can also set up a fatal tetanus in
animals by inoculating, along with sterilised earth, spores deprived of
their toxin by means of heat. The particles of soil protect the spores
against the aggression of the phagocytes, allow them to germinate and
then to poison the organism. Lactic acid produces an analogous effect,
by destroying or weakening the phagocytes. Micro-organisms, most often
inoffensive in themselves, also prevent the phagocytosis of the tetanus
spores and thus aid the intoxication.
[Sidenote: [181]]
The facts above summarised have been demonstrated to be the rule for
several species of anaerobic pathogenic bacteria. Thus, Besson[240]
showed that the septic vibrio is, by itself, incapable of setting up
septicaemia; in order to do this it needs the co-operation of other
micro-organisms. Leclainche and Vallée[241] have extended the same rule
to the bacillus of symptomatic anthrax (_Bacillus chauvaei_), so
important as being the cause of an epizootic disease of the Bovidae. The
spores of this bacillus when heated to 80°–85° C. lose the preformed
toxin and at once become incapable of producing infection.
In this case also, these spores soon after injection become the prey of
phagocytes, which seize them, prevent their germination and check their
pathogenic action. If to these heated spores, however, we add a small
quantity of toxin, they are enabled to germinate in the tissues and set
up a typical infection. If heated spores are mixed with sterile sand,
and the mixture introduced into guinea-pigs, these animals almost
invariably acquire a fatal symptomatic anthrax. The spores in the
superficial part of the sandy mass are readily devoured by the
phagocytes; but those which are included within the central part of the
mass, being protected for some time against these cells, germinate as
soon as they become permeated with the fluids of the animal organism. If
we envelope the sand in a paper sac the protection against the
phagocytes is still more complete and allows almost all the spores to
germinate and in every case to set up a fatal infection. Leclainche and
Vallée conclude from their experiments “that we only require to protect
the spore _mechanically_ in order to see an infection produced; here we
cannot allege an increase of its virulence, as when we associate a
chemical substance with the virus, and the exclusive part played by the
phagocytosis in the protective process stands out clearly” (p. 221).
The history of these three anaerobic organisms clearly proves that the
natural immunity against them cannot be made dependent on either the
bactericidal power of the fluids, or on any antitoxic property, or on
the incapacity of the micro-organism to secrete its toxin in the fluids
of the refractory animal. The cause of this immunity resolves itself
into the reaction of the phagocytes which prevent the micro-organisms
from producing their poisons.
[Sidenote: [182]]
All that has been said on the subject of the natural immunity of the
Vertebrates has had reference to cases of resistance against Bacteria.
But may not the immunity against micro-organisms belonging to other
groups depend on other factors with which the reader has not yet been
made sufficiently acquainted? Amongst the lower plants there are
Blastomycetes (_Torulae_ and Yeasts) which are capable of producing
infections, e.g. the disease amongst the _Daphniae_.
[Sidenote: [183]]
Some observers, no doubt, have come to the conclusion that the various
Blastomycetes, when introduced into a refractory organism, undergo
complete destruction within a few hours without any intervention of
phagocytosis. Thus Jona[242] explains the disappearance of yeast-cells
injected into the veins or peritoneal cavity of the rabbit as due to the
sole influence of the microbicidal property of the bloodfluid.
Gilkinet[243] looks at it from the same point of view. He injected beer
yeast (_Saccharomyces cerevisiae_) into a rabbit and observed that it
had disappeared shortly afterwards. The destruction of the yeast-cells,
according to this observer, “is effected by means of plasmatic juices”
and “is due to a specific property of the organic fluids” whose nature
is “quite unknown as regards its essential principle.” Phagocytosis is
said to play no part in this phenomenon. Let us hasten to say that
before the publication of the two works just cited, a memoir by
Schattenfroh[244] had appeared on the same subject. This observer, who
carried out his experiments in Buchner’s laboratory at Munich,
accurately observed and described the destruction of injected yeasts by
phagocytes, whilst his experiments on the microbicidal power of the
blood and serum failed. This testimony is the more important that it
emanates from a school by whom the microbicidal power of the “humours”
is regarded as the principal factor in the defence of the animal
organism. The facts described by Schattenfroh are perfectly accurate and
have been confirmed in my laboratory by Skchiwan[245], who did not
restrict himself to injecting ordinary yeasts (pink yeast,
_Saccharomyces pastorianus_) but inoculated guinea-pigs with pathogenic
yeast-cells, isolated by Curtis[246] from a case of myxomatous tumour in
man. The guinea-pig is refractory to small doses of this yeast but
succumbs to injections of larger quantities: Skchiwan convinced himself
that the ingestion of the non-pathogenic yeast-cells takes place with
great rapidity. Thus the _Saccharomyces pastorianus_, in the peritoneal
cavity of the guinea-pig, is ingested almost exclusively by microphages
at the end of two hours. Some (3–4) hours after injection, “sowings” of
the peritoneal exudation no longer yield growths. On the other hand
Curtis’ pathogenic yeast-cells resist the action of the phagocytes for a
much longer time. After a period of phagolysis in the peritoneal cavity,
the leucocytes that have just arrived in large numbers begin to seize
the yeast-cells. Usually several macrophages fuse around the same yeast
globule forming a very characteristic kind of rosette. Sometimes the
macrophages run together to produce a giant cell, whose centre contains
the yeast-cell. This latter defends itself against phagocytosis by
secreting a fairly thick membrane. The struggle between the two living
elements is a fairly prolonged one; 24 to 48 hours after inoculation all
the yeasts are surrounded by phagocytes, amongst which microphages are
exceptional. But the parasites remain alive for 4–6 days after their
injection into the peritoneal cavity, as proved by the cultures that are
obtained from the exudation when the fluid is “seeded” out. It must be
concluded, therefore, that the yeast-cells were surrounded by the
phagocytes whilst still presenting all the signs of life. Skchiwan was
no more successful than Schattenfroh in demonstrating any kind of
microbicidal action of the fluids on the Blastomycetes.
There is, consequently, no doubt whatever that the resistance of the
animal organism against yeasts follows the same rules that hold in the
defence against bacteria.
[Sidenote: [184]]
The animal micro-organisms are much rarer in infective diseases than are
the microphytes; moreover the impossibility of obtaining cultures of
them renders their investigation much more difficult. Yet there exist
facts that are capable of affording us information as to the means made
use of by the refractory organism against certain parasitic Protozoa.
Amongst these latter the _Trypanosomae_ play a most important part. One
species of this genus (_T. lewisi_) produces an infective disease in
rats, especially in the grey rat (_Mus decumanus_), the blood of these
rodents often containing a very large number of them, whilst the small
flagellated organisms flourish well in the serum prepared from the blood
of affected animals. Laveran and Mesnil[247], in their studies on the
_Trypanosomae_, injected defibrinated blood containing numerous
_Trypanosomae_ into the peritoneal cavity of guinea-pigs, which exhibit
a natural immunity against this parasite. The parasites remained alive
for some days and then disappeared completely. Here again it is the
phagocytes of the peritoneal exudation which rid the animal of the
_Trypanosomae_ by ingesting them. Laveran and Mesnil were able, by the
examination of hanging drops of the peritoneal exudation of their
guinea-pigs, to detect leucocytes in the act of devouring _Trypanosomae_
which showed, by their active movements, that they were still alive.
Once the parasites were completely enclosed within the macrophages,
their final disappearance took place with extraordinary rapidity.
In this chapter we have attempted to place before the reader a complete
series of the phenomena observed in natural immunity in animals. We have
passed in review the resistance of the animal organism against the
principal groups of bacteria, and we have dwelt on certain of them which
are most capable of adapting themselves to various media, and on others
which present examples of microorganisms more exacting and more
delicate. We have examined the immunity against Blastomycetes and
parasitic animalcules. Above all, in the lower animals, just as in the
Vertebrata of all classes, we have always observed this general
phenomenon: phagocytic resistance as the principal and constant factor
in natural immunity.
CHAPTER VII
THE MECHANISM OF NATURAL IMMUNITY AGAINST MICRO-ORGANISMS
The destruction of micro-organisms in natural immunity is an act of
resorption.—Part played by inflammation in natural
immunity.—Importance of microphages in immunity against
micro-organisms.—Chemiotaxis of leucocytes and ingestion of
micro-organisms.—Phagocytes are capable of ingesting living and
virulent micro-organisms.—The digestion of micro-organisms in
phagocytes is most often effected in a feebly acid
medium.—Bactericidal property of serums.—Phagocytic origin of the
bactericidal substance.—Theory of the secretion of the
bactericidal substance by leucocytes.—Comparison of the
bactericidal power of serums and of blood plasmas.—The
bactericidal substance of blood serums must not be considered a
secretion-product of leucocytes; it remains within the phagocytes,
so long as they are intact.—The cytases.—Two kinds of cytases:
macrocytase and microcytase.—Cytases are endo-enzymes, allied to
trypsins.—Changes in the staining properties and in the form of
micro-organisms in the phagocytes.—Absence or rarity of fixatives
in the serums of animals endowed with natural immunity.—The
agglutination of micro-organisms does not play any important part
in the mechanism of natural immunity.—Absence of antitoxic
property of the body fluids in natural immunity.—The phagocytes
destroy the micro-organisms without their ingestion being preceded
by neutralisation of the toxins.
[Sidenote: [185]]
The facts we have set forth in the preceding chapter clearly justify us
in concluding that the destruction of the micro-organisms in natural
immunity is reduced to their resorption by the phagocytes.
We have now, therefore, returned to the point arrived at and already
studied in Chapter IV, where we attempted to establish certain
fundamental laws. It remains to be seen up to what point these laws
apply to the phenomena of natural immunity against infective
micro-organisms.
[Sidenote: [186]]
The introduction into the animal organism of foreign blood, of
spermatozoa belonging to the same or a different species, or of any
other cells, as in the case of the penetration of micro-organisms into
the tissues or cavities of the body of a refractory animal, determines,
primarily, a localised inflammation, associated with which is a
diapedesis of many white corpuscles. Instead of aseptic inflammation, as
in the case of the resorption of cells, there is produced, in
antimicrobial immunity, a septic inflammation at the point of invasion
of the micro-organisms. In this inflammation the redness and heat are
slight, the fluid part of the exudation is insignificant, but what is
especially characteristic is the large number of leucocytes which come
up towards the point menaced. This constancy of the inflammatory
reaction in natural immunity is one of the best proofs of the accuracy
of the view that inflammation is a phenomenon useful to the animal
organism, especially in its struggle against microbial invasion. As we
have devoted a whole volume to the discussion of the comparative
pathology of inflammation it is here unnecessary to discuss it further.
Since the publication of this book numerous articles on inflammation
have appeared, but none of them have undermined, in the least degree,
the fundamental bases of the phagocytic theory of inflammation. The view
that this phenomenon really constitutes a healing reaction of the
organism is at present accepted by many investigators in all countries.
It is therefore needless to re-defend it.
Although there still remain a certain number of points that are not
sufficiently cleared up in the essential mechanism of inflammation, it
is now proved beyond doubt that the sensitiveness of the cell elements
which here play a part, is one of the essential factors in the process.
The nerve cells which govern the vascular dilatation, the endothelial
cells which allow of the passage of leucocytes, and the leucocytes
themselves which escape from the vessels in order to reach the point of
entrance of the micro-organisms, all must be influenced in a special
fashion. In natural immunity the phagocytes exhibit a positive
chemiotaxis and this form of sensitiveness is a condition indispensable
to a state of immunity and to the disappearance of the micro-organisms.
In my eighth lecture on inflammation I have already set forth the
fundamental facts upon which rests the doctrine of the chemiotaxis of
leucocytes. During the last ten years numerous data corroborating these
results, obtained first by Leber, Massart, and Charles Bordet, and since
confirmed by numerous other observers, have been accumulated.
[Sidenote: [187]]
In the resorption of blood corpuscles and of animal cells in general, it
is especially the macrophages which intervene, but in natural immunity
against micro-organisms positive chemiotaxis is exhibited by the
microphages more than by the macrophages. When we examine an
inflammatory exudation and find a preponderance of microphages we are
satisfied that there has been an intervention of micro-organisms. Even
in the examples where it is, at first, principally the macrophages which
destroy the micro-organisms (as in the case of the resistance of the
animal organism against the tubercle bacillus), there is also a great
afflux of microphages. The sensitiveness of the two chief categories of
phagocytes often exhibits a marked difference. We need merely recall to
the reader the example of the spirilla, ingested and destroyed
exclusively by the macrophages of the guinea-pig, which alone exhibit
the necessary positive chemiotaxis. In many other examples of natural
immunity the part played by the macrophages is masked by that of the
microphages.
In natural immunity the motile phagocytes, having come up to the
invaders, perform a second physiological function; they ingest the
micro-organisms. Sometimes the leucocytes devour at one swoop whole
masses of these organisms, and carry out their work in a very short
time. In other cases, especially when actively motile micro-organisms,
such as the spirilla of Obermeyer or of Sacharoff, have to be dealt
with, the ingestion takes place with more difficulty and requires
special conditions. Thus, in order to ingest a spirillum, the
macrophages of the guinea-pig throw out long conical processes. Never in
the ingestion of micro-organisms have I observed methods comparable to
that by which the macrophages seize upon the red corpuscles of birds or
upon other animal cells.
[Sidenote: [188]]
Some observers have expressed the opinion that micro-organisms make
their way into the cells spontaneously and do not need to be drawn in by
means of protoplasmic processes thrown out by the phagocytes. It is of
course indisputable that certain micro-organisms may pass into the
interior of the cell independently of any act of phagocytosis. Such is
the case with the malaria parasite and allied species which make their
way into the red blood corpuscles. But here we are dealing with amoeboid
organisms, quite capable of perforating the wall of the red blood
corpuscle by means of their own pseudopodia. Bacteria, which do not
possess amoeboid movements, are deprived of this power of invasion.
There are, however, very rare cases in which such penetration does take
place. For example, Bizzozero[248] has described spirilla in the stomach
of the dog; these may be found inside epithelial cells. But here these
actively motile bacteria make their way into the interior of vacuoles
which open on the free surface. Attracted, probably, by the epithelial
secretions the spirilla first draw near to the cells and then take
advantage of small openings through which they pass into the secretory
vacuole. In almost all cases, however, living and even actively motile
bacteria are incapable of penetrating into cells. Thus, when we observe
the spirilla of recurrent fever or of goose septicaemia in the
neighbourhood of leucocytes, we often see them exhibit very brisk
corkscrew movements on the surface of these cells without ever being
able to invade them. On the other hand, when the leucocyte sends out a
process towards the spirillum ingestion rapidly takes place. In anthrax
exudations, or in the spleen of animals that have succumbed to anthrax,
large numbers of bacilli may often be observed in the immediate
neighbourhood of the leucocytes or of the cells of the splenic pulp,
without a single bacillus being found within these cells. Nor do we ever
see any bacteria (which develop abundantly in a drop of exudation
withdrawn from the organism) invade the dead leucocytes, lying alongside
them. Whilst on the other hand we see the micro-organisms swarming
outside the neighbouring leucocytes and occupying the free spaces
between these cells.
[Sidenote: [189]]
Almquist[249] has recently described a method by means of which
micro-organisms can be taken into the substance of dead leucocytes. He
collects leucocytes from mammalian blood, mixes them with bacteria, and
centrifugalises the mixture for some time. He convinced himself that
after a not very prolonged contact the bacteria are found within
leucocytes. Here Almquist excluded phagocytosis, properly so-called,
that is to say, the ingestion of the bacteria by the active movements of
the leucocytes; but he does not give sufficient proof that the cells, in
his experiments, were actually dead. He thinks that the relatively low
temperature (below 15° C.) excluded the possibility of amoeboid movement
in the leucocytes of warm-blooded animals. This argument, however, does
not accord with actual fact, for it is indisputable—and we have often
convinced ourselves of this—that the leucocytes of man and warm-blooded
vertebrates maintained at even a lower temperature than 15° C. are quite
capable of motion and of ingesting foreign bodies. In all cases, the
data as a whole, some of which we have cited above, leave no doubt that
the ingestion of micro-organisms unprovided with amoeboid powers takes
place by means of active movements of the living protoplasm of the
leucocytes. To dissipate any remaining doubt on the part of the reader I
need only recall Bordet’s investigations, cited in the preceding
chapter, of the behaviour of leucocytes in the peritoneal cavity of
guinea-pigs inoculated with streptococci and _Proteus_ bacilli. The
leucocytes of the peritoneal cavity allow the virulent streptococci to
develop freely, not ingesting a single one, whilst the _Proteus_
bacilli, injected later, are quickly devoured and at the end of a very
short time are all found in the substance of these same phagocytes. This
example, so demonstrative, of the chemiotaxis (positive as regards
_Bacillus proteus_ and negative as regards the streptococcus), is at the
same time the best proof of the fact that the ingestion of the
micro-organisms is a vital, physiological act and not merely a simple
phenomenon of mechanical penetration of micro-organisms into the soft
protoplasm of the leucocytes.
It was formerly thought that leucocytes, charged with micro-organisms,
provide the latter with a good culture medium and serve also as vehicles
of transport for them from one place to another in the living organism.
This view has often been affirmed without any proof whatever being given
of it. It has now been demonstrated to be erroneous. The
micro-organisms, with some rare exceptions, find within the leucocytes a
very unfavourable medium. Usually they perish there, or, in the case of
very resistant micro-organisms, such as the tubercle bacilli in
refractory animals or the endospores of certain bacteria, without being
actually destroyed, they are prevented from germinating and multiplying.
[Sidenote: [190]]
Later, another view has been advanced that phagocytes are capable of
ingesting only those micro-organisms that have been previously killed by
some substance which is found outside the defensive cells. This view is
quite as erroneous as the one we have just analysed. The phagocytes are
perfectly capable of seizing and devouring living micro-organisms. We
have only to recall on this point the facts cited in the preceding
chapter on the subject of living bacteria ingested by the leucocytes of
various animals, or the history of the very active spirilla which retain
their motility up to the moment when they become completely enclosed by
the protoplasmic processes of the leucocytes of the guinea-pig.
Observations _in vitro_ have, as already described in the same chapter,
afforded a demonstration of the ingestion of living flagellated
Infusoria by the leucocytes of refractory animals.
These facts, fairly numerous in themselves, are not, however, the only
ones that might be cited in favour of the fundamental thesis that
phagocytes possess all the means for incorporating living
micro-organisms. In my first works on phagocytosis I cited the example
of amoeboid cells, in the Invertebrata, containing motile bacteria[250],
and that of leucocytes of the frog charged with motile bacilli[251] of
an artificial septicaemia. Since then the number of similar cases has
increased considerably. Nothing is easier than to observe the
phagocytosis of living micro-organisms _in vitro_. Take a drop of frog’s
lymph and add to it a few of the _Bacilli pyocyanei_, we soon observe
the struggle between the leucocytes and the very motile bacteria, and
inside the digestive vacuoles bacilli executing very pronounced and
active movements.
[Sidenote: [191]]
The same result may be obtained by another method, by which at the same
time we gather information as to the virulence of the micro-organisms
ingested by the phagocytes. The view has often been expressed that
phagocytes seize only those bacteria that have been deprived of their
virulence by a previous action of the fluids of the animal organism;
consequently search has been made for some attenuating property of these
fluids. We have already answered this objection in the previous chapter
by the citation of cases in which the exudations of refractory animals,
containing only micro-organisms ingested by the phagocytes, were,
nevertheless, very virulent for susceptible animals. This question has
been especially discussed in relation to the anthrax of frogs, on which
subject several investigations have been carried out, the result of
which is completely convincing. Bacilli ingested by the leucocytes of
these Batrachians retain their full virulence for a long time.
Exudations which contain only intraphagocytic bacilli, the majority of
which have already lost their normal staining by aniline dyes, produce
fatal anthrax in susceptible animals, such as the mouse and the
guinea-pig. Mesnil has demonstrated the same fact by using the
exudations of fresh-water fishes that are refractory to anthrax. The
same rule applies equally to the exudations of dogs and fowls that have
been inoculated with the bacillus.
Long before these experiments on anthrax were made, Pasteur[252] had
shown that the virus of fowl cholera, which in the guinea-pig sets up a
mild affection and gives rise to the formation of abscesses, retains its
virulence for a considerable time in the pus of these abscesses. When he
injected rabbits with a small quantity of guinea-pig’s pus developed at
the point of inoculation of the cocco-bacillus of fowl cholera, the
animals succumbed to a generalised and rapid infection. The conviction
has since been arrived at that, in the guinea-pig, these micro-organisms
readily become the prey of the leucocytes that are present in the
exudations.
The rule, therefore, is general that in animals endowed with natural
immunity the phagocytes seize and ingest even living micro-organisms
that have retained their initial virulence.
[Sidenote: [192]]
[Sidenote: [193]]
Once within the phagocytes, the micro-organisms are surrounded by a
clear fluid, which accumulates in vacuoles, or they are lodged directly
in the protoplasm. In both cases the micro-organisms are subjected to a
digestive action which usually dissolves them completely. It is not
always easy to form an idea of the conditions under which the
intracellular digestion takes place. At first[253] I used a weak
solution of vesuvin for the purpose of gaining some idea as to the
condition of the micro-organisms that have been ingested by the
leucocytes and demonstrated that the living bacteria remain unstained in
this solution, whilst the dead bacteria take on a somewhat deep brown
stain. Thanks to this reaction I was able to furnish one of the proofs
of the fact that in immunised animals ingested bacteria are killed
inside the phagocytes. The use of Ehrlich’s neutral red (_Neutralroth_)
gives us further valuable indications. This colour, quite innocuous for
living elements, is an excellent indicator of acid or alkaline reaction.
Plato[254], in Breslau, has carried out numerous researches on the
staining of micro-organisms by a weak aqueous solution (1%) of this
substance. He has shown that “free” micro-organisms remain alive in this
solution without taking on any tinge of colour. On the other hand, the
same micro-organisms, when ingested by the phagocytes, are stained
brownish-red. Most of these stained organisms no longer exhibit any sign
of vitality; but amongst those within the phagocytes are some which, in
spite of being deeply stained, are certainly alive. Plato insists on the
fact that ingested micro-organisms remain stained as long as the
phagocytes are alive, for, shortly after the death of these cells,
decoloration of the micro-organisms and of the intracellular granules
takes place. When neutral red is added to an exudation in which the
leucocytes are dead, the staining of the ingested micro-organisms—dead
or living—does not take place. I have myself verified these
observations, and Himmel[255], who has carried out an elaborate
investigation on this subject in my laboratory, has confirmed them in
numerous cases. In the third and fourth chapters of this work I have
already brought forward arguments in favour of the view that the
staining of the ingested elements indicates a feebly acid reaction
inside the phagocytes. Sometimes this reaction manifests itself in the
digestive vacuoles; in other cases it is exhibited only in the
micro-organisms directly lodged in the protoplasm (Fig. 38). Whilst the
phagocyte is still living the acid juice which fills the vacuoles or
permeates the ingested organisms does not mix with the protoplasm which
is always alkaline. But shortly after the death of the phagocytes this
mixture is effected without difficulty, and the alkalinity of the
protoplasm is then amply sufficient to neutralise or even render
alkaline the feebly acid juices. This interpretation of the facts is in
complete harmony with all the data, collected up to the present, on the
staining by neutral red of phagocytised micro-organisms.
[Illustration:
FIG. 38.—Peritoneal macrophage of guinea-pig that has ingested a
number of _Bacilli coli_. Stained _intra vitam_ with neutral red.
]
All ingested bacteria do not, however, stain in the way we have
indicated. Tubercle bacilli, even in cases of natural immunity, remain
unstained inside the phagocytes or take on only a very slight
straw-yellow tint. Himmel made this observation on the bacilli of avian
tuberculosis that had been ingested by the peritoneal leucocytes of the
guinea-pig, a species resistant to this micro-organism. It might be
thought that such a resistant membrane as that of the tubercle bacillus,
with its waxy layer, would prevent the penetration of the acid
leucocytic juice; but several bacilli which resist decoloration by
acids, as do the tubercle bacilli, notably the bacilli of Moeller and
their allies, are stained a bright red by neutral red as soon as they
are ingested by the phagocytes. It is, therefore, more probable that, in
the case of true tubercle bacilli, the reaction in the cells is no
longer acid, but alkaline. This conclusion is confirmed by what is
observed in the giant cells of the Algerian gerbil (_Meriones shawii_),
a species of rodent which exhibits a great natural resistance against
the bacillus of human tuberculosis[256]. The bacilli, ingested by these
phagocytes, secrete a series of concentric membranes which become
impregnated with phosphate of lime (Fig. 5). The process causes the
death of the bacilli, of which there remain only the calcified
membranes. The precipitation of the lime salt around bacillary membranes
itself indicates the alkaline reaction of the medium. The use of certain
staining substances fully confirms this conclusion. Thus, with alizarin
sulpho-acid the giant cells stain deep violet, this affords clear proof
of a very distinct alkaline reaction.
[Sidenote: [194]]
We arrive then at the general conclusion that phagocytic digestion
usually takes place in a medium weakly acid, but that it can also go on
in an alkaline medium. It is impossible, in the present state of our
knowledge, to define the nature of the acid secreted by the phagocytes.
H. Kossel[257] has expressed the view that the intracellular digestion
of micro-organisms is effected by the nucleic acid, secreted by the cell
nucleus and accumulated in the vacuoles of the contents of the
phagocytes. He has brought forward in support of this view the fact that
nucleic acid is distinctly bactericidal, killing certain pathogenic
micro-organisms, and giving a precipitate composed of albumen and
nucleic acid. Later H. Kossel pointed out the presence in these formed
elements of albuminoid substances which have an alkaline reaction but
which also destroy bacteria. Thus he has isolated from the spermatic
fluid of the sturgeon a protamine, “Sturin,” which, even in very weak
solutions, exhibits a strong bactericidal action on the typhoid
bacillus, staphylococcus, etc. It is possible that these substances play
a part in intracellular digestion. On the other hand, however, we must
regard it as well established that in phagocytes there is a soluble
ferment which kills and digests micro-organisms. We have already seen,
in connection with the resorption of animal cells, that it is the
ferment alexine, or cytase, which plays the principal part in the
digestive function. We must now ask ourselves whether the same substance
acts also on micro-organisms.
For more than fifteen years a study of the bactericidal power of the
blood and other fluids drawn from the animal organism has been carried
on. Based on the not very definite results of Traube and
Gscheidlen[258], Fodor[259] drew attention to the property of the
defibrinated blood of the rabbit to destroy the bacteria sown in it.
Under the inspiration of Flügge[260], Nuttall[261] carried out a whole
series of experiments on this bactericidal property of defibrinated
rabbit’s blood, of the aqueous humour, and of some other fluids. After
confirming Fodor’s general result, Nuttall went further and showed that
the bactericidal power of the fluids is due to a substance of
undetermined nature which is destroyed by heating to 55° C. for one
hour. This discovery was confirmed by a large number of observers, and
soon became an accepted fact.
[Sidenote: [195]]
Flügge now considered that he could base a theory of immunity on the
presence of the bactericidal substance of the body fluids. Bouchard[262]
and his school adopted and developed this view, especially with
reference to researches on the microbicidal power of blood serum.
Buchner[263] soon came forward as the chief advocate of this theory, and
enriched it by numerous investigations carried out by himself or along
with collaborators in his school at Munich. It is to him that we owe the
suggestion of the term _alexine_ (protective substance) to designate the
bactericidal substance of blood serum and other fluids of the animal
organism which are capable of killing micro-organisms. Buchner
determined the conditions under which alexine acts best as a bacterial
poison and developed the humoral theory of natural immunity, according
to which the latter is reduced to the bactericidal property of the body
fluids.
[Sidenote: [196]]
As the postulates of this theory are often not in accord with the real
facts, as Lubarsch[264], especially, has demonstrated in many of his
papers, we[265] expressed the opinion that a portion at least of the
bactericidal power might come from substances that had escaped from the
leucocytes during the preparation of the defibrinated blood and of the
blood serum. This hypothesis remained for several years unnoticed, but
later several observers have, quite independently, arrived at the
conclusion that alexine is nothing but a leucocytic product. Denys and
Havet[266] were the first to show that exudations rich in white
corpuscles exhibited a bactericidal power much higher than that of the
corresponding blood serums. Shortly afterwards H. Buchner[267] showed
the same thing on comparing the bactericidal power of exudations rich in
leucocytes with the blood serum of the same animals. As this property
disappeared from both fluids after they had been heated to 55° C.,
Buchner concluded that the bactericidal substance of the exudations must
be identical with the alexine of the blood serum. Several other
observers, amongst whom Bail, Schattenfroh, Jacob and Löwit, may be
cited, obtained results more or less in accord with the above, though
obtained by different methods, so that it has now for some time come to
be recognised that the leucocytic origin of the alexines is generally
accepted, especially since Jules Bordet[268], in an investigation
carried out in my laboratory, arrived at the same result from various
very demonstrative experiments.
Nevertheless several authoritative voices have been raised against this
interpretation of the facts. R. Pfeiffer especially, with his school,
has pronounced against the leucocytic origin of the bactericidal
substance found in the blood serum. Pfeiffer and Marx[269] and
Moxter[270] have insisted on the fact that the fluids of exudations rich
in leucocytes are often much less bactericidal than is the serum of the
blood of the same animals.
For some years, struck by the marked difference between the phagocytic
function of the macrophages and that of the microphages, I have thought
that the contradictory results of the observers cited might be explained
by some difference in the nature of the leucocytes of the various
exudations and of the blood which served for the preparation of the
serums. I therefore asked Gengou to devote his attention to this
particular point and to compare the bactericidal power of exudations,
rich in microphages, with that of others containing many macrophages and
also with the blood serum of the same animals. Gengou[271] has carried
out his experiments with remarkable exactness and care, and as I have
followed them closely I am in a position to speak as to their extreme
accuracy.
[Sidenote: [197]]
In order to obtain exudations very rich in microphages Gengou injected
gluten-casein by Buchner’s method into the pleural cavity of dogs and
rabbits. Usually at the end of 24 hours he was able to collect a large
quantity of fluid containing numerous leucocytes, almost exclusively
microphages. To obtain macrophagic exudations Gengou injected washed red
blood corpuscles of the guinea-pig into the pleural cavity of his
animals; two days afterwards he withdrew from the pleural cavity a very
viscid fluid, containing, as regards formed elements, macrophages almost
exclusively. After isolation of the leucocytes by centrifugalisation of
the exudations, Gengou washed the cells with physiological salt solution
and then added to them an equal volume of broth. This mixture was frozen
by Buchner’s method, and was then submitted to a temperature of 37° C.
Under these conditions the leucocytes, killed by cold, gave up to the
fluid their bactericidal substance.
Studied in this way, the bactericidal power of the extract of
microphages showed itself always superior to that of the corresponding
blood serum. The greatest difference was observed in the dog, where, as
already mentioned in the preceding chapter, the serum of the blood has
no bactericidal property as regards the anthrax bacillus, whilst the
extract of microphages manifests this property very strongly. The
microphagic extract of the exudations of rabbits was more active in the
destruction of the bacilli of anthrax and typhoid, _Bacillus coli_ and
the cholera vibrio, than was the blood serum.
The result of these experiments leaves no room for doubt. The
microphages, collected in the aseptic exudations of the dog and rabbit,
contain more bactericidal substance than does the blood serum of the
same animals. Nor can there be a doubt that this bactericidal substance
is the same whether it appears in the microphages or in the blood serum:
in both cases it is destroyed by heating to 55° C. and, in all other
respects, it behaves in the same manner.
The experiments of Gengou with the extracts of macrophages have
demonstrated, on the other hand, that this fluid exerts no bactericidal
power. Let it be understood at the outset that this fact is in no way an
indication of the absence of the bactericidal ferment in the
macrophages. Direct examination of the phenomena which are manifested
inside these cells demonstrates most clearly that the macrophages kill
and digest micro-organisms. But this process usually goes on much more
slowly in the macrophages than in the microphages, owing probably in the
former to the presence of a smaller quantity of the bactericidal
substance. Under these conditions we can readily understand that this
substance does not pass, or passes only in small amount, into the
extracts. There is nothing remarkable in the fact that, with so
imperfect a method of preparing the extracts, the greater part of the
bactericidal substance should remain in the bodies of the cells.
The facts just set forth afford a sufficient explanation of the marked
difference in the results obtained by various observers as to the
bactericidal power of the exudations. When the latter are rich in
microphages, the bactericidal property is very marked: when, on the
other hand, the exudations contain a large number of macrophages, the
bactericidal power may be very weak or even _nil_.
[Sidenote: [198]]
The experiments above summarised confirm the conclusion that the
microphages must be regarded as the source of the bactericidal substance
of the body fluids. But here arises the question: Do the microphages
secrete the substance during life, giving it up to the blood plasma, or
does this substance escape only after the death of the leucocytes and
the damaging of the cells, due to various external causes? We here touch
on a problem which has been the subject of much discussion and one of
very great importance in connection with the question of Immunity in
general.
After the discovery of the bactericidal power of serums, several
investigators set to work in search of the source of the bactericidal
substance. Hankin[272], and shortly afterwards Kanthack and Hardy[273],
expressed the view that this substance is the secretion-product of the
eosinophile leucocytes which would thus appear to be a kind of motile
unicellular glands. This theory could not be supported by solid
arguments and must be regarded as generally abandoned, because it is now
completely out of accord with well-established facts. Thus, various
osseous fishes, in spite of the total absence of eosinophile or
pseudo-eosinophile granules are none the less capable, thanks to their
leucocytes, of destroying a large number of pathogenic micro-organisms
(Mesnil, _l. c._).
[Sidenote: [199]]
A similar theory was enunciated by H. Buchner[274], though he holds that
it is not the eosinophile leucocytes only that secrete the bactericidal
substance, but the leucocytes in general. Being attracted to the point
menaced by the micro-organisms, these cells secrete their bactericidal
product, which diffuses into and along with the plasma of the exudations
and of the blood. In these fluids the micro-organisms undergo a more or
less complete destruction, or at least severe injury which renders them
more susceptible to the attack of the phagocytes. At the International
Congress of Hygiene, held at Budapest in 1894, Buchner proclaimed the
thesis that “the leucocytes fulfil an important function in the natural
defence of the organism ... by means of soluble substances which they
secrete.” Later, his pupils, Hahn[275] and Schattenfroh[276],
endeavoured to support this theory by exact experiments, but they found
it impossible to do this at all satisfactorily. Later, another of
Buchner’s pupils, Laschtschenko[277], published a paper in which he
maintains that he has found a convincing argument. It is as follows. A
blood serum, by itself void of bactericidal property, some minutes after
white corpuscles from another species of mammal have been added to it
acquires this property. Thus the rabbit’s leucocytes added to dog’s
serum immediately give to it the bactericidal power, so long as a large
number of cells remain alive and motile. But when the leucocytes of the
same species are added to rabbits’ serum the fluid becomes no more
bactericidal than before. The same result may be obtained by mixing
rabbits’ leucocytes with the blood serum of the horse, pig and other
species. Laschtschenko concludes from these observations that the vital
secretion of the bactericidal substance by the leucocytes of the rabbit
takes place when they are irritated by the serum of a different species.
As an analogous effect has been observed with mixtures of rabbits’
leucocytes with the serum of a different species heated to 60° C.,
Laschtschenko believes himself safe from the objection that the giving
up of the bactericidal substance results from the death or injury of the
white corpuscles. According to him this injurious effect on the white
corpuscles can only be produced by an unstable substance which is
destroyed by heating to 60° C. Laschtschenko forgets that the leucocytes
are in general delicate cells, capable of being affected even by fluids
which do not actually kill them. Now we know that serums, when heated to
60° C., still retain their power of agglutinating the leucocytes, a
power which must hamper these cells in their normal function.
[Sidenote: [200]]
Trommsdorff[278], in an investigation carried out in Buchner’s
laboratory, endeavoured to supplement Laschtschenko’s results and to
support them by new and more convincing experiments. But he only
succeeded in a few cases in obtaining a bactericidal serum after adding
rabbits’ leucocytes to the blood serum of other animals. “In a great
number of my experiments,” says Trommsdorff, “I very often did not
succeed in extracting the alexines from the rabbit’s leucocytes by the
use of Laschtschenko’s method” (p. 385). On the other hand, Trommsdorff,
wishing to establish the living condition of the leucocytes mixed with a
foreign serum, arrived at the following result: “In the majority of the
cases, as in fresh exudations, the number of living leucocytes after
their treatment with active horse’s serum, as well as with inactive
serum (heated to 60° C.) of dog, ox and horse, varied between 60 and
80%” (p. 391). In spite of these verifications, Trommsdorff comes to the
conclusion that the presence of alexine in those serums to which
leucocytes had been added, must “in all probability” be attributed to
its secretion by the living leucocytes. We regard it as much more
probable that the alexine, in those cases where it passed into the
serum, was due to the breaking up of the dead leucocytes, whose numbers
rose to 40 %, that is to say, almost half their total number. Our
conclusion is, in any case, much more in accord with the more constant
and more exact results obtained by other methods.
[Sidenote: [201]]
In spite of the insufficiency of proofs in favour of the theory of
bactericidal secretions by the leucocytes it has been very favourably
received by many investigators. As, however, it came into collision with
the general fact that, in the refractory animal, the microorganisms
remain alive in the plasmas of the exudations and are, in this
condition, ingested by the phagocytes, it was therefore very important
that this fundamental contradiction should be settled by decisive
experiments. The attempt has often been made to obtain blood plasma and
to compare its bactericidal action with that of serum from the same
animal. In the preceding chapter we have already mentioned an attempt in
this direction made by Sawtchenko. Hahn[279] had previously attempted to
prepare plasma by adding histon to blood. As this “plasma” was found to
be just as bactericidal as the blood serum Hahn concluded that the
bactericidal substance, secreted by the living leucocytes, circulates in
the living blood. In all the experiments carried out by this method it
was impossible to avoid certain sources of error, and in my laboratory
Gengou[280] undertook a new series of researches, endeavouring to obtain
from blood a fluid resembling normal plasma as closely as possible. The
method he employed has been described in detail in a memoir, on an
anticoagulating serum, which he published along with Bordet[281]. The
blood was drawn into paraffined tubes and centrifugalised at once in
other tubes whose walls were likewise covered with a layer of paraffin.
The fluid thus prepared is certainly more allied to circulating plasma
than is the blood serum obtained after the coagulation of the blood.
Nevertheless, it is still far from being identical with true normal
plasma; it still coagulates, though tardily. Gengou compared, in their
bactericidal action, the blood serum and the serum, decanted after the
tardy coagulation of the fluid analogous to plasma. He carried out a
great number of experiments with the two fluids, obtained from dogs,
rabbits and rats, making a comparative study of their bactericidal power
as regards the anthrax bacillus, the typhoid bacillus, and the cholera
vibrio. I have closely followed all these experiments and can confirm
the results described by Gengou, namely, that the fluid, in this plasma
serum, possesses an insignificant bactericidal power or none at all,
whilst the blood serum almost always exhibits this property to a marked
degree.
As a result of the researches just summarised it is no longer possible
to maintain the theory of bactericidal secretions by leucocytes or by
any other category of cells. The bactericidal substance does not
circulate in the blood plasma nor in that of the exudations, and this is
a sufficient reason for refusing to it the title of a secretion-product.
Its presence in the blood serum is due, like that of the fibrin-ferment,
to the destruction or more or less grave injury of the phagocytes.
[Sidenote: [202]]
This fact, upon which we must insist most strongly, is in flat
contradiction to the view recently formulated by Wassermann[282]. In a
work devoted to natural immunity against micro-organisms, this author
describes how he submits his animals (guinea-pigs) to the action of an
anticytase (or anti-alexine) serum whose preparation, described in the
fifth chapter of this work, offers no difficulties. Under the influence
of this serum, the guinea-pigs, into the peritoneal cavity of which a
strong dose of typhoid cocco-bacilli is inoculated, die from infection,
whilst the control animals, inoculated in a similar manner, but which
have received in addition some normal rabbit’s serum, heated to 60° C.,
entirely resist the infection. Wassermann concludes that the first
series of guinea-pigs succumbed because of the impossibility of fighting
against the typhoid bacillus by means of the free cytase, this being
neutralised by the anticytase serum. The fact pointed out by Wassermann
is perfectly accurately stated and has been confirmed by Besredka[283],
in an investigation carried out in my laboratory. Nevertheless, it is
impossible to accept Wassermann’s view as to the part played by
anticytase in his experiment. As clearly demonstrated by Besredka, the
anticytase serum acts not merely by neutralising the bactericidal
ferment, but also by its other properties, especially by one which
prevents the stimulation of the phagocytes.
In the struggle of the guinea-pig’s organism against a strong dose of
typhoid cocco-bacilli (in Wassermann’s experiments 40 times the lethal
dose), the free cytase plays a part so infinitely small that even the
injection into a guinea-pig of a large quantity of serum (3 c.c.) from a
normal guinea-pig (containing much cytase) does not prevent the death of
the animal. It is only the blood serum of other species (rabbit or ox)
that is capable of protecting a guinea-pig against such a large quantity
of typhoid bacilli.
Wassermann was in error in supposing that his experiment was a case of
natural immunity. It comes entirely within the range of the phenomena of
acquired immunity. In fact, the natural immunity of the guinea-pig is
only exhibited against a dose 40 times less than that employed by
Wassermann. Consequently the control guinea-pigs which received such a
huge quantity of the typhoid cocco-bacilli, going beyond 40 times the
limit of their natural immunity, require to be preserved from death by
the injection of a large quantity of blood serum heated to 60° C. from
the normal rabbit. This serum, deprived of its cytase, retains its other
properties, by which the organism of the guinea-pig profits, especially
exercising a stimulating action on the phagocytes of the guinea-pig. The
immunity of Wassermann’s control animals was, then, really an acquired
immunity, the result of the introduction into their organism of the
stimulating serum of the rabbit. For this reason an analysis of the work
of this observer must be postponed until we treat of the phenomena of
acquired immunity under the influence of normal serums.
We must, then, persist in the opinion that the plasmas of the normal
animal, containing no cytases, cannot play a bactericidal part in
natural immunity, a part which devolves upon the cytase contained within
the phagocytes.
[Sidenote: [203]]
This result accords well, also, with the whole of the facts bearing on
the destruction of micro-organisms in the animal body. The
transformation into granules of the attenuated cholera vibrios that is
sometimes observed in the peritoneal cavity during the period of
phagolysis, and the absence of this transformation under conditions
where the peritoneal leucocytes are protected against this injury, is
clearly explained. In the first case, Pfeiffer’s phenomenon is set up by
the bactericidal substance which has escaped from the leucocytes that
have been altered by the foreign substances injected into the peritoneal
cavity; in the second case, this phenomenon is not produced because the
leucocytes remain intact. The absence of this granular transformation in
the anterior chamber of the eye and in the subcutaneous tissue is also
readily explained by the fact that the bactericidal substance, not being
present in the blood plasma, cannot pass into the exudations of the eye
and subcutaneous tissue[284].
The bactericidal substance, then, is essentially some substance which
remains inside the uninjured phagocytes in the living animal but which
escapes from these cells when they are injured, either in the body of
the animal or outside in the blood withdrawn from the organism. Buchner
has given to this substance the name of alexine and we must now
determine whether this substance is the same cytase which digests the
formed elements on their resorption.
[Sidenote: [204]]
Since his first researches on the power of one normal blood serum to
dissolve the red corpuscles of another species, Buchner[285] has
maintained the identity of the haemolytic substance with the
bactericidal substance of the same serum. In both cases we have to do,
according to him, with one and the same substance of an albuminoid
nature, with the same “alexine.” In his later work, Buchner attempted to
confirm and develop this thesis. Bordet[286] has, on several occasions,
brought forward arguments in favour of the same view; but against this
Ehrlich and Morgenroth[287] have declared themselves. According to these
observers a single serum may contain several alexines or “complements.”
The same serum may even contain two complements, one of which is
destroyed by heating to 55° C., whilst the other, much more stable as to
the action of heat, resists this temperature. In one of their most
recent memoirs, Ehrlich and Morgenroth lay special stress on the
importance of an experiment which has enabled them, by means of
filtration, to separate two complements from the normal serum of the
goat, one of them attacking the red corpuscles of the guinea-pig, the
other those of the rabbit.
Max Neisser[288] has adopted this view of the plurality of alexines.
According to Ehrlich and Morgenroth, the same serum may possess several
complements which attack the red blood corpuscles of various species and
other complements which attack micro-organisms. In favour of this thesis
Neisser gives a summary of his experiments on the absorption of
complements which, in his opinion, prove the plurality of alexines. By
centrifugalising rabbit’s blood serum to which he had previously added a
certain number of anthrax bacilli, he obtained a fluid which no longer
destroyed this bacillus but which still dissolved the red corpuscles of
goat and sheep. There are then, according to Neisser, in the normal
serum of the rabbit, at least two different complements; one for the
bacilli and one for the red corpuscles.
[Sidenote: [205]]
With the object of explaining the discrepancy between these results and
those of his previous experiments, Bordet[289] undertook a new series of
researches on the absorption of cytases. He first made it clear that the
normal red corpuscles, when plunged into a normal haemolytic serum, are
incapable of fixing all the cytase. When such a serum is
centrifugalised, after a prolonged contact with red corpuscles of a
different species, the fluid no longer dissolves normal red corpuscles.
But if these latter be sensibilised by means of a specific fixative, the
red corpuscles are dissolved in large numbers. It must be admitted that
in this experiment we have to do with a single cytase because, before
centrifugalisation, as after it, the red corpuscles of the same species
are added. In the first case, however, these corpuscles were normal,
whilst in the second they were sensibilised by the fixative.
When, after the first part of this experiment, that is to say, after the
fixation of a certain quantity of cytase by the red corpuscles, we
centrifugalise the mixture and add, not the sensibilised red corpuscles
of the same species but the normal red corpuscles of a different
species, we find that the latter still dissolve and fix a certain
quantity of cytase. As the first experiment (with sensibilised red
corpuscles) has shown that the whole of the cytase has not been absorbed
by the red corpuscles, we readily understand that the portion remaining
in the fluid will act on the normal red corpuscles of another species.
[Sidenote: [206]]
But when we fix the cytase to the sensibilised red corpuscles the
absorption becomes complete and the addition of other species of red
corpuscles no longer produces any solution. It is easy, therefore, by
means of sensibilised red corpuscles, to take out the whole of the
cytase from a serum. When to such a serum, thus deprived of the whole of
its haemolytic cytase, we add bacteria, these latter show no sign of
disintegration; whilst previously, that is before the absorption of the
cytase by the sensibilised red corpuscles, the same serum was highly
bactericidal. Let us take a concrete example so that the reader may form
some definite idea of the phenomena observed. Take a normal rat’s serum
which, in a very short time, transforms cholera vibrios into granules or
deforms and dissolves anthrax bacilli. The same serum dissolves the red
corpuscles of a different species. We will first leave this serum in
contact with these red corpuscles sensibilised by the specific fixative.
After the solution of a quantity of these red corpuscles, let us add to
the serum a few cholera vibrios or anthrax bacilli. The vibrios, in this
serum, are no longer transformed into granules and the anthrax bacilli
undergo no change at all; they stain in the normal fashion by basic
aniline dyes, they present neither deformations nor solution of their
contents. In other words, no bactericidal action takes place in a serum
that is deprived of its cytase by sensibilised red corpuscles.
Is it necessary to conclude from this and other analogous experiments
that the cytase, fixed by the sensibilised formed elements (red blood
corpuscles or micro-organisms), is always one and the same cytase? May
it not be that these elements, impregnated with specific fixatives,
become so greedy for cytases that it is easy for them to absorb not only
one variety but several species of cytases?
The facts we have summarised in Chapter IV concerning the cytases,
indicate that very probably there exist two kinds of cytases, connected
with the two great groups of phagocytes. Extracts of the mesenteric
glands, of the omentum and of the exudations, which are composed for the
most part of microphages, do not dissolve the red corpuscles, but are,
on the other hand, specially bactericidal. Sarassewitch has carried out
numerous experiments on this point in my laboratory and has brought
forward a large number of data in favour of this theory of two
phagocytic cytases. He found that, even when specific fixative is added
to the extract of microphagic exudations (of rabbit), the sensibilised
red corpuscles are not dissolved. It must then be accepted that
microcytase, so active against bacteria, is entirely powerless against
animal cells.
As the microphages seize, though rarely, and digest red blood
corpuscles, spermatozoa and other cells of animal origin, it must be
admitted that they also contain a small quantity of macrocytase, or that
the microcytase, given time, is capable of dissolving these elements. On
the other hand, the macrophages, in spite of their marked predilection
for animal cells, also ingest and digest certain bacteria. This is due
perhaps to the presence of a little microcytase or to the power that the
macrocytase has of attacking micro-organisms. These questions are too
subtle to be definitely resolved at present.
[Sidenote: [207]]
The duality of the cytases does not clash with the experiments of Bordet
summarised above. We have only to admit that the formed elements, once
they are impregnated with specific fixatives, become capable of
absorbing not only the cytase which digests them, but also another
which, without dissolving them, is simply fixed to them. Here we should
have a phenomenon analogous to the fixation by fibrin of diastases,
other than trypsin and pepsin, or to the fixation by silk threads of all
kinds of soluble ferments.
It may be accepted, then, that the phagocytes elaborate two cytases:
macrocytase, active for animal cells, and microcytase, which digests
bacteria. This result up to a certain point has been anticipated by
Schattenfroh’s[290] experiments and foreseen by Max Neisser (_l.c._).
It has already been noted that the reaction inside the phagocytes is
usually feebly or very feebly acid, and only rarely distinctly alkaline.
On the other hand, it is well known that cytases, in serums, act in an
alkaline medium. It is certain therefore that these soluble ferments can
carry on the process of digestion under varied conditions. Hegeler[291],
working in Buchner’s laboratory, has studied the influence of the
alkalinity and acidity of the medium on the bactericidal action of
serum. He comes to the conclusion that the destruction of
micro-organisms can take place in a serum to which has been added small
quantities of alkali (carbonate of soda) and also in a weakly acid serum
(from the addition of small quantities of sulphuric acid). Once the
serum becomes distinctly acid the bactericidal power disappears at once.
Our knowledge of the cytases, as a whole, leads us to approximate these
diastases to the group of trypsins, papain, amoebodiastase and
actinodiastase. The cytases are elaborated by the phagocytes, but are
not secreted into the plasmas and they remain inside the cells so long
as these cells remain uninjured.
[Sidenote: [208]]
In this respect the cytases must be placed in the group of the
“Endo-enzymes,” according to the nomenclature of Hahn and Geret[292].
These observers have carefully studied the proteolytic diastase of the
yeast of beer which likewise acts inside the cells without ever being
excreted. This diastase, to which they give the name of “yeast
endotrypsin” (Hefeendotrypsin), presents in general an undeniable
relationship with the phagocytic cytases, from which it is distinguished
however by a greater sensitiveness to alkalis. Kutscher[293] in his
researches on autodigestion in yeast has established analogous facts.
The cytases and endotrypsin are consequently endo-enzymes, as are also
amoebodiastase, actinodiastase, plasmase (fibrin ferment) and the zymase
of E. Buchner. All remain confined within the cells which have
manufactured them and are not secreted or excreted, as are the sucrase
and invertin produced by yeasts or Mucedinae.
Our present knowledge on the cytases is as yet far from perfect, which
is not astonishing, seeing how recently the question has been brought
forward. The cytases found in the serum of the same animal are the same,
for we have seen that the macrocytase which dissolves red blood
corpuscles is the same which digests spermatozoa; whilst the same
microcytase digests bacilli, spirilla, and cocci. But in the serums of
different species, the cytases differ. Thus the cytases of the dog are
not the same as are those found in the serums of the rabbit or horse.
Whilst the majority of the cytases are very sensitive to heat and are
destroyed at a temperature of 55°–56° C., some, _e.g._ the microcytase
of rat’s serum, resist this temperature and are only destroyed at 65°
C., presenting, consequently, an example of cytase stable to heat
similar to that discovered by Ehrlich and Morgenroth.
[Sidenote: [209]]
It is as yet very difficult to establish whether, besides the cytases,
there exist other endo-enzymes within phagocytes, that is to say,
soluble ferments which do not pass into the serums on the destruction of
the phagocytes, but continue within these cells. Our present methods of
investigation do not enable us to come to any conclusion on this point.
We know only that the digestion of the formed elements is more complete
inside the phagocytes than in the serums. Thus, as we have seen in
Chapter IV, the best spermotoxic and haemolytic serums never digest
either spermatozoa or the nuclei of the red corpuscles of birds. And yet
these elements are completely dissolved in the phagocytic contents. Does
this difference depend on the fact that, in the serums, we get only a
very small part of the macrocytase, or upon the injurious influence of
the alkalinity of the serums on the macrocytase which acts better in
weakly acid media, or on the presence in the phagocytes of other
endo-enzymes still unknown? These are questions to which at present no
definite answer can be given.
Just as animal cells, when ingested by phagocytes during resorption (see
Chap. IV), immediately become permeable to stains, so in natural
immunity do micro-organisms taken into phagocytes acquire the same
property. Very often, under the influence of the phagocytic action, the
ingested micro-organisms become stainable by eosin (fig. 36). This
eosinophile transformation has been observed in the cholera vibrio, the
anthrax bacillus and in _Proteus vulgaris_. It is probably widely
diffused among the phagocytised bacteria. This fact demonstrates clearly
that at least some of the eosinophile granules are derived from foreign
bodies ingested by the phagocytes. Others of these granules are probably
the result of the transformation of soluble substances absorbed by the
phagocytes. In fact, during inflammation, many microphages which contain
no foreign solid body, may often be seen charged with a quantity of
small pseudo-eosinophile granules.
Certain vibrios and bacilli, when ingested by microphages, become
transformed, almost immediately, into spherical granules. The cholera
vibrio undergoes the same transformation in the peritoneal exudation at
the moment of phagolysis, as also in blood serum. The _Bacillus coli_,
the typhoid bacillus, and certain other cocco-bacilli do not change in
the least, or change very slightly in serum, but exhibit the
transformation into granules when inside microphages. The macrophages,
on the other hand, digest the same bacteria (vibrios and cocco-bacilli)
without these bacteria presenting any signs of this change of form. The
bacterial membrane resists the influence of the phagocytic digestion
longer than do the contents, but is in the long run also completely
digested. After the ingestion and destruction of micro-organisms by the
phagocytes, débris of indeterminate form may, for long, be found in the
cells, but I have never been able to demonstrate any solid excreta from
them. We must suppose, then, that the undigested portions are not thrown
out from the phagocytes.
[Sidenote: [210]]
When describing the solution of red blood corpuscles by normal serums,
we have mentioned Ehrlich and Morgenroth’s view that the cytases are
incapable of fixing themselves to these cells without the help of
fixatives. They cite in support of their opinion several examples of
fixatives (intermediary substances or “Zwischenkörper”) discovered by
them in the serums of various species of mammals. Is this so with
microcytase in respect to micro-organisms? If this soluble ferment is
incapable alone of fixing itself upon the bodies of these parasites, the
help of fixatives would be indispensable to it. The bactericidal
property of the microcytase, then, would depend on the existence of
another body (fixative) which, perhaps, might not owe its origin to
phagocytes. The problem, then, has a wide general range.
In one of his memoirs, Bordet[294] had already raised the question of
the existence of this sensibilising (or fixative) property in normal
serums. By mixing two normal serums coming from different species, he
was sometimes able to demonstrate the existence of such fixatives. Thus
the cholera vibrios, which do not undergo granular transformation in
either the normal serum of the horse (which is capable only of arresting
their movements and agglutinating them into a mass) or in that of the
normal guinea-pig, readily become transformed into granules when placed
in contact with a mixture of the two serums. Bordet, however, refrains
from any hasty generalisation on this observation and proposes to make
fresh researches on this subject. Independently, Moxter[295] has
attempted to demonstrate the presence of fixative in the normal serum of
the guinea-pig. When deprived of cytases by heat, this serum is
incapable of transforming the cholera vibrios into granules; but when
fluid from the peritoneal exudation of the same guinea-pig is added, the
transformation takes place very rapidly. Nevertheless, as this exudation
was already, by itself, capable of producing Pfeiffer’s phenomenon,
Moxter’s conclusions on the presence of the fixative in the normal
guinea-pig’s serum cannot be accepted without a fuller analysis of the
facts, and this demands fresh researches.
[Sidenote: [211]]
A recent investigation, carried out by Bordet[296] in collaboration with
Gengou, devoted to the study of the absorption of cytases by
micro-organisms that have been sensibilised by means of fixatives, also
gives us information on the question which now occupies us. It was easy
to demonstrate the presence of fixative in the serums in the case of the
cholera vibrio and its allies, by reason of their transformation into
granules, appreciable on microscopical examination. When a serum, which
of itself is incapable of setting up this transformation, produces it
directly we add another serum heated to 55° C., we must conclude that
the latter fluid contains the cholera fixative, whilst the former
contains only cytases. But, as the majority of bacteria do not undergo
any analogous transformation in serums, we are, in these cases, without
any criterion as to the presence of fixative. Bordet and Gengou have
eliminated this inconvenience in determining the fixation of alexine by
bacteria which undergo neither granular transformation nor any other
visible change. A normal unheated serum, which always contains a
sufficient quantity of cytases, is mixed with any micro-organism, _e.g_.
with the anthrax bacillus or the cocco-bacillus of plague. The serum,
decanted after a prolonged contact with these bacteria, remains quite as
capable of dissolving the red corpuscles of a determined foreign species
as it was originally. This proves that cytases remain in the serum and
that they have not been absorbed by the bacteria. Repeat the same
experiment with this difference, that instead of normal anthrax bacilli
or plague cocco-bacilli we introduce into the unheated normal serum
these bacteria after they have been sensibilised by the corresponding
fixatives (that is to say, previously submitted to the influence of
specific serums heated to 55° C.). After contact for a certain length of
time with these bacteria the serum is no longer capable of dissolving
the red corpuscles of a determined foreign species, thus demonstrating
that the cytases have, thanks to the help of the fixatives, been linked
to the bacteria. We see, therefore, that it is easy to determine whether
a serum, whose properties are unknown, contains fixatives or not. It is
heated to 55° C. and mixed with normal unheated serum to which bacteria
are added. If, after contact with these latter the normal serum has lost
the power of dissolving the red corpuscles (which it was capable of
dissolving previously), it is because its cytases, thanks to the
fixative which must be present in the heated serum, have been absorbed
by the bacteria. In the other case, we conclude the non-existence of the
fixative.
[Sidenote: [212]]
In their researches, Bordet and Gengou often employed normal unheated
serums to which they added several species of bacteria. They
demonstrated that in these mixtures the cytases remained intact or
nearly so. These soluble ferments were scarcely, if at all, absorbed by
the bacteria, which proves that in the normal serums there are no
fixatives in any appreciable quantity. Of all their experiments the one
that interests us most was carried out with _Proteus vulgaris_. This
organism placed in prolonged contact with normal guinea-pig’s serum
showed itself incapable of absorbing or fixing anything beyond the most
minute quantities of the cytases. There is consequently no fixative for
_Proteus_ in normal guinea-pig’s serum, or, if any exists, it is only in
negligible quantity. And yet this same _Proteus vulgaris_, when injected
into guinea-pigs, was in a short time ingested and destroyed by the
phagocytes which assure to the animal a natural immunity of the most
stable character. The facility with which the leucocytes of the
guinea-pig devour the _Proteus_ follows, among others, from an
experiment by Bordet[297] carried out with quite another object. A
guinea-pig, very ill as the result of the injection into its peritoneal
cavity of a very virulent streptococcus, contained in the peritoneal
exudation a quantity of empty microphages incapable of ingesting these
streptococci. At this critical moment there was injected into the same
position a mass of _Proteus vulgaris_. “At the end of a very short time,
it is seen that the leucocytes which energetically refuse to ingest
streptococci greedily seize upon the new organism offered to them; and
at the end of half-an-hour the whole of these organisms are found inside
phagocytes.”
Here, then, we have an actual proof of the fact that the phagocytes, in
order to rid the animal organism of a microbe and assure to it a natural
immunity, have no need of any previous help from an extraphagocytic
fixative. The phagocytes act, so to speak, _motu proprio_, and
themselves bring about the resorption of the intruders. The question of
fixatives in normal serums, then, loses its importance for us and their
origin no longer presents any essential interest for the problem with
which we are at present occupied.
[Sidenote: [213]]
Can we conclude, from the data just summarised, that the cytases, which
in several respects approximate to the trypsins, have this further
feature in common with them that they can act without the help of any
fixative? It is known, as mentioned in Chapter III, that trypsin can
digest alone, or in collaboration with enterokynase, that ferment of the
intestinal juice which acts as such a powerful adjuvant to the
pancreatic ferments. Is this also the case with the cytases? The fact
that when _Proteus vulgaris_ is placed in contact with normal unheated
guinea-pig’s serum, it is incapable of absorbing cytases, although it is
so readily digested by phagocytes, indicates rather that, for the
fixation of cytases, the help of the fixative is indispensable. But, as
this fixative is absent from the serum, and since, nevertheless, it must
exist for the needs of digestion, it must clearly be concluded that it
is found inside the phagocytes. Its quantity is perhaps so small that
when it has passed into the serum its action is entirely lost or nearly
so. Fresh researches are necessary to elucidate this delicate point.
But perhaps the phagocytes which, as we have just seen, can engage in a
struggle with and ingest the micro-organisms without the latter being
previously modified by the fixative, may be incapable of fulfilling
their functions without the help of some other substance circulating in
the blood plasma? Amongst these substances is one which manifestly acts
upon the micro-organisms by rendering them motionless and agglomerating
them into masses. This agglutinative property is met with in the normal
fluids of many species of animals and is exercised upon many bacteria.
It may be demonstrated not only in the blood serum, but also in the
fluids of transudations and exudations and in certain secretions such as
milk, tears, and urine. Little is known as yet of the mechanism of this
agglutinative action, and we can the more readily refrain from entering
into details concerning it as it is of no great importance from the
point of view of natural immunity.
In the preceding chapter we have already spoken of the ingestion of
cholera vibrios in the peritoneal cavity of guinea-pigs. In those cases
in which the animals exhibit an effective resistance, the phagocytes
devour the vibrios whilst they still exhibit very active movements. Even
when a large majority are already seized by the leucocytes and only a
few isolated free vibrios remain, these latter still continue to exhibit
normal movements. These facts, repeatedly observed, clearly demonstrate
that phagocytosis may take place without any previous agglutinative
action; this does not, however, prevent the micro-organisms, when united
into motionless masses, from being ingested by the leucocytes with
greater ease.
[Sidenote: [214]]
In the case of the typhoid bacillus, one of the most active of bacteria,
the same facts may be observed as in the case of the cholera vibrio. In
animals that remain unaffected we often see the last free bacilli moving
about actively between the leucocytes filled with microbes. In many
other examples of natural immunity we constantly meet with phagocytes
containing but a single or a small number of micro-organisms
(streptococci, yeasts, etc.).
The presence of motile micro-organisms inside phagocytes proves also
that it is possible for these cells to do without the help of
agglutinative substance in carrying on their protective work. The most
carefully studied case of the relations between natural immunity and
agglutination is that met with in the anthrax bacillus. We owe it to
Gengou[298], who at the Liège Bacteriological Institute carried out a
very detailed investigation on this question. He showed that the
bacillus of Pasteur’s first anthrax vaccine is agglutinated by the blood
serum of a great number of animals. But he also showed that the serums
which have the greatest agglutinative action on this bacillus do not
come from the most refractory species. Human serum agglutinates most
strongly the bacillus of the first vaccine (in the proportion of one
part of serum to 500 parts of culture) but man is far from being exempt
from anthrax. Pigeon’s serum, on the other hand, is completely without
any agglutinative power, although this species resists not only the
first vaccine but very often even virulent anthrax. The serum of the ox,
a species susceptible to anthrax, is more agglutinative (1 : 120) than
that of the refractory dog (1 : 100). There are, however, exceptional
cases in which the agglutinative property corresponds to the degree of
susceptibility. Thus the serum of the mouse has not the slightest
agglutinative action on the bacillus of the first vaccine. But alongside
this example is that of the rat, a species of moderate susceptibility to
anthrax, whose serum possesses the least agglutinating power of all,
acting only in the proportion of 1 : 10. All these facts fully justify
the conclusion formulated by Gengou that “we cannot establish any
relation between the agglutinating power and the refractory state of the
animals to anthrax” (p. 319). This conclusion may be extended to the
phenomena of the agglutination of micro-organisms and to those of
natural immunity in general.
[Sidenote: [215]]
Amongst the properties of humours, there exists one which might play a
part in natural immunity against micro-organisms. I mean the power
possessed by the blood and certain other fluids of the animal body to
neutralise the action of microbial poisons. Perhaps, it may be
suggested, the phagocytes are not capable of commencing to do their work
except after a previous action of antitoxins. After the neutralisation
of the principal means possessed by the micro-organisms to injure the
organism, these parasites, having been rendered innocuous, might be
readily destroyed by the phagocytic cells. We have already had occasion
to treat this fundamental question. Thus, we have insisted in the
preceding chapters on the absence of any parallelism between immunity
against micro-organisms and that against their toxins, taking as our
examples anaerobic bacteria (tetanus bacillus, septic vibrio, bacillus
of symptomatic anthrax) in connection with which phagocytosis takes
place without any help from the antitoxic function.
We must now pass directly to the examination of the question of
antitoxins in the fluids of animals naturally refractory to the
micro-organisms and of the ultimate part played by them in this
immunity.
[Sidenote: [216]]
Examples of the presence of antitoxic serum in normal animals are very
rare. It might be supposed that animals endowed with natural immunity
against micro-organisms and at the same time against their toxins,
present an appreciable natural antitoxic power. Let us examine some of
the more typical examples. The fowl enjoys a very marked immunity
against the tetanus bacillus and its toxin; its blood and its serum,
however, as demonstrated by Vaillard[299], exhibit no antitoxic power;
this observation has been confirmed by several other workers. The rat is
very refractory to diphtheria; it resists subcutaneous inoculation of a
large quantity of diphtheria bacilli and vigorously withstands
diphtheria toxin when injected anywhere but into the brain. It has been
demonstrated by Kuprianow[300], in an investigation carried out under
Loeffler’s direction, that the blood serum and the emulsion of the
organs of the grey rat (_Mus decumanus_) possess no antitoxic property.
This fact has been confirmed by other observers. Von Behring[301], in a
review of the phenomena of immunity in general, sums up this question as
follows: “we find no antitoxin in the blood of individuals that are
naturally refractory.” There are, however, certain exceptions, perhaps
only apparent, to this rule. Thus Wassermann[302] has shown that blood
serum from healthy human beings is sometimes antitoxic to the diphtheria
poison. The individuals who furnished this antitoxin maintained that
they had never suffered from diphtheria. We know, however, that this
disease is sometimes present in so benign a form that it may pass
unnoticed. More conclusive appears the example of normal horses whose
blood serum, as demonstrated by Meade Bolton[303] and Cobbett[304], is
very often antitoxic for the diphtheria toxin. This property, however,
is not found in every horse; in certain individuals it is entirely
absent. This last fact affords an indication that the antitoxic property
in the blood of horses has been acquired as the result of some affection
produced by a bacillus allied to the diphtheria bacillus. This view has
not yet been sufficiently examined and consequently cannot claim to be
accepted as settled. Recently, Max Neisser and Wechsberg[305] have
discovered an antitoxin in human blood which is capable of preventing
the solution of the red corpuscles by the toxin of staphylococci. This
antitoxic power varies considerably in different individuals and is
probably to be accounted for by the fact that the staphylococcus is one
of the most widely diffused organisms among the bacterial flora of the
human body. The small lesions produced by these micro-organisms (acne,
boils, etc.) are so frequent in man that they may readily bring about
the production of an antitoxin. In this case, however, we have again an
example of acquired antitoxic power.
The examples just summarised can in no way affect the general thesis
that the phagocytes, in order to fulfil their microbicidal function in
an animal endowed with natural immunity, have no need of any previous
action of the body fluids to neutralise the corresponding toxins.
[Sidenote: [217]]
The facts and views analysed in these two chapters afford us a general
picture of the phenomena exhibited in natural immunity against
micro-organisms. The dominant feature is represented by the phagocytic
reaction that is observed throughout the animal series and that is
exercised against parasites belonging to all the microbial groups.
Phagocytosis is exhibited not only by the macrophages but also, in a
high degree, by the microphages which stand out as the defensive cells
_par excellence_ against micro-organisms. Their action is divided into a
series of vital physiological acts, such as sensitiveness to the
micro-organisms and their products, amoeboid movements which serve to
ingest the micro-organisms, and into chemical and physico-chemical
processes, such as the destruction and digestion of the devoured
organisms.
The phagocytes enter into a struggle against the micro-organisms and rid
the animal organism of them without requiring any previous help on the
part of the body fluids. Phagocytosis, exercised against living and
virulent micro-organisms, is sufficient to ensure natural immunity. The
bactericidal power of the serum, which for a long time served as the
basis for a humoral theory of immunity, represents merely an artificial
property, developed in consequence of the setting free of the
microcytase of the leucocytes that have become disintegrated after the
blood has been drawn. The agglutinative power of the normal fluids of
the body plays no important part in natural immunity.
The phagocytes, in order to fulfil their function, can attack
micro-organisms that are capable of producing toxins. Any antitoxic
action against these bacterial poisons is in no way necessary to allow
of phagocytosis coming into action.
Taken as a whole, the data collected on natural immunity against
micro-organisms clearly demonstrate that the destruction of these
parasites in the refractory animal organism represents merely a special
phase of the resorption of formed elements.
CHAPTER VIII
SURVEY OF THE FACTS BEARING ON ACQUIRED IMMUNITY AGAINST MICRO-ORGANISMS
The discovery of attenuated viruses and its application to vaccination
against infective diseases.—Vaccination by microbial
products.—Vaccination with serums.—The acquired immunity of the
frog against pyocyanic disease.—The acquired immunity against
vibrios.—Extracellular destruction of the cholera vibrio.—Part
played by two substances in Pfeiffer’s phenomenon.—Specificity of
fixatives.—Phagolysis and its relation to the extracellular
destruction of vibrios.—Part played by phagocytosis in the
acquired immunity against vibrios.—Fate of the spirilla of
recurrent fever in the organism of immunised guinea-pigs.—Acquired
immunity against the bacteria of typhoid fever and pyocyanic
disease.—Acquired immunity against swine erysipelas and
anthrax.—Acquired immunity against the streptococcus.—The acquired
immunity of rats against _Trypanosoma_.
[Sidenote: [218]]
Certain of the hypotheses on acquired immunity are of as ancient origin
as are those on natural immunity. For example, it has for long been
known that man is constitutionally refractory to certain diseases which
are very fatal to cattle. It has also been recognised that after a first
attack of a contagious disease, such as small-pox, measles, scarlatina,
typhoid fever, etc., man acquires a lasting immunity; and that the same
rule applies to domestic animals, for example, cattle that have
recovered from cattle plague or sheep that have got better from
sheep-pox, become refractory against these diseases.
[Sidenote: [219]]
The discoveries of variolisation and vaccination, as methods of
conferring on man a resistance to small-pox, have notably advanced our
knowledge upon acquired immunity. The researches on the properties of
vaccine had already led to some important results. But it is only since
the publication of Pasteur’s investigation, carried out with his
collaborators Chamberland and Roux, in the first place, and with
Thuillier later, that we have been able to take up the study of acquired
immunity in a really scientific manner. The first in this series of
discoveries, which have opened up a path so fruitful to science and
medical art, was the discovery of the attenuation of micro-organisms.
The small cocco-bacillus of fowl cholera after several weeks’ culture in
broth was found to have become markedly attenuated in virulence. To
Pasteur the idea occurred of testing whether fowls that had resisted the
inoculation of these attenuated organisms had acquired any real immunity
against virulent fowl cholera. Experiment confirmed his expectation and
led to the discovery of the vaccine against this disease. The method was
at once applied to other infective epizootic diseases and shortly
afterwards Pasteur, Chamberland and Roux found a method of preserving
sheep and cattle from anthrax. To attain this end it was found necessary
to prevent the bacillus from producing spores (in this they succeeded by
cultivating it in broth at a temperature of 42°·5 C.), because these
spores fix the virulence and prevent attenuation. Having overcome this
main obstacle, Pasteur and his collaborators found that their cultures,
thus deprived of spores, become attenuated on exposure to the air and so
become transformed into vaccines. They were thus able to prepare their
two anthrax vaccines which soon found such wide application in practice.
A few years later, Pasteur and Thuillier discovered the vaccines against
swine erysipelas and, in collaboration with Roux and Grancher, Pasteur
made the first application of his discoveries to the vaccination of man
against rabies.
[Sidenote: [220]]
The path thus opened up was traversed by many other investigators and
led to many very remarkable discoveries. Vaccination with
micro-organisms became a recognised method and in the hands of Arloing,
Cornevin and Thomas, soon found its application to symptomatic anthrax.
The next step in this onward progress of science was taken when Salmon
and Smith, working at hog-cholera, demonstrated the possibility of
vaccinating not only with hog-cholera bacilli, but also with culture
fluids in which these organisms had developed. These fluids, when
completely deprived of micro-organisms by filtration, protected the
experimental animals from virulent hog-cholera. This discovery, at first
sceptically received, was soon confirmed and extended by the work of
other investigators. Beumer and Peiper extended it to the experimental
disease set up by the typhoid bacillus in small laboratory animals;
Charrin applied it to the disease that he produced by means of the
bacillus of blue pus; and Chamberland and Roux prepared vaccines from
the soluble products of the septic vibrio and of the bacillus of
symptomatic anthrax. And now, as the result of these investigations,
vaccinations by microbial products are in everyday use in all research
laboratories. The vaccinations now used (anthrax, symptomatic anthrax,
swine erysipelas and rabies) are still being carried out by means of the
inoculation of living viruses.
The comparative history of acquired immunity is still very incomplete.
The facts known concerning the adaptation of unicellular organisms to
all kinds of injurious influences of a physical or chemical nature
enable us to perceive that acquired immunity is just as general in
living beings as is natural immunity; but it is impossible, in the
present state of our knowledge, to confirm this hypothesis by exact and
experimental data. The reason for this lies in the great difficulty we
have in carrying out experiments on the lower animals. The majority of
the Invertebrata in captivity do not remain alive long enough and can
not be sufficiently often inoculated for us to obtain in them a well
marked acquired immunity against micro-organisms. Kowalevsky[306], the
celebrated Russian zoologist, has tried to overcome these various
difficulties by making use of Myriapods. He found first that
_Scolopendrae_, when inoculated with anthrax bacilli, die therefrom
during the heats of summer, the blood containing a number of anthrax
bacilli. But when the temperature does not exceed 17°–18° C., a fairly
large number of these myriapods survive. The same survival was observed
when Pasteur’s first vaccine was injected. Kowalevsky utilised the
_Scolopendrae_ that had resisted the first injection of anthrax bacilli
to ascertain whether they had contracted an acquired immunity. The
results were not absolutely demonstrative and Kowalevsky sums up his
results in the following words: “I cannot say, therefore, that I have
succeeded in solving this question of vaccination, but it appears to me
very probable” (p. 607).
[Sidenote: [221]]
In view of this doubt, I asked Mesnil to make a fresh attempt, making
use of _Scolopendrae_ and inoculating them with anthrax bacilli. These
creatures, however, were so delicate and so little capable of remaining
alive under the artificial conditions of their captivity, that the
attempt soon had to be abandoned. I tried to obtain better results with
the larvae of _Oryctes nasicornis_; here again the difficulties were too
great. These insects exhibit a perfect natural immunity against certain
micro-organisms, but for others they showed an insurmountable
susceptibility. It is very evident, then, that it is not an easy matter
to set up an acquired immunity in the Invertebrata.
It was necessary, therefore, to go higher up the animal scale and have
recourse to cold-blooded vertebrates. The choice naturally fell on the
frog. I asked Dr Gheorghiewski[307], who was working in my laboratory,
to try to vaccinate the Batrachians against pyocyanic disease. I ought
first to state that the bacillus of blue pus is pathogenic for the frog,
which it kills both at the ordinary laboratory temperature, and at that
of the incubator, 30°–37° C. In the first case the fatal dose is much
greater than in the second, but it is always easy to induce a fatal
infection. In this respect, therefore, the _Bacillus pyocyaneus_ is much
better adapted for study than the anthrax bacillus or many other
micro-organisms. Gheorghiewski vaccinated green frogs (_Rana
esculenta_), which had been accustomed to the incubator temperature, 30°
C., by injecting every 4 to 7 days considerable doses of cultures of
_Bacillus pyocyaneus_ heated to 80° C. in order to kill all the
micro-organisms. Some (3–4) weeks afterwards the prepared frogs became
more resistant to the _Bacillus pyocyaneus_ than were the controls
placed under the same conditions. The frogs, inoculated with a fatal
dose of the bacilli, clearly exhibited a certain, though slight, degree
of acquired immunity. They withstood a dose that was always fatal to the
controls or even a dose and a half, but died when injected with double
the fatal dose. The lymphatic fluid of the vaccinated frogs feebly
agglutinated (1 : 20–1 : 30) the _Bacillus pyocyaneus_ although it still
formed an excellent culture medium for this organism. Gheorghiewski
satisfied himself that the agglutination was insufficient to assure
immunity to the frog. The bacilli agglutinated into clumps were very
virulent.
[Sidenote: [222]]
A detailed examination of the phenomena observed in the immunised frogs
revealed the following facts. During the earliest stage the bacilli,
injected into the dorsal lymphatic sac, were found free in the fluid,
retained their form and were not transformed into granules. The bacilli
carried by the lymphatic current spread rapidly throughout the body.
Very shortly after inoculation, however, some of the leucocytes began to
ingest the bacilli which became transformed into spherules within these
cells. Later, the phagocytic reaction increased and at the end of 15 to
20 hours all the bacilli were found inside leucocytes. It was easy to
demonstrate that these bacilli had been ingested in a living condition.
Forty-eight hours after inoculation, no bacilli were to be found in the
lymph of the dorsal sac, either inside or outside the cells. But this
fluid when sown on nutrient media gave colonies of the _Bacillus
pyocyaneus_ up to 15 and even 18 days after inoculation.
We may conclude from these facts that the cold-blooded vertebrata are
capable of acquiring immunity to a slight degree and that, in this
acquired immunity, a marked phagocytosis may be observed, but no
bactericidal action of the fluids.
In order to gain a more complete idea of the mechanism of acquired
immunity, it is necessary to observe it in higher vertebrates in which a
well developed immunity of this type is readily obtained. Here we must
have recourse to mammals and pass in review an ample number of examples,
before we attempt to give to our readers a general summary of the
question.
For long, researches on acquired immunity were confined almost
exclusively to the analysis of the facts observed in animals submitted
to anti-anthrax vaccinations by means of the two vaccines of Pasteur. A
large number of important facts were thus collected, the more weighty of
which must be presented to the reader. But, before entering on the
subject, a general orientation on acquired immunity in laboratory
animals against vibrios is indispensable as this example dominates, so
to speak, the whole of the chapter on acquired immunity against
micro-organisms.
[Sidenote: [223]]
Von Behring and Nissen[308], in their researches on the bactericidal
power of serums, examined, amongst others, several specimens of serums
coming from animals that had been vaccinated against various
micro-organisms. In the majority of the examples given by them the
acquired immunity produced no increase in this power, but the blood
serum of guinea-pigs that had been immunised against Gamaleia’s vibrio
(_Vibrio metchnikovi_) was found to be much more bactericidal as regards
this micro-organism than the serum of normal susceptible guinea-pigs.
These authors came to the conclusion that in acquired immunity, at least
as regards the vibrio mentioned, the chief part is played by a
bactericidal substance which is developed in the fluids of the
vaccinated animals. They were content with the mere demonstration of
this fact without making any attempt to follow the course of events in
the destruction of the vibrios as it occurs in the organism of the
vaccinated guinea-pig. R. Pfeiffer[309] in collaboration with Issaeff
sought to fill this gap. But, instead of taking Gamaleia’s vibrio, these
observers concentrated their attention on the study of the acquired
immunity of guinea-pigs against the cholera vibrio. As this vibrio is as
a rule less virulent than Gamaleia’s vibrio, it was necessary, in order
to obtain a fatal infection, to inject it, not into the subcutaneous
tissue but into the peritoneal cavity. We have already seen (Chapter VI)
that the cholera vibrio when inoculated into the peritoneal cavity of
the guinea-pig, there meets with a vigorous resistance on the part of
the leucocytes which seize the living and virulent vibrios and digesting
them rid the animal of their presence. But when the dose of the vibrios
is increased, they multiply in spite of the phagocytic reaction; they
are found swarming in the peritoneal cavity, whence they invade the
lymphatic and blood vessels and cause the death of the animal. It is
easy, then, to induce a fatal infection of the guinea-pig with the
cholera vibrio. But it is also easy to vaccinate these animals against
this experimental disease. We have only to inoculate them with a
non-fatal quantity of living cholera vibrios, or to inject into them a
culture in which the vibrios have been killed by heat, or some of the
culture fluid from which the vibrios have been removed by filtration.
All these methods soon produce an acquired immunity in guinea-pigs. If,
when this has been brought about, a little blood is withdrawn and to the
serum a small quantity of cholera vibrios is added, _in vitro_, we can
readily demonstrate their disappearance, under the influence of the
bactericidal substance dissolved in the fluid. In this respect there is,
then, a marked analogy with the fact established by v. Behring and
Nissen as regards Gamaleia’s vibrio.
[Sidenote: [224]]
When into the peritoneal cavity of vaccinated guinea-pigs a certain
quantity of cholera culture containing virulent and very motile vibrios
is injected, we find that in the peritoneal fluid drawn off by means of
a fine pipette, the vibrios have undergone profound changes in the
refractory organism. Even a few minutes after the injection of the
vibrios, the leucocytes disappear almost completely from the peritoneal
fluid; and only a few small lymphocytes and a large number of vibrios,
the majority of which are already transformed into granules, are found
(fig. 39); and there is presented a most typical case of Pfeiffer’s
phenomenon. Alongside the round granules may be seen swollen vibrios,
and others which have kept their normal form, but all are absolutely
motionless. Some of these granules are gathered into small clumps,
others remain isolated in the fluid. When to the hanging drop containing
these transformed vibrios a small quantity of a dilute aqueous solution
of methylene blue is added, we observe that certain granules stain very
deeply, whilst others take on merely a very pale tint, scarcely visible.
Many of these granules are still alive, because it is easy to watch them
develop outside the animal and elongate into new vibrios. A large number
of the granules, however, no longer exhibit any sign of life and are
evidently dead. R. Pfeiffer and certain other observers affirm that the
granules may be completely dissolved in the peritoneal fluid just as a
piece of sugar dissolves in water. We have repeatedly sought for this
disappearance of the granules in hanging drops of the peritoneal fluid,
without being able to find any diminution in the number of these
transformed vibrios, even after several days; nor have we been able to
observe the phenomenon of the solution of the granules. It is at any
rate indisputable that this granular transformation is a manifestation
of very profound lesions undergone by the cholera vibrios under the
influence of the peritoneal fluid of the immunised animal.
[Illustration:
FIG. 39. Cholera vibrios in the peritoneal cavity showing Pfeiffer’s
phenomenon.
]
[Sidenote: [225]]
An attempt has been made to define the mechanism of Pfeiffer’s
phenomenon more exactly, and Fischer[310] has sought to refer it to
osmotic action, exercised by the salts of the fluids in which the
vibrios are suspended. These vibrios, under the action of media richer
or poorer in salts than is the fluid in which they had developed, are
said to present an increase of their internal pressure, in consequence
of which the vibrios swell up or allow a spherical droplet of protoplasm
to escape at one of their poles. This explanation was inadequately
supported by its author and cannot be regarded as proved. On the other
hand, one is compelled to the conclusion that the granular
transformation is due, as we shall see later, to a fermentative action
of the peritoneal exudation.
Whilst the vibrios are undergoing this transformation in the peritoneal
cavity of an immunised guinea-pig, the animal recovers from a _malaise_
that is quite transitory and continues to live, whilst normal
unvaccinated guinea-pigs die, an enormous quantity of vibrios swarming
in the peritoneal exudation. The difference between the two animals is
most striking, and we can readily understand that Pfeiffer was so
impressed by it that he was led to attribute the acquired immunity of
his guinea-pigs solely to the granular transformation set up by a
bactericidal substance contained in the fluids of the immunised animals.
[Sidenote: [226]]
The ease with which we can gain an idea of the change of form in the
vibrios under the influence of the fluids of the body, greatly aids the
study of the bactericidal substance. Before passing to the question of
the part played by this substance in acquired immunity we must consider
for a moment the principal properties of this acquired immunity. Very
manifest in the peritoneal fluid, the power of causing Pfeiffer’s
phenomenon is equally evident in the blood serum of immunised
guinea-pigs, as has been demonstrated by Bordet. A drop of this serum,
when quite fresh, readily and rapidly transforms a number of vibrios
into granules. When the serum is kept for several days or has been
heated to 55° C. for an hour, the total disappearance of the substance
which produces Pfeiffer’s phenomenon is brought about. This at once
betrays the presence of microcytase in the fluids of guinea-pigs that
have acquired immunity against the cholera vibrio. Yet the blood serum
and the peritoneal fluid of these animals, having been deprived of their
microcytase by heating to 55° or 56° C., still retain a remarkable power
over the vibrios. These organisms no longer undergo granular
transformation, under the influence of the heated body fluids, but they
are deprived of all power of motion, agglutinate into clumps and acquire
a special susceptibility to the action of cytase. Soon after the
discovery of Pfeiffer’s phenomenon, I[311] was able to demonstrate that
this granular transformation can be obtained _in vitro_ under the
following conditions. Prepare a hanging drop with the blood serum of a
guinea-pig vaccinated against the cholera vibrio, a serum which has lost
the power of transforming, by itself, the vibrios into granules. Add to
it a drop of the peritoneal lymph of a normal unvaccinated guinea-pig;
this lymph contains dead or living leucocytes and is, by itself, also
incapable of producing Pfeiffer’s phenomenon. When, to the mixture of
these two fluids, which are inactive when they are employed separately,
a few cholera vibrios are added, they are quickly transformed into
granules. This transformation, obtained _in vitro_, is remarkably like
that produced in the peritoneal cavity of the vaccinated animal.
Jules Bordet[312], working in my laboratory, made a very complete
investigation of Pfeiffer’s phenomenon outside the animal body and found
that, in my experiment, the peritoneal lymph can be replaced by the
blood serum of the vaccinated guinea-pig without thereby in the least
hindering the granular transformation. After making a specially thorough
study of the question he has come to the conclusion that Pfeiffer’s
phenomenon is the result of the action of two substances. One of these
is found in the blood serum and in the peritoneal fluid of guinea-pigs
vaccinated against cholera, heated to 55°–56° C. or deprived by some
other means of their individual power of transforming vibrios into
granules. This substance resists this temperature and only loses its
activity on being heated to 68°–70° C. The second of the two substances,
that found in the peritoneal lymph or in the blood serum of the normal
guinea-pig, is, on the other hand, destroyed at 55°–56° C. and is
nothing but the ordinary cytase (or alexine) present in the fluids of
normal animals.
[Sidenote: [227]]
The facts we have described with regard to Pfeiffer’s phenomenon in the
body fluids of immunised animals must, then, be interpreted as follows.
The fresh peritoneal exudation or blood serum of these animals readily
produces the granular transformation, because in these fluids both the
two necessary substances are found. But as microcytase is a very
unstable substance which, under the influence of time or heating to
55°–56° C., is destroyed, the fluids of immunised animals are very
readily deprived of it. The blood serum then, after being some time
outside the body, becomes incapable of transforming vibrios into
granules; but when to it is added a small quantity of the cytase, found
in the blood serum or in the peritoneal lymph of the normal guinea-pig,
the transformation takes place with great rapidity. To the serum of the
immunised animal, which has become inactive, is restored its property of
setting up Pfeiffer’s phenomenon. This interpretation, formulated by
Bordet, corresponds to the whole of the known data and is now generally
accepted.
[Sidenote: [228]]
As the fluids of immunised animals, that have become incapable of
transforming vibrios into granules, still retain their power of
rendering these organisms motionless and of uniting them into clumps, it
might be asked whether this agglutinative substance might not be the
substance, stable under heat, which is necessary for the production of
Pfeiffer’s phenomenon. For some time, indeed, it was believed that this
phenomenon is due to the microcytase acting on vibrios which have first
been modified by the agglutinative substance. This latter substance
resists heating to 55°–56° C., is only destroyed at higher temperatures,
and is retained in the blood serum long after the cytase has entirely
disappeared. The analogy between the agglutinative substance of the
fluids of animals that have acquired immunity and the substance in the
same fluids that is stable under heat is undeniable, and yet these two
substances are not identical. A whole series of observations, which we
shall presently describe, demonstrate this thesis clearly. A serum may
be highly agglutinative without being capable of bringing about the
transformation of vibrios into granules; the converse also holds good.
The substance which sets up Pfeiffer’s phenomenon, and which is found in
the fluids of immunised guinea-pigs, is a “fixative substance” analogous
to those we have already met with in the serums of animals so adapted
that they are able to resorb the various cell elements. As in the
resorption of cells, so also in the destruction of micro-organisms, the
fixatives are specific. The substance which aids the transformation into
granules is not only distinct from the fixatives which sensibilise red
blood corpuscles or spermatozoa, but also from the fixatives which
sensibilise bacteria. This specificity has been demonstrated and
carefully studied by Pfeiffer, who has shown that it may even serve to
distinguish species of bacteria. The serum of a guinea-pig which has
been immunised against the cholera vibrio, will render sensitive these
vibrios, and these only, to the action of the microcytase. Even allied
vibrios, such as various water vibrios, for example, are not sensitive
to the fixative of anticholera serum. On the other hand, the serums
obtained after the inoculation of these aquatic vibrios are incapable of
producing granular transformation in the cholera vibrio.
When we inject into one and the same animal several species of vibrios
we obtain a serum or a peritoneal fluid which produces Pfeiffer’s
phenomenon with the vibrios of all the species which have been used to
make the inoculations. This antivibrio serum contains only a single
cytase for the vibrios, but contains as many different fixatives as
there were species inoculated.
[Sidenote: [229]]
The transformation of vibrios into granules, when produced in a high
degree against virulent vibrios, under the influence of the body fluids
of immunised animals, affords a very valuable indication of the
simultaneous presence of cytase and of specific fixative. As we have
already stated, at the commencement of this account of the acquired
immunity of guinea-pigs against the cholera vibrio, Pfeiffer’s
phenomenon is manifested in the peritoneal fluid of these animals in a
very short time (5 to 20 minutes) after the inoculation of the vibrios.
This proves that in this fluid the two substances really occur together,
and that the fixative and the cytase are in solution in the plasma of
the exudation. Is it the same in every part of the body of immunised
guinea-pigs? If, instead of introducing the cholera vibrio into the
peritoneal cavity, we inject it into the subcutaneous tissue or into the
anterior chamber of the eye of these animals, Pfeiffer’s phenomenon does
not make its appearance. The vibrios, isolated or collected into small
clumps, do not undergo granular transformation; they keep their normal
vibrio form and remain alive much longer than in the peritoneal cavity.
Some of them may be found still living 24 hours after subcutaneous
injection and several (4–6) days in the anterior chamber of the eye. Nor
can Pfeiffer’s phenomenon be observed when the cholera vibrio is
introduced into the oedema of the foot, produced in consequence of the
slowing of the circulation, the vibrios remaining alive for a fairly
long time. These facts clearly indicate that in the fluid thrown out in
passive oedema, just as in the aqueous humour of the eye or in the
subcutaneous tissue, the two substances necessary to set up the granular
transformation are not present. Are both of them absent or only one?
This question is easily answered on adding to the fluids mentioned
normal guinea-pig’s serum, a serum which, by itself, is incapable of
producing Pfeiffer’s phenomenon. Bordet[313] has made these experiments
and found that when to the fluid of the passive oedema of the immunised
guinea-pig normal serum is added, this fluid transforms the cholera
vibrio into granules, but does so in less degree than does the serum of
the same immunised guinea-pig, when heated to 55°–56° C., to which
normal serum has likewise been added. There is, then, reason to conclude
that the fluid of the oedema does not contain any cytase, but contains a
certain quantity of cholera fixative, less, however, than that which is
found in the blood serum. As to the aqueous humour of the eye of
immunised animals, analogous experiments have demonstrated that it
contains neither of the two substances necessary for the production of
Pfeiffer’s phenomenon.
With the help of the facts I have here summarised, we arrive at the
following conclusion. In the animal that is immunised against the
cholera vibrio, microcytase is found in the peritoneal exudation; it
does not pass, however, either into the fluid of the passive oedema or
into the aqueous humour of the eye; the cholera fixative is found in the
peritoneal fluid and passes into the oedema, but does not penetrate into
the fluid of the eye. This indicates that microcytase is found in fluids
rich in leucocytes, but is absent from those which contain very few or
none of these cells.
The introduction of vibrios into the peritoneal cavity of immunised
guinea-pigs at once produces Pfeiffer’s phenomenon, and at the same time
causes the disappearance of the majority of the leucocytes from the
peritoneal lymph. We have already had occasion, several times, to speak
of this phagolysis, because it is produced as a sequel to the injection
into the peritoneal cavity of blood, spermatic fluid, and all kinds of
fluids. The greater the quantity of fluid injected and the greater the
difference of the temperature between it and the contents of the normal
peritoneum the more vigorous is phagolysis.
[Sidenote: [230]]
Pierallini[314], working in my laboratory, studying phagolysis in the
peritoneal cavity of the guinea-pig, has obtained several results worthy
of attention. Of all the fluids used by him, such as water, broth,
filtered cultures of micro-organisms and physiological saline solution,
the last of these caused the least intense phagolysis, yet one
sufficiently well marked. Immediately after the injection of any of the
above fluids the number of leucocytes in the peritoneal lymph diminishes
very considerably, the cells being found collected in clumps on the
omentum. Many of them exhibit signs of enfeeblement and of partial
destruction. Alongside the leucocytes are found fibrinous masses, this
affording evidence that some of the leucocytes have been greatly damaged
and have given up the fibrin-ferment which induces coagulation of the
fibrin. When Pierallini injected fluids containing coloured powders in
suspension, such as Indian ink and vermilion, he observed that these
substances accumulated on the greater omentum, which became stained
black or red. Microscopical examination revealed the existence of a not
very intense phagocytosis and a number of free coloured granules in the
midst of filaments of fibrin.
[Sidenote: [231]]
The leucocytes which, during this phagolysis, allowed the fibrin-ferment
to escape might also give up a certain amount of their microcytase. This
microcytase would pass into the peritoneal fluid and give rise to
Pfeiffer’s phenomenon. If this hypothesis be correct, the suppression of
phagolysis would result in the absence of the transformation of the
vibrios into granules. It is not a difficult matter to verify this
hypothesis as we have a means of preventing phagolysis or at least of
reducing it very considerably. Issaeff[315], in an investigation carried
out in Pfeiffer’s laboratory, demonstrated that an intraperitoneal
injection of physiological salt solution, broth, urine, etc., reinforces
the leucocytes and brings them up in large numbers into the peritoneal
cavity. It is easy to foresee that such an injection would serve to
diminish the intensity of the phagolysis. In fact, if we first inject a
few cubic centimetres of physiological salt solution or of fresh broth
into the peritoneal cavity of a guinea-pig, and if, on the following
day, we repeat the same operation, we shall find that after the second
injection phagolysis is much less powerful than after the first.
Pierallini, who repeated these experiments, observed that the
phagocytosis of the coloured granules is much more complete in the
guinea-pigs that were treated by a preliminary injection into the
peritoneal cavity. The amount of fibrin on the omentum is in this case
much less, and the phenomena as a whole show that in these guinea-pigs
the damage to the leucocytes is very considerably attenuated.
We have been able to demonstrate that in the case where phagolysis is
thus diminished, Pfeiffer’s phenomenon is not produced or is manifested
in a very feeble degree. If the experiment succeeds, the fluid taken
from the peritoneal cavity of a guinea-pig prepared the day before and
then injected with a culture of cholera, is opaque and thick, like pus.
It contains a mass of leucocytes in good condition, a large number of
which gorge themselves in a few minutes with a number of vibrios. The
plasma of this exudation contains few vibrios, and these retain their
normal form and do not exhibit, save exceptionally, a granular change. A
little later there remain no free vibrios; they are all contained within
leucocytes. Pfeiffer[316] declared himself against the facts I have just
summarised, because he was never able to prevent the granular
transformation of the vibrios, in spite of the preparatory injection of
sodium chloride. Abel[317], who repeated the experiments, expressed an
intermediate view: in guinea-pigs prepared by injections the day before,
he observed that one portion of the vibrios became transformed into
granules, whilst another became the prey of the leucocytes. The fact is,
the suppression of phagolysis demands special conditions: the broth that
is injected must be freshly prepared, and before its introduction into
the peritoneal cavity it must be heated to 37°–39° C. Even when these
precautions are taken it sometimes happens that the experiment is not
very successful. In making the experiment we must be guided by the
appearance of the peritoneal fluid withdrawn into the small glass
pipettes. If the fluid which enters the tube is clear or scarcely
clouded, it indicates that phagolysis has taken place, in spite of the
preparatory injection. The experiment is successful in those cases where
the peritoneal exudation is very cloudy and resembles pus.
[Sidenote: [232]]
As the demonstration of the suppression of Pfeiffer’s phenomenon as well
as that of phagolysis is of fundamental importance, I asked M.
Garnier[318] to carry out further experiments with the object of setting
the question at rest. Using a whole series of fluids for the preparatory
injection he found that fresh broth gives the best results. In
guinea-pigs in which the phagolysis had been reduced to a minimum,
phagocytosis commenced immediately after the injection of the vibrios.
In from two to five minutes many vibrios are found inside the
leucocytes, the free vibrios now being few in number and not exhibiting
Pfeiffer’s phenomenon. Garnier in his memoir gives photographic
reproductions of leucocytes crammed with vibrios; these should convince
the veriest sceptic. Since the publication of this paper no objection
has been advanced, and this question of the suppression of the granular
transformation of vibrios may now be regarded as definitely settled. I
have since demonstrated this feature to many observers, all of whom have
assured themselves of its accuracy. It must, then, be accepted that
Pfeiffer’s phenomenon is not produced in the peritoneal cavity except
when there is phagolysis. As this fact renders it very probable that the
microcytase, which is necessary for the transformation of the vibrios,
escapes from the injured leucocytes, it becomes necessary to verify this
hypothesis by a series of other experiments. If this hypothesis be well
founded, Pfeiffer’s phenomenon should not be observed in those
situations in the body where there are no, or almost no, leucocytes
already present. These conditions can be realised by injecting cholera
vibrios into the subcutaneous tissue or into the anterior chamber of the
eye of guinea-pigs that are well vaccinated against the cholera vibrio.
Under these conditions, as I have demonstrated in my work on the
extracellular destruction of cholera vibrios, the vibrios retain their
normal form and are never transformed into granules. Pfeiffer has
questioned this result, stating that beneath the skin of vaccinated
guinea-pigs the granular transformation is always produced, though in a
more feeble fashion and after more delay than in the peritoneal cavity.
The contradiction between Pfeiffer’s experiments and my own can,
however, be explained. When inoculating the vibrios into the
subcutaneous tissue, or during the withdrawal of the exudation formed at
the point of infection, small haemorrhages are sometimes produced and a
certain amount of microcytase is set free from the leucocytes found in
the effusion of blood; these cells also give up to the extravasated
blood a portion of their fibrin-ferment. When the experiment is
successful, that is to say when no haemorrhage is produced during the
operations involved, the subcutaneous exudation contains normal vibrios
only, without the appearance of any trace of Pfeiffer’s phenomenon in
the fluid.
[Sidenote: [233]]
If the extracellular transformation of the vibrios into granules were
the real cause of the acquired immunity, the absence of this phenomenon
in the subcutaneous tissue of the vaccinated guinea-pig should lead to
the death of the animal. As a matter of fact this does not take place
and the animal resists the inoculation of the vibrios. This conclusion
is open to one serious objection. As the cholera vibrio in the great
majority of cases is incapable of producing a fatal infection when
inoculated subcutaneously, even in normal unvaccinated guinea-pigs, this
example of immunity must be placed in the category of natural immunity,
a kind of immunity which may depend on causes other than those on which
acquired immunity depends. To answer this objection it was necessary to
select a race of vibrios capable, when injected subcutaneously, of
causing death. Mesnil[319], chief of my laboratory staff, undertook to
carry out experiments with the Massowah vibrio, which is regarded by
some authors as belonging to the true cholera species. When inoculated
subcutaneously into unprotected guinea-pigs, it induces local oedema, in
which the vibrios swarm; the infection rapidly becomes generalised and
causes the death of the animal in 24 hours. Yet this vibrio, when
injected into the subcutaneous tissue of well vaccinated guinea-pigs, is
completely resisted by these animals and not the least trace of
Pfeiffer’s phenomenon is produced. Under these conditions, a certain
number of the vibrios are at first united into masses, but a
considerable number remain isolated and motile. Some hours after
inoculation the number of clumps diminishes and the isolated vibrios
become more numerous, a fact which indicates a certain amount of
adaptation of the vibrio to the medium in which it now finds itself. But
never, so long as the vibrios remain free in the subcutaneous exudation,
do they become transformed into granules.
[Sidenote: [234]]
Salimbeni[320], in an investigation carried out in my laboratory,
endeavoured to satisfy himself whether or no Pfeiffer’s phenomenon is
produced in the subcutaneous tissue of a horse that had been
hyperimmunised against the cholera vibrio. This animal had, during a
period of 14 months, received considerable quantities of these
microorganisms, and the serum of its blood transformed the vibrios into
granules with great rapidity and intensity. In spite of such favourable
conditions for the manifestation of Pfeiffer’s phenomenon, it was never
produced beneath the skin of this horse. The vibrios when injected in
this position became completely motionless in a very short time, but
they kept their vibrio form and remained alive for a number of hours.
The exudation drawn off up to 24 hours after inoculation still gave
growths of the cholera vibrio.
As it is more easy to introduce, without effusion of blood, the cholera
vibrio into the anterior chamber of the eye than beneath the skin, and
as the aqueous humour contains no fixative, the absence of the granular
transformation in the first of these two situations has been observed
even by Pfeiffer himself. The demonstration of this fact presents no
difficulty, and for a considerable period we may observe free and
perfectly motile vibrios moving about in the aqueous humour. The
exudation from the eye contains many of these living organisms, which
when sown on culture media made their appearance as colonies even when
the fluid has been withdrawn from the eye several days after
inoculation.
[Sidenote: [235]]
These carefully established facts show very clearly that the microcytase
is only met with in the fluids of the living animal in those situations
in which there are many pre-existing leucocytes and under conditions in
which the cells undergo a more or less marked phagolysis. This may be
corroborated by a decisive experiment. When we inject a suspension of
the cholera vibrio directly into the veins of a guinea-pig, well
vaccinated against these organisms, and whose serum produces _in vitro_
Pfeiffer’s phenomenon with great rapidity, this phenomenon is not
manifested. This experiment has been performed and described by
Bordet[321]. Having injected a suspension of this vibrio into the
jugular vein of a guinea-pig vaccinated against the cholera vibrio, he
killed the animal an hour later and found, in the blood of the heart,
vibrios that had kept intact their form and their property of staining
with methylene blue. Cultivation of the blood of the heart, liver and
spleen gave growths of vibrios. In another guinea-pig, hypervaccinated
against the same organism and inoculated by the same method, the blood
drawn off shortly (4–15 minutes) afterwards showed, in preparations
treated with methylene blue, well-stained vibrios, of normal form and
quite intact. This is the most direct proof of the absence of Pfeiffer’s
phenomenon in the blood fluid of a living animal that enjoys a very
marked acquired immunity. The intact vibrios were lodged inside the
leucocytes.
Levaditi[322] repeated these experiments in my laboratory and varied the
conditions under which the vibrios were injected into the blood vessels.
He was sometimes able to observe phagolysis of the leucocytes of the
blood and their almost complete disappearance from the peripheral
circulation. In these cases the injured leucocytes accumulated in the
pulmonary capillaries and masses of them were seen surrounding groups of
vibrios that were transformed into granules. It was, however, easy to
exclude phagolysis by preparing the animals by means of injections of
physiological saline solution or broth. Under these conditions the
leucocytes remained in the blood current and very soon ingested the
vibrios. Whilst the vibrios that were still free in the blood plasma
retained their form and staining power intact, those found inside
microphages were already, in great part, transformed into granules. The
rapidity with which these phagocytes ingest the vibrios and set up the
changes in them is really extraordinary.
In this case, which affords a typical example of the reaction of the
animal organism in acquired immunity, we see a very marked and immediate
phagocytosis. It is this same process that has already been described as
occurring in the peritoneal cavity of vaccinated guinea-pigs in which
phagolysis was absent as the result of preparatory injection. In the
subcutaneous tissue and in the anterior chamber of the eye, where
Pfeiffer’s phenomenon is regularly absent, the phagocytosis follows its
ordinary course and causes the destruction of the vibrios. This result
has been confirmed repeatedly—see works by Bordet, Mesnil and Salimbeni
already quoted.
[Sidenote: [236]]
We need only compare the extension of Pfeiffer’s phenomenon and that of
phagocytosis in animals that are immunised against the cholera vibrio,
to satisfy ourselves that the former phenomenon is a limited one whilst
the latter is general. There might be advanced against the latter
conclusion the fact of the absence of any ingestion of the vibrios in
the peritoneal fluid of guinea-pigs that are immunised but are not
preserved against phagolysis. When a little of the peritoneal fluid is
drawn off with small tubes shortly after the injection of vibrios into
the peritoneal cavity, as a matter of fact, only a very intense
Pfeiffer’s phenomenon is seen, phagocytosis being completely or almost
entirely absent. But this procedure is insufficient. If we are to get an
idea of what really takes place in the abdominal cavity, the animal must
be killed and the peritoneum and especially the omentum very carefully
examined. As first demonstrated by Max Gruber[323] and later by
Cantacuzène[324], the greater omentum is, in these cases, covered with a
thick layer which contains a large number of leucocytes, of which some
are filled with vibrios; further, this layer contains a mass of vibrios,
in part transformed into granules, in part agglutinated or isolated and
retaining their vibrionic form intact. As time goes on, the phagocytosis
becomes more and more marked, and it is impossible to deny its existence
or to attribute to it merely a secondary part.
We have seen that the suppression of Pfeiffer’s phenomenon in the
peritoneal cavity and in the blood, or its total absence in the anterior
chamber of the eye, does not in the least deprive the vaccinated
guinea-pig of its acquired immunity. The animal resists the vibrios
perfectly, without these requiring to be transformed into granules in
the body fluids. This transformation does take place undoubtedly, but
only inside the phagocytes. As already stated in the discussion on
natural immunity (Chaps. VI, VII) the vibrios, after being ingested by
the microphages, almost immediately undergo within these cells a change
in form, very similar to that observed in the real Pfeiffer’s
phenomenon. The microphages are often full of a quantity of granules,
derived from the ingested vibrios, which in a short time are completely
digested. This fact, of such constant occurrence in the phagocytosis of
the vibrios, furnishes us with still another proof of the microphagic
origin of microcytase.
[Sidenote: [237]]
If Pfeiffer’s phenomenon is merely a particular instance of the
transformation of vibrios into granules in fluids containing
microcytase, it is quite natural that its suppression should not involve
a fatal infection of the vaccinated animals. On the other hand, if the
phagocytic reaction, so widely different, really plays an important part
in acquired immunity, everything that interferes with phagocytosis must
at the same time compromise the refractory condition. With the object of
solving this question Cantacuzène[325], working in my laboratory,
undertook a detailed investigation of this point. He showed that the
injection of opium, in a non-fatal dose, narcotised the guinea-pig and
at the same time prevented the movements of the leucocytes. Small glass
tubes containing cholera vibrios and introduced beneath the skin of
vaccinated guinea-pigs, became filled with numbers of leucocytes in the
non-narcotised animal; in the guinea-pig that had received tincture of
opium, the tubes left for several hours contained no leucocytes and
later only did they begin to enter the tubes. When a strong dose of
cholera vibrios was injected into the peritoneal cavity of thoroughly
vaccinated guinea-pigs, the animals easily resisted the inoculation.
When, however, similarly vaccinated guinea-pigs were submitted to the
influence of tincture of opium, the same dose of vibrios caused their
death. In these narcotised animals, in spite of the considerable
dilatation and hyperaemia of the vessels and in spite of the marked
hyperleucocytosis of the blood, diapedesis was not produced during the
first few hours after the injection of the opium, and it was not till
later (5 to 6 hours after injection) that the leucocytes began to appear
in the peritoneal cavity. The vibrios profit by the period of inactivity
of the phagocytes to multiply, retaining their motility and also the
property of staining with basic aniline dyes. When the retarded
leucocytes make their appearance in the peritoneal cavity, they find it
already invaded by a multitude of vibrios. In spite of this the
leucocytes, especially the microphages, ingest an enormous number of the
organisms; this does not prevent the death of the guinea-pigs, though it
takes place some hours later than in the unvaccinated control animals.
At the moment of death, free vibrios are no longer found in the
exudation; they have all been ingested by the microphages, inside which
they have undergone granular transformation. On making a post-mortem
examination of the animal a large number of small heaps of vibrios, such
as are never met with in animals that have not been submitted to the
action of opium, are found on the omentum.
[Sidenote: [238]]
All that is necessary, then, is to retard the phagocytic reaction for a
few hours in order to cause well-vaccinated guinea-pigs to succumb to
the action of the vibrios. One can readily understand that, with this
result before us, there can be no hesitation in attributing to
phagocytosis a much more important part in assuring acquired immunity
than to Pfeiffer’s phenomenon.
The study of other diseases produced by vibrios only serves to
corroborate the general conclusions that follow from the detailed study
of the essential processes in acquired immunity against the cholera
vibrio. It is here necessary to recall the discovery by v. Behring and
Nissen of the very marked bactericidal power of the blood serum of
guinea-pigs that have been vaccinated against Gamaleia’s vibrio. When
this fact was first demonstrated we were justified in thinking that the
vibriocidal property of the blood might by itself explain this acquired
immunity; but a comparative study of the phenomena which take place _in
vitro_ with those which take place in the living animal, soon
demonstrated how slight was the foundation for this hypothesis. Whilst
the vibrios, when sown in the blood serum of hypervaccinated
guinea-pigs, there perished in large quantities or even the whole of
them, these same organisms, when inoculated into the subcutaneous tissue
of the same animals, remained alive for several days. Gamaleia’s vibrio
is much less capable of being transformed into granules than is the
cholera vibrio, and we find it retaining its normal form even inside the
leucocytes. There is no occasion in this case, therefore, to look for
Pfeiffer’s phenomenon.
[Sidenote: [239]]
The rapid and marked destruction of Gamaleia’s vibrio, _in vitro_, in
the blood serum of vaccinated guinea-pigs, and the prolonged survival of
these organisms in the living animal, afford additional evidence that
the two groups of phenomena cannot be identical. On the other hand, it
furnishes a further proof that, during the preparation of the serum,
there is produced, parallel with the coagulation, another process which
confers bactericidal power on the serum. It is quite evident that, as in
the case of the cholera vibrio, we have here to do with the liberation
of microcytase at the expense of the destroyed or injured leucocytes.
Acting along with the specific fixative of the body fluids, this cytase
causes the death of the vibrios introduced into the serum. In the living
organism, the microcytase not being free, these vibrios, although
influenced by the fixative, resist until they have become the prey of
the phagocytes. In an investigation which was the subject of a
communication to the International Congress of Hygiene in London in
1891[326], I demonstrated that the phagocytic reaction is produced with
great intensity in guinea-pigs that have been vaccinated against
Gamaleia’s vibrio. The inoculation of this organism into the
subcutaneous tissue, an inoculation which sets up a rapidly fatal
infection in untreated guinea-pigs, gives rise in immunised animals to
the formation of an abundant exudation, in which the numerous vibrios
soon meet with resistance from the phagocytes. These phagocytes ingest
the living vibrios, retaining them for some considerable time in their
interior, but in the long run always digesting them completely. During
the last phase of this struggle, we sometimes find, inside the
leucocytes, vibrios that are transformed into spherical granules. It was
with these cells, filled with ingested vibrios, that I was able first to
carry out an experiment that has since been repeated again and again,
always with the same result. When from a well-vaccinated guinea-pig a
drop of subcutaneous exudation is withdrawn, at a stage when all the
vibrios have for some time been ingested by the leucocytes, and
transferred, in the form of a hanging drop, to the incubator at 35°–37°
C., it is found that the ingested vibrios develop inside the phagocytes
which have died outside the animal. The vibrios first fill the leucocyte
and, continuing to multiply, cause the cell to burst when they
distribute themselves in the fluid of the hanging drop (figs. 40 and
41). This experiment proves, in the first place, that the vibrios have
been ingested alive, and, secondly, that the plasma of the exudation was
incapable of preventing their later development.
[Illustration:
FIG. 40. Vibrios (_V. metchnikovi_) developed inside a microphage from
a vaccinated guinea-pig.
]
[Illustration:
FIG. 41. Vibrios (_V. metchnikovi_) developed in a drop of exudation
from a vaccinated guinea-pig. The vibrios have ruptured the
microphage and scattered themselves in the fluid.
]
[Sidenote: [240]]
[Sidenote: [241]]
Having summarised the principal phenomena exhibited by vibrios in an
animal possessing acquired immunity, we must now enquire whether the
mode of destruction and disappearance taking place in these vibrios is
of general application. Naturally, we commence this study with the
spirilla, which in many respects present a great analogy to the vibrios.
The task is an easy one, thanks to a very careful work recently
published by Sawtchenko[327], on the _Spirochaete obermeyeri_ of
recurrent fever. We know, from what has been said in Chapter VI, that
the spirochaetes found in the serum of persons attacked by this
organism, are, in the peritoneal cavity of guinea-pigs, destroyed by the
intervention of the macrophages. These phagocytes guarantee the natural
immunity of the guinea-pig against the parasite of recurrent fever. In
guinea-pigs, into which blood or serum containing spirilla has been
injected on several occasions, the destruction of these micro-organisms
is effected in a different way. When Sawtchenko introduced a number of
_Spirochaete obermeyeri_ into the peritoneal cavity of guinea-pigs so
prepared, he noted that they underwent a transformation resembling that
observed in Pfeiffer’s phenomenon. In a short time the majority of these
micro-organisms assumed the form of very delicate spirilla to which were
attached round granules. There was not a complete transformation of the
spirilla into granules, but a portion of their contents exuded in the
form of spherical drops. The spirilla that exhibited these changes lost
their motility and collected into clumps. There was undoubtedly an
extracellular transformation of the spirilla, but this took place only
in the peritoneal cavity. When injected into the subcutaneous tissue of
a prepared guinea-pig the spirilla brought about the formation of a firm
but scanty exudation in this situation. In this exudation were found
leucocytes containing spirochaetes which retained their normal form.
These micro-organisms were found exclusively in macrophages and gave no
evidence of the occurrence of Pfeiffer’s phenomenon. A like absence of
this phenomenon was observed in normal guinea-pigs which had been
injected subcutaneously with the same quantity of spirilla. In these
animals, however, the oedema that appeared at the seat of inoculation
was abundant and soft, and the disappearance of the spirilla, that is to
say their ingestion by the macrophages, took place at a very much later
period than in the prepared guinea-pigs. We have, therefore, in this
respect a complete analogy with the vibrios: in both cases there is an
absence of granular transformation below the skin and an ingestion by
the leucocytes of the exudation; on the other hand, we have Pfeiffer’s
phenomenon appearing in the peritoneal fluid. This analogy extends even
further. Thus, in guinea-pigs prepared by repeated injections of human
serum rich in spirilla, Sawtchenko could suppress Pfeiffer’s phenomenon
in the peritoneal cavity just as easily as in the case of the vibrios.
All he had to do was to inject a certain quantity of broth into the
peritoneal cavity of his immunised guinea-pigs. Twenty-four hours later,
on introducing spirilla into the animals at the same site, they retained
their motility for hours, did not exhibit any granular transformation
and were ultimately completely ingested by the macrophages.
[Sidenote: [242]]
These facts lead us to conclude that the fate of the spirochaetes of
recurrent fever in the organism of guinea-pigs prepared by previous
injections is governed by laws the same as those established for
acquired immunity against vibrios. The spirilla are ingested and
destroyed by the phagocytes, except where phagolysis occurs, in which
case the cytase, being set free, attacks the micro-organisms outside the
leucocytes.
[Sidenote: [243]]
After his discovery of the granular transformation of vibrios, R.
Pfeiffer, in collaboration with several of his pupils, set himself to
discover how far this phenomenon was general in acquired immunity. He
directed his attention to the typhoid cocco-bacillus, upon which he had
already published[328] a very detailed account of work carried out in
conjunction with Kolle. These observers availed themselves of the
discovery made by Beumer and Peiper[329], and Chantemesse and Widal[330]
and confirmed by other observers, that laboratory animals, especially
mice and guinea-pigs, could be easily vaccinated against the fatal
disease set up by the micro-organism of typhoid fever. As in the
experimental infection of the guinea-pig by the cholera vibrio, the
vaccination of the animals against the typhoid bacillus could be carried
out very easily, either by using sterilised cultures or the fluids of
cultures deprived of their organisms by filtration. In the small
laboratory animals a most marked acquired immunity may thus be obtained,
and the study of the phenomena which appear in the vaccinated organism
afforded evidence of a general analogy with those which have been
observed when vibrios are used. In the peritoneal cavity of the
immunised guinea-pigs, Pfeiffer’s phenomenon proper does not appear,
that is to say, only a few of the bacilli are transformed into granules,
the large majority retaining their bacillary form; still they are
evidently greatly damaged: they become motionless and agglutinate more
or less completely into clumps. If, however, a few of these
micro-organisms are sown on nutritive media, they multiply freely and
give abundant growths. The peritoneal fluid, then, acts most
unmistakably upon the typhoid bacillus, but in a much less degree than
does the peritoneal exudation of guinea-pigs upon the cholera vibrio
when immunised against that organism. In both cases we have a pronounced
phagolysis which sets free the microcytase, whose action on the vibrio
is more marked than on the bacillus of typhoid fever. This extracellular
action on the typhoid bacillus in the peritoneal cavity can be easily
prevented by a previous injection, twenty-four hours before, of broth,
physiological salt solution, or normal serum. The suppression of
phagolysis is, as in the case of vibrios and spirilla, followed by the
suppression of extracellular action on the typhoid bacilli.
The same analogy is observed in the phenomena which appear beneath the
skin. The bacillus of typhoid fever, when introduced into the
subcutaneous tissue of vaccinated guinea-pigs, although not appreciably
injured by the fluid of the exudation, undergoes some agglutination. The
injurious action of the fluids of the body is here still less effective
than in the peritoneal cavity. But, as in the peritoneal cavity of
vaccinated guinea-pigs previously treated with broth, so in the
subcutaneous exudation it is the phagocytes which destroy the
micro-organisms. In both cases there is a very great afflux of
leucocytes, mainly microphages. These cells ingest and digest the
bacilli, which ultimately disappear. The micro-organisms ingested by the
microphages, once inside these phagocytes are transformed into granules
very like those observed in the cholera vibrio similarly treated. In
this respect the analogy between the two micro-organisms is complete.
[Sidenote: [244]]
Oppel, working in my laboratory, has repeated Cantacuzène’s work on the
retarding action of opium upon the phagocytic process. He obtained the
same results: under the influence of the narcotic, the leucocytes
intervened only at a late stage, with the result that the vaccinated
guinea-pigs succumbed to the typhoid infection. The same conclusion must
be drawn from the experiments made by A. Wassermann[331]. Guinea-pigs
that had been immunised against the bacillus of typhoid fever were
completely resistant to a dose that was always fatal to the control
animals. When, however, along with this dose of bacilli, a certain
quantity (3 c.c.) of a serum which hinders the phagocytic reaction is
injected, the guinea-pigs lose their immunity and die from typhoid
infection. The serum employed by Wassermann was obtained from rabbits
that had been treated with the blood serum of guinea-pigs. Rabbit’s
serum, thus prepared, neutralises the action of the guinea-pig’s cytase,
but, as demonstrated by Besredka[332], it also exercises several other
functions, one especially, that of preventing phagocytosis. In
Wassermann’s experiments it was the antiphagocytic function, then, that
was the important factor in the suppression of the acquired immunity of
the guinea-pigs. These experiments supply a fresh proof of the great
importance of the phagocytic reaction in this kind of immunity, and
afford further confirmation of the analogy between the mechanism of
resistance of the animal’s organism against the typhoid bacillus and
that against the cholera vibrio.
In presence of this striking analogy, it is unnecessary to insist
further on the details of the acquired immunity of animals against the
experimental disease set up by the micro-organism of typhoid fever. It
will be better to select another example from the group of bacilli. Let
us first take the acquired immunity against the bacillus of blue pus
(_Bacillus pyocyaneus_) which for many years has been regarded as the
best example in which to study this kind of immunity. Charrin, who was
the first to obtain disease with this bacillus experimentally, published
several notes[333] on the acquired immunity of the rabbit against it. He
demonstrated the possibility of vaccinating this animal not only with
living bacilli, but also with the products of their culture; he studied
the blood serum of vaccinated animals, comparing it with the serum of
normal rabbits, especially as to its action on the development of the
_Bacillus pyocyaneus_. Although unable to find any bactericidal power
properly so called in the serum of immunised rabbits, Charrin was the
first to draw attention to certain modifications undergone by the
bacilli when grown in this medium. He noted that under these conditions
no pyocyanin was produced, and, in collaboration with Roger, he
demonstrated that, in the serum of the vaccinated rabbit, the Bacillus
pyocyaneus forms packets composed of little chains of greater or less
length, whilst in the serum of the normal, susceptible rabbit, it
develops in the form of normal rods, the rods for the most part being
isolated.
[Sidenote: [245]]
From his experiments _in vitro_ Charrin concluded that there was marked
enfeeblement of the functions of the _Bacillus pyocyaneus_ when
submitted to the action of the vaccinated animal organism. Bouchard[334]
has gone so far as to develop a theory of acquired immunity, in which
the principal part is attributed to the impossibility of the
micro-organism, after it has invaded the refractory animal, secreting
its fluid products; there is no vascular dilatation and diapedesis does
not take place. A comparative observation of the phenomena observed in
rabbits that are susceptible to the pyocyanic disease and of those met
with in vaccinated rabbits, most clearly, however, demonstrates the
impossibility of accepting Bouchard’s interpretation. The inoculation of
the bacillus of blue pus below the skin of the ear of the normal
(unvaccinated) rabbit sets up extensive inflammatory reaction with
marked hyperaemia; the diapedesis of the white corpuscles takes place at
a comparatively late stage of the process and phagocytosis is neither
set up nor completed until very late. On the other hand, in vaccinated
rabbits, infected in the same way, the hyperaemia of the ear is
insignificant, but diapedesis occurs very early and phagocytosis
commences at once. It is not, therefore, the impossibility for the
leucocytes to traverse the vessel wall, owing to the absence of the
dilatation of the veins, which prevents them from making their way
rapidly to the field of battle; it is their imperfect positive
sensitiveness that is accountable for the tardy and incomplete
phagocytosis. This interpretation is confirmed in other cases of
acquired immunity.
[Sidenote: [246]]
More recently, Paul Müller[335] has laid special stress on the part
played by the bactericidal action of the serum of animals that have been
vaccinated against the pyocyanic disease. For him the negative results
obtained by his predecessors lose their significance, since all their
experiments were carried out under conditions of aërobiosis, whilst it
is only in the absence of free oxygen that this bactericidal power can
be exerted at all freely. Müller, therefore, set himself to compare
under anaerobic conditions the bactericidal action on the _Bacillus
pyocyaneus_ of serums coming from normal and from vaccinated animals. He
succeeded in demonstrating that the blood serum of vaccinated animals is
more bactericidal than that of normal rabbits. Before, however, drawing
any conclusion from this observation, the following question must be
answered: Are the phenomena observed _in vitro_ comparable with those
seen in the living animal? In preceding chapters it has been shown so
often that the blood serum obtained after the separation of the
extravascular clot, can in no way be identified with the plasma of the
circulating blood, that it is unnecessary to argue this matter further.
If we wish to gain a clear idea of the mechanism of immunity in the
living animal we must observe the course of events in the vaccinated
animal and not draw conclusions from observations _in vitro_ except
after strict examination. All the works on pyocyanic immunity above
summarised lie under the reproach that in them this rule has not been
adhered to.
Since the discovery of Pfeiffer’s phenomenon in animals that have been
vaccinated against the cholera vibrio, much greater care has been taken
to attend to the changes that occur in the animal that enjoys acquired
immunity. Wassermann[336] was the first to attempt to apply Pfeiffer’s
discovery to the _Bacillus pyocyaneus_. With a race of this bacillus
rendered more virulent he succeeded in producing a fatal experimental
malady in the guinea-pig against which he was able by various methods to
vaccinate these animals.
[Sidenote: [247]]
He thus describes the phenomena observed in the peritoneal cavity of
immunised guinea-pigs. Soon after injection the bacilli of blue pus
become motionless, then “the rods swell up and melt, like wax in hot
water. The formation of granules, such as occur in the cholera vibrio,
has been observed but rarely. The process recalls rather that which
takes place in experimental typhoid fever, as described by R. Pfeiffer.
In all cases the phenomenon of solution takes place entirely in the
fluid of the exudation, without any co-operation on the part of the
leucocytes” (p. 284). We see that we have still to do with a kind of
attenuated Pfeiffer’s phenomenon, without any granular change, but with
an immobilisation of the bacilli. As Wassermann has remained satisfied
with the examination of the peritoneal content which, as we know, gives
but an imperfect picture of acquired immunity, Gheorghiewsky[337] set
himself to study the question more thoroughly under my direction. With
this object he vaccinated a series of guinea-pigs with living bacilli of
blue pus, a sure method of obtaining acquired immunity. On examining the
peritoneal fluid (withdrawn shortly after the injection of the bacilli)
of the vaccinated guinea-pigs, he found that the bacilli were motionless
and had undergone a certain degree of agglutination. They were not
transformed into granules but became thicker and somewhat more dumpy.
These changes are observed during the period of phagolysis, when only a
few scattered leucocytes are to be found in the fluid of the peritoneal
cavity. About two hours after the injection of the bacilli the
leucocytes begin to reappear in the peritoneal exudation, more
especially the microphages, which lose no time in seizing the bacilli,
some of which become transformed into granules. A few hours later the
exudation, containing a multitude of leucocytes, no longer contains any
free bacilli: all are found inside the microphages. Nevertheless, if a
drop of the exudation now be withdrawn and kept for some time at a
temperature of 37° C., it will be found that the bacilli multiply inside
the dead phagocytes outside the animal. We thus obtain colonies of
bacilli, a fact which clearly proves that these bacilli whilst still
alive have been ingested by the leucocytes. This experiment is,
therefore, very similar to the one we have described in connection with
Gamaleia’s vibrio.
[Sidenote: [248]]
Even at a later period, 24 or 30 hours after the injection of the
bacilli, that is to say at a period when an examination of the exudation
no longer reveals the presence of bacilli, the sowing of a drop of this
exudation on a nutrient medium still gives isolated colonies of the
_Bacillus pyocyaneus_ capable of producing the characteristic pigments.
At a still later period, when the peritoneal exudation remains sterile,
a post-mortem examination of the animals enables one to recognise,
beneath the peritoneal surface, small white points made up of
leucocytes. The sowing of these masses almost invariably gives colonies
of the _Bacillus pyocyaneus_ which form blue pigments. We see from this
account that, even in the peritoneal cavity of vaccinated animals,
matters by no means go on in a uniform fashion, as would appear from
Wassermann’s statements. Some bactericidal action in the peritoneal
fluid there certainly is, but it is quite transient, and is limited to
the period of phagolysis. The majority of the bacilli resist this attack
of the body fluids to continue their struggle with the phagocytes,
which, however, ultimately get the upper hand. In the subcutaneous
tissue the part played by this phagocytic reaction is still more
general. Gheorghiewsky has studied it not only in vaccinated guinea-pigs
but also in a goat which had received several large injections of the
_Bacillus pyocyaneus_. He observed that shortly after the subcutaneous
injection of these bacilli, the fluid which accumulates at the seat of
inoculation renders them motionless and in part agglutinates them. This
fluid is clear and contains a few leucocytes and a number of bacilli
which still retain their usual form. Some time later the leucocytes
begin to come up to the seat of inoculation and to ingest the bacilli.
At the end of 10 to 15 hours all the bacteria have been seized by the
microphages and we no longer find any of them free. A hanging drop of
this exudation, transported to the incubator, soon swarms with bacilli
which have sprung from the organisms ingested by the leucocytes.
The exudation becomes more and more abundant at the seat of inoculation
and ends in the formation of an abscess, from the contents of which
cultures of the _Bacillus pyocyaneus_ may be obtained for a fortnight.
The bacilli, however, finally disappear, this being due to the
destructive action of the phagocytes and not to that of the fluid of the
exudation.
This fundamental part played by phagocytosis in acquired immunity
against the _Bacillus pyocyaneus_ has been confirmed by Gheorghiewsky by
experiments on guinea-pigs vaccinated and then submitted to the action
of opium. As in the analogous experiments of Cantacuzène on the cholera
vibrio, the opium narcosis retards diapedesis and this, for some time,
increases the chances of the bacilli. A tardy diapedesis and
phagocytosis, no doubt, is produced which ends in the ingestion of the
bacilli, but the animal loses its acquired immunity and finally succumbs
in spite of the fact that the dose of _Bacillus pyocyaneus_ was
insufficient to kill a control guinea-pig vaccinated to the same degree,
but not submitted to the action of opium.
[Sidenote: [249]]
The example we have just analysed relates, then, to a micro-organism
which is more resistant than are the vibrios, Obermeyer’s spirilla or
even the typhoid bacillus, to the action of the microcytase which has
escaped from the cells during phagolysis. The _Bacillus pyocyaneus_
undergoes, in the fluids of the vaccinated animal, the action of the
specific fixative and can thus be rendered motionless and become
agglutinated. But this action is insufficient to ensure immunity and
should phagocytosis not take place in time to ingest the bacilli, the
vaccinated animal succumbs. The reaction of the phagocytes is,
therefore, indispensable if the acquired immunity is to be effective. In
this respect the analogy is very great between the resistance of the
vaccinated animal against the various bacteria (vibrios, spirochaetes,
typhoid cocco-bacilli, bacilli of blue pus) that we have so far studied
in this chapter. These bacteria have, however, this in common;—they are
all endowed with a considerable power of motion. Pursuing our
examination of the principal data on acquired immunity against
micro-organisms, we must now choose examples from the group of
non-motile bacilli; amongst these we assign the first place to the
micro-organism of swine erysipelas. This small bacillus has been the
subject of several important researches on acquired immunity, one of
which at a certain period caused quite a sensation in the
bacteriological world. Emmerich[338], in an investigation carried out in
collaboration with di Mattei, made an unexpected announcement. He said
he believed that he was justified in affirming that the acquired
immunity of rabbits against the bacillus of swine erysipelas is due to
the formation, in the fluids of the body, of an antiseptic substance
which very quickly destroys this organism. This substance, secreted by
the cells of the vaccinated animal, was supposed to act after the
fashion of a solution of bichloride of mercury and to kill a large
number of bacilli, introduced subcutaneously, in from 15 to 25 minutes.
This discovery was not confirmed. In a series of experiments that I
carried out[339] with the object of clearing up this question, and made
under conditions as favourable as possible for the demonstration of the
supposed bactericidal secretion, this action was never manifested. Not
only did the virulent bacilli of swine erysipelas, when injected
subcutaneously into well vaccinated rabbits, remain alive in the
subcutaneous exudation for hours and even days, but the attenuated
bacilli of Pasteur’s vaccines likewise remained intact. These bacilli
when introduced into the anterior chamber of the eye survived for even a
longer period. Here, as beneath the skin, the injection of the bacilli
induced an exudation rich in leucocytes, amongst which microphages
predominated. These phagocytes at once began to seize the bacilli which
were destroyed not in the fluid of the exudation but within the
leucocytes. Long after all the bacilli had been ingested, 24 hours and
more after inoculation, the sowing of the exudation frequently gave
growths in appropriate media.
[Sidenote: [250]]
Emmerich[340] sought by new experiments to remove these objections, but
he found that the bacilli of swine erysipelas did not disappear from the
vaccinated animal until some 8 or 10 hours after they had been
introduced. There is, therefore, no longer any question of a rapid
bactericidal action at all comparable to that of corrosive sublimate,
which would destroy the introduced bacilli in less than an hour. The
limit of 8 to 10 hours, accepted by Emmerich, is still too short and is
not in accordance with my experiments; but even this was quite
sufficient for the appearance of a free phagocytosis, a condition that
really occurs. Emmerich has not directed his researches in this
direction, and his theoretical conclusions did not in the least weaken
the value of my arguments drawn from the demonstration of the ingestion
and intracellular destruction of the bacilli by phagocytes.
[Sidenote: [251]]
The researches on immunity against swine erysipelas then languished for
some time, until the discovery of Pfeiffer’s phenomenon gave a fresh
stimulus to the study of this problem. One of Pfeiffer’s pupils,
Voges[341], sought to apply the results obtained in the case of the
cholera vibrio to the acquired immunity against the bacillus of swine
erysipelas. He studied the blood serum of animals vaccinated against
this bacillus and believed himself justified in affirming the existence
of an acquired bactericidal power. Under no condition, however, did he
observe anything comparable to Pfeiffer’s phenomenon, and he was
compelled to admit that the bactericidal action of the serum is very
feeble and only takes effect on young bacilli whose membranes are as yet
very delicate and not very resistant. Mesnil[342] repeated these
researches in my laboratory, but his results were very different from
those obtained by Voges. The blood serum of rabbits, fully vaccinated
against the bacillus of swine erysipelas, proved to be a good culture
medium for this bacillus, and Mesnil affirms, as the result of numerous
well-established observations, that “_in vitro_, the serum of rabbits
immunised against the erysipelas has no bactericidal power or a very
insignificant one.” On the other hand, the same fluid had a very marked
agglutinative power. The bacillus of swine erysipelas, being non-motile,
does not present the abrupt change that is observed in vibrios or in the
typhoid bacillus when submitted to the influence of specific
serums—under which conditions these organisms at once lose their
motility. But the bacilli of swine erysipelas, when introduced into the
specific serum of vaccinated animals, run together into masses which
become more and more voluminous and fall to the bottom of the vessel,
leaving a limpid supernatant fluid. When this bacillus is sown in the
serum of vaccinated animals, it is seen to develop in the form of
chains, composed of a large number of segments, which fall to the bottom
of the tube. These bacilli, however, whether agglutinated or developed
in chains, never show any attenuation in virulence. When the serum which
bathes them is got rid of by washing, they are just as virulent as are
the bacilli developed in the serum of normal unvaccinated rabbits. It is
important to show that this virulence is kept up in spite of the fact
that the bacilli, when placed in contact with the serum of immunised
animals, become permeated with the specific fixative, as shown by the
experiments of Bordet and Gengou[343]. These observers, indeed, have
demonstrated that the bacilli of swine erysipelas, when kept for 24
hours in the specific serum heated to 55° C., acquire the property of
absorbing the cytases contained in the unheated serum of normal animals.
The study of acquired immunity against the bacillus of swine erysipelas
teaches us that this immunity is not due to any extracellular
destruction comparable with Pfeiffer’s phenomenon; and that this
immunity causes the production of a specific fixative and of a specific
agglutinative substance, whose action on the resistance of the animal,
to judge from the complete virulence of the bacilli when agglutinated
and impregnated by fixative, is feeble or _nil_. It is the phagocytic
reaction which is dominant in the immunised animals and which brings
about the intracellular destruction of the bacilli.
The history of the anthrax bacillus, another representative of the group
of non-motile bacilli, is particularly interesting, the more so that for
some time the researches on acquired immunity have been concentrated
almost entirely on the analysis of the facts observed in animals that
have been vaccinated with the two Pasteur vaccines. In this way a large
number of valuable facts have been collected; of these the more
important may be presented to the reader.
[Sidenote: [252]]
In my first work on this subject[344] I called attention to the fact
that in the rabbit vaccinated against anthrax, the bacilli, when
inoculated subcutaneously, soon become the prey of leucocytes which
accumulate at the spot menaced. In the unvaccinated control rabbits,
however, the anthrax bacilli remain in a free state in the fluid of the
subcutaneous exudation, only a few isolated rods being found inside
phagocytes. I have since been able to confirm this fact[345], which must
now be regarded as fully established. In the vaccinated rabbits the
leucocytes exhibit a very marked positive chemiotaxis against the
anthrax bacilli, whilst in normal unvaccinated rabbits the chemiotaxis
of the leucocytes in the anthrax of the subcutaneous tissue is
distinctly negative. When a small quantity of anthrax culture is
inoculated subcutaneously into vaccinated and into unvaccinated rabbits
there may be observed, even within a few hours, a very great difference.
In the former there is found at the seat of inoculation an infiltration
which swarms with leucocytes in the act of devouring bacilli. In the
normal, susceptible rabbit, on the other hand, the exudation produced is
soft, rich in fluid, and very poor in leucocytes. The vessels in the
vicinity are distended with blood, and the fact that the leucocytes do
not come up to the seat of inoculation is in no way due to the absence
of vascular dilatation which might prevent diapedesis. The vessels are
much more dilated than in the vaccinated rabbit, and yet in the latter
the emigration is incomparably greater. This essential difference must
be attributed to the sensitiveness of the leucocytes, which exhibit a
negative chemiotaxis in the normal rabbit but a very marked positive
chemiotaxis in the vaccinated rabbit.
It has been shown repeatedly that the subcutaneous exudation, very rich
in leucocytes which have had time to ingest all the bacilli, when
inoculated into guinea-pigs, ensures the appearance in them of a
generalised and fatal anthrax; this affords evidence that the
phagocytosis is exercised against virulent and therefore living bacilli.
Marchoux[346], in Roux’s laboratory, has carried out numerous
experiments on the vaccination of rabbits and has observed that the
inoculated anthrax bacilli cause an exudation very rich in leucocytes,
and that these cells ingest and destroy the bacilli. The phagocytes
easily rid the refractory animal of the bacilli in the vegetative state,
but the spores are much more resistant. After being devoured by the
leucocytes they may remain inside them for months without germinating.
Marchoux obtained cultures of anthrax from the subcutaneous exudation
taken from vaccinated rabbits 70 days after inoculation.
[Sidenote: [253]]
The fact that the bactericidal action of the blood serum on anthrax
bacilli is specially well marked in the rat, suggested the idea of
trying to obtain, in this rodent, an augmentation of this property as a
result of vaccination. Sawtchenko[347] attempted to do this in an
investigation already cited in Chapter VI, carried out in my laboratory.
He succeeded in thoroughly vaccinating white rats against virulent
anthrax and in showing that the blood serum of these animals rendered
refractory “is bactericidal in the same degree as that of non-immunised
rats.” In the vaccinated rats “the subcutaneous exudation was as free
from bactericidal substances as was the lymph of the control animals.”
Sawtchenko was unable to demonstrate any increase of bactericidal power
except in the peritoneal exudation of rats vaccinated by injection of
cultures into the peritoneal cavity.
In spite, however, of the absence of any increase in the bactericidal
property of the blood serum and of the subcutaneous exudation in
vaccinated rats, the cell reaction obtained in them is very different
from that met with in normal, susceptible rats. In a very short time (3
to 5 hours) after the subcutaneous injection of anthrax bacilli into the
control rats (susceptible), an evident oedema is produced; in the
vaccinated rat there is none. The exudation, not very abundant in the
latter, already contains a number of leucocytes which are actively
phagocytic, whilst in the control animal, examined simultaneously,
“leucocytes are rarely met with, and few of them contain bacilli.”
Later, the difference becomes still more marked. Pronounced oedema
occurs in the control animal, it is poor in leucocytes but rich in
bacilli, which continue to multiply; but “in the immunised rat, we find
not a clear exudation but a thick and purulent fluid, full of
leucocytes.” These cells devour all the bacilli; not a single one
remains free. “Even after 14 hours bacilli ingested by the leucocytes
are present and a culture of anthrax bacilli may be obtained from fluid
taken from the seat of inoculation. Further, guinea-pigs or rats, when
inoculated with a drop of this exudation (which contains no anthrax
spores), succumb to anthrax.”
[Sidenote: [254]]
Even before these researches on the immunity of rats had been carried
out, an attempt had been made to gain some idea of the differences
presented by the vaccinated fluids of animals as compared with those
presented by the fluids of control animals susceptible to anthrax. In
1886 I was able to demonstrate[348] that the anthrax bacillus develops
abundantly in the defibrinated blood of sheep that had acquired immunity
as the result of vaccination by Pasteur’s method. When these bacilli
contain spores and are inoculated into rabbits they rapidly produce a
fatal anthrax; but when no spores are present the injection of bacilli
does not produce a fatal disease, and such infection is well supported
by the rabbits. From this I concluded at that time that the anthrax
bacillus must, in the blood of the vaccinated sheep, undergo a real
attenuation in virulence, an interpretation which, as will be seen in
the next chapter, was found to be erroneous.
Nuttall[349] showed that the defibrinated blood of refractory sheep
acted as a nutrient medium for the anthrax bacillus. Making comparative
investigations, by the plate method, on the bactericidal power of the
blood of vaccinated and normal sheep, he observed that, in both cases,
there was, at first, a certain decrease in the number of bacilli sown,
more marked in the blood of the vaccinated than in that of the control
animals. Nevertheless, 8 hours after the commencement of the experiment
the anthrax bacteria had produced innumerable bacilli in the blood of
the refractory sheep. Nuttall satisfied himself that this feeble
bactericidal power was not to be compared with the very much greater
power of the blood of the rabbit, an animal specially susceptible to
anthrax.
More recently the properties of the serum of sheep which have been
vaccinated against anthrax have been studied very carefully by
Sobernheim[350]. He also was able to show that this serum allows of an
abundant development of the bacillus, and that, outside the animal, it
does not exercise any more appreciable bactericidal power than does the
serum of the normal sheep. The serum of the best vaccinated sheep was
found to be incapable of destroying even very small quantities of
anthrax bacilli. The only change that Sobernheim could make out was with
regard to the thickening of the bacterial membrane. This modification,
however, was not constant and could not be seen in the serum of certain
vaccinated sheep.
[Sidenote: [255]]
The serum of the sheep vaccinated by Sobernheim exhibited no increase of
agglutinative power as regards virulent bacilli. Gengou[351], however,
made it clear that repeated injections of cultures of the first vaccine
of Pasteur into dogs produced a marked augmentation of this
agglutinative power; but it was only produced when the attenuated
bacillus was used. The virulent anthrax bacillus, developed as isolated
rods, was not affected in the least by serum that was highly
agglutinative for the bacillus of the first vaccine. Gengou also made
the converse experiment with the serum of a dog into which he had
previously injected a number of virulent anthrax bacilli. The dog,
naturally refractory to anthrax, resisted the inoculation perfectly, but
its serum did not acquire any agglutinative power against the first
vaccine. He concluded therefrom that “the part played by agglutinins in
the defence of the animal must be regarded as extremely problematical”
(p. 339). On the other hand the phagocytic reaction in the vaccinated
sheep is always very pronounced and constant. Von Behring[352], in one
of his most recent publications, expresses the opinion that this example
of acquired immunity must be placed in the category of phagocytic
immunity.
In the group of bacilli, several examples of which we have studied, the
typhoid bacillus approaches still more closely to the vibrios and
spirilla in its relation to humoral properties. Here may be observed a
kind of attenuated Pfeiffer’s phenomenon and somewhat profound
modifications taking place under the influence of the serum of
vaccinated animals. The _Bacillus pyocyaneus_ is more resistant to the
injurious influence of fluids taken from immunised animals. This
resistance is still more marked in the bacillus of swine erysipelas and
again still greater in the anthrax bacillus. Whilst, however, these
properties of the fluids of the body are found to be very variable and
of unequal power, the phagocytic reaction is constantly manifested and
always very actively. The leucocytes which, in susceptible animals,
exhibit a very marked negative chemiotaxis or only a tardy and
incomplete positive chemiotaxis, have, in the vaccinated animal, this
positive susceptibility developed in a very high degree.
[Sidenote: [256]]
Before quitting the group of bacteria we must cast a glance at the
mechanism of acquired immunity against representatives of the group of
spherical micro-organisms. Amongst the cocci the streptococci have been
especially studied as regards this immunity. For long great difficulties
were encountered in vaccinating animals against these chain cocci, but
Roger[353], Marmorek[354], Denys and Leclef[355] overcame these
obstacles and succeeded in immunising the rabbit, one of the most
susceptible species, to their pathogenic action. More recently the
larger mammals, notably the horse, have been successfully immunised. A
certain number of important facts, the knowledge of which is useful to
complete the survey of the phenomena of acquired immunity, have thus
been collected.
[Sidenote: [257]]
Roger set himself to study the properties of the blood serum of rabbits
vaccinated against the streptococcus, and established the fact that this
fluid had not the slightest appreciable bactericidal action; the
streptococcus grew in it just as well as in the serum of fresh
unvaccinated rabbits. When, however, he injected cultures grown in the
serum of immunised animals into rabbits, these rabbits did not die and
presented only transient and insignificant lesions. From this fact Roger
concluded that there must be an attenuation of the streptococcus by the
immune serum, a view which was shared by several other observers. In
formulating this view, however, he had not taken into account the
possibility that this serum acted not upon the coccus that had developed
in it but upon the organism of the animal into which it was injected.
Bordet[356], indeed, was able to show that the streptococcus which grows
in the serum of immunised animals is in no way weakened in virulence.
When he took a race very virulent for the rabbit (Marmorek’s
streptococcus) and injected a minimal dose of a culture grown in the
serum of immunised animals, the rabbits died just as did the control
animals, because the amount of serum introduced was too small to exert
any influence. So also, when he filtered this culture and got rid of the
serum bathing the streptococci, it was found to be just as virulent as
that grown in the serum of susceptible unvaccinated animals.
In confirmation of the discovery made by Roger with the serum of
vaccinated rabbits, Bordet showed that the blood serum of horses highly
immunised against the streptococcus did not exhibit any bactericidal
action. Moreover, he found that this serum caused the development of
somewhat agglutinated streptococci and that it was capable of throwing
streptococci grown on the ordinary media into clumps. Summing up his
researches on the properties of this serum Bordet concludes that it
“causes no profound change in the streptococcus. The vegetative
character of the coccus is not appreciably diminished, and its
morphology remains the same save for certain variations in the length of
the chains. Even the agglutinative power, recognised in numerous serums
by recent researches, is, in the antistreptococcic serum, developed but
slightly” (p. 196).
More recently von Lingelsheim[357] has studied the properties of the
serum of animals which he had thoroughly vaccinated against the
streptococcus. He observed a certain slowing of the development of the
coccus in this serum as compared with the growth in cultures made in the
serum of normal, susceptible animals. But this retardation was slight
and transient, and exhibited itself especially in serums to which von
Lingelsheim, following Denys, had added leucocytes.
[Sidenote: [258]]
Von Lingelsheim also noted a certain degree of agglutination of the
streptococcus by the serum of vaccinated animals, although this was much
more feeble than in the case of the cholera vibrio or the typhoid
bacillus, when agglutinated by their corresponding serums. Speaking
generally, he regarded the direct action of the body fluids as
insufficient to bring about the rapid destruction of the streptococci in
the vaccinated organism. “Since the action of the bactericidal
substances is limited in time, the streptococci are able to adapt
themselves to these substances and recover their former energy. As the
phenomena of extracellular solution, of such a form as those observed
under the influence of the cholera antibodies, are absent in the case of
the streptococcus and as, on the other hand, a considerable ingestion of
these organisms by the leucocytes is observed ... we must seek in the
activity of these cells a second important element of the defence of the
animal organism” (p. 78).
[Sidenote: [259]]
To Salimbeni[358], who has carried out in my laboratory an investigation
on this subject, we are indebted for the most reliable information on
the phagocytic reaction in acquired immunity against the streptococcus.
He studied specially the phenomena in the subcutaneous tissue of a
horse, hypervaccinated against Marmorek’s streptococcus; this animal
received in all, at several injections, about five litres of living
culture. In spite of this refractory condition, an oedema at the point
of inoculation was soon produced; in this the micro-organisms remained
free and the leucocytes were sparse. But the cellular reaction, at first
insignificant, developed with great rapidity and many leucocytes,
amongst which the macrophages were much the more numerous, were
attracted. The phagocytosis was still delayed for some time, but it
continued to increase and 20 to 24 hours after the inoculation it was
complete. As soon as the phagocytosis was well established the oedema
began to disappear. In the thick exudation, containing a mass of
leucocytes, the macrophages are filled with a very large number of
streptococci packed together. These cocci develop inside the cells,
cause them to burst and again become free. A fresh arrival of
leucocytes, however, takes place, this time mainly microphages. These
microphages seize the free streptococci that have struggled so
victoriously against the macrophages; this second phagocytic phase is
final. The streptococci still remain alive inside the microphages for
some days, but ultimately are killed and digested by the phagocytes. At
a period when, 5 or 6 days after injection, insignificant or isolated
traces of streptococci are to be found in the microphages, the exudation
when sown in nutritive media still gives abundant cultures. The
incidents of this struggle between the streptococcus and the animal
organism demonstrate the important part played by the phagocytes. The
fact that the macrophages perish and allow the cocci to escape, clearly
proves that these cocci have been ingested alive and virulent, and
consequently that the fluid of the exudation was incapable of destroying
or even of attenuating them. The macrophages, also, were powerless to
bring about this result and the intervention of the microphages was
necessary to cause the disappearance of the cocci. It is, however,
always the phagocytes which ensure the final resistance of the animal.
In presence of these very precise results obtained from the work of
Salimbeni, a work which I followed very closely, the previous researches
by Denys and Leclef (_l.c._) made under less favourable conditions on
vaccinated rabbits are deprived of their importance. These observers
wished to get an idea of the difference between the reactions of the
animal organism (_a_) after the injection of streptococci into the
pleural cavity of immunised rabbits, and (_b_) after injection into that
of normal susceptible rabbits. They killed the inoculated animals and
found a very marked diminution of micro-organisms in the pleuritic
exudation of the former. This diminution could not be attributed to a
lysis of the streptococci by the body fluids, because there were never
any signs of such destruction. Nor could the phagocytosis, very feeble
at first, be considered as the cause of the disappearance of a large
number of the streptococci. Denys and Leclef put forward a third
hypothesis, which attributed this disappearance to the rapid resorption
by the lymph stream of the injected fluid containing the organisms.
Going over the record of their experiments it will be seen that in
vaccinated rabbits the quantity of pleuritic exudation was always very
much less than in normal rabbits. In presence of this feature there is
reason to ask whether, in the case of the streptococci, a large number
of these organisms were not fixed, along with the leucocytes, on the
walls of the pleura, as in guinea-pigs that are inoculated
intraperitoneally? Instead of being satisfied with merely examining the
fluid exudation, the surface of the pleura should have been scraped in
order to ascertain whether the phagocytic reaction was localised in this
region.
In any case such incomplete results on the active immunity of rabbits in
no way weaken the positive results obtained in the subcutaneous tissue
of the horse, in which the phagocytic reaction plays a really
preponderant part.
This example of the streptococci completes our series of bacteria in
which we have studied their relations with the properties of the animal
organism that has acquired immunity. We have still to see whether the
acquired immunity against micro-organisms of animal origin is subject to
the same law as that against bacteria.
[Sidenote: [260]]
For some years past a zealous study of the infectious diseases produced
by animal micro-organisms has been carried out. Besides malaria, which
occupies a most important position, attention has been directed to
certain diseases in domestic animals that are set up by endoglobular
haematozoa and by flagellata, and a fairly large number of accurate data
have been collected with regard to Texas fever and its parasite the
_Piroplasma bigeminum_, as well as upon the epizootic diseases due to
_Trypanosomata_ (Tsetse fly disease or Nagana, “Dourine,” etc.).
We are indebted to Smith and Kilborne[359] for the earliest information
concerning the acquired immunity of Bovidae against Texas fever. R.
Koch[360] then added some very precise observations on the immunity of
calves which had been inoculated with parasites attenuated in the body
of the tick (_Boophilus bovis_). Lignières[361], who devoted much
attention to this question in the Argentine Republic, has discovered a
sure method of vaccinating the Bovidae against the “Tristeza,” the local
name for Texas fever. He brought to Alfort specimens of attenuated
haematozoa, and in Nocard’s presence performed successful vaccination
experiments. Lignières is now engaged in devising a practical method of
ensuring immunity under the special conditions found in the home of the
“Tristeza.” Up to the present, however, there are no sufficient data as
to the mechanism of the acquired immunity in this case. We have fuller
information as to the essential phenomena observed in the organism of
the rat vaccinated against _Trypanosoma lewisi_. We owe to Mme. L.
Rabinowitsch and Dr Kempner[362] the first important data as to the
possibility of immunising white or piebald rats against the disease
produced by the flagellated infusorian. They noted that these animals
when inoculated with the blood of grey rats containing _Trypanosomata_
acquire a very transitory disease which, however, confers an immunity
against any subsequent infection. The flagellated organisms disappear
from the blood within a few weeks, after which fresh injections of these
parasites have no pathogenic effect.
[Sidenote: [261]]
Laveran and Mesnil[363] confirmed these observations, and in addition
made careful observations on the mechanism of this acquired immunity.
After making several inoculations with blood containing _Trypanosomata_
into white rats, they made a study of the properties of the blood serum
of these immunised animals. First they established the fact that this
serum exerts no microbicidal action on the _Trypanosomata_, but it
agglutinates them without, however, rendering them motionless:—“The
masses may be resolved into rosettes in which the _Trypanosomata_,
united merely by their posterior extremities, have their flagella free
and motile at the periphery.”
Laveran and Mesnil then studied the phenomena evolved in the refractory
organism. When injected into the peritoneal cavity of immunised rats the
_Trypanosomata_ are not acted upon injuriously by the body fluids. They
are, however, devoured by the leucocytes. Laveran and Mesnil thus
express themselves on this subject: “... we have demonstrated clearly
and repeatedly that the _Trypanosomata_ are ingested alive, perfectly
isolated and very motile, by phagocytes, and we have followed the
details of this process of ingestion which recalls that of the ingestion
of spirilla by the leucocytes of the guinea-pig. We consider, therefore,
that the immunity is phagocytic in character.”
[Sidenote: [262]]
The main facts on acquired immunity established in connection with the
most diverse micro-organisms, facts just described, may already be said
to lead to certain general conclusions. They indicate in the first place
that acquired immunity is accompanied by phenomena more complicated than
those observed in natural immunity. In the two categories of processes
observed in acquired immunity the phagocytic reaction is the only one
that can be said to be constant. We find it in those examples in which
the influence of the fluids of the body is most manifest, as in the
experimental cholera peritonitis of the guinea-pig, as well as in those
cases where the humoral action is most feeble, as in anthrax or in the
_Trypanosoma_ disease of rats. We have, however, still to establish the
relations that exist between phagocytosis and the part played by the
fluids of the immunised animal, in order that we may, as far as
possible, present a general picture of the inner mechanism of acquired
immunity against micro-organisms. To attain this result we must place
the reader in possession of further well-established facts, and we must
postpone its discussion to the following chapter, which will be entirely
devoted to the above-mentioned problem.
CHAPTER IX
THE MECHANISM OF ACQUIRED IMMUNITY AGAINST MICRO-ORGANISMS
Cytases and fixatives.—Only the latter are augmented in the immunised
organism.—Properties of the fixatives.—Difference between them and
the agglutinative substances.—The part played by the latter in
acquired immunity.—Protective property of the fluids of the
immunised organism.—Stimulant action of the body fluids.—The
protective power of serum cannot serve as a measure of acquired
immunity.—Examples of acquired immunity in which the serums
exhibit no protective power.—Phagocytosis in acquired
immunity.—Negative chemiotaxis of leucocytes.—Theory of
attenuation of micro-organisms by the fluids of immunised
animals.—Refutation of this theory.—Phagocytosis acts without
requiring any previous neutralisation of the toxins.—The origin of
the fixative and protective properties of the body fluids.—The
relation between these properties and phagocytosis.—The side-chain
theory of Ehrlich and the theory of phagocytes.
[Sidenote: [263]]
Whilst, in natural immunity against micro-organisms, humoral phenomena
play no prominent part, in acquired immunity these phenomena assume a
much greater importance. The bactericidal power of the fluids of the
body is, in natural immunity, reduced to a mere trace, for it has been
demonstrated that the power of normal serums to destroy bacteria
corresponds to no natural phenomenon of the living organism, but is
dependent upon the presence of cytases which have escaped from the
phagocytes at the time of the formation of the clot _in vitro_ and
separation of the serum. The presence of the fixative, that other
important element in immunity, has been demonstrated in the normal
fluids only in rare cases and in small quantity. The agglutinative
property of these fluids has likewise shown itself to be little
developed and without any importance in natural immunity.
[Sidenote: [264]]
[Sidenote: [265]]
In acquired immunity against micro-organisms, on the other hand, we find
that the bactericidal and agglutinative powers of the fluids of the body
are very greatly increased. With the discovery that the bactericidal
property was so highly developed in the serums of animals that had been
vaccinated against vibrios arose the belief in the acquisition of a new
and purely humoral property. R. Pfeiffer, especially, insisted on the
fundamental difference between the power of the serum of immunised
animals to transform the cholera vibrios into granules and the
corresponding property of normal serums. In the first case Pfeiffer’s
phenomenon exhibited marked specificity; in the second, it was much more
general. A normal serum transforms into granules, indifferently, vibrios
that are very distinct from one another; whilst the serum of an animal
vaccinated against a particular species or race of vibrios gives
Pfeiffer’s phenomenon with this species or race only. Bordet’s[364]
researches have definitely settled this question. This investigator has
shown that Pfeiffer’s phenomenon is produced, with all the usual serums,
by means of the same substances, the cytases (alexine, or complement of
Ehrlich). But in the serum of vaccinated animals there is added to these
cytases the fixative (sensibilising substance of Bordet, immunising body
or amboceptor of Ehrlich) which exhibits specific properties. Having
thus carefully distinguished the two substances that set up the granular
change in vibrios, Bordet shows that in vaccinated animals it is the
fixative which increases in quantity, whilst the cytase remains pretty
much in the same proportions as in the normal animal. He demonstrated,
in fact, that when we take a very small dose of the serum of a
vaccinated animal which by itself is incapable of transforming the
vibrios into granules, about the same quantity of immunised serum or of
normal serum must be added to it in order that Pfeiffer’s phenomenon may
appear. The quantity of cytase, that soluble ferment which is necessary
for the production of the phenomenon, is, therefore, about the same in
the serum of a normal animal as in that of a well-vaccinated animal.
Whilst the cytase does not increase as a result of vaccinal injections,
the fixative, on the other hand, becomes more and more abundant.
Consequently it is this second soluble ferment that impresses its
characters on the blood serum and on some of the other fluids of the
vaccinated animal. It has been pointed out in the preceding chapter that
the fixative is found in the fluid of the oedema of vaccinated animals,
although in less quantity than in their blood serum. It has also been
mentioned that no fixative is found in the aqueous humour of
well-vaccinated animals. It must be admitted that this ferment is not
inseparably bound to the cells which produce it, as is the case with the
cytases. I have already developed, at some length, the thesis that the
cytases remain, in the normal animal, within the phagocytes, and only
escape from them when these cells are destroyed, whether in the living
animal (during phagolysis) or outside the animal (during the preparation
of the serum). Gengou’s experiments with the plasma and the blood serum
of normal animals have completely confirmed the fundamental observations
that the cytases are not found free in the circulating blood. It is
evident that the same law applies also to an animal that has acquired
immunity. For this reason neither Pfeiffer’s phenomenon nor any
analogous process that demands the action of cytases is ever produced in
the anterior chamber of the eye, or in the subcutaneous tissue, or in
oedema either active or passive. Further, it is in virtue of this same
law that Pfeiffer’s phenomenon does not manifest itself even in the
peritoneal cavity or in the blood vessels of vaccinated animals in which
the phagocytes have been protected from phagolysis by previous
injections of various fluids (physiological saline solution, broth,
etc.). It would be very interesting to be able to demonstrate the
absence of cytases in the fluids of immunised animals by experiments of
the same order as those carried out by Gengou with the fluids of normal
animals, but the obstacles to the realisation of this postulate are too
great. We saw when discussing Gengou’s experiments that it is impossible
to obtain _in vitro_ a fluid identical with the plasma of living blood.
The greatest precautions in collecting the blood and in its after
treatment are insufficient to prevent coagulation taking place sooner or
later. It follows that, as there is always a considerable quantity of
free fixative in the plasma of immunised animals, an infinitesimal
quantity of microcytase, set free from the leucocytes, is sufficient for
the production of Pfeiffer’s or any other analogous phenomenon. There
must be a great improvement in the methods of preparation of plasmas
outside the body before it will be possible to undertake successful
researches on the above problem. For the present we must rest satisfied
with other proofs, already numerous and very demonstrative, of the
absence of free cytases in the normal plasmas of vaccinated animals.
[Sidenote: [266]]
The cytases being found in about the same quantity and presenting the
same properties in all animals that enjoy immunity whether natural or
acquired, it must be the fixative which specially distinguishes these
two categories of immunity. Now, the fixative is found in the serum of
perhaps all cases of acquired immunity. Bordet and Gengou have studied
it by the method already mentioned (Chap. VII.). A certain quantity of
micro-organisms of various species is introduced into the serum. If the
cytases, present in the serum when the experiment was commenced,
ultimately disappear from it, it indicates that this ferment has been
absorbed by the bacteria, thanks to the fixative, which consequently
should be found in the serum under observation. The presence or absence
of the cytases can be demonstrated by the production or absence of
Pfeiffer’s phenomenon with vibrios.
The application of this method enabled Bordet and Gengou[365] to satisfy
themselves that the serum of animals immunised against several species
of bacteria (plague bacillus, typhoid bacillus, bacillus of swine
erysipelas, first anthrax vaccine, and _Proteus vulgaris_), really
contains an appreciable quantity of fixative. It may, then, be accepted
that the production of this substance is fairly constant in acquired
immunity against bacteria, and that it constitutes one of the most
important factors in such immunity.
The question has been raised: What is the nature of the substance to
which the name of fixative is given? Pfeiffer and Proskauer[366] have
attempted to solve this question by making use of a serum which acts
against the cholera vibrio and which they obtained by vaccinating
animals with this vibrio. They carried out a long series of experiments
which led them to the conclusion that this substance, which they term
“cholera antibody,” cannot be identified with any of the albuminoid
substances of the serum. Further, the fixative is not represented by any
of the salts or extractive substances of the serum, because these
substances dialyse easily, whereas the cholera antibody does not pass
through the dialysing membrane. The fixative is wholly precipitated by
alcohol, and is regarded by Pfeiffer and Proskauer as belonging to the
category of soluble ferments, an opinion which is certainly shared by
many other investigators.
[Sidenote: [267]]
[Sidenote: [268]]
What is it that communicates to this ferment its remarkably specific
character? Without being able to give a precise answer to this
question, the authors just cited point out the analogy that exists
between the cholera antibody and the soluble ferments of yeasts which
have been studied by Emil Fischer. Some of these act only upon certain
special sugars in a manner equally specific. From a logical point of
view it might be permissible to attribute the specificity of fixatives
to something borrowed from the species of micro-organism that has
played a part in their production. It has long been recognised that in
old cultures of the cholera vibrio these micro-organisms are
transformed into spherical granules, the arthrospores of Hueppe, which
closely resemble the granules produced in Pfeiffer’s phenomenon. There
are, then, undoubtedly, vibrionic products which act much as do the
microcytases, and it would be very interesting if we could find them
in the bactericidal ferments of the animal body. An attempt of this
kind was undertaken by Emmerich and Löw[367], who attribute the
acquired immunity to a particular substance which they term
“Nuclease-Immunproteïdin.” According to their hypothesis the microbial
products which are produced in the animal during the period of
vaccination—the nucleases—combine with proteid substances of the blood
and organs to furnish the substance to which these authors have given
such an elaborate name. In their most recent publication Emmerich and
Löw even describe a method of producing this substance outside the
animal body, by the action of ox blood, or better still pounded
spleen, on the nuclease produced by the bacteria found in old
cultures. To it they attribute the property of dissolving the various
bacteria, of conferring immunity against and even of curing several
infective diseases. But these authors do not say whether this
remarkable substance is identical with, or analogous to, the
antimicrobial ferments composed, as we have seen, of microcytase and
fixative. It must be concluded that they look upon it as being similar
to the alexine of Buchner, which is nothing more than a mixture of the
two substances just named. Unfortunately the whole account given by
Emmerich and Löw will do anything but gain over the reader, and in
their publications no proof of their assertions can be found. Several
of the facts advanced by them do not fall in with well-established
data. Thus they speak of the complete lysis of the bacilli of swine
erysipelas by their soluble “Erysipelase-Immunproteïdin” in vaccinated
animals, a process that has never been demonstrated by them and which
in no way accords with conscientious and carefully carried out
observations. On the other hand, they cite facts which contradict one
another. The “Pyocyanase-Immunproteïdin” is a substance which
possesses an extraordinary bactericidal power, not only against the
_Bacillus pyocyaneus_ but also against several other bacteria, _e.g._
the bacilli of anthrax, diphtheria, typhoid, and plague. This
substance rapidly breaks up these bacteria, and cures diphtheria and
experimental anthrax. But it is, at the same time, so affected by the
invasion of the most common bacteria, such as _Bacillus subtilis_,
that it is necessary to add antiseptics in order to preserve it. To
these contradictions, inaccuracies, and uncertainties must be added
further the advice, given by Emmerich and Löw to bacteriologists, not
to attempt to reproduce their experiments, because they may easily
fail, and I think that, in spite of the seductiveness of the attempt
to attribute to bacterial products a share in the elaboration of
antimicrobial substances, we must conclude not to follow these authors
further. It is better to confess our ignorance of the chemical
composition of these substances in general and of the fixatives in
particular.
[Sidenote: [269]]
As the fixatives resist temperatures much higher than those which
destroy the cytases, in this respect resembling the agglutinative
substances so frequently found in the fluids of vaccinated animals,
there has long been a tendency to identify them with these latter. It is
indisputable that between the fixatives and the agglutinative substances
the analogies are fairly numerous. Both are produced in quantity during
the process of immunisation, and are found not only in the blood serum
but also in the fluids of the living animal, especially in the fluids of
exudations and transudations. Both dialyse through parchment more
readily than do the cytases. Buchner[368] has demonstrated that his
alexines (bactericidal substances of normal serum) will dialyse only
where the lower fluid is pure water; dialysis is _nil_ when the
distilled water is replaced by physiological saline solution. The
fixatives and agglutinins, as demonstrated by Gengou[369] for the
latter, pass almost completely through the dialyser in the case of pure
water, and one-half still passes when the lower fluid approaches as
nearly as possible to normal serum.
In spite of these analogies, however, the agglutinative property must be
sharply distinguished from the fixative power of serums. In this fluid,
derived from normal animals, the agglutinative property is often very
marked when the power of fixing the cytases is totally, or in great part
absent. Bordet and Gengou[370] have demonstrated also that feebly
agglutinative serums of persons convalescent from typhoid fever may
exhibit a great capacity for fixing the cytases. Other facts, to be
mentioned later, confirm the real difference between the fixative and
the agglutinative properties.
[Sidenote: [270]]
The agglutination of bacteria was noted during the course of a series of
researches on the acquired properties of the blood serum of vaccinated
animals. Charrin and Roger[371], seeking to obtain a clear idea of the
difference between the serum of normal animals and that of animals
vaccinated against the _Bacillus pyocyaneus_, observed that this
bacillus developed in the normal fashion in the former, but in the
latter gave rise to special forms of growth. Instead of growing in the
form of rods, it elongates into segmented filaments which become
entangled and fall to the bottom of the tubes, leaving a supernatant
limpid serum. I was able not only to confirm the accuracy of this
observation for the _Bacillus pyocyaneus_, but to extend it to
Gamaleia’s vibrio and to the pneumococcus[372]. In all these instances
we have a modification of the bacteria developed in specific serums
coming from vaccinated animals. Later, Bordet[373], during his
researches on the bacteriolysis of vibrios _in vitro_, observed that
these vibrios, when introduced into the blood serum of vaccinated
animals, lose their movements and soon unite into more or less
voluminous masses. This observation was confirmed by Gruber and
Durham[374], who were the first to apply it in the specific diagnosis of
bacteria. They showed that the agglutinating power of vaccinated
animals, although not rigorously specific, might, nevertheless, be
utilised for the differentiation of certain bacteria, especially the
cholera vibrio and the typhoid bacillus. But, independently of this
result, Gruber[375] essayed to formulate a theory of acquired immunity
based on the agglutinative property of the serum. He accepted, in
connection with the phenomenon of the destruction of the bacteria,
Bordet’s hypothesis of the concurrent action of two substances, of which
one, the bactericidal substance proper, is nothing but the alexine of
Buchner, the second being that which agglutinates the bacteria. This
agglutination, according to Gruber, results from the swelling of the
bacterial membrane which becomes viscous and so leads to the cohesion of
the bacteria and the formation of clumps. Thus transformed and rendered
motionless, the bacteria succumb more readily to the destructive action
of the alexine. It is supposed that the phagocytes do not intervene at
all in these cases of acquired immunity, except in a purely secondary
fashion when they ingest the bacteria already greatly weakened by the
united action of the agglutinin and the alexine. The principal _rôle_ in
this theory of immunity is thus given to the agglutinative substance,
which is regarded as being a microbial product, modified by the
macrophages and thrown into the blood.
[Sidenote: [271]]
The discovery of this agglutination of bacteria has acquired great
importance, especially in connection with its application to the
diagnosis of typhoid fever. Widal[376] succeeded in showing that typhoid
bacilli agglutinate readily under the influence of blood serum and other
fluids (milk, transudations, tears, etc.) derived from patients
suffering from typhoid fever. As this phenomenon could be utilised for
the early recognition of the disease, it began to be studied with great
care and many interesting data concerning it have been collected. The
general outcome of these researches accords with the conclusions drawn
by Widal, and the serum-diagnosis of typhoid fever has taken an
important place among the methods used for the recognition of this
disease. This aspect of the question, however, does not interest us from
the point of view of the problem of immunity which we now have under
consideration, and we cannot here enter upon the study of the
serum-diagnosis of typhoid fever and certain other diseases (cholera,
tuberculosis, pneumonia). Moreover, we must refrain from any analysis of
the hypotheses advanced to explain the mechanism of agglutination. A
lively discussion has been carried on between the partisans of the
chemical theory—according to whom the agglutinin acts directly on the
agglutinable substance of the bacteria—and the advocates of the physical
theory, led by Bordet[377], who attribute the agglutination to
modifications in the molecular attractions which unite the agglutinable
elements, be it between each other or with the surrounding fluid. At one
time it was thought that Roger’s[378] observation that the cell
membranes of _Oïdium albicans_, when cultivated in the specific serum of
immunised animals, increased in volume and became greatly swollen,
settled the question in favour of Gruber’s theory. But the objection
formulated by Kraus and Seng[379], on the one hand, and by Bordet, on
the other, dealt a severe blow to this view. As the serum employed by
Roger was not deprived of its cytases (alexine), the viscosity of the
membrane of the fungus could not be attributed to the agglutinin. When
Bordet[380] demonstrated that the red blood corpuscles, under the
influence of the serums, undergo an agglutination as marked as that seen
in bacteria, it enabled us to study this phenomenon in the large red
corpuscles of birds, in which no one has ever been able to demonstrate
any viscosity of the corpuscular stroma. In a mixture of red corpuscles
of bird and mammal, submitted to the action of a serum which
agglutinates the former only, the red corpuscles of the mammal never
unite with those of the bird, although this should undoubtedly take
place if the membrane of the agglutinated corpuscles had really become
viscous. All the facts collected up to the present are, therefore, in
favour of Bordet’s physical theory in which an analogy between the
phenomena of agglutination and of coagulation is traced.
[Sidenote: [272]]
The point that interests us more particularly in regard to agglutination
is the relation of this phenomenon to immunity. We have already given
(Chapter VII) the arguments which render it impossible for us to
attribute to the agglutinative property of the fluids of the body any
_rôle_, however unimportant, in natural immunity against
micro-organisms. We must now study the importance of this property in
the condition of acquired immunity, in which the agglutination of
micro-organisms by the fluids of the body is much more frequent and
active than in natural immunity.
[Sidenote: [273]]
The first question to be settled is the following: Is the agglutinative
property really constantly present in the fluids of vaccinated animals?
The blood serum of animals that have acquired immunity is unquestionably
usually agglutinative as regards the corresponding micro-organism. This
agglutination may be more or less pronounced, but it certainly exists in
the great majority of cases. Nevertheless, examples can be cited in
which, in spite of the refractory condition acquired as the result of
immunisation, the serum exhibits not a trace of agglutinative power.
Having demonstrated that several bacteria (_Bacillus pyocyaneus_,
_Diplococcus pneumoniae_, _Vibrio metchnikovi_) develop in the serum of
vaccinated animals in the form of elongated filaments more or less
interlaced, I was quite prepared to allow that this fact might be of
general import. But the study of a cocco-bacillus which produces the
pneumo-enteritis of swine and which was isolated by Chantemesse during
an epizootic at Gentilly, led me to believe that this was not the case.
As this bacillus is characterised by great motility, I concluded[381]
that it was identical with that of the hog cholera of American writers.
Theobald Smith[382], to whom I sent a specimen and who is a competent
authority on this question, refers it, however, to the species which
produces swine plague. Knowing that the question of these two bacteria
is not finally settled, it is impossible to come to an absolute decision
in the matter. Fortunately, from the point of view of immunity, this is
of no great importance. The point upon which I must lay stress is that
the serum of rabbits vaccinated against the Gentilly bacillus, when sown
with this cocco-bacillus, gave very abundant and uniformly turbid
growths. In my researches, undertaken at a period when the rapid
agglutination of micro-organisms added directly to the specific serum
had not yet been recognised, I noted merely that the cocco-bacilli which
grew in the blood serum of vaccinated rabbits presented their normal
form and gave rise to a general turbidity of the fluid. Since then,
however, it has often been observed that the mode of development of a
micro-organism in a serum gives an even more delicate indication than
does the agglutination properly so called, produced by the serum to
which has been added an organism cultivated on its usual medium. Thus
Pfaundler[383] saw that _Bacillus coli_ and _Proteus vulgaris_, which
were not agglutinated by certain serums, developed in them in an unusual
fashion and produced very long and interlacing filaments. When a serum
is incapable of revealing its properties by agglutinative reaction
properly so called, it is sown with the corresponding micro-organism and
the development is then compared with that observed in a normal serum.
Frequently a very marked difference is noted, the same organism growing
into filaments in the specific serum and forming rods only in the normal
serum. The first mode of development is sometimes designated
“Pfaundler’s reaction.”
In the serum of rabbits vaccinated against the Gentilly cocco-bacillus,
no filaments corresponding to those met with in the agglutinative
reaction are formed, but bacilli are produced. In spite of this the
animals that furnish the serum show a distinct resistance to infection.
More recently, Karlinski[384] has studied the properties of the serums
of animals treated with the cocco-bacilli of hog cholera and swine
plague. He was able to demonstrate that blood serum from oxen that had
received repeated injections of cultures or toxin of hog cholera, was
not only incapable of killing the cocco-bacilli of the two swine
diseases but it even “gave rise to no agglutination” of the two bacilli
and did not arrest the motions of those of hog cholera. On the other
hand, serums have been obtained from other species of animals (dog, pig)
which brought about the typical agglutination of the cocco-bacillus of
hog cholera[385].
[Sidenote: [274]]
In the preceding chapter, Gengou’s experiment on the serum of a dog that
had been treated with a virulent culture of anthrax has already been
cited. This serum did not agglutinate the bacillus, even of the first
vaccine of Pasteur. Nevertheless, a second dog treated with an
attenuated culture of this bacillus furnished an agglutinative serum.
The immunisation of the first dog was carried very much further than
that of the second, but the agglutinative properties were in inverse
order. Sawtchenko, in his study of immunity against anthrax,
demonstrated that the subcutaneous exudation from vaccinated rats does
not agglutinate the bacillus which usually exhibits such a great
tendency to collect into clumps.
Agglutination has been studied particularly carefully in typhoid fever.
We know that after an attack of this disease, an acquired refractory
condition is produced which lasts for a considerable period. In most
cases the agglutinative power of the blood diminishes very rapidly, and
disappears a few weeks after the commencement of convalescence. It is
only in rare cases that it persists for years[386]. On the other hand,
during the period of apyrexia which precedes the relapse in typhoid
fever and during the period of relapse, the agglutinative power may
manifest itself in a very marked degree. In an observation made on a
case reported by Widal and Sicard[387], the agglutinative power was
raised, two days before the relapse, to a ratio (1 : 150) it had never
attained during the first attack. “The appearance of the relapse, two
days after this observation”—these authors add—“renders it evident that
the agglutinating reaction is independent of the state of immunisation.”
Analogous cases have been pointed out repeatedly by several observers.
[Sidenote: [275]]
The examples cited show, on the one hand, that the serum of individuals
endowed with acquired immunity may be without any agglutinative
property, but, on the other, that this power may be highly developed in
the serum of susceptible individuals. The argument based on these data
may be corroborated by several other series of facts. Thus,
Salimbeni[388] has pointed out that the cholera vibrio is not
agglutinated in the fluids of immunised animals. The subcutaneous
exudation of a horse treated with a large quantity of these vibrios does
not agglutinate Koch’s vibrio except outside the body. When this
exudation is drawn off shortly after the injection of the vibrios, the
organisms render the fluid uniformly turbid. But a short exposure to the
air is sufficient to bring about the agglutination of the vibrios in the
same exudation. Guided by this observation, Salimbeni carried out
comparative experiments on the action of the serum of vaccinated animals
outside the body, in tubes deprived of oxygen and in others exposed to
the air. In the former agglutination did not take place or was very
incomplete, in the latter it soon came on. This fact accords perfectly
with the observation of Pfeiffer’s phenomenon in the peritoneal cavity
of guinea-pigs from which we withdraw a fluid containing granules that
have resulted from perfectly isolated vibrios. In other micro-organisms
a difference has been noted in this respect. Thus Gheorghiewsky has seen
the agglutination of the _Bacillus pyocyaneus_ produced under the
influence of the serum of vaccinated animals, even in tubes deprived of
oxygen. Durham has made a similar observation in the case of the typhoid
bacillus. When, however, Trumpp[389] wished to satisfy himself as to the
agglutination of the same organism in the body of well-vaccinated
guinea-pigs, he obtained only imperfect results. He concluded from his
experiments “that the formation of typhoid clumps may precede the
breaking down of the bacteria in the animal body itself, but only under
certain conditions—when the degree of immunity of the animal is
sufficiently high and when the bacilli introduced are not too numerous”
(p. 130). In the case of the typhoid bacillus, a certain degree of
agglutination is produced inside the animal body, but it is markedly
increased in the fluids that have been withdrawn and exposed to the
action of the air.
[Sidenote: [276]]
It has been demonstrated, repeatedly, that the agglutination of
micro-organisms by their specific serums does not prevent their growth
and multiplication. These agglutinated organisms lose none of their
virulence. Issaeff[390], working in my laboratory, carried out an
investigation on this point in the case of the pneumococcus. He
vaccinated rabbits against this organism and satisfied himself that the
organism still grows well in the blood serum of such rabbits; but,
instead of presenting the typical form of lanceolate diplococci, the
pneumococcus, under these conditions, forms very long chains of true
streptococci. Having filtered the cultures in order to get rid of the
serum, he injected them into rabbits and mice and demonstrated that the
pneumococci had retained to the full their initial virulence.
Sanarelli[391] carried out corresponding experiments with Gamaleia’s
vibrio, which, as we know, also forms chains in the serum of vaccinated
animals. When filtered on a paper filter and washed with physiological
saline solution, the vibrios were found to be just as virulent as were
the control vibrios grown in the serum of susceptible animals. More
recently, Mesnil[392] demonstrated the same point in connection with the
bacillus of swine erysipelas. He experimented on cultures that were
agglutinated after their formation and also on others agglutinated as
they were growing. The fluid of the culture was decanted and replaced by
fresh broth until the elimination of the serum was complete. Mice,
inoculated with the washed clumps, died in the normal period, thus
affording proof that “agglutination in no way alters the vitality and
virulence of the bacillus of swine erysipelas” (p. 492).
We can readily understand, after the demonstration of these various
facts, that it is impossible to maintain Max Gruber’s theory that the
agglutinative power constitutes the fundamental basis of acquired
immunity. Hence this writer, after publishing several preliminary notes
in 1896, has not yet decided to give to his hypothesis a more extended
development. Nor has any one else attempted to defend it.
[Sidenote: [277]]
It is probable that in certain special cases the immobilisation of very
motile bacteria and their agglutination into clumps may facilitate the
reaction of the animal organism, especially the rapidity of
phagocytosis. Thus, Besredka[393] observed that guinea-pigs when
inoculated with typhoid bacilli that had previously been mixed with the
blood serum of normal animals survived. The most active amongst these
serums was ox serum heated to 60° C. Guinea-pigs furnished a serum which
was much less active. The resistance of guinea-pigs, inoculated into the
peritoneal cavity, was in direct ratio to the agglutinated condition of
the bacilli. Besredka lays stress on the facility with which the
bacilli, when agglomerated into large clumps, were ingested by the
phagocytes, and suggests that there is a certain stimulating action of
the serums on the leucocytes. When he injected into guinea-pigs a
mixture of typhoid bacilli and guinea-pig’s serum, made immediately
before injection, his animals died from infection. But when he left the
bacilli for some time in contact with the guinea-pig’s serum outside the
body, and did not inject the mixture until after agglutination was
complete, the inoculated animals usually survived. This experiment
indicates the part played by agglutination in the resistance offered by
the animal, and at the same time proves that in the body of the
guinea-pig the agglomeration of the micro-organisms into clumps does not
take place to the same degree as in the serum prepared in, and left in
contact with, the air.
In any case, the data collected by Besredka cannot be put forward as an
argument in favour of the essential part played by agglutination in
acquired immunity, nor can they weaken the facts indicated as to the
absence of agglutinative power in examples of acquired immunity and as
to the virulence of the agglutinated micro-organisms. The part played by
agglutination in this immunity is merely accidental and subordinate.
[Sidenote: [278]]
Special researches have been carried out with the object of defining,
exactly, the origin of agglutinins in the body of an animal that has
acquired immunity. Observers are unanimous in recognising that, of all
parts of the organism, the blood is richest in agglutinin. This
substance is found in the blood serum as well as in the plasma. From
this (corroborated by the agglutinative property of other fluids, such
as the pericardial fluid, oedemas very poor in formed elements, etc.) it
follows that the agglutinin circulates in the blood and lymph of the
living animal. Several observers, amongst whom I may cite Achard and
Bensaude[394], Arloing[395], and Widal and Sicard[396], put to
themselves the question whether, before passing into the blood, the
agglutinin is not formed in the exudation developed at the seat of
inoculation of the micro-organisms. Their conclusions were invariably
negative; they were never able to find more agglutinins in these
exudations than in the blood. Pfeiffer and Marx[397] had occasionally
observed that their animals, inoculated with the cholera vibrio, early
exhibited an agglutinative power in the spleen; but this result was not
met with sufficiently constantly to enable them to draw a positive
conclusion. A little later, van Emden[398] studied in detail the
distribution of the agglutinative property in the body of an animal
inoculated with _Bacillus aërogenes_. His researches led him to the
conclusion that the spleen and the lymphoid organs must be regarded as
the source of the agglutinins. Shortly after the inoculation of the
bacilli, an extract of the spleen was more agglutinative than the blood
or any of the other organs. In rabbits from which the spleen had been
removed, the same _rôle_ was filled by the bone marrow and probably also
by the lymphatic nodules. But this preponderance of the haematopoietic
organs did not continue long, the blood soon becoming the most important
seat of the agglutinative power.
The proof that this question of the origin of the agglutinins is a very
delicate and difficult one is afforded by an investigation very
carefully carried out by Gengou[399] on the agglutination of the
attenuated anthrax bacillus (Pasteur’s first vaccine) by the fluids and
organs of normal and prepared guinea-pigs. This observer was never able
to obtain any confirmation of the results obtained by van Emden with
another micro-organism. In Gengou’s guinea-pigs it was always the blood
fluid which showed itself most agglutinative, the organs exhibiting
merely a feeble and inconstant agglutinative power. As the extracts of
leucocytes were always found to be markedly less active than the blood
and the fluids of the exudations, Gengou was obliged to come to the
conclusion that the agglutinins cannot be regarded as products of the
cells of the animal body; this he sums up by saying that “in the
increase of the agglutinative power of its blood the organism of the
animal plays only a relatively passive part” (p. 337).
[Sidenote: [279]]
I think that, in spite of the facts established by Gengou, his
conclusion can scarcely be regarded as final. The agglutinative
property, developing in the animal body, must be attributed to some
cellular influence, because we know that the prolonged sojourn of
micro-organisms in the animal fluids is incapable of conferring on them
this power. As Gengou’s experiments did not permit him to attribute the
formation of agglutinin to any formed element, it must be concluded
that, although perfectly exact, they were insufficient to solve the
problem. Gengou killed his animals at a stage when their blood was
already pretty strongly agglutinative. At this stage the organs only
possessed it to a much more feeble degree. Perhaps, if he had examined
his animals at an earlier stage, when the blood possessed a much less
marked agglutinative power, he might have obtained a more powerful
agglutination with an extract of the organs. In my researches on the
resorption of cells, I observed, on several occasions, that the
abdominal fluid of guinea-pigs which had received an injection of
goose’s blood became agglutinative before the blood serum. Later,
however, the blood exhibited a greater agglutinative power than did the
peritoneal fluid. If to this fact we add the results of van Emden’s
experiments, we shall be tempted to assign to the cells found in the
peritoneal exudation and in the lymphoid organs a share in the
production of the agglutinin. This question of the origin of the
agglutinative power is, however, a very difficult one, and it is
impossible, in the imperfect state of our knowledge, to express oneself
in a more positive fashion. Fortunately, according to the whole of our
data on this phenomenon, the part played by agglutination in immunity
can only be very inconsiderable, and we may be allowed to consider our
general problem without concerning ourselves over much about the origin
of the agglutinative property.
Among the definite results obtained from the study of the agglutinins,
it may be specially pointed out that these substances can in no way be
identified with the fixatives. These latter were, for long, spoken of as
_preventive substances_. They are so termed in the early papers of Jules
Bordet treating upon this question. The explanation of this designation
is that, for a series of years, the presence of the fixatives was
revealed chiefly by the preventive or protective property of the media
which contained them.
[Sidenote: [280]]
To gain a clear conception of this protective property, which occupies
so important a place in the study of acquired immunity, we must go back
to an epoch in our science when it was sought to prove that the fluids
of the body played a part in the production of immunity. Shortly after
the earliest researches on the bactericidal power of the blood had been
made, the idea of applying the results obtained in this direction to the
production of immunity in animals by means of injections of blood
occurred. The first step in this direction was taken by Richet and
Héricourt[400], who succeeded in vaccinating rabbits against a variety
of staphylococcus by means of defibrinated dog’s blood. The dog is
naturally refractory against this organism, and the blood of a normal
dog exercised a certain vaccinal or protective influence on rabbits
inoculated with the staphylococcus. But this action was much more marked
when Richet and Héricourt employed the defibrinated blood of dogs which
had previously received inoculations of the staphylococcus. Shortly
after this observation, von Behring[401] made his discovery of
antitoxins in the blood serum of animals immunised against tetanus and
diphtheria toxins. In collaboration with Kitasato he demonstrated that
the serum of these animals, when injected into normal animals, protected
them against intoxication by the poisons of diphtheria and tetanus. This
great discovery, which has been confirmed on all sides and extended to
other poisons, gave rise to the view that a serum exerting any
protective power depends solely on its property of impairing the action
of the toxins. A more careful study of the phenomena which appear under
the influence of the serums has, however, demonstrated the inaccuracy of
this view. I was able to furnish the proof[402] that the blood serum of
rabbits vaccinated against the micro-organism of the Gentilly
pneumo-enteritis prevented normal rabbits from contracting a fatal
infection. Nevertheless, the serum exerted no influence on the toxin of
this micro-organism; the rabbits that received the minimal lethal dose
of this toxin, mixed with serum from vaccinated rabbits, died, as did
the control animals, from rapid poisoning. It was evident then that this
serum, which prevented infection without in any way hindering
intoxication, could not be classed in the category of antitoxic serums.
We find ourselves, therefore, in the presence of a new property of the
fluids of the body to which we have given the name of _protective_ or
_anti-infective power_. We are driven to this conclusion the more as the
serum in question was neither bactericidal nor agglutinative.
[Sidenote: [281]]
This discovery was soon confirmed by R. Pfeiffer[403] for the cholera
vibrio. Animals vaccinated against this organism furnished Pfeiffer with
a serum which, whilst not at all antitoxic, was distinctly protective
when injected into normal guinea-pigs. It protected these animals from a
fatal infection by the vibrio and, when injected into the peritoneal
cavity, it set up the granular transformation of the cholera
vibrios,—Pfeiffer’s phenomenon. Pfeiffer, for this reason, gave to the
protective antivibrio serum the name of bactericidal serum. As the
granular transformation was produced, under the influence of this serum,
with cholera vibrios only and never with other species of vibrio,
Pfeiffer gave to the active substance in the serum the name of _specific
cholera antibody_. This substance, according to his theory, was formed
in the animal body at the expense of an inactive antibody which became
transformed into an active substance under the influence of the
peritoneal endothelium.
The possibility of thus vaccinating susceptible animals by means of the
serums of immunised animals, quite apart from any antitoxic power, was
easily confirmed and extended to several other infective diseases.
Pfeiffer and Kolle[404], Funck[405], Chantemesse and Widal[406]
demonstrated it in connection with the experimental disease produced in
animals by the typhoid bacillus; Loeffler and Abel[407] for the
_Bacillus coli_, etc. The protective or anti-infective power of the
serum and other fluids of immunised animals was soon recognised as a
general property.
[Sidenote: [282]]
Pfeiffer and his collaborators, as well as many other investigators,
laid special stress on the bactericidal character of these protective
fluids. It was seen that the serums of immunised animals were often
almost or completely incapable of killing the corresponding
micro-organisms, but they were still regarded as bactericidal, because,
when injected into the peritoneal cavity of normal animals, they set up
the transformation of vibrios into granules, or, in the case of other
bacteria, determined certain phenomena of extracellular destruction.
Whilst carrying on researches in this direction, Fränkel and
Sobernheim[408] discovered a fact of great importance. They found that
the protective substance of the serum of animals vaccinated against the
vibrios resisted heating to 70° C. When submitted to the influence of
this temperature, the serum lost its bactericidal power completely, but
remained quite as protective as the unheated serum, when injected into
susceptible animals. This experiment, which has since been confirmed
repeatedly, furnished us with a means of separating the bactericidal
power from the protective power in cases where both were present in the
same serum. Later, in the hands of Bordet, it proved to be of great
service in connection with his researches on the concurrence of two
substances in acquired immunity.
The possibility of obtaining Pfeiffer’s phenomenon outside the body by
“reactivating” the protective serum with peritoneal fluid or blood serum
of normal unvaccinated animals has still further facilitated the study
of the action of the two substances in acquired immunity. It was with
the help of this method that Bordet was able to furnish so much valuable
information on the subject of anticholera serums and, later, on that of
haemolytic serums. The discovery by Ehrlich and Morgenroth[409] of the
fixation by the sensitive elements of the heat-resisting (thermostabile)
substance (that which resists a temperature of 65°–70° C.) constitutes a
new and important acquisition to the study of acquired immunity. The
discovery has been applied by Bordet to micro-organisms, and since then
it has been found possible to study much more precisely the mode of
action of specific protective serums.
[Sidenote: [283]]
Even before this last scientific advance had been made it was possible
to determine the relations between the protective power and the
agglutinative power of the fluids of animals that had acquired immunity.
Both resist about the same temperatures; both are found in the blood
plasma and pass into the fluids of exudations and transudations. But it
may be affirmed with certainty, as already stated, that the two
properties are quite distinct. Pfeiffer has laid great stress on the
fact that highly protective serums often exhibit only a feeble
agglutinative power and _vice versa_. During an investigation[410] into
an epidemic of typhoid fever, he had occasion to study the serum of
patients convalescent from this disease. The exact dosage of the two
properties demonstrated that a slightly marked agglutinative property
might be associated with a very powerful protective property.
Gheorghiewsky[411] made similar observations on animals vaccinated
against the _Bacillus pyocyaneus_. The serum of a goat, although more
agglutinative, invariably proved to be less protective than that of a
rabbit. A similar result was obtained with the serum of immunised
guinea-pigs. “This shows distinctly”—concludes Gheorghiewsky—“that the
property possessed by serums of agglutinating the _Bacillus pyocyaneus_
does not march parallel with the protective property” (p. 304).
Analogous examples are sufficiently numerous to justify us in accepting
the distinctiveness of the two properties of specific serums.
The protective or anti-infective substance is, therefore, not the same
as the agglutinin. But are we justified in regarding it as identical
with the fixative substance, or fixative (sensibilising substance,
immunising or intermediary substance, or amboceptor)? From the fact that
the fixative was at first rightly designated by Bordet as protective
substance we should conclude in the affirmative. The question is an
important one and merits close examination. The discovery of an exact
method of determining the presence of fixatives has rendered it possible
to ascertain whether these substances are always found in the protective
fluids and also whether the presence of fixatives necessarily implies
the protective power of the serums.
The first of these questions has been answered in the affirmative. All
the protective serums studied from this point of view, by Bordet and
Gengou, were found to be endowed with very distinct fixative properties.
They also found the specific fixative in the serum of guinea-pigs
immunised with the attenuated bacilli of the first vaccine of Pasteur.
Now this serum is powerless to prevent the production of fatal infection
in mice into which is simultaneously injected the bacillus of the first
vaccine. Consequently a fixative fluid is not necessarily protective.
This is in accordance with the fact that the micro-organisms that have
absorbed the fixative may, nevertheless, retain their virulence. We have
already cited the experiment of Mesnil that the bacilli of swine
erysipelas, mixed with the specific serum and then deprived of this
fluid, produce a fatal infection in mice. We have also drawn attention
to the fact, demonstrated by Sawtchenko, that anthrax bacilli, obtained
from the exudation of immunised rats, give rise to a fatal anthrax in
normal guinea-pigs and rats. The experiments of Bordet and Gengou proved
that there is absorption of the fixative substance by the bacilli of
swine erysipelas and of anthrax when placed in contact with the specific
serums of the immunised animals. In order that the protective power may
manifest itself adequately, therefore, besides the fixative substance,
some other factor capable of acting is also necessary.
[Sidenote: [284]]
In connection with my work on immunity against the micro-organism of
swine pneumo-enteritis I was able to demonstrate that the serum of
vaccinated rabbits, incapable of preventing the multiplication of the
specific cocco-bacillus, is also powerless to deprive it of its
virulence; it is without the power of causing its agglutination or of
neutralising its toxin. In short, this serum appears to exercise no
direct action on the micro-organism, yet, in spite of that, it prevents
its pathogenic action. With these results before me, I was led to assume
a certain stimulating action of the serum on the defensive elements of
the animal organism and especially on the phagocytic system. The
discovery of the fixative property of serums would lead us to believe
that this stimulation was entirely useless, and that the permeation of
micro-organisms by the fixative was amply sufficient to bring about
their destruction and removal from the animal. A living micro-organism
in its normal form, endowed with full virulence and provided with its
fighting weapon, the toxin, but at the same time permeated by the
fixative substance, might behave in the animal in some special way. It
might excite a strong positive chemiotaxis of the leucocytes and be
ingested and destroyed by these cells with greater facility. _A priori_,
there would be nothing to object to in this view, but certain facts are
opposed to it. Thus, in the case of micro-organisms just cited, we see
bacteria, permeated not only with the fixative but also with cytases,
capable of producing a fatal infection. We are thus compelled to accept
the theory of an influence of protective serums not only on the
micro-organisms but also on the organism of the animal into which they
are introduced. As this influence manifests itself in the form of a
strong phagocytosis, it is only natural that we should attribute it to
the existence of a _stimulating action_ of the serums of vaccinated
animals on the phagocytes of the normal animals. The detailed analysis
of the mechanism of the immunity acquired as the result of the injection
of these serums, as we shall attempt to prove in the following chapter,
in many cases confirms this view.
[Sidenote: [285]]
The important part played by the stimulation of the phagocytic reaction
in acquired immunity is supported by yet another series of facts and
from a different side. It has been clearly established that not only the
serum of immunised animals but also that of normal man and normal
animals, themselves susceptible to the pathogenic action of the
micro-organisms, protects the animal organism against infection. This
fact was first demonstrated in connection with researches on the
vaccination of guinea-pigs against the experimental peritonitis produced
by the cholera vibrio.
G. Klemperer[412] was the first to observe that the blood of several
individuals who had never had cholera was, nevertheless, in the case of
guinea-pigs, protective against peritoneal infection by the cholera
vibrio. He concluded therefrom that the individuals who had furnished
this protective blood possessed immunity against cholera. Soon
afterwards I[413] was able to extend analogous researches over a large
number of persons and to show that the protective power of the blood is
of very wide distribution in human beings. But, instead of assuming that
all these individuals, whose fluids protect the guinea-pig from
peritoneal infection, possess a natural immunity against cholera, I came
to the conclusion that the protective power of the blood cannot be taken
as a measure of the immunity of the individual from whom the blood was
drawn. Here again I assumed a stimulant action of the human blood on the
phagocytic reaction of the guinea-pig, looking upon it as quite natural
that the blood, capable of exciting the reaction in an alien animal,
might remain inactive in the body of the animal which furnished it.
[Sidenote: [286]]
R. Pfeiffer[414] has given much attention to the protective action of
serums; he has laid special stress on the essential difference between
the influence of normal serums and of those obtained from animals that
have acquired immunity. Whilst, in order to obtain a protective effect
with the normal blood or serum of man and animals, it is necessary to
inject a considerable quantity (from 0·5 c.c. upwards), the specific
serum, i.e. serum obtained from persons recovered from cholera or from
animals vaccinated against the cholera vibrio, is active in a very
minute dose. Sometimes the cholera peritonitis of the guinea-pig is
prevented by a fraction of a milligramme of such serum[415]. Based on
these facts, Pfeiffer has expressed the view that the normal serum acts
by stimulating the natural powers of defence of the animal, whilst the
specific serum exercises its influence in virtue of the property of
causing the formation of a special secretion which acts only against the
micro-organism which served for the production of the immunity. Pfeiffer
and his collaborators have demonstrated that normal serums are
protective, not only against the cholera vibrio, but also against
several other micro-organisms, e.g. the typhoid bacillus. One of his
pupils, Voges[416], believed that, in certain infections, the protective
power of normal blood might be greatly exaggerated, and that, in these
cases, the limit between the activity of normal and of specific serums
might be almost completely effaced. He affirmed, especially, that very
small doses (0·1 c.c.) of blood serum from a normal guinea-pig was quite
sufficient to prevent, in other guinea-pigs, a fatal infection by the
micro-organism of hog cholera and its allies. As this fact might be of
general application I asked M. Saltykoff[417], who was working in my
laboratory, to verify the statements of Voges. Several series of
experiments demonstrated the incorrectness of the contention. The small
doses of normal serum of guinea-pigs, indicated by Voges, were found to
be absolutely incapable of protecting against the virus used by him in
his experiments.
The fact that normal serums, injected in sufficiently large doses,
exhibited an undoubted protective property, affords additional proof
that this property cannot be identified with the fixative power. The
latter was present in serums which were not protective; here, then, we
have the inverse phenomenon and we see normal serums exercise their
protective action although they contain no fixative. This follows from
Bordet and Gengou’s experiments already described, according to which
the cytases, placed in contact with micro-organisms in normal serums,
remain free, simply because of the absence of fixatives.
We are led, then, from these demonstrations to recognise the presence of
stimulins not only in specific serums, but also in normal serums.
Between the two there is this difference that, when applied with the
normal fluids, the stimulins alone act, whilst when injected with the
serum of the animal enjoying acquired immunity the action of the
stimulins is facilitated and reinforced by the fixatives or sometimes,
perhaps, by the agglutinins.
[Sidenote: [287]]
[Sidenote: [288]]
The stimulating influence of certain normal serums may be so
considerable that it may prevent infection by the micro-organism,
injected at the same time in a dose many times more than lethal.
Wassermann[418] protected guinea-pigs by injecting into the peritoneal
cavity a quantity as great as 40 times the lethal dose of typhoid
bacilli, by introducing at the same time and at the same place 3 c.c. of
normal rabbit’s serum, heated to 60° C. Besredka[419], who confirmed
this observation, has analysed its special mechanism. He showed that the
serum exercises a very marked stimulating influence on the guinea-pig’s
leucocytes, which then exhibit a truly extraordinary phagocytic
activity. They are seen to act in the peritoneal fluid, but they are
much more active in the region of the omentum, where the leucocytes
gorge themselves with micro-organisms, devouring them by dozens. The
stimulating action of the heated rabbit’s serum is exercised in a
similar fashion if, instead of micro-organisms, grains of carmine be
injected. Very shortly after the commencement of the experiment very
little carmine is found outside the cells; it is all either ingested by
individual leucocytes, if the grains are small, or surrounded by
numerous leucocytes when the grains are massed together; this
phagocytosis is most developed in the region of the omentum, exactly as
in the case of typhoid bacilli.
These facts, which so clearly demonstrate the stimulating action of the
normal rabbit’s serum, prove in another way that the stimulin resists
heating to 60° C., and that, in this respect, it resembles the
agglutinins and fixatives. This may afford us an indication as to the
nature of the stimulating substance. The possibility of obtaining an
antistimulin gives us another valuable indication. Wassermann, in the
work we have just cited, showed that the serum of a rabbit, previously
treated with guinea-pig’s serum and injected under the same conditions
as in the experiment with normal rabbit’s serum, has completely lost its
protective power. The typhoid bacilli multiply freely in the peritoneal
cavity and the organism of the guinea-pig is incapable of opposing a
sufficient resistance. Wassermann thinks that, in this case, the disease
becomes grave because of the anticytase found in the serum of rabbits
treated with guinea-pig’s blood. There is no doubt that this serum is
really anticytasic. But as the free cytases found in the peritoneal
cavity of a guinea-pig inoculated at the moment of phagolysis, become
inactive under the influence of the anticytase and play merely a minor
part, it is impossible to accept the German investigator’s
interpretation. Indeed, Besredka has proved that, in this case, it is
the antiphagocytic or antistimulant action of the rabbit’s serum which
brings about the fatal issue in the case of the typhoid inoculation.
We have laid stress on the point that an animal, whose serum is
protective when introduced into another animal, may itself not be
refractory against the specific micro-organism. As regards the serum of
normal unvaccinated animals this has been so fully demonstrated that
nowadays no one doubts it. The question is more complicated in the case
of animals that have acquired immunity. As in the great majority of
cases the serum of these animals is found to be endowed with a very
great protective power, it has been accepted as proved that the animal
which furnishes it must itself possess great immunity. The degree of
protective power has even been taken as the measure of the acquired
immunity. Thus, the numerous attempts to vaccinate the human subject
against typhoid fever, undertaken in consequence of the researches of
Pfeiffer and Kolle[420], were based on the fact that in these cases the
serum of vaccinated individuals acquires a great protective power. It
was argued that if this power is present it can only be due to the
acquired immunity of the individuals who furnish such a serum.
Undoubtedly the protective property of the fluids and the resistance are
often equal; but it is none the less true that there are cases where, in
spite of this property being markedly developed, the animal that
furnishes the protective serum is susceptible to the action of the
micro-organism and may even succumb to infection therewith.
[Sidenote: [289]]
As the hypothesis just mentioned is of importance from a general point
of view it must be supported by adequate proof. It was during the course
of the vaccination of rabbits against the micro-organism of the
pneumo-enteritis epidemic at Gentilly that I was first able[421] to
assure myself of its accuracy. I noticed that some of these rabbits,
although vaccinated, ultimately succumbed to pyaemia, set up solely by
this micro-organism. They were consequently not refractory against the
disease, and yet their blood serum, when injected into normal rabbits
along with an absolutely fatal dose of micro-organisms, was found to be
highly protective. This observation drove me to the conclusion that the
protective power is not a function of immunity and cannot be received as
a measure of this immunity. Analogous facts have since been demonstrated
in certain other cases. Thus, Pfeiffer[422] on several occasions has
found that guinea-pigs, highly immunised against the cholera vibrio,
have succumbed after the injection of a moderate quantity of these
organisms. “On post-mortem examination of these cases living vibrios
were found in the peritoneal cavity, sometimes in considerable numbers;
and yet minimal doses of the heart blood given to normal guinea-pigs
caused in these animals a very marked breaking down of the vibrios.”
Alongside these facts may be placed others, described in the preceding
chapter, of well immunised animals dying from infection, after they had
been weakened by opium, cold, or other lowering agent. It is clearly
seen, then, that for the manifestation of acquired immunity it is
necessary that the reaction of the living cell elements should take
place without let or hindrance. When this reaction fails, the possession
of even great protective power is insufficient to prevent the immunised
animal from contracting a fatal infection.
[Sidenote: [290]]
[Sidenote: [291]]
If, in acquired immunity against micro-organisms, it is really the cell
defence which plays the most important part, we can readily imagine
cases where it by itself can confer immunity without calling in the
co-operation of the protective power of the fluids. When in this
connection we study the resistance of an animal against various
pathogenic organisms, we note, first of all, the very great variability
that exists in the production of the acquired humoral properties. In
certain cases, as in vaccination against vibrios or typhoid bacilli, the
serum very readily becomes not only protective, but agglutinative and
fixative. In other cases these properties develop with difficulty and
are only manifested after a long period of vaccination. Such is the case
with anthrax. After the discovery of protective serums, numerous
attempts were made to obtain a serum protective against the anthrax
bacillus. Several observers failed in their attempts, others were more
fortunate. Sclavo[423] and Marchoux[424] were the first to succeed in
obtaining a protective serum from animals hyperimmunised against
anthrax. They were able to show that the serum of sheep, treated first
with vaccines and then repeatedly with anthrax virus, would protect
rabbits against a fatal dose of the bacillus. Marchoux even obtained,
with hyperimmunised rabbits, a serum which prevented normal rabbits from
contracting fatal anthrax. Sobernheim[425] was less fortunate in his
first experiments. He satisfied himself that the blood serum of cattle
that had recovered spontaneously from anthrax or that had been
vaccinated according to Pasteur’s method, was absolutely unable to
protect small animals against the anthrax bacillus, and his
hypervaccinated rabbits furnished serums of doubtful activity. It was
only later that he succeeded[426] in obtaining better results;
especially when he used sheep. Even then he found that in the production
of the anti-infective property the individuality of the immunised
animals had a dominant influence. Thus, in two sheep, treated in exactly
the same way, the serum of one was found to be incapable of protecting a
rabbit, whilst that of the other exhibited an undoubted, although
feeble, protective power.
But what is of greater interest to us, from our point of view, is that
guinea-pigs which have been vaccinated against anthrax and which enjoy a
considerable immunity against this disease, exhibit no protective power.
In a letter from Behring I learnt that this fact had for the first time
been demonstrated by Wernicke in experiments carried out in the Hygienic
Institute at Marburg. After repeated and painstaking attempts this
observer succeeded in vaccinating guinea-pigs against enormous doses of
virulent anthrax bacilli. The serum from the animals so immunised was,
however, quite incapable of protecting normal guinea-pigs against a
fatal infection. This result was the more extraordinary since Wernicke’s
pigeons, likewise vaccinated against anthrax, gave a serum whose
protective power was quite distinct. Realising the great importance of
these facts I asked M. de Nittis[427] to repeat these experiments in my
laboratory. The vaccination of pigeons is an easy matter, but that of
guinea-pigs presents great difficulties. He succeeded, nevertheless, in
vaccinating some of these rodents very highly, and this enabled him to
compare the protective power of the blood serum in the two species. That
of the vaccinated pigeon was found to be endowed with this power and
protected guinea-pigs and mice against virulent anthrax. The serum of
the immunised guinea-pigs, on the contrary, exhibited no protective
property, just as in Wernicke’s experiments. The guinea-pigs and mice,
into which this serum was injected at the same time as the anthrax
bacilli, died even when attenuated anthrax was used. We have, then, in
this case, an example of acquired immunity, independent of any
protective power of the fluids of the body.
In the course of their researches on the bacillus isolated by R.
Pfeiffer from persons attacked by influenza, Delius and Kolle[428] tried
to vaccinate susceptible animals (guinea-pigs) against this minute
organism and to immunise animals naturally refractory (dog, sheep, goat)
against fairly large doses of cultures. They succeeded in vaccinating
guinea-pigs against ten times the lethal dose, but never obtained any
protective serum. Nor did the other animals that were treated furnish a
protective serum. “From the whole of our experiments carried on for
several years”—conclude Delius and Kolle—“it is quite evident that we
were unable to produce any appreciable change in the blood by the use of
those methods which have produced specific immunising serums against
other bacteria such as the bacilli of diphtheria, cholera, typhoid
fever, and ‘blue pus’” (p. 345). Slatineano undertook a detailed study
of Pfeiffer’s bacillus in my laboratory, but he found it impossible to
demonstrate any unquestionable protective effect exerted by the blood
serum of vaccinated guinea-pigs upon normal guinea-pigs inoculated with
a fatal dose of this organism. We are not justified, therefore, in
classing this bacillus with the anthrax bacillus; we may, however, cite
it as an argument illustrating the difficulty that is met with, in
certain examples of acquired immunity, of discovering the protective
power, when feeble and masked.
The inoculation with micro-organisms of animal nature causes the
development of acquired immunity, but in this case the properties of the
fluids of the body are but little in evidence or they may be even _nil_.
Let us return to the example of the _Trypanosoma_ of the rat which
excites in vaccinated animals a protective and weakly agglutinative
power of the serum. This fluid, however, is usually found to be
incapable even of rendering the flagellated parasites motionless.
[Sidenote: [292]]
The question of immunity against malaria has been much discussed. It is
well known that a first attack of this disease, so far from conferring
any immunity of the slightest durability, leaves a certain
predisposition to another attack. In spite of this the study of malaria
in various countries and in individuals belonging to different races has
demonstrated that there does indeed exist a certain degree of acquired
immunity against this disease. During recent years Koch[429] has paid
special attention to this subject and has furnished us with very
valuable data, based especially on a comparative study of the blood of
children and adults. The frequency of Laveran’s parasite in the former
and its rarity in the latter, have led him to the conclusion that
infantile malaria sets up an immunity which persists in the adult.
Moreover, it has been established that in malarial countries the
indigenous inhabitants exhibit an attenuated form of the disease,
unaccompanied by acute attacks, but with phenomena that are chronic and
very slow in development.
In spite of the existence of a certain degree of acquired immunity
against malaria, all attempts to demonstrate any protective action of
the serum have been fruitless. Celli[430], indeed, injected, as a
preventive, the blood serum of individuals who had recovered from
malaria or of others who were bled during the period of defervescence
following an acute crisis of this disease, but in every instance these
injections were found to be useless in preventing an attack of malaria.
We can readily understand that in a disease which is exclusively human,
such as malaria, it has not been possible to perform a sufficient number
of experiments to decide the question of the protective property of the
blood. In this respect we shall have greater chance of obtaining
satisfactory data if we direct our attention to some analogous disease
attacking one of the lower animals. Such a disease we have in Texas
fever, occurring in the Bovidae, as the result of the action of an
animal parasite, _Piroplasma bigeminum_, which invades the red blood
corpuscles much as Laveran’s parasite invades those of the human
subject.
[Sidenote: [293]]
[Sidenote: [294]]
As mentioned in the preceding chapter, Smith and Kilborne and Koch have
demonstrated that the Bovidae may acquire a real immunity against Texas
fever. Nicolle and Adil Bey[431] at Constantinople found indigenous
races that exhibited a remarkable immunity against the _Piroplasma_.
Having demonstrated this fact the idea occurred to them to inoculate
these refractory cattle with very large quantities of virulent blood and
to make use of the serum from animals so treated for the prevention of
infection in susceptible races of Bovidae. This experiment gave negative
results. Lignières[432] elaborated a special method of vaccinating
susceptible Bovidae and was successful in obtaining very encouraging
results. A commission of veterinary surgeons from Alfort[433] appointed
to verify these observations came to the conclusion that “the
vaccination as carried out by Lignières was absolutely effective.”
Lignières also carried out researches on the protective power of the
blood serum of his immunised cattle. In a communication to the
International Congress of Medicine, held in Paris in 1900, he stated
that the injection of several hundred cubic centimetres of this fluid
did not protect normal animals against infection. We must conclude,
therefore, that, here also, we have another example of acquired immunity
unaccompanied by the presence of any protective property of the blood
fluid.
These results have received confirmation from a most authoritative
source. Nocard has kindly communicated to me the fact that he has tried
in vain to confer immunity on normal dogs into which he has injected
blood serum coming from dogs that had recovered from the disease
produced by a haematozoon closely allied to that of Texas fever or serum
from sheep immunised with blood from the affected dogs.
Looking at the data we have just summarised as a whole, we are compelled
to recognise that, on the one hand, the protective power of the body
fluids may coincide with a susceptibility to the corresponding
micro-organism, and that, on the other, real acquired immunity may exist
without any manifestation of this humoral property, especially as, even
in immunised animals, the acquired immunity often persists longer than
does this property. It must be accepted then, that, in this immunity,
there exists something other than the powers of the fluids of the body,
that is to say, the factor which plays the predominant part is to be
sought for in the cellular elements. We need only recall the many facts
collected in the preceding chapter to be convinced that in acquired
immunity phagocytosis is the most constant and most general phenomenon.
We find it in cases where the humoral properties are the most marked, as
well as in those in which they are only slightly developed or are
entirely absent. We need not again discuss Pfeiffer’s phenomenon
analysed in the preceding chapter. It is sufficient to mention that this
example of the extracellular destruction of micro-organisms only occurs
under limited and special conditions. It is observed only in cases where
the injection is made into a situation rich in leucocytes which undergo
phagolysis as a result of the sudden change brought about in their
conditions of existence. Further, this phenomenon is observed only in
connection with micro-organisms that are slightly resistant to the
influence of the microcytases. In those cases in which we meet with
Pfeiffer’s phenomenon, we also meet with a widely extended phagocytic
reaction.
[Sidenote: [295]]
This reaction is most pronounced where the properties of the body fluids
are only slightly developed or are absent. The study of acquired
immunity against anthrax provides us with a very convincing proof of
this. We have already cited the example of vaccinated rabbits and rats
in which phagolysis is incomparably greater than in the susceptible
control animals which contract a fatal anthrax. This rule is general. It
is confirmed in the vaccinated sheep and guinea-pig. The absence, or
feeble development, of the protective power of the blood or of the other
humoral properties in no way, then, prevents the considerable change
which is set up in the phagocytes of animals that have acquired immunity
against anthrax. The negative chemiotaxis of the leucocytes, so marked
in susceptible animals, is modified into positive chemiotaxis as the
result of vaccination. This fact, one of fundamental importance, was
first demonstrated for the immunity against anthrax, later being
extended to other micro-organisms. Massart[434] studied the general
subject and collected a series of data which led him to say that
“vaccination effects an education of the leucocytes; these latter become
so adapted that they can approach the virulent micro-organisms.” The
best method of forming an estimate of the change which the leucocytes
undergo is by injecting subcutaneously very virulent micro-organisms
capable of setting up a generalised infection. The anthrax bacillus,
Gamaleia’s vibrio, the streptococci and the cocco-bacilli of swine and
fowl cholera are very suitable for such study. These micro-organisms,
when inoculated subcutaneously into susceptible animals, set up a very
slight local reaction or none at all, in the form of an exudation of
transparent fluid almost entirely without leucocytes. The
micro-organisms grow freely in these exudations and soon invade the
animal. In vaccinated animals the local reaction is more marked and the
exudation, very rich in leucocytes, is poor in fluid; the
micro-organisms remain free for a very short time, being soon ingested
by the leucocytes. Their destruction, inside these cells, takes a longer
or shorter time according to circumstances; but in the end it is always
complete.
[Sidenote: [296]]
The difference as regards phagocytic reaction between susceptible and
vaccinated animals, such as I have just described, has been generally
recognised by many observers. A few opponents are still found, however,
who consider that they are justified in affirming that the negative
chemiotaxis of the susceptible animal does not exist and that,
consequently, vaccination can in no way change it into positive
chemiotaxis. Werigo made himself the spokesman of this view, which he
has maintained in several papers[435]. Instead, however, of introducing
the virulent micro-organisms into the subcutaneous tissue of susceptible
animals he injected them directly into the veins. Using cultures of the
anthrax bacillus and of the cocco-bacillus of fowl cholera he injects
these into the venous system of normal rabbits. The animals soon die
from general infection. If, however, these animals are killed shortly
after inoculation, it is found on examination of sections that many of
the micro-organisms have been ingested by the leucocytes. Werigo
concludes from these facts that in the higher animals the chemiotaxis is
always positive; but that it ends in the destruction of the
micro-organisms in the vaccinated animals, never bringing about this
result in susceptible animals. Taking all the data on this question into
consideration, it is easy to convince oneself that this view cannot be
accepted as correct, for not only the definite phenomena observed below
the skin but also the no less demonstrative process appearing in the
peritoneal cavity prove most clearly the existence of this negative
chemiotaxis of the leucocytes. I need only recall Bordet’s experiment on
the fate of streptococci and _Proteus vulgaris_ when injected together
into the peritoneal cavity of guinea-pigs. Whilst the _Proteus_ bacilli
at the end of a very short time are all ingested by the leucocytes, the
streptococci remain free in the peritoneal fluid up to the death of the
animal. The leucocytes which exhibit a positive chemiotaxis as regards
the former, manifest a negative chemiotaxis as regards the streptococci.
[Sidenote: [297]]
In spite of the great force of these arguments, the discovery of a means
of reconciling the results obtained from the inoculation of
micro-organisms subcutaneously or into the peritoneal cavity, with those
observed after they had been injected into the blood vessels would be of
great interest, and Zilberberg and Zeliony[436] have undertaken a series
of experiments with this object. Following Werigo they made use of the
cocco-bacilli of fowl cholera, and found, in accordance with his
observations, that the intravenous injection of these organisms,
obtained from cultures in nutrient media, causes a very marked
phagocytosis of the cocco-bacilli. When, however, they injected into the
veins of rabbits cocco-bacilli that had been grown in the peritoneal
fluid of other rabbits, they found the micro-organisms free in the blood
plasma and observed only a very restricted phagocytosis in the
microphages of the liver. It follows from these experiments that the
ingestion of the cocco-bacilli, in Werigo’s experiments, was dependent
on the presence of a large number of attenuated micro-organisms which
were present in the cultures that he employed for his injections.
Alongside these organisms, slightly or not virulent, were others,
endowed with their normal pathogenic activity and quite numerous enough
to set up a fatal infection. When Zilberberg and Zeliony replaced
cultures on agar by the peritoneal exudation which contained virulent
cocco-bacilli almost exclusively, the phagocytosis in rabbits, injected
into the veins, was found to be almost suppressed. With the object of
establishing whether the absence of the phagocytic reaction, in this
case, really depended on negative chemiotaxis on the part of the
leucocytes, the above cited observers performed the following
experiment. They injected into the vein of a rabbit, already affected
with a generalised infection by the cocco-bacillus of fowl cholera, an
innocuous culture of a saprophytic staphylococcus. Post-mortem
examination showed that these cocci were almost entirely ingested by the
same phagocytes which refused so energetically to seize the
cocco-bacilli. This experiment, analogous to that of Bordet on
streptococcus and _Proteus_, compels us to reject Werigo’s conclusions
as to the absence of negative chemiotaxis in the phagocytes of the
higher animals. I ought to add that the work of Zilberberg and Zeliony
was in part executed in my laboratory so that I was able to convince
myself by ocular demonstration of the complete accuracy of their
statements.
Independently of these observers and even before their work appeared,
Th. Tchistovitch[437] published an interesting study on the same
question. He injected very virulent streptococci into the ear vein of
rabbits. These micro-organisms set up a generalised and fatal infection
in which phagocytosis was completely absent or nearly so. Here again was
manifested a negative chemiotaxis of the phagocytes, which, henceforth,
could no longer be questioned.
[Sidenote: [298]]
In certain infective diseases terminating fatally a very marked
phagocytosis is observed even in susceptible animals. The most typical
example of this is furnished by swine erysipelas and mouse septicaemia.
We know from the researches of Koch[438], followed by those of
Loeffler[439], Schütz[440] and others, that in animals which have died
from these two diseases the leucocytes are gorged with small specific
bacilli. A method of vaccinating animals against the micro-organism of
swine erysipelas was worked out by Pasteur and Thuillier[441] and was
afterwards studied by many observers. Thanks to this method it has been
possible to demonstrate the phenomena which may be observed in
vaccinated animals (especially rabbits). Here also a phagocytosis takes
place, even more rapid and more complete than in susceptible animals.
What is more important, the intracellular digestion of the ingested
bacilli is followed by the total destruction of the micro-organisms in
the vaccinated animals, though in the normal animals this digestion is
very imperfect.
[Sidenote: [299]]
The acquisition of immunity against micro-organisms is, therefore, due
not only to the change from negative to positive chemiotaxis, but also
to the perfecting of the phagocytic and digestive powers of the
leucocytes—a general superactivity and adaptation of the phagocytic
reaction of the immunised animal is produced. This conclusion, based
upon a large number of well-established facts and in complete harmony
with the whole of the data at our disposal concerning acquired immunity,
has been attacked by Denys and Leclef[442] in their work on the
streptococcus. They base their opposition upon experiments made _in
vitro_ on the action of serums and leucocytes on this micro-organism.
They have compared the bactericidal power of mixtures of the serums of
normal and of vaccinated rabbits with leucocytes isolated from
exudations from these two groups of animals. The leucocytes, whether
derived from normal or from vaccinated rabbits, when mixed with normal
serum were equally incapable of ingesting and destroying the
streptococci. When mixed with blood serum from vaccinated rabbits,
however, the two kinds of leucocytes exhibited a very marked phagocytic
reaction. Denys and Leclef conclude from this that phagocytosis,
although an important factor in immunity, plays merely a secondary part
and is dependent on the humoral properties. The experiments and views of
these two observers have been generally received by the partisans of the
bactericidal theory of the body fluids as an actual proof of this
theory. We cannot agree. Researches extending over a long period have
shown us that the study of phagocytosis _in vitro_ can give only a very
inexact and imperfect idea of the course of the phenomena in the living
animal. Usually the leucocytes taken from the exudations, although
amoeboid, no longer fulfil their phagocytic functions at a time when in
the animal they would ingest micro-organisms with the greatest rapidity.
As a general rule, existence outside the living body weakens them very
considerably. But in some cases, rare it is true, the leucocytes
although inactive in the animal exhibit intense phagocytosis when
introduced into a hanging drop of fluid from an exudation or even of
urine. In any case it is very hazardous to infer from phenomena which
appear under these artificial conditions what takes place in the living
animal. The value of the experiments of Denys and Leclef is still
further marred by the fact that they mixed the leucocytes with blood
serum. They appear to have lost sight of the fact that this fluid is far
from corresponding to that which bathes the leucocytes in the living
animal. The serums contain leucotoxin in greater or less quantity and it
is not to be wondered at that the leucocytes when mixed with normal
rabbit’s serum should perish very rapidly. Further, the serum of
vaccinated rabbits is agglutinative (this fact, however, was not
sufficiently elucidated in 1894 when the researches of Denys and Leclef
were made) and the clumping of streptococci might simulate their
destruction. In a word, the experiments of these observers have been
carried out under such conditions that it is impossible to base upon
them a refutation of data obtained in the living animal. Moreover, in
the description of the phenomena which appear in the subcutaneous tissue
of rabbits inoculated with the streptococcus, Denys and Leclef provide
us with arguments against their own view.
[Sidenote: [300]]
These observers introduce the same quantity of streptococci below the
skin of the ear of normal and of vaccinated rabbits. In the first there
is soon produced a very marked oedema of the ear, in which may be seen a
number of streptococci and of leucocytes that have not ingested any
micro-organisms. In the second the oedema does not develop, but at the
seat of invasion a number of leucocytes come up and these soon ingest
the streptococci. As we see, the phenomena manifest themselves here just
as they do with the anthrax bacillus and many other micro-organisms when
under analogous conditions. Denys and Leclef, indeed, recognise that,
below the skin of the ear of vaccinated rabbits, the small quantity of
exudation fluid is not sufficient to enable us to accept it as capable
of exerting any considerable influence as regards humoral properties.
Nevertheless, they think that the “serum” of this fluid may exercise a
certain action, but they furnish no proof of this, and seem to ignore
the fact that the plasma of the subcutaneous exudation is far from being
identical with blood serum obtained outside the animal. At present it is
well known that this latter fluid contains cytases which are absent from
the plasmas. Now, the feeble bactericidal action, if this really exists
as regards the streptococcus, must be attributed to the microcytase
which has escaped from the leucocytes at the time of the preparation of
the serum.
To sum up, the example studied by Denys and Leclef clearly comes under
the general law of phagocytic reaction in acquired immunity against
micro-organisms. It is impossible to deny that the superactivity of the
phagocytes which is always found in this immunity, although readily
observed, cannot be demonstrated in a rigorous fashion outside the
fluids which bathe the cells. There are, however, very important
analogies which may be invoked in favour of this thesis. We have already
cited in our fifth chapter Delezenne’s experiments on the digestion of
gelatine by the leucocytes of the dog, which show in the most
demonstrative fashion that these cells accustom themselves to bring
about this digestion more and more quickly and this quite independently
of any humoral influence.
For some time past there has been no doubt as to the fundamental fact
that the phagocytes in immunised animals seize and destroy living
micro-organisms. Several attempts have been made to show that such
destruction of these bacteria takes place solely by the body fluids, and
that the phagocytes intervene only as “scavengers” to carry off the dead
bodies of the micro-organisms. The numerous observations, described in
the preceding chapter, absolve us from again entering into a discussion
of this question. Moreover, the majority of these opponents now
recognise that micro-organisms are ingested in a living state by the
phagocytes of immunised animals. Some, however, have expressed the
opinion that these living micro-organisms, before becoming the prey of
the phagocytes, must undergo some preliminary attenuation of virulence
through the action of the body fluids. Hence the theory of the
attenuating power of the fluids of the body, maintained especially by
Bouchard and his pupils. During the course of our exposition of the
facts concerning acquired immunity, we have several times had occasion
to speak of the virulence of micro-organisms in the immunised animal.
Here, therefore, we may confine ourselves to a brief summary of the
observations collected on this point.
[Sidenote: [301]]
Having observed that the anthrax bacillus, when developed in the blood
of immunised sheep, was incapable of giving fatal anthrax to rabbits, I
expressed[443] the opinion that under these conditions its virulence had
become attenuated. Later, analogous changes were shown by Charrin[444]
in the _Bacillus pyocyaneus_ when cultivated in the serum of immunised
animals. Bouchard[445], generalising on these data, arrived at the
following theory of vaccination. “The inoculation of a strong virus into
a vaccinated animal is equivalent to the inoculation of an attenuated
virus. The attenuation, however, instead of being done beforehand in the
laboratory, is brought about in the tissues of the vaccinated animal”
(p. 18). Charrin and Roger[446] upheld this view, and the latter offered
several new arguments in support of it. He observed that animals
inoculated with pneumococci and streptococci grown in the blood serum of
vaccinated animals, contracted a transient and benign disease merely,
whilst the control animals, inoculated with the same micro-organisms,
cultivated in normal serum, always died from generalised infection.
[Sidenote: [302]]
The discovery of the protective property of serums has thrown a new
light upon these experiments. We must now ask ourselves: Does the
innocuousness of micro-organisms depend not on the attenuation of the
virus, but rather on the protective action of the serum itself? When, in
the course of my researches on the Gentilly cocco-bacillus, I found that
this organism, cultivated in the serum of vaccinated rabbits, became
much less pathogenic than when it was grown in the serum of normal
rabbits, I set myself to answer this question. Simple filtration through
paper was sufficient to rid the organism of the serum in which it had
grown. The inoculation of cocco-bacilli thus treated proved at once that
their virulence was in no degree modified, and that it was the
intervention of the serum that prevented the micro-organism from setting
up the rapidly fatal disease. Issaeff[447], who, in my laboratory,
carried out the investigation, was able to extend this to the
pneumococcus. He obtained agglutinated cultures in the serum of
vaccinated rabbits, and he compared their activity by injecting them (1)
with, and (2) without their culture medium. The difference was very
marked. In the first case the infection produced was much slower in its
course than in the second. The virulence of the washed pneumococci was
found to be the same whether they came from a culture in normal serum or
from one in immunised serum. Sanarelli[448] obtained the same result
with Gamaleia’s vibrio. The vibrios when grown in the serum of
vaccinated guinea-pigs proved to be very virulent so soon as they were
freed from the fluid in which they were grown. Later, similar
demonstrations were given by Bordet[449] and Mesnil[450] with respect to
streptococci and to the bacilli of swine erysipelas. We must, then,
conclude that we have here to do with a general law. Some experiments
made by de Nittis[451] might seem to indicate an exception to such a
law. He observed that anthrax bacilli when grown in the serum of
vaccinated pigeons lost a part of their virulence. It must not be
forgotten, however, that he grew his cultures under special conditions;
the bacillus was grown for several days at 42° C., this in itself being
quite sufficient to bring about a certain attenuation of virulence.
The theory of the attenuating action of the body fluids, based on the
attenuation of the virus in the serum of vaccinated animals, can no
longer be maintained, as it is a well-established fact that the serum,
obtained outside the body, is a fluid differing in character and
properties from the plasma of the living animal. We have seen up to what
point this demonstration has shaken the theory of the bactericidal
action of the body fluids.
[Sidenote: [303]]
It cannot be doubted that a micro-organism may undergo a certain
weakening in virulence, as well as in certain other functions, in the
body of the animal that has acquired immunity. But the question must be
put: Is this effect obtained as the result of humoral or of cellular
action? As a general rule, exudations obtained from vaccinated animals,
and containing living micro-organisms, are found to be virulent when
inoculated directly into susceptible animals. This fact was established
by Pasteur[452] when he first carried out his researches on acquired
immunity against fowl cholera. He showed that the exudations of
vaccinated fowls set up a fatal disease in normal fowls, without there
being the least evidence of any attenuation of the micro-organism. The
same applies to the Gentilly cocco-bacillus and to the anthrax bacillus
in a very great majority of examples. De Nittis observed that the
exudations of immunised pigeons produced a fatal infection in the
guinea-pig and in the mouse. In the immunised guinea-pig, on the other
hand, he found that the exudations soon became innocuous for these
animals. This alteration, however, must be attributed not to the body
fluids (which exhibit no protective or attenuating power) but to the
action of the cells.
With the object of gaining some idea of the changes that the
micro-organisms undergo in the immunised animal, Vallée[453] carried out
a series of experiments on rabbits vaccinated against the bacillus of
swine erysipelas. He enclosed these bacilli in sacs of collodion which
he introduced into the peritoneal cavity of susceptible rabbits and of
others that were hyperimmunised. The bacillus developed well in both
cases. It gave homogeneous non-agglutinated cultures in the sacs placed
in normal animals, whilst in the sacs introduced into the peritoneal
cavity of hyperimmunised rabbits the bacilli grew into agglutinated
filaments. This proves that the wall of the sacs permitted of the
passage of the active substances elaborated in the immunised animal.
Different from the point of view of agglutination, the cultures likewise
exhibited a considerable difference in their pathogenic activity. The
cultures developed in the sacs in hyperimmunised rabbits were found to
be much more virulent than those grown in the sacs in control animals.
This augmentation of virulence depends, probably, on the influence of
the active substances which pass through the walls of the sacs. In any
case, this experiment affords further confirmation of the impossibility
of maintaining the theory of the attenuation of micro-organisms by the
fluids of an animal enjoying acquired immunity.
[Sidenote: [304]]
Since the discovery of the antitoxic property of the fluids of the body,
it has been accepted that its manifestation was indispensable for the
acquisition of immunity. It was thought that in order to get rid of
pathogenic micro-organisms the animal had first to develop the means of
neutralising their toxins. These substances once prevented from exerting
their toxic action, the micro-organisms were left without their weapon
of attack and found themselves reduced to the condition of simple
saprophytes. It was accepted, therefore, that an effective antitoxic
power was always to be found in the fluids of animals that had acquired
immunity. Against this explanation, however, are certain established
facts. Chauveau[454] had observed that Algerian sheep, whose natural
immunity was further strengthened by considerable doses of anthrax
bacilli, exhibited a susceptibility to injections of anthrax blood quite
as marked as that of normal sheep. The immunity against the virus, then,
did not progress _pari passu_ with that against the poison. Later,
Charrin and Gamaleia[455] furnished important data on this subject. They
showed that animals vaccinated against the _Bacillus pyocyaneus_ and the
vibrios of Koch and Gamaleia were even more susceptible to intoxication
by the soluble products of these micro-organisms than were normal
animals which had acquired no immunity against the corresponding
bacteria. Shortly afterwards this observation was confirmed by
Selander[456], in his work on hog cholera, carried out under Roux’s
direction. Rabbits vaccinated against the cocco-bacillus of this disease
resisted infection by the virus, but died as a result of the exhibition
of the same doses of toxin that killed normal rabbits. I[457] was able
not only to verify this, but to add to it the further fact that the
blood serum of vaccinated rabbits, although markedly protective against
infection, exercised not the slightest antitoxic action.
When, later, R. Pfeiffer set himself to study the immunity of animals
against the cholera vibrio, he, along with his collaborators, was able
to furnish numerous data confirming the hypothesis that animals
thoroughly vaccinated against this vibrio had not thereby become more
resistant to its toxin and that their anti-infective serum exhibited no
antitoxic power. These results have been confirmed repeatedly and must
be regarded as fully established.
[Sidenote: [305]]
Von Behring here recognised a general law which, with the aid of his
collaborators, he attempted to develop. We owe to him the knowledge that
the susceptibility, augmented as regards the toxins, of animals
vaccinated against micro-organisms, might even serve in doubtful cases
to reveal the presence of their bacterial poisons. Culture products when
deprived of micro-organisms often set up no poisoning in normal animals
susceptible to infection. From this fact it is generally concluded that
the toxin is not present in the products in question. But animals of the
same species when immunised against infection by the micro-organism,
owing to their “hypersusceptibility,” react much more delicately and
allow of the demonstration of the presence of bacterial poisons in
fluids inactive for unvaccinated animals.
In collaboration with Kitashima[458], von Behring immunised guinea-pigs
against the diphtheria bacillus, and demonstrated that two or three
injections of diphtheria toxin were quite sufficient to render these
animals refractory to infection by the diphtheria bacillus though they
became more susceptible to intoxication. Von Behring considers that this
augmentation of susceptibility to the diphtheria poison may be a means
of rendering the local reaction of the living elements at the point of
introduction of the bacilli more active.
In any case, it is beyond question that acquired immunity against
microbial infection is quite independent of the resistance against the
toxins of the corresponding micro-organism. An antitoxic manifestation
of any kind, therefore, cannot be regarded as necessary for the
development of immunity against the micro-organism.
Of all the humoral properties developed in acquired immunity against
micro-organisms, the fixative power and the protective power are the
most constant. It might naturally be suggested, as a result of this
observation, that these two powers are indispensable for the
manifestation of phagocytosis for the purpose of destroying and of
ridding the animal of the pathogenic organisms. It is quite possible to
understand how, under these conditions, the idea has been put forward
that anti-infective acquired immunity is the result of two different
factors: in the first place, a humoral property independent of the
phagocytes and, in the second place, the phagocytes themselves. But the
part played by these cells cannot be accepted as purely secondary—a view
which has been advanced and defended again and again. This question is
of such importance that it is reasonable to ask whence come the humoral
properties, such as the fixative power and the protective power, factors
of such far-reaching influence in anti-infective immunity?
[Sidenote: [306]]
Thanks to the work of several investigators this question may now be
answered. Pfeiffer and Marx[459] first supplied important information
concerning the origin of the protective property. Into rabbits they made
subcutaneous inoculations of cholera vibrios, killed by heat (70° C.),
and then examined, most minutely, the protective power of the blood and
of extracts from various organs. Examining, separately, the protective
power of the serum and that of the layer of leucocytes deposited in
tubes, Pfeiffer and Marx were unable to find any marked difference. Nor
did they ever obtain any definite effect with leucocytes collected from
pleuritic exudations. From these observations they concluded that the
leucocytes of the blood could not be regarded as the source of the
protective substance (or “cholera antibody”). At a period when the serum
as yet exhibited an insignificant protective power or none at all, the
extract from the spleen often exerted an action of the most marked
character. In an experiment in which the rabbit was killed 48 hours
after the injection of the vibrios, 0·3 c.c. of the serum was incapable
of preventing fatal infection of a guinea-pig, whereas 0·03 c.c. of an
extract of the spleen exerted a marked protective effect. From this and
similar experiments, Pfeiffer and Marx conclude that the spleen is the
principal source of the protective substance. In order to verify this
observation they injected killed cholera cultures into rabbits which had
previously been deprived of their spleens, but the asplenic rabbits
still produced the same amount of protective substance, and these two
observers were led to conclude that the lymphatic glands and the
bone-marrow might also serve as the sites of origin of this substance.
It is only during the first few days, however, that these organs exhibit
a protective power greater than that of the blood. Three or four days
after the injection of the vibrios the blood serum becomes richer in
protective substance; the organs contain much less of it. This condition
is maintained for some time, after which the blood in turn begins to get
impoverished.
[Sidenote: [307]]
Pfeiffer and Marx put to themselves the question: Is the marked
protective power of the spleen due to the production of preventive
substance by this organ, or is it to be explained by an accumulation in
the spleen of this substance manufactured elsewhere? With the object of
obtaining an answer to this question they injected protective serum from
other individuals into rabbits, when they found that the protective
substance showed not the slightest tendency to accumulate in the spleen.
These authors were compelled to conclude, therefore, that the spleen and
other haematopoietic organs (lymphatic glands and bone-marrow) are the
real seats of the production of the protective substance. We may add
that these organs are also the phagocytic organs _par excellence_, that
is to say, the centres which serve not only for the development of
phagocytes but which contain a large number of the adult elements
capable of exercising the phagocytic function.
Almost simultaneously with Pfeiffer and Marx, A. Wassermann[460], in
collaboration with Takaki, undertook similar researches on the origin of
the substance protective against the typhoid cocco-bacillus. The outcome
of this work was that “it was the bone-marrow, the spleen, and the
lymphatic system, including the thymus gland, which exhibited immunising
power against the bacillus of typhoid fever, whilst the other organs,
the blood, brain, spinal cord, muscles, liver, kidney, etc., did not at
this stage show any marked specific property.”
[Sidenote: [308]]
As these observations on the production of protective substance in the
phagocytic organs was one of essential importance in connection with the
problem of acquired immunity, I asked M. Deutsch[461], working in my
laboratory, to carry out a series of experiments on this subject. Using
guinea-pigs, he injected into the peritoneal cavity cultures of the
typhoid bacillus killed by heat (66° C.). A few days later the serum had
become distinctly protective. At this stage, and even before the
appearance of this property in the blood, Deutsch killed some of his
animals and carefully measured the protective power of the extract of
the various organs. He began by confirming the result obtained by
Pfeiffer and Marx as to the non-production of the protective substance
in the peritoneal exudation. Usually this fluid was insufficient to
protect normal guinea-pigs against typhoid infection. In a few
experiments only was the exudation found to be as protective as the
blood serum; in most of the others, the blood serum was much more active
than the fluid of the exudation. The spleen was the organ which
exhibited the greatest protective power, and in nearly one-half of the
cases it was more active than was the blood. The bone-marrow sometimes
gave analogous though much less marked results. The spleen consequently
must be looked upon as the principal seat of the production of the
protective substance.
Having confirmed this observation of Pfeiffer and Marx and of Wassermann
and Takaki, Deutsch tried to obtain the protective property in
guinea-pigs deprived of their spleens. The experiment was quite
successful, and here again his result agreed with that obtained by
Pfeiffer and Marx. Guinea-pigs from which the spleen had been removed
developed the protective property just as well as did the control
animals; in the former the bone-marrow was found to be specially active.
When Deutsch, instead of removing the spleen from his guinea-pigs before
the injection of the micro-organisms, did so some (3–5) days afterwards,
there often occurred a marked diminution in the amount of the protective
substance produced. We must conclude, therefore, that soon after
inoculation there appears in the spleen a phenomenon which is associated
with the development of the protective power. The most simple
explanation of these facts is that the micro-organisms injected into the
peritoneal cavity and soon afterwards seized by the phagocytes (for the
most part by the microphages), are carried to the phagocytic organs,
particularly the spleen, lymphatic glands, and bone-marrow. In those
animals whose spleens are left intact a large number of these
microphages loaded with micro-organisms make their way into this organ,
a fact confirmed by direct observation. When the spleen is removed the
microphages must necessarily betake themselves to other phagocytic
organs. As the micro-organisms undergo intracellular digestion in the
phagocytes, it is very difficult, if not impossible, to follow them for
any length of time after they have been ingested, but the analogy with
the phenomena of the resorption of red blood corpuscles, described in
Chapter IV, justifies us in concluding that in the case of
micro-organisms matters go on in much the same way. These organisms,
seized at the seat of inoculation by the phagocytes, are transported by
these cells, in their peregrination through the organs, into the general
circulation. The interpretation I have just given has been accepted by
Deutsch.
[Sidenote: [309]]
This observer wished also to come to some conclusion as to the origin of
the agglutinative property so well developed in the fluids of animals
inoculated with the typhoid cocco-bacillus. He did not succeed in
solving this question, but he was able to demonstrate the undoubted
difference between this property and the protective power. The facts
brought forward by Deutsch must, therefore, be ranged alongside the many
others, reported on above, which demonstrate in the most conclusive
fashion that these two powers of the body fluids are essentially
distinct.
Such concordant results obtained by all investigators who have studied
the origin of the protective power warrant the conclusion that it is the
elements of the phagocytic organs, that is to say, the phagocytes
themselves, which produce the protective substance. But it will be
asked: Can we therefore accept the fixative substance or fixative as
being derived from the same source? When the experiments I have just
summarised were carried out the fixatives were not as yet sufficiently
known and were confounded with the protective substances. Nevertheless,
there can be no doubt as to what the answer to the question just put
must be. In the account of the experiments of Pfeiffer and Marx we find
very precise statements as to the granular transformation of the
vibrios. Thus, they observed on several occasions that an extract of the
spleen set up this transformation in a particularly distinct and rapid
fashion at a period when the blood and serum, used in a much stronger
dose, were incapable of producing the same effect. Now, as Pfeiffer’s
phenomenon is a visible manifestation of the action of the specific
fixative, it cannot be doubted that the spleen is really the principal
seat of development of the fixative substance before it makes its
appearance in the blood.
Before concluding this chapter we must review very briefly the principal
phenomena associated with acquired immunity against micro-organisms. The
extracellular destruction of these parasites takes place in the living
animal under special conditions only, when the phagocytes suffer a
temporary injury (phagolysis) and allow their microcytases to escape.
These latter by no means represent attributes of the body fluids, as is
even yet maintained by some writers. These soluble ferments are
connected with the phagocytes and represent the ferments of
intracellular digestion. The cytases undergo no modification during the
process of immunisation and correspond to those which act in natural
immunity.
[Sidenote: [310]]
The agglutinative substance often present in the normal fluids of the
body becomes much more developed in those of immunised animals. It is
truly humoral, as it circulates in the plasmas and passes into the fluid
exudations and transudations. But the part played by it in immunity is
very restricted.
The protective and fixative properties, most often closely connected
with each other, are very markedly developed in an animal enjoying
acquired immunity. They may act upon the micro-organisms which become
permeated by the fixative substance, or upon the infected animal by
stimulating its defensive reaction, but they are incapable of affecting
the vitality or virulence of the micro-organism. The two properties
(protective and fixative) reside in the fluids of the body, but they are
functions of the cell products. The elements of the phagocytic organs
(spleen, bone-marrow, lymphatic glands), or phagocytes, produce the
specific protective and fixative substances which pass thence into the
plasmas.
The phagocytic reaction is very general in acquired immunity. The
phagocytes which have a very imperfect antimicrobial function or none at
all, become, as the result of vaccination, much more active. They
exhibit a very marked positive chemiotaxis and acquire the faculty of
digesting micro-organisms in a greatly intensified degree. It is with
the increase of this digestive power that we have connected the
over-production by the phagocytes of the fixative and protective
substances which are excreted in large quantities by these cells and
pass into the fluids of the animal. As these substances are phagocytic
products it may be readily conceived that in certain examples of
acquired immunity the animal overcomes the micro-organisms without the
protective substances being found in the fluids. It is sufficient that
it is in the possession of the phagocytes, which may retain it within
themselves and not throw it off into the circulation.
[Sidenote: [311]]
From this account it will be seen that the phenomena, in acquired
immunity against micro-organisms, are merely a more or less stereotyped
copy of those that are presented in the animal after the resorption of
cells. There, also, we have intracellular digestion with over-production
of specific fixatives, part of which are excreted and thus pass into the
plasmas. In the resorption of cells there is also a double action of
cytases and fixatives; but in this case the macrocytases intervene,
whilst in the resorption of micro-organisms this function is performed
by the microcytases. The fixatives in the two cases are very different
from the point of view of their action, for they are specific; but the
cells which act in their production belong, in both cases (resorption of
animal cells and of micro-organisms), to the category of phagocytes.
It is often maintained that the theory I have just summarised is
fundamentally opposed to the theory of side-chains or receptors
formulated by Ehrlich[462]. This view I cannot accept. Applied to
acquired immunity against micro-organisms this theory may be summed up
as follows. The micro-organisms, when inoculated in a non-lethal but
immunising dose, combine with certain cells of the animal. The receptors
of the micro-organisms find corresponding receptors in these cells, but,
when once combined, the receptors of the cells become incapable of
fulfilling their normal nutritive function. The cells, thus deprived of
their receptors, reproduce such an enormous quantity of them that a
portion is excreted into the surrounding medium and passes into the
plasmas. These receptors, originating from cells, but which have become
constituent parts of the body fluids, are nothing but the fixatives or
intermediary bodies, or the amboceptors of Ehrlich. On a fresh arrival
of the same micro-organisms, they meet with, in the fluid of the
exudations, numerous amboceptors which combine with the corresponding
receptors of the micro-organisms, without, however, destroying them or
interfering with their vitality. As these amboceptors possess still a
second affinity, that for the molecules of the cytases, or the
“complements” of Ehrlich, the micro-organisms can be placed in contact
with these soluble ferments. Without the intervention of the fixatives,
the combination of the body of micro-organisms with the cytase can never
take place, because the receptors of the micro-organisms are not adapted
to those of the cytases. When the molecules of these ferments are found
in the plasmas in a free state, they can be attacked by the
corresponding group of the amboceptors.
[Sidenote: [312]]
Let us compare the theory we have just sketched with that described
further back. The micro-organisms, inoculated with a non-lethal but
immunising dose, are, as we have seen, ingested by the phagocytes and
afterwards digested within them. This intracellular digestion is
followed by the over-production of the specific fixative, of which a
part is excreted and passes into the plasmas. These are the results of
the well-established experimental data described in this chapter.
Ehrlich’s theory is in no way in opposition to this; it simply attempts
to penetrate more deeply into the mechanism of the phenomena observed as
taking place between the micro-organism and the cell. The act which we
simply term intracellular digestion is divided by Ehrlich into its
constituent parts. According to him there is a combination of the
fixative, on the one hand, with the molecule of the micro-organism, on
the other, with that of the soluble ferment or cytase. According to
Ehrlich it is the amboceptors of the cells which become detached in
order to furnish the fixative that circulates in the plasmas. For us
there is simply an over-production of one of the two ferments of
intracellular digestion, without defining more exactly what constituent
part of this ferment passes into the circulation. The two theories may
supplement each other but are in no way contradictory in principle.
There is only a single important point wherein they do not accord.
Ehrlich thinks that the cytases are always free in the body fluids and
that the cells, in order to exert a digestive action on the
micro-organisms, must previously seize their molecules by means of one
of the groups of their amboceptors. We, on the contrary, have developed
the idea that the cytases are only free in the animal during phagolysis
and that under normal conditions the cytases remain closely bound up
with the phagocytes. This statement is based upon a large number of
well-established experimental facts and must therefore be accepted as
proved. It does not, however, affect any fundamental principle of
Ehrlich’s theory. On the other hand the bases of Ehrlich’s theory affect
none of the main features of the theory I have developed. The doctrine
which regards acquired immunity as a particular case of resorption may
be reconciled with the conception of amboceptors. But it accords equally
well with Bordet’s conception, according to which the fixatives act not
as intermediary substances between the micro-organism and the cytase,
but as substances which sensitise the micro-organisms for the
penetration of the digestive ferment. This delicate question has not yet
been definitely settled, but Bordet’s experiments described in Chapter
IV are greatly in favour of this view.
[Sidenote: [313]]
Neisser and Wechsberg[463] have tried to obtain some idea of the manner
in which the fixatives act on the micro-organisms and have recorded a
series of very interesting facts. They have shown that these substances
only bring about the destruction of bacteria when they are in certain
relations with the cytase. Mixtures of fixatives and cytases in which
the former are found in excess not only do not kill the micro-organisms
but even allow them to develop abundantly. To attain this result Neisser
and Wechsberg mixed constant quantities of bacteria and normal serum
containing cytase with variable quantities of the serum of immunised
animals heated to 56° C. As we know, this specific serum, as the result
of being thus heated, is deprived of its cytases, but may be readily
made active again by the addition of normal, unheated serum. This
paradoxical fact, demonstrated by Neisser and Wechsberg can, in their
opinion, be explained only by Ehrlich’s theory of amboceptors. When
these bodies with double affinities are found in too large quantity as
regards the cytase, it may happen that one part only of those which
combine with the receptors of the micro-organisms succeed in linking to
themselves the molecules of the active ferment. The amboceptor being by
itself incapable of destroying the micro-organism, can be injurious to
it only on condition that it brings cytase. Consequently as the amount
of this cytase is too small for the much larger number of amboceptors we
can readily conceive that the micro-organisms may profit thereby and
remain alive. This interpretation is certainly very ingenious, but
nothing proves that it corresponds with the real state of things.
Neisser and Wechsberg have themselves observed that the serum of the
normal goat can also prevent the bactericidal action of the cytase. In
this case, however, they suggest the intervention of an anticytase of
this normal serum. The same explanation might perhaps serve also to
explain the preventive action of the serum of immunised animals. We know
that anticytases are found frequently enough in the various serums and
that they undergo great variations, according to the conditions present
in the animals furnishing the blood.
[Sidenote: [314]]
In any case, it is evident that the theory of receptors must in no way
be regarded as the antithesis of the theory of phagocytosis. This latter
quite retains its right to affirm that, in acquired immunity against
micro-organisms, phagocytes play the most general and important part.
They hold back the cytases which are capable of ridding the animal of
micro-organisms from destroying them. It is further these same cells
that produce and excrete the fixative and protective substances. The
free fixatives may attack the micro-organisms in the body fluids but
they are incapable of depriving them of life or even of virulence. The
cytases, after escaping from the phagocytes, may certainly, in
collaboration with the fixatives, destroy a certain number of the
micro-organisms, but only in special cases met with, no doubt, but only
rarely, under natural conditions. On the other hand, the phagocytes in
the animal which enjoys acquired immunity constantly fulfil the function
of seizing the micro-organisms and of submitting them in their interior
to the combined action of fixatives and cytases.
Acquired immunity, like natural immunity against micro-organisms,
presents merely special phases of intracellular digestion.
CHAPTER X
RAPID AND TEMPORARY IMMUNITY AGAINST MICRO-ORGANISMS, CONFERRED BY
SPECIFIC AND NORMAL SERUMS, OR BY OTHER SUBSTANCES, OR BY
MICRO-ORGANISMS OTHER THAN THOSE AGAINST WHICH IT IS DESIRED TO PROTECT
AN ANIMAL
Immunity conferred by specific serums.—Analogy of the mechanism of
this immunity with that observed in immunity obtained with
pathogenic micro-organisms and their products.—Part played by
phagocytosis in the immunity conferred by specific
serums.—Influence of opium on the course of immunisation by these
serums.—Stimulant action of specific serums.—Protective and
stimulant action of normal serums.—Influence of fluids, other than
serums: broth, urine, physiological saline solution, etc.
Antagonism between anthrax and certain bacteria.
[Sidenote: [315]]
We have seen how important in the study of acquired immunity against
micro-organisms is the demonstration of the protective power of the body
fluids. Without being absolutely general, this power is, nevertheless,
widely diffused and is found even in certain examples of acquired
immunity against micro-organisms belonging to the animal kingdom. Up to
the present I have refrained from doing more than point out the
presence, in the fluids of the immunised animal, of this protective
property and have studied it exclusively in relation to the animal that
produces it. We must now pass to the question: How do the protective
substances act in the animal which receives them ready formed? This
immunity, which has received from Ehrlich the name of _passive immunity_
against micro-organisms, must now be examined.
[Sidenote: [316]]
The study we now propose to enter upon is rendered much easier from our
study of the data acquired on the phenomena exhibited in the animal
vaccinated with micro-organisms or their products, data already given in
the preceding chapter. There is, indeed, a very striking analogy between
the two kinds of immunity, and though we draw a sharp line of
distinction between them, this is due to the fact that the immunity
conferred by micro-organisms or their products requires some time for
its development and endures for a long period, whilst the immunity due
to the introduction of specific serums into an animal is set up
immediately, but endures for a very short time only.
The diseases of the Invertebrata being seldom due to the micro-organisms
that produce infections in the higher animals, the question as to
whether the Invertebrata can be immunised by means of protective serums
has not yet been studied. Still, we already have certain ideas on the
protection of lower vertebrates by specific serums. Gheorghiewsky[464],
in my laboratory, carried out some experiments on this point. He found
that the serum of mammals (guinea-pig, goat) immunised against the
_Bacillus pyocyaneus_, was under certain conditions capable of
protecting the green frog against a dose of this organism that was
always fatal to the control animals. When injected along with the
_Bacillus pyocyaneus_, the serum did not prevent a fatal infection;
sometimes this infection developed even more rapidly than in the control
frogs. It was only when the protective injection was made 24 or, better
still, 48 hours before the inoculation of the bacilli, that the
protective action became evident. The serum used in these experiments
was not bactericidal for the _Bacillus pyocyaneus_ which grew most
luxuriantly; but it agglutinated a large proportion of the bacilli.
Gheorghiewsky pointed out, however, that frogs injected with cultures
agglutinated by the goat’s serum died just as readily as did the control
animals. As the phagocytic reaction was invariably very active in those
frogs which resisted the virus, after the injection of protective serum,
it is very probable that this fluid exercises a stimulant influence on
the phagocytes.
[Sidenote: [317]]
This idea of stimulation by anti-infective serums in cases of temporary
immunity conferred by these fluids, has already been set forth in my
researches on the immunity of rabbits against the Gentilly
cocco-bacillus, induced by the serum of vaccinated rabbits. This view,
however, has not been favourably received, especially in view of the
discovery of the phenomenon of the transformation of cholera vibrios
into granules. Pfeiffer himself noted that this transformation took
place not only in the peritoneal cavity of vaccinated guinea-pigs but
also in the peritoneal cavity of normal guinea-pigs, into which he had
injected small quantities of specific serum. As this latter fluid, in
Pfeiffer’s hands, was incapable of transforming the vibrios into
granules _in vitro_, he concluded that the cellular elements of the
normal animal were endowed with the power of modifying the inactive
substance of the specific serum into bactericidal substance. According
to this conception the immunity conferred by this serum was not entirely
passive since, in order to prepare the substance which transforms and
kills the vibrios, the co-operation of the living cells was necessary.
My demonstration of the possibility of obtaining Pfeiffer’s phenomenon
_in vitro_ at once turned the balance in favour of the theory that the
immunity induced by the specific serum is due to a direct humoral action
upon the micro-organism. Under these conditions such immunity could only
be interpreted as being purely passive. This view seemed to be finally
established by Bordet’s discovery that a specific serum, inactive by
itself, became capable of producing Pfeiffer’s phenomenon, as soon as a
small quantity of normal, non-specific serum was added to it.
Bordet[465] thus sums up his theory of the immunity conferred by
specific serums: “Passive immunity is due, in part at least, to a
chemical action exerted on the vibrios by two pre-formed substances, the
one present in the animal before any injection is made, the other found
in the serum that is injected; this phenomenon is purely chemical in the
sense that it can go on without the aid of a vital reaction, of any new
cell secretion: indeed it is found to take place in fluids from which
the cells have been entirely removed” (p. 217). These demonstrations led
up to the belief that the organism of the animal remained absolutely
passive when it was subjected to the action of protective or
anti-infective serums, and that the case of the cholera vibrio
represented a kind of schema, which was applicable to the whole of the
group of phenomena met with in passive immunity.
As in the study of the immunity obtained as the result of vaccinations
with micro-organisms or their products, so in “passive immunity” there
was seen only the direct chemical action of two substances on the
micro-organism, and efforts were made to extend this demonstration to a
series of infective diseases.
[Sidenote: [318]]
[Sidenote: [319]]
Pfeiffer and Kolle[466] having observed that the blood serum of persons
convalescent from typhoid fever, as well as that of animals vaccinated
with the typhoid bacillus, exhibited a great protective power for the
guinea-pig, wished to get some idea of the mechanism of this immunity.
They found that in the peritoneal cavity of guinea-pigs, inoculated with
the typhoid cocco-bacillus and simultaneously subjected to the action of
protective serums, the micro-organisms lose their mobility almost
immediately. A little later, they exhibit a degeneration of form, become
less refractile and disintegrate. After the injection of large doses of
specific serum the bacilli, much as in the case of the cholera vibrio,
become transformed into granules. “But,” say these authors, “this last
mode of destruction, that is to say the formation of granules at the
expense of the injected bacteria, does not occur with such remarkable
regularity as it does in Pfeiffer’s phenomenon in the cholera vibrio”
(p. 219). Whilst these changes are going on in the peritoneal fluid, the
leucocytes begin to come up and to ingest the bacilli and their
_débris_. “Phagocytosis, therefore, undoubtedly plays a part in the
destruction of the bacteria. Nevertheless, as most of the injected
bacteria die in the fluid of the exudation, phagocytosis can not be
regarded as the cause of the protective action of the serum” (p. 220).
We see from this description that even in the case of the typhoid
cocco-bacillus the direct action of the fluids of the body is
perceptibly less marked than in the case of the cholera vibrio. Even in
the latter, however, it is necessary to make many reservations. The same
laws apply to the immunity against this micro-organism, conferred by the
serum of immunised animals, as to the immunity due to vaccinations by
the vibrios or their products. To treat this subject fully one would
have to repeat almost textually the two preceding chapters, but I will
simply recall the fact that this transformation, almost general and very
rapid, as we observed _in vitro_ in vibrios placed in contact with fresh
specific serum or with the mixture of this serum, heated to 55°–56° C.,
and normal unheated serum, is only met with in the animal body where
phagolysis appears. Pfeiffer first observed the phenomenon which bears
his name in the peritoneal cavity, and it is best seen in that situation
during the period of the phagolysis of the white corpuscles. Vibrios,
mixed with small doses of specific serum which by itself is able to
render them motionless and agglutinate them, but which is absolutely
unable to transform them into granules, present this transformation
immediately they are introduced into the peritoneal cavity of normal
guinea-pigs. In this case the vibrios, permeated by the fixative of the
specific serum, are affected by the microcytase which has escaped from
the injured phagocytes and is found in the peritoneal fluid. The
preparation of the peritoneal cavity of normal guinea-pigs by means of
an injection of broth or physiological saline solution the day before,
prevents the production of Pfeiffer’s phenomenon, in spite of the
protective serum, just as in vaccinated guinea-pigs. In both cases the
vibrios, without being transformed into granules by the fluid part of
the peritoneal exudation, are ingested by the phagocytes _en masse_ and
with extraordinary rapidity. This experiment was repeated by
Garnier[467] with the typhoid cocco-bacillus. He first injected into the
peritoneal cavity of young guinea-pigs several c.c. of physiological
salt solution, of fresh broth or of some other fluid. The next day he
introduced into the same situation typhoid cocco-bacilli mixed with
blood serum from a donkey that had been for a long time immunised
against this organism. A few minutes (2–4) after this latter injection
the leucocytes, whose phagolysis had been prevented by the previous
day’s preparation, were found crammed with cocco-bacilli. Some of these
bacilli, like those still free in the peritoneal fluid, retained their
normal form, but a very large number of those ingested by the
microphages were already transformed into granules. This experiment
affords fresh confirmation of the hypothesis that the substance which
transforms the cocco-bacilli or the vibrios into granules is the
microcytase. In the phagocytes in their normal condition this
microcytase is found in the microphages, but during phagolysis a portion
of it escapes into the surrounding fluid. In the control experiments
made by Garnier with young normal guinea-pigs not prepared by
preliminary injection, the simultaneous injection of typhoid
cocco-bacilli and specific donkey’s serum set up this attenuated and not
very typical Pfeiffer’s phenomenon described in Pfeiffer and Kolle’s
memoir.
[Sidenote: [320]]
Soon after the discovery of Pfeiffer’s phenomenon I[468] was able to
bring forward a proof that it was produced neither in the subcutaneous
tissue, in the oedemas set up by the arrest of the circulation, nor in
the anterior chamber of the eye of animals when cholera vibrios mixed
with anti-infective specific serum were injected into these situations.
Here the micro-organisms retain their normal form, remain quite alive
and in this condition are ingested by the leucocytes which are brought
up to the points invaded. These cells, attracted by the vibrionic
products, do not undergo any phagolysis and, untrammelled, fulfil their
phagocytic function. Inside them are found vibrios which have kept their
elongated form and others which have become transformed into granules.
The exudations containing these elements still give cholera cultures on
nutrient media, a proof that some at least of the intracellular vibrios
are alive. Here we have no destruction of the micro-organisms in the
fluids of the body, consequently no direct action of the bactericidal
substance. This substance, enclosed in the phagocytes, can only act
through the intervention of these elements.
[Sidenote: [321]]
Mesnil[469] made analogous experiments with the Massowah vibrio, which,
unlike the cholera vibrio, is peculiarly virulent when injected
subcutaneously into guinea-pigs. In spite of this difference, this
micro-organism, when injected along with protective serum into normal
animals, behaves much as does the cholera vibrio proper. Mesnil injected
the Massowah vibrios at the same time as the anti-infective specific
serum, into the subcutaneous tissue of young and adult guinea-pigs and
of young rabbits. In every case he observed the same reaction phenomena
in the animal organism. The vibrios caused the formation of oedema at
the point of inoculation and remained isolated in the fluid. The
majority of these micro-organisms became motionless, but a few remained
motile. Pfeiffer’s phenomenon was never observed. There was sometimes an
aggregation of the vibrios, but this was not comparable with the marked
agglutination brought about by the specific serum _in vitro_. The
vibrios retained their power of reproduction, and Mesnil was able to
observe them in all phases of division. Some hours (6–8) after
inoculation the leucocytes began to come up to the seat of injection and
set to work at once to ingest the vibrios. This phagocytosis became more
and more marked, and finally there was ingestion of the whole of the
micro-organisms. Drops of the exudation containing only intraphagocytic
vibrios, when placed in the incubator, gave abundant cultures. The
leucocytes died outside the animal body, whilst the vibrios continued to
live and grow well under the new conditions. Certain leucocytes became
three times their original size, and their contents were seen to be made
up of vibrios closely packed together. The subcutaneous exudation, when
withdrawn even eight days after the injection of the micro-organisms and
sown on nutrient media, still gave colonies of vibrios.
It is evident, therefore, that the direct action of the protective serum
on the vibrios was reduced to a mere trifle. It rendered them motionless
and brought about a very slight clumping, but it was incapable of
transforming the vibrios into granules or of destroying them. We see,
then, that even in the case of the vibrios, the part played by
Pfeiffer’s phenomenon is very limited. The destruction of the vibrios is
effected with certainty, and completely, under the influence of the
specific serums, not by a direct action of the two antibacterial
substances but through the mediation of the phagocytes. Before the
fixative, introduced with the protective serum, can bring about this
result, the leucocytes, impressed with a special sensitiveness, must
come up to the seat of inoculation, seize the micro-organisms and
secrete around them their cytase. It is only as a result of these
actions, purely vital, that the chemical or physico-chemical reaction of
the substances which intervene in the destruction of the vibrios is
brought about.
[Sidenote: [322]]
Under these conditions it can easily be understood that if the vital
action of the phagocytes is retarded or depressed the injection of
protective serum cannot preserve the life of the animal.
Cantacuzène[470], who had already made a similar demonstration on
guinea-pigs vaccinated against the cholera vibrio by these organisms or
by their products, carried out numerous experiments on the action of
opium on normal guinea-pigs simultaneously inoculated with vibrios and
specific serum. Before injecting this mixture Cantacuzène narcotised his
animals by means of tincture of opium. The great majority (⅘) of the
guinea-pigs so treated died at the end of one or several days. The
transformation of the vibrios into granules, under the influence of the
serum, took place in the peritoneal cavity, but the leucocytes, on
account of the narcotic action of the opium, were tardy in coming up. On
their arrival in the peritoneal cavity they were capable of ingesting
the granules, but absolutely refused to seize entire vibrios, always
fairly numerous in the exudations. In spite of the appearance of a large
number of leucocytes, these cells were still too weak to offer any
adequate opposition to the vibrios, which increased in number and
continued to multiply up to the death of the animal, when the exudation
simply swarmed with very motile vibrios. Sometimes the struggle was
prolonged. The weakened leucocytes allow the vibrios to develop, but,
after a greater or less length of time, they regain their strength and
begin to ingest the micro-organisms vigorously. Complete phagolysis
follows, but the guinea-pig, attacked by the toxic products of the
vibrio, finally succumbs in spite of the absence of free vibrios from
its body.
An analysis of the phenomena observed in the body of an animal treated
with antivibrionic serum, demonstrates that, in spite of a certain
direct action of the substances contained in this fluid, there still
remain a whole series of processes, amongst which the carriers of the
cytases, that is to say the phagocytes, fill the most important _rôle_.
Nevertheless, the cholera vibrio with its allied forms is still the most
sensitive of all the micro-organisms to the bactericidal action of the
fluids of the body. It may, therefore, readily be conceived that the
more resistant micro-organisms are even less subject to the direct
influence of the specific serums. Thus we have seen that the
coccobacillus of typhoid fever presents, in the phagolysed peritoneal
fluid, merely an attenuated form of Pfeiffer’s phenomenon. The other
representatives of the group of bacilli are still less subject to the
direct action of the serums, and Gheorghiewsky[471], in his studies on
the _Bacillus pyocyaneus_, found that normal guinea-pigs, injected
subcutaneously with anti-infective specific serum, and inoculated into
the peritoneal cavity with this organism, present the same phenomena as
those described in Chapter VIII. He never noticed either lysis of the
bacteria in the fluids of the animal or their total transformation into
agglutinated masses outside the phagocytes. The resistance offered by
the animal was always in direct relation to the rapidity of the
appearance and extent of the phagocytic reaction.
[Sidenote: [323]]
In order to determine the relative importance of each of the factors
which act in the preservation of animals subjected to the influence of
the specific serum, Gheorghiewsky repeated Cantacuzène’s experiments on
the effect of narcotisation by tincture of opium. This alkaloid retards
diapedesis, but does not affect the tactile sensibility or the motility
of the leucocytes. The humoral properties, on the other hand, are not in
the least affected by the narcosis. In spite of the fact that in
guinea-pigs, narcotised and treated with anti-infective serum, the
direct action was not interfered with, the animals always died because
the retarded and incomplete phagocytic reaction was insufficient to
destroy the bacilli rapidly enough.
Mesnil[472] studied the action of the specific serum against swine
erysipelas on normal animals into which he had injected it some time
before inoculation of the corresponding bacillus into the peritoneal
cavity. This serum exercises a most marked protective action on the
mouse, an animal very susceptible to the pathogenic action of this
micro-organism. In mice so prepared complete and rapid phagocytosis
takes place. These micro-organisms before being ingested by the
phagocytes show no appreciable change; they are always stained very
uniformly and intensely by Gram’s method, and they never swell up. The
bacilli undergo no agglutination in the body of the mouse, a fact of
which we can convince ourselves by examining hanging drops of the
exudation. The phenomenon which strikes the observer most is the very
pronounced phagocytosis, due principally to the activity of the
microphages. Some hours after inoculation these cells are found to be
crammed with bacilli, a large number of which no longer stain in the
normal fashion. Without being transformed into granules, these
micro-organisms undergo intracellular digestion which at the end of a
few days is complete. This destruction is more rapid and complete in the
microphages, slower in the macrophages. Drops of exudation collected
from these mice, at a stage when the ingestion is completed, produce
fatal septicaemia in untreated mice. This is proof that at the moment
when they were seized by the phagocytes the bacilli still retained their
virulence. Mesnil, as the result of his experiments, concludes that “the
effect of the serum is to stimulate the phagocytes and especially the
polynuclear forms; they ingest more quickly, they digest more quickly.
The serum is, therefore, a stimulant of the cells charged with the
defence of the animal” (p. 496).
[Sidenote: [324]]
We need not describe the phenomena produced in mice inoculated
subcutaneously and treated with protective serum, for even in the
peritoneal cavity neither Pfeiffer’s phenomenon nor any extracellular
destruction of the bacilli can be observed. The micro-organisms, when
subjected to the influence of the specific serum, readily absorb the
fixative, as demonstrated by Bordet and Gengou[473]. This absorption
must certainly favour the action of the intraphagocytic cytases. It is
not, however, sufficient to explain the protective, anti-infective
action of the serum. Such explanation was given by the experiments which
Gengou, at my request, was good enough to make. He inoculated mice with
the bacilli of swine erysipelas, mixed with specific serum heated to 55°
C., to which was added some normal guinea-pig’s serum. The mice so
treated resisted the infection but controls died in a few days. Being
thus assured of the protective action of the serum, Gengou prepared the
same mixtures of swine erysipelas bacilli and of the two serums; but,
instead of injecting the whole of the mixture, he removed the bacilli
from the serums, after a prolonged contact, and injected the bacilli
alone into the mice. The bacilli had become permeated with fixatives,
but, in spite of this, they killed the mice almost as quickly as the
controls. Consequently, it is not the fixative adherent to the
micro-organisms which determines the protective action of the specific
serum. This fluid must contain another substance, one that will
stimulate the phagocytes.
[Sidenote: [325]]
The analysis of the mechanism of the immunity termed passive, that is to
say, communicated to normal animals by the introduction of an
anti-infective specific serum, teaches us that, even when the direct
action of the humoral substances is very limited, the protective effect,
thanks to the stimulant action which brings about the destruction of the
micro-organisms through the mediation of the phagocytic reaction, is
still produced. The result at which we have thus arrived is confirmed by
the examination of the phenomena observed in animals subjected to the
action of anti-anthrax serum. Marchoux[474] first supplied us with
precise details as to the mode of action on the rabbit of the serum of
animals treated with anthrax bacilli. He found that, in the peritoneal
cavity of rabbits injected the day before with anti-anthrax serum, the
inoculated anthrax bacilli almost immediately become the prey of
phagocytes. Within a couple of minutes after the introduction of bacilli
into the peritoneal cavity, the great majority of them are ingested by
the leucocytes; ten minutes later, there are no free bacilli. Not only
the ingestion but also the destruction of these micro-organisms takes
place with great rapidity, and even a few hours after the injection, the
peritoneal exudation, when sown on nutrient media, remains sterile. In
the subcutaneous tissue the phagocytic reaction requires a longer time
than in the peritoneal cavity, nevertheless, it goes on very rapidly.
Thus, when inoculated into the subcutaneous tissue of the ear of rabbits
treated with specific serum, the bacilli are in great part ingested at
the end of half-an-hour. At the end of an hour phagocytosis is usually
complete.
In Marchoux’s experiments the importance of the part played by the
phagocytes becomes still more prominent when it impedes their function
in any way. Rabbits injected with anti-anthrax blood and 24 hours later
inoculated below the skin of the ear with anthrax bacilli always resist
infection, exhibiting the well-marked phagocytosis just mentioned. In
other rabbits, however, prepared in the same way with the serum, but
inoculated the following day into an ecchymosis excited by tapping the
ear lightly, a certain number of the bacilli escape the phagocytes and
succeed in setting up an abundant oedema followed by a fatal anthrax at
the end of a few days. On making a post-mortem examination of these
animals the bacilli were not numerous, but they were found in all the
organs. The same result was obtained in another experiment in which
Marchoux inoculated subcutaneously with anthrax blood which coagulated
_in situ_ rabbits prepared with specific serum. The blood clot attracted
only the macrophages, as pointed out in Chapter IV. The microphages did
not come up until late and then in small numbers. Now, as these are the
phagocytes that are chiefly instrumental in destroying the anthrax
bacillus, their absence allowed the bacilli to multiply and to set up a
fatal anthrax. The rabbits prepared with the same serum but injected
with anthrax blood diluted with broth (which prevents the formation of
clot) completely resisted infection, thanks to the phagocytic reaction
which went on without hindrance.
[Sidenote: [326]]
Sclavo[475] also, who made numerous investigations on the action of the
anti-anthrax serum, is of opinion that this action is not a direct one
upon the bacillus but is produced indirectly through the action of the
animal organism. He maintains that the serum stimulates the function of
the phagocytes and augments the bactericidal action of the body fluids.
But since this bactericidal power enters the cytase as a substance
destroying the micro-organisms, and this cytase is contained in the
phagocytes, we can readily understand what a dominant part in the
process these elements play.
Sobernheim[476], also, has paid much attention to the question now under
discussion. As the result of his researches he comes to the conclusion
that the anti-anthrax serum “cannot exert any effect on the virus by a
direct action of the protective specific substances.” In order that the
serum may be effective, the active intervention of the organism of the
animal is necessary, otherwise, it is impossible to explain why the
serum, used in the same proportion against the same quantity of anthrax
bacilli, should protect one species of animals (the rabbit) and allow
another (guinea-pig, mouse) to succumb. When Sobernheim tried to apply
to anthrax the discovery of the transformation of cholera vibrios into
granules, he got only negative results. There was nothing produced
comparable to Pfeiffer’s phenomenon and the anthrax bacilli usually
underwent no apparent modification. Sobernheim affirms also that the
rapid phagocytosis under the influence of the serum, described by
Marchoux, “does not appear to be produced under all circumstances” (p.
117). As, however, his researches on this subject were made on
guinea-pigs which, in spite of the treatment with specific serum, always
ended by succumbing to anthrax, we readily understand that his results
cannot be compared with those obtained by Marchoux. I was present at the
experiments of this observer and convinced myself of the accuracy of the
facts recorded in his memoir.
[Sidenote: [327]]
Most of the examples here studied justify fully the hypothesis of the
stimulant action of protective serums, a view that I formulated as the
result of my researches on the immunity of rabbits against the Gentilly
cocco-bacillus[477]. In this the first case of anti-infective immunity,
due to the serum elaborated by an immunised animal, I could not find
either a bactericidal action, however slight, or any agglutinative or
attenuating property of the fluids of the body. As, on the other hand,
this serum had no antitoxic power, everything indicated that we must
look for its action, which was _nil_ or very slight on the
micro-organism, as being exerted on the organism of the animal into
which it was injected for protective purposes. A comparative examination
of the course of the phenomena in the subcutaneous tissue of the ear in
rabbits, some of which received an injection of the specific serum into
the veins whilst others were kept as controls, at once showed how widely
different were the two cases. In the control animals, the cocco-bacilli
immediately began to multiply without meeting with any opposition on the
part of the organism of the animal; on the other hand, in the rabbits
treated with serum, the serum became rich in leucocytes which at once
set to work to ingest the micro-organisms. In course of time the latter
gradually diminished in numbers, whilst the leucocytes went on
increasing. The phagocytosis, also, became more and more marked. This
struggle was continued for more than 24 hours, after which the purulent
exudation, containing masses of leucocytes, no longer included any
cocco-bacilli visible under the microscope either outside or inside
cells. Nevertheless, this pus was still capable of producing a fatal
septicaemia in untreated rabbits, clearly proving that it still
contained some living and virulent micro-organisms. These cocco-bacilli
persisted for a long time inside the phagocytes; their presence being
demonstrated by injecting the exudation into unprotected rabbits and
thus setting up a fatal infection. Finally, however, they disappear
completely. On consideration of such facts as these I considered that I
was justified in formulating the following conclusion at the end of my
memoir: “From the facts I have described, taken collectively, we may
draw the conclusion that the preservation of unvaccinated rabbits
treated with serum is due to a superactivity of the phagocytic defence;
and it is allowable to express the opinion that the protective serum of
hog cholera acts in rabbits by stimulating the phagocytes, rendering
them less sensitive to the toxins, and by stimulating them in their
struggle against the bacteria” (p. 310). The facts since collected by
various observers fully justify this hypothesis. Amongst the other
micro-organisms against which a rapid immunisation has been obtained by
means of serum, we must cite the cocco-bacillus of bubonic plague.
Numerous experiments, carried out on several species of animals, have
shown that antiplague serum markedly augments the phagocytic reaction.
[Sidenote: [328]]
In the group of the cocci, the streptococci have been specially fully
studied from the point of view now under discussion. As already stated
in another chapter, success has been attained not only in thoroughly
immunising several species of animals against this dreaded
micro-organism but active serums have been obtained capable of
conferring distinct and certain immunity. The protective action of
Marmorek’s serum, prepared at the Pasteur Institute, has been specially
carefully studied. This serum is obtained from horses that have received
numerous injections of various races of streptococci pathogenic for
animals and for man[478]. At Louvain, Denys and his pupils prepared
several other antistreptococcic serums and studied their protective
effect on laboratory animals.
[Sidenote: [329]]
In collaboration with Leclef, Denys[479] began by vaccinating rabbits
against streptococci and studied the mechanism of the immunity obtained
in these animals. A summary of their researches will be found in the
eighth chapter. Denys and Leclef considered that the serum of vaccinated
rabbits intervenes in two ways, first by directly hindering the
multiplication of the streptococcus and then by exalting the activity of
the leucocytes. They applied these results to the case in which immunity
is conferred upon normal rabbits by the intervention of the serum of the
vaccinated rabbit, but they were unable to furnish any data bearing
directly on this immunity. Somewhat later, Denys[480], in collaboration
with Marchand, published another memoir in which he describes
experiments on the mechanism of the immunity conferred on rabbits by
injections of the blood-serum of vaccinated horses. From these
experiments they draw the conclusion that “the serum of the horse
immunised against the streptococcus possesses no bactericidal
properties, properly so called, against this micro-organism; it does not
affect it directly; but it contains a substance which renders the
phagocytic power of the leucocytes extremely active. Even in the
presence of small quantities of this serum, the white corpuscles rapidly
ingest the streptococci and are capable of stopping all development so
long as they retain their amoeboid movements.” “The action of the serum
upon the leucocyte in its conflict with the streptococcus, is really
derived from the horse immunised against this organism. It exists
neither in the ordinary horse nor in the horse vaccinated against
diphtheria” (p. 15). Against these experiments of Denys and Marchand we
might bring the same objection that we raised against the analogous
experiments of Denys and Leclef, because, in both cases, these writers
lay too much stress on the presence or absence of the phenomena of
phagocytosis in preparations kept outside the body of the animal. Under
these conditions phagocytosis is effected in a fashion too artificial to
be capable of furnishing exact information.
Von Lingelsheim[481] met Denys and Marchand with the fact that, in their
researches, the serum of the horse immunised against the streptococcus
was only feebly bactericidal. After a prolonged contact (6–12 hours)
with a specific serum, the streptococci, when transferred to rabbit’s
blood, showed retarded development as compared with streptococci
subjected to the influence of the antidiphtheritic and antitetanic horse
serum. Von Lingelsheim himself, however, points out that the
bactericidal action of the antistreptococcic serum was feeble and
transient, and required the intervention of the reaction of the animal
cells within the body.
[Sidenote: [330]]
The researches carried out by Bordet[482] in my laboratory are not open
to the objections that we were justified in putting forward against the
experiments made by Denys and Marchand, since he carefully watched the
phenomena of immunity as they developed in the body of the animal
subjected to the action of antistreptococcic horse serum. Bordet began
by studying the properties of this serum and accepted Denys’ and
Marchand’s statement that bactericidal power, however small, was absent.
The streptococcus grows as well in this serum as it does in that of the
untreated horse. In the specific serum, however, markedly longer chains
are produced than in normal serum. This difference is found only in the
earliest period of the growth. The agglutinative power of the
antistreptococcic serum is but feebly marked. The injection of a large
quantity of this serum into a normal rabbit confers no bactericidal
power upon the serum of this animal. “The serum extracted 24 hours after
injection is quite as suitable for use as a culture medium as that
furnished by the blood before the introduction of the serum. In both the
micro-organism grows rapidly and vigorously” (p. 195). Consequently, in
the antistreptococcic serum there is nothing comparable to what we
obtain so readily with antivibrionic serum: nothing which recalls
Pfeiffer’s phenomenon, even of an attenuated nature. We have already
noted the result obtained by Bordet, according to which the
streptococci, developed in the specific horse serum, were found to be
endowed with their normal high virulence. The antistreptococcic serum,
injected into the peritoneal cavity of the rabbit the day previous to
the microbial inoculation, protects the animal absolutely, provided that
the micro-organisms be not too numerous or the quantity of serum not too
small. Under these conditions the virus is ingested pretty rapidly and,
so far as we know at present, completely. The micro-organism is thus
prevented from developing and the animal remains in good health, whilst
the control animal, which has received no serum, dies in a very short
time.
When the number of streptococci is increased the effort of the animal
organism to get rid of them becomes, in spite of the protective serum,
more severe and much more prolonged. Some of the micro-organisms
certainly become the prey of phagocytes, but a sufficient number remain
free in the peritoneal cavity to multiply rapidly. When the number of
streptococci has become sufficiently great a phenomenon, to which Bordet
gives the name of “phagocytic crisis,” is suddenly observed. In the
peritoneal exudation, which has become thick and has taken on the aspect
of a homogeneous and white pus, a most rapid phagocytosis is evidently
set up. In a short time the whole of the streptococci, which were
swarming outside the cells, are ingested by the leucocytes. “The
essential condition for recovery is always this complete ingestion” (p.
203). If the ingestion is not general, the rabbit may die, although much
later than the control animal.
[Sidenote: [331]]
The phases of the struggle between the animal organism, when subjected
to the influence of the protective serum, and the streptococcus, recall
Salimbeni’s experiments on immunised horses. The rabbit, in which
phagocytosis could not take place at once owing to the presence of too
large a number of micro-organisms, exhibits first a stage of free
development of the streptococci, after which the phagocytes begin to
fulfil their antibacterial function. Here it is especially the
macrophages which act; the microphages, although present in fairly large
numbers, are entirely inactive. This first stage of phagocytic reaction
is insufficient. It is followed by a period when the streptococcus
appears to gain the upper hand. Many small chains, having escaped the
phagocytes, multiply and give birth to quite a new generation of
micro-organisms. If a fresh impulse to phagocytosis does not take place
the animal dies from infection. When, however, the protective serum has
been of sufficient strength, a new army of leucocytes arrives on the
scene and these become masters of the situation. Phagocytosis becomes
complete and microphages as well as macrophages devour a large number of
streptococci.
Bordet, who, through his previous investigations, was well acquainted
with the direct action of the protective serum on vibrios, could find
nothing resembling it taking any part in the struggle of the organism of
the animal treated with antistreptococcic serum against the
streptococcus. The most that he could find was that the streptococci
which again begin to swarm in the exudation are smaller in size than the
normal streptococcus. It must be accepted, as indicated by the most
recent researches, that this micro-organism becomes permeated by the
fixative substance of the specific serum. We know already, however, that
this fixation cannot deprive the micro-organisms of their virulence. In
any case, then, a large share in the process must be attributed to the
action of the phagocytes, stimulated by the protective serum, in the
struggle of the animal against the streptococcus.
[Sidenote: [332]]
Having considered this series of examples of immunity against bacteria
conferred by specific serums, we are in a position to form some idea of
the mechanism of this immunity. Before we come to any general
conclusion, it may be useful to glance at an example of this so-called
passive immunity against a micro-organism belonging to the animal
kingdom. Such examples are not numerous, as, in the majority of cases of
acquired immunity against Protozoan parasites, the serum is inactive and
incapable of communicating immunity to normal individuals. We have only
a single example, the _Trypanosoma_ of rats, against which Dr Lydia
Rabinowitch and Dr Kempner[483] have demonstrated the possibility of
immunisation by the blood serum of vaccinated white rats. The mechanism
of this immunity has been studied by Laveran and Mesnil[484], who found
that it was like that described (Chap. VIII) in connection with the
immunity in white rats, conferred by the inoculation of living
_Trypanosomata_. The specific serum does not affect these infusoria
except that it brings about slight agglutination. _Trypanosomata_ placed
in contact with it retain their pristine vitality and motility. This
fact led Mme Rabinowitch and Dr Kempner to advance the hypothesis that
the protective action of the serum must depend upon its antitoxic power.
Since, however, in the infection of rats by the _Trypanosomata_, the
toxic action is very feeble if not nil, it is very difficult to accept
this view. It certainly appears to be much more probable that the serum
acts in this case, as in many others, by stimulating the phagocytic
reaction. The rapidity with which the living _Trypanosomata_ are
ingested by the phagocytes has been shown by Laveran and Mesnil.
[Sidenote: [333]]
Reviewing the whole of the data on immunity produced under the influence
of anti-infective or protective serums, it is evident that they fall
under two main categories. On the one hand there is a direct action of
these serums on the micro-organisms, an action that is either
microbicidal properly so called, agglutinative, or fixative. On the
other hand, a stimulation of the phagocytic defence which leads to the
final destruction of the micro-organisms is set up. This last factor is
general; even in the case where the direct action is most marked
(vibrios in the phagolysed peritoneal cavity), its importance is
considerable. The micro-organisms which can be deeply injured by the
direct action of the specific serum are few in number. In most cases
this action is a feeble one and needs, for its completion, effective
co-operation on the part of the phagocytes. In this respect
micro-organisms present a whole gamut which begins with the cholera
vibrio, the micro-organism most sensitive to the action of the body
fluids, and ends with the _Trypanosoma_ of the rat, a flagellated
Infusorian which cannot have even its motility affected by the direct
action of the fluid elements. In all these cases, of course, the
immunity conferred by the serums is due to the final destruction of the
micro-organisms which invariably resolves itself into the same
fundamental act—digestion by the cytases, a phenomenon which can only be
produced at all quickly by the action of cytases contained in the
protective serums or that have escaped from the phagocytes during
phagolysis. The digestion by the cytases may also, and this is usually
the case, be effected only after the manifestation of a regular series
of vital phenomena on the part of the defensive elements of the body. As
this factor fills such an important _rôle_, it is readily understood
that we can not accept the term passive immunity by which to designate
the immunity conferred by the specific serums. The action of the
cytases, which is necessary to bring about the final result in this
immunity, depends too much on the activity of the cells which contain
the bactericidal ferment. For this reason, when the functional activity
of the phagocytes is in abeyance or is retarded, the animal succumbs, in
spite of the presence in its organism of a more than sufficient quantity
of cytases. In this connection Wassermann’s[485] suggestion of adding
normal serums rich in cytases to the specific serums must be regarded as
very apposite. When protective serums poor in cytases or which have lost
them as the result of heating, of the use of antiseptics, or simply from
the influence of time, are injected, no immunising effect is ever
obtained, simply because of the inactivity of the phagocytes, the cells
in which the cytases are found. If at the same time normal serum rich in
cytases ready prepared be injected, better results should be obtained.
We may recall here an analogous example—the anthrax of the rat. Although
possessing a large quantity of cytase, very effective against the
bacillus, the organism of the rat can make no use of it, because the
phagocytes which contain it do not manifest a sufficient activity. But
the injection into a rat of blood serum from the same species containing
a certain amount of cytase that has escaped during the formation of the
clot, is sufficient to preserve the animal against a fatal infection.
[Sidenote: [334]]
To support his view, sound in principle, Wassermann made an experiment
the interpretation of which presents certain difficulties. He injected
guinea-pigs with protective antityphoid serum, in a dose insufficient to
protect them against a fatal infection. By introducing along with this
serum a certain quantity of normal ox serum which, by itself, is also
incapable of averting a fatal issue, Wassermann obtains an absolute
immunity of his animals. This immunity is due, according to Wassermann,
to the cytase of the ox serum acting along with the fixative of the
specific serum. The united action of the two ferments causes the death
of the micro-organisms. Besredka[486] has justly observed that normal ox
serum contains, in addition to cytases, a substance which exerts a
distinct agglutinative action on the typhoid cocco-bacillus and another
which stimulates the phagocytic action. These two substances resist a
temperature of 55°–60° C., and Besredka shows that with normal ox serum,
deprived of its cytases by heating as above, we can obtain the same
protective effect as with the same serum unheated.
As the result of another series of experiments, Wassermann[487]
recognises the immunising action of normal serum heated to 60° C. and so
entirely deprived of its cytases. Into the peritoneal cavity of
guinea-pigs he injects, mixed with heated normal rabbit’s serum, a dose
of typhoid cocco-bacilli several times greater than the lethal dose. The
guinea-pigs resist this completely. Analysing the mechanism of this
immunity, Besredka (_l.c._ p. 229) attributes it to the combined action
of the agglutinin and of the substance which stimulates the phagocytes.
We have here another proof that the stimulins which play such an
important part in immunity conferred by serums, are found not only in
the specific serums, but also in normal serums, whether unheated or
heated to 55°–60° C.
The protective property of the normal serums of man and animals against
the cholera vibrio has already been referred to. We may now go a little
more deeply into the mechanism by which these serums act. This task is
an easy one thanks to the important work by Issaeff[488] carried out in
R. Pfeiffer’s laboratory. Having confirmed the observation, made by
other investigators, that blood serum from the human subject, whether in
health or affected by any disease, is capable of protecting the
guinea-pig against the cholera vibrio provided that it is injected 24
hours before the micro-organisms, Issaeff studied the phenomena observed
in the peritoneal cavity of the animals experimented upon. By means of
small capillary pipettes he drew off at intervals a small quantity of
fluid from the peritoneal cavity and examined it in hanging drop or in
stained preparations. Some time after the injection this fluid became
more and more rich in leucocytes which seized the vibrios, ingested and
destroyed them. To obtain this protective effect it was necessary to
inject from 0·1 to 5 c.c. of human blood serum. With these doses he
could prevent, not only infection of the guinea-pigs by the cholera
vibrio, but also the lethal effects of other vibrios. The protective
action of normal human serum is general, therefore, and not specific,
such as is the immunity conferred by the serums of vaccinated animals or
of the human subject who has suffered from an attack of cholera.
[Sidenote: [335]]
Shortly afterwards Funck[489] confirmed this result in the case of the
typhoid cocco-bacillus. He observed that normal horse’s serum, injected
as a protective agent in the dose of half a c.c. into the peritoneal
cavity of the guinea-pig, preserved this animal from a fatal infection.
Pfeiffer and Kolle and Chantemesse and Widal obtained the same results
with human serum. The former observers lay special stress on the
non-specific character of this protective action of normal serums. As to
its mechanism, Funck sums it up as follows: “the specific serum brings
about a rapid lysis of the bacilli, normal serum acts in a much more
limited fashion; if the dose is very large and if the animal resists
infection, the phenomena of extracellular degeneration are rarely
appreciable, and it seems that here the specially important factor is
the intracellular destruction of the bacteria, in the phagocytes” (p.
70).
Wassermann has shown the protective action of normal serum against the
experimental disease produced by the staphylococcus. This action,
although not absolutely general, is nevertheless widely distributed.
Wassermann[490], from comparative investigations on this subject, came
to the conclusion that “the serum of a different species of animal acts
by greatly increasing the resistance, whilst the serum of the same
species produces an effect which is not nearly so marked.” As in these
normal serums a stimulating influence on the phagocytes is specially
marked, it may readily be understood that the serum of the same animal
or of the same species does not produce so energetic an effect as the
serum of a different species. As these normal serums possess, not only
the property of exciting phagocytosis, but often also that of rendering
motionless and of agglutinating certain micro-organisms, there might be
some difficulty in interpreting the part played by these serums. It may
be useful, therefore, to pass in review the protective action of fluids
less complicated than blood serums.
[Sidenote: [336]]
Issaeff, in the work already cited, demonstrated that not only normal
serums but a whole series of fluids, such as urine, broth, etc., exert a
protective effect against microbial infections. These fluids must be
injected about 24 hours before the introduction of the bacteria. The
best method consists in injecting them directly into the peritoneal
cavity, after which the animals acquire an immunity against absolutely
fatal doses of cholera vibrios. Funck verified this observation for the
infection caused by the typhoid cocco-bacillus, and Bordet confirmed it
for the streptococcus. The injection of peptonised broth into the
peritoneal cavity of the normal guinea-pig, made 24 hours before an
inoculation of double the fatal dose of the streptococcus, exerts a
distinct protective action; the infection does not kill the animal. This
broth is neither bactericidal, attenuating, nor agglutinative; it forms
a good culture medium for the streptococcus and possesses no fixative
power. Consequently it does not act directly on the vitality or
virulence of the micro-organism; nevertheless, it is distinctly
protective.
According to Issaeff’s researches, the protective substances used by him
must be arranged in the following order as regards their action against
the cholera vibrio. Tuberculin is the most effective; then comes a 2%
solution of nuclein, followed by normal human serum, broth, and urine,
whilst physiological saline solution is the least active. All prevent
infection by the vibrios, but the protection is effective for some days
only; this protective action is exerted against various kinds of
bacteria, being in no sense specific.
Pfeiffer lays so much stress on the great difference between the
protective power of normal serums, as well as of the other fluids
mentioned, and that of the anti-infective specific serums, that he even
proposes to classify the first group as giving rise to _pseudo-immunity_
or _resistance_. This view is certainly an exaggerated one, because it
is difficult to draw a very distinct line between the two groups of
phenomena. There are normal serums, of which 0·1 c.c. is quite
sufficient to confer the protective effect, just as there are specific
serums of which it is necessary to make use of a much greater dose to
attain the same result.
[Sidenote: [337]]
Protective fluids, other than the serums, only manifest their influence
by exciting a great phagocytic “superactivity.” As the result of their
injection into the peritoneal cavity of normal guinea-pigs, first a
transitory phagolysis is induced, this being soon replaced by a very
considerable afflux of leucocytes, which is maintained for 24 hours or
longer, and then gives place to the normal condition. It is during the
period of the greatest leucocytosis of the peritoneal fluid that the
animal exhibits the most marked resistance against infective
micro-organisms. The vibrios are rapidly ingested by the phagocytes,
without having previously been acted upon by the “humours.” Bordet shows
that the same thing happens in the case of the streptococcus inoculated
into guinea-pigs after a protective injection of peptonised broth.
[Illustration:
FIG. 42. Culture of the plague bacillus developed within a macrophage
from guinea-pig.
]
[Illustration:
FIG. 43. Macrophage from guinea-pig filled with plague bacilli.
]
[Illustration:
FIG. 44. Macrophage from guinea-pig containing plague bacilli which
are commencing to escape from the protoplasm.
]
[Illustration:
FIG. 45. Macrophage from guinea-pig which has burst as the result of
the development of plague bacilli within it.
]
[Sidenote: [338]]
We have observed the same phenomenon in guinea-pigs and white rats
inoculated with the cocco-bacillus of plague. Treated with freshly
prepared peptonised broth the day previous to inoculation, these animals
oppose to the micro-organism a much more marked resistance than do the
control animals. The injection of the cocco-bacillus of plague sets up a
marked phagocytosis on the part of the macrophages. These cells ingest
large numbers of micro-organisms which, after a time, have all passed
into the phagocytes. If a drop of the peritoneal exudation is now
withdrawn, we find only intracellular cocco-bacilli (fig. 43). If the
drop be kept for some time outside the animal and at a suitable
temperature the macrophages may be seen to perish and the
micro-organisms to develop in their contents. We thus obtain abundant
cultures which pass from the interior of the macrophages into the fluid
of the exudation (figs. 42, 44, 45). When the animals are not
sufficiently protected the same phenomenon is observed in the peritoneal
cavity of the living animal. The macrophages, crammed with
cocco-bacilli, burst, allowing the micro-organisms to escape. These
multiply in the peritoneal fluid and spread through the animal, which
soon dies.
Wassermann affirms that “the artificially increased resistance is
nothing but an active and reinforced afflux of the complements (cytases)
towards one point in the animal, for the purpose of digestion.”
(_Ztschr. f. Hyg._, Leipzig, 1901, Bd. XXXVII, S. 199.) Wassermann does
not explain how this afflux of cytases is produced. The entirely
concordant researches on this point by Issaeff, Funck, Bordet, and
ourselves, prove that this afflux takes place not through the mediation
of the fluids, but solely through the phagocytes, the carriers of the
cytases. Consequently it is beyond dispute that in the immunity
conferred by physiological saline solution, broth, and several other
fluids, we have to do solely with an augmentation of the phagocytic
reaction. In the immunity conferred by normal or specific serums, this
same stimulating factor still plays the more important part. Along with
it, however, there is an intervention more or less pronounced, according
to circumstance, and more or less frequent, of cytases, brought by the
serums prepared outside the body or that have escaped during phagolysis,
as well as of substances truly humoral, such as the fixatives or the
agglutinins.
[Sidenote: [339]]
Amongst the non-specific substances which are capable of conferring an
immunity more or less stable, must be placed the products of
micro-organisms other than those against which we wish to protect the
animal. Pasteur[491] noted that when the anthrax bacillus, mixed with
other micro-organisms, in themselves inoffensive, is inoculated into
animals, anthrax does not develop and the animals remain well. Later,
Emmerich[492] showed that the streptococcus of erysipelas exerts an
antagonistic influence against the anthrax bacillus. He succeeded in
immunising and even in curing rabbits inoculated with anthrax, by
submitting them to the action of this streptococcus.
These experiments served as the starting-point for several works on the
vaccination of animals against anthrax by means of various
micro-organisms, as well as by their products. Pawlowsky[493],
Watson-Cheyne[494], and Bouchard[495] have proved that bacteria not very
pathogenic and even saprophytes, such as the _Coccobacillus
prodigiosus_, Friedländer’s bacillus, and the _Bacillus pyocyaneus_,
were also capable of preventing infection by the anthrax bacillus.
Freudenreich[496] showed that not only did the bacillus of blue pus
exert an antagonistic action but that the same effect could be obtained
with sterilised cultures of this organism. Woodhead and Cartwright
Wood[497] studied the vaccinating action of these products on rabbits
inoculated with virulent anthrax bacilli. The animals resisted
completely or survived for some time. Analysing the phenomena produced
under such conditions, these two authors came to the conclusion that the
action of sterilised cultures of _Bacillus pyocyaneus_ is “indirect and
as taking place either by opposing itself to the action of the poison
upon the tissues, or by stimulating certain tissues and increasing their
functional activity.” With the object of obtaining an exact
interpretation of this antagonistic influence I suggested to M.
Blagovestchensky[498] that he should investigate in detail the phenomena
which take place in the organism of rabbits inoculated with the anthrax
bacillus and submitted to the action of sterilised cultures of the
_Bacillus pyocyaneus_. At the very outset this observer was met by the
fact that these cultures act directly upon the vitality of the anthrax
bacillus. Thus the association of the former with the anthrax bacillus
_in vitro_ was sufficient to interfere with the development of the
latter. Under these conditions he had to renounce the investigation of
the part played by the cellular elements of the rabbit in the antagonism
of the two bacteria.
[Sidenote: [340]]
Friedländer’s bacillus has been found to be much more suitable for this
line of research as is shown by work carried out by Freiherr von
Dungern[499] in my laboratory. This observer convinced himself that
“anthrax bacilli are weakened neither by the encapsuled bacilli nor by
the substances which they contain.” These micro-organisms do not
interfere in the slightest with the anthrax bacilli either outside or
within the animal, and “when the anthrax infection does not become
generalised it is due to the fact that the anthrax bacilli are ingested
by the phagocytes at the seat of inoculation and destroyed within these
cells” (p. 183).
In this action of foreign micro-organisms upon micro-organisms against
which we wish to protect the animal we have to deal with something
analogous to the condition we obtain when immunising with normal serums
or with any other kind of fluid. In both cases immunity is rapidly
established, but it is very transient and is confined to a stimulation
of the phagocytic resistance. Direct action may also intervene, as in
the case of _Bacillus pyocyaneus_, but it is not indispensable. The
animal whose phagocytes are in a condition of superactivity can do
without this direct action, its own resources being sufficient to arrest
anthrax.
Following the same lines of investigation as those on the antagonism
between the anthrax bacillus and several other micro-organisms,
Klein[500] has demonstrated that, in order to prevent a guinea-pig from
contracting experimental cholera peritonitis, it is only necessary to
inject into it, the day before infection, a culture of Finkler and
Prior’s vibrio or of certain other bacteria. These experiments by Klein
served as the point of departure for Issaeff’s work which led to the
discovery of the stimulating influence of all kinds of fluids injected
into the peritoneal cavity of guinea-pigs.
In this transient immunity obtained with products foreign to the
micro-organism against which one is vaccinating, the most constant and
consequently most important part is again played by the phagocytes. But
there is associated with it an influence, greater or less in degree, of
substances present in the serums, such as the microcytases and
fixatives, which are able to exercise a direct action on the pathogenic
micro-organisms. In all cases known and analysed up to the present, the
intervention of the living organism of the animal is indispensable,
consequently this form of acquired immunity against micro-organisms
cannot be regarded as being really passive.
CHAPTER XI
NATURAL IMMUNITY AGAINST TOXINS
Examples of natural immunity against toxins.—Immunity of spiders and
scorpions against tetanus toxin.—Immunity of the scorpion against
its own poison.—Antivenomous property of the blood of the
scorpion.—Immunity against tetanus toxin in the larvae of
_Oryctes_ and in the cricket.—Immunity and susceptibility of frogs
against this toxin.—Natural immunity of reptiles against tetanus
toxin.—Antitetanic property of the blood of alligators.—Immunity
of snakes against snake venom.—Immunity of the fowl against
tetanus toxin.—Immunity of the hedgehog against poisons and
venoms.—Immunity of the rat against diphtheria toxin.
[Sidenote: [341]]
As in this book we are dealing specially with the immunity against
infective diseases, the question of the resistance of the animal to
poisons interests us only in so far as it is related to immunity against
micro-organisms. Consequently the reader must not expect a treatise on
intoxications properly so called nor one on immunity against all kinds
of poisons. To perform such a task we should have to far overstep the
bounds of the subject that we have chosen and enter upon an examination
of questions which are beyond our sphere. Our chief aim is to present to
the reader a summary of our present knowledge on immunity against
microbial toxins and to establish the relations between this kind of
immunity and immunity against infective micro-organisms. In order to do
this, however, we shall have now and again to go beyond the limits of
our programme and discuss certain problems bearing on the resistance of
the animal organism against poisons not of microbial origin.
[Sidenote: [342]]
The immunity against toxins, like that against the micro-organisms
themselves, may be either natural or acquired. As many poisons have been
known from time immemorial, we are able to collect numerous observations
on the resistance of the animal organism to such substances made when
there was no idea of immunity against infective diseases. The etiology
of intoxications is often much more evident and simple than is that of
infections; this is one of the reasons that the older conceptions on the
subject of immunity against poisons were more advanced than were those
on immunity against infective diseases.
Several examples of natural immunity in the lower animals have already
been cited. Thus, we have seen in previous chapters that the Infusoria
are resistant to poisons that exert a powerful action on a large number
of the higher animals, such as the tetanus and diphtheria toxins and
especially the ichthyotoxin of eel’s serum. We have mentioned the case
of the larva of _Oryctes nasicornis_ which is unaffected by large doses
of the toxins of certain bacteria and which at the same time is very
subject to fatal infections by very small doses of the bacteria that
form the poisons. These larvae, like those of the cockchafer, are,
however, fairly susceptible to the poison of the scorpion. Several other
species of Arthropoda, which have been studied from the point of view of
immunity against toxins, have exhibited analogous features. Thus spiders
and scorpions are refractory to tetanus toxin. In one experiment I
injected into the abdominal cavity of a _Mygale_ from the Congo (which
weighed 7 grm. 5) 1 c.c. of tetanus toxin on two several occasions. This
dose is sufficient to kill, with the symptoms of tetanus, 1000 mice of
double the weight. The spider, kept in the incubator at 36° C., remained
quite well during the two months that the experiment lasted. It
exhibited no symptom, not even transient, of muscular stiffening, nor
any change in its habits and natural functions. The tetanus toxin
disappeared from the blood of the _Mygale_, but this blood at no time
showed the slightest antitoxic power against this poison. This example
of natural immunity cannot, therefore, be ascribed to any antitoxic
property of the fluids and must be regarded as a case of immunity of the
tissues—von Behring’s histogenic immunity. In the present imperfect
state of our knowledge it is impossible to describe precisely the
mechanism of this immunity. When we say that the spider is refractory to
the tetanus toxin because its susceptible elements have no receptors
capable of seizing the haptophore group of this poison, we simply give
expression to a hypothesis which we are not in a position to verify by
experiment.
[Sidenote: [343]]
The scorpion, a well-known representative of the Arachnida with
segmented abdomen, shares with the _Mygale_ in the immunity against
tetanus toxin. The Algerian and Tunisian scorpions (_Scorpio afer_ and
_Androctonus occitanus_) withstand the action of doses of this poison
which are fatal for 1000 mice and more. Taking weight as our standard we
may inject into them, with impunity, more than 5000 times as much toxin
as into mice, without setting up a single morbid symptom. Scorpions,
like the _Mygale_, live well in the incubator at 36° C., where they are
kept whilst submitted to the action of the tetanus poison. Here again we
have to do with a case of histogenic immunity. The fluids of the
scorpion exert no antitoxic action. When blood from the normal scorpion
is mixed with various doses of tetanus toxin and injected into mice
these animals contract tetanus and die just as do the control animals.
In certain exceptional cases some slight retardation was observed, but
the blood of the scorpion is, in most cases, incapable of preventing
tetanus in animals susceptible to this disease.
Scorpions, injected with tetanus toxin, do not retain it in their blood
for long. A few days after the injection of the tetanus poison such
blood, when injected subcutaneously into mice, excites no trace of
tetanus. The preparation of extracts of the different organs of
scorpions treated with tetanus toxin demonstrates that the liver and the
liver only absorbs the poison. It is found there a few days after the
injection of the toxin and it remains there unaltered for some
considerable time. The exudation of the liver of scorpions, killed a
month or more after the introduction of the toxin into the general
cavity, injected into mice sets up a typical and fatal tetanus.
The presence of the tetanus toxin in the organism of scorpions does not
give rise to the production of antitoxin. At any rate a whole series of
experiments on this point carried out by us never gave a positive
result. The scorpions resisted repeated doses of the tetanus toxin and
lived without any difficulty at 36° C., but their blood was never at any
period capable of preventing mice from contracting fatal tetanus.
Nevertheless the scorpion may possess antitoxic power.
[Sidenote: [344]]
Everyone has heard of the supposed suicide of the scorpion. We are told
that when this animal finds itself under conditions in which its death
is inevitable, it stings itself with the end of its tail and dies from
the effect of its own poison. A simple method of reproducing this
experiment is actually described:—Surround the scorpion with a circle of
fire. The animal rushes in all directions to find a way out, and finding
none, deliberately commits suicide. Bourne[501] at Madras carefully
investigated this question in a large species of Indian scorpion and
demonstrated the absolute erroneousness of the story of suicide which,
had it been true, would have afforded a unique example of voluntary
death in animals. On carrying out the classic experiment he observed
that within this ring of fire the scorpion is subjected to a very high
temperature. When the temperature reaches 40° C. the scorpion begins to
grow weak and as the temperature approaches 50° C. it passes into a
comatose condition. Moreover Bourne showed that the scorpion’s poison,
which is fatal for large spiders, insects, and vertebrates, was
innocuous for individuals of the species furnishing it.
I can confirm all the statements of this English observer. When I was
studying the embryology of the scorpion I repeatedly tried the
experiment but the animal never committed suicide. Further, I repeatedly
assured myself of the innocuousness of the scorpion’s poison when
injected into individuals of the same species, and I was able to
demonstrate most conclusively that the blood of the scorpion is endowed
with undoubted antitoxic power. The addition of 0·1 c.c. of this blood
to a dose of poison which kills mice in half-an-hour is sufficient to
enable a mouse injected with the mixture to resist it completely. This
antitoxic power is the same in the _Scorpio afer_ and in the Algerian
_Androctonus_. An emulsion of the liver of the scorpion, however, is
absolutely incapable of preventing fatal intoxication of mice.
[Sidenote: [345]]
This case of antitoxic action is the only one I have been able to
demonstrate in an invertebrate. Must we regard it as a case of natural
innate antivenomous power or as something acquired during the life of
the animal? It is not easy to settle this question by experiment. We can
certainly procure new-born scorpions and rear them for some time, but
the quantity of blood that can be got from them is insufficient for
injection for protective purposes. Scorpions do not love one another and
when kept together we often find them engaged in fierce and mortal
combat, the stronger killing the weaker and sucking their blood. It is
therefore possible that, in some stage of their life, the scorpions find
means of vaccinating themselves against their own poison either through
the intestine or as the result of punctures caused by the point of the
tail. It would be very interesting to study this question under
favourable conditions, because it is capable of throwing light on the
problem of the origin of antitoxins, from a general point of view.
Whichever view be taken, the acquisition of any antitoxic property by
the blood in the Invertebrata must take place slowly and with great
difficulty as is shown by our want of success with tetanus toxin.
Insects are, as a rule, very tolerant of this latter poison. As,
however, the tetanus toxin (we shall illustrate this later) only acts
well and in small doses at a high temperature (about 30° C.) and as most
insects do not readily adapt themselves to this temperature, it was
necessary to choose species capable of living at these high temperatures
and for this line of study the larva of _Oryctes_ is most suited. It
flourishes well at a temperature of 30°–36° C., and under these
conditions exhibits a much greater resistance to infection by _Isaria_
than at lower temperatures. It can be kept in the incubator for months
if placed in glass jars filled with earth mixed with tanner’s bark. The
injection of enormous quantities of very active tetanus toxin directly
into the blood has not the slightest effect on these larvae. Whilst,
however, the blood fluid of the Arachnida rapidly gets rid of the
poison, that of _Oryctes_ retains it for a very long period. If a small
quantity of blood be taken from larvae several months after injection
and then injected into mice, these animals contract typical tetanus and
quickly succumb.
The toxin, however, finally disappears from the blood though a certain
portion of it may still be found in the pericardial cells and especially
in the fat-bodies.
Never, under any circumstances, was I able to observe that the blood of
the larvae of _Oryctes_ exerted any antitoxic action. At the stage when
this fluid no longer gives tetanus to mice, it is absolutely incapable
of preventing intoxication when mixed, before injection, with tetanus
toxin.
Amongst adult insects the cricket is best adapted for researches on
tetanus. The field cricket will bear a temperature even higher than 30°
C. It is completely resistant to injections of tetanus toxin, but it
showed no more capacity than did the larvae of _Oryctes_ or the
Arachnida of producing any tetanus antitoxin.
[Sidenote: [346]]
All the Invertebrata that I have been able to study have exhibited a
remarkable resistance against the known bacterial toxins, but the
mechanism of this natural immunity could not be exactly made out owing
to the difficulty met with in investigating the toxins in the organs and
following their modifications. The idea of making use of these lower
animals for the purpose of solving the problem of the origin of
antitoxins is not realisable, from the fact that the Invertebrata that
have been studied have never, in my experience, produced any of these
substances as the result of injections, whether single or repeated, of
toxins.
The natural immunity of the Invertebrata against bacterial toxins cannot
therefore be regarded as an example of humoral immunity. It must be
placed in the category of histogenic immunity, although we are not in a
position to define accurately the part played by the cellular elements
in the defence of the animal against these poisons. We must, therefore,
go higher up in the animal scale if we are to solve the principal
questions in regard to antitoxic immunity.
[Sidenote: [347]]
The lowest Vertebrata, the fishes, are not well-suited for this kind of
research. The best known bacterial toxins act specially on warm-blooded
animals and require the co-operation of high temperatures. Fishes do not
live well in captivity except at relatively low temperatures and soon
die if placed in an incubator kept at 30° C. or higher. It is necessary,
therefore, to have recourse to the Amphibia, which are much more easily
acclimatised to these temperatures. The Axolotl, coming from Mexico, is
naturally capable of withstanding great heat. These animals will live
for long at a temperature of 30°–37° C. They possess the drawback,
however, of being very susceptible to the tetanus toxin, very small
doses of it being fatal. The green frog (_Rana esculenta_) is the most
suitable for our purpose. It readily adapts itself to optimum
temperatures (30°–36° C.) and exhibits at least a certain degree of
immunity against various bacterial toxins. We have stated in a preceding
chapter that the green frog is unaffected by considerable quantities of
diphtheria toxin. It is resistant also to tetanus toxin, but this
natural immunity appears to be connected with special conditions.
Courmont and Doyon[502] were the first to draw attention to the fact
that beyond 20°–25° C. green frogs may contract tetanus. Refractory in
winter they become susceptible in summer. These observers afterwards
found that of frogs inoculated with the same dose of toxin and divided
into two sets, one set kept at a temperature of about 10° C. remained
quite well whilst the other set subjected to one of 30°–39° C.
contracted tetanus after five days’ incubation. This experiment has been
confirmed by several observers, and indicates that the tetanus poison
demands, for the manifestation of its toxic action, a favourable and
fairly high temperature. This result must, however, be accepted with
some reserve. Undoubtedly the doses of tetanus toxin which induce fatal
tetanus in frogs kept at a high temperature are innocuous when these
animals are living at low temperatures. But we can, by increasing the
dose, produce tetanus in frogs even when the temperature is not very
high. Thus Marie[503] was able, during the whole of the winter, to
tetanise both green and brown frogs living in water the temperature of
which oscillated between 13° and 18° C. The incubation period in this
case is very much longer (sometimes extending to 25 days) than in frogs
kept at higher temperatures.
Temperature, therefore, is an important factor in the poisoning by the
tetanus toxin and in the resistance of the frog, but, in the long run,
this poison can exert its specific action even at relatively low
temperatures.
[Sidenote: [348]]
Morgenroth[504] endeavoured to analyse the mechanism of this resistance
and of the susceptibility of the green frog when maintained at various
temperatures. He demonstrated that the tetanus toxin is fixed in the
central nervous system, even at low temperatures, near 8° C.; under
these conditions, however, it is incapable of causing the slightest
tetanic symptom. When placed in an incubator kept at 32° C. the frogs
contract tetanus after a period of incubation of some (2 to 3) days.
During the first 24 hours of this period the frogs manifest no sign of
tetanus, and if they are again put in a cool place they continue in good
health. If, however, after a not too prolonged stay in the cold, these
animals are subjected a second time to the higher temperature, they
become tetanic, after a shortened incubation period. Cold, therefore,
may arrest tetanus even at a stage when the toxin has already produced
certain latent but permanent modifications of the nervous system.
Frogs injected with tetanus toxin and kept in a cold place finally get
rid of the poison. When transferred to a warm chamber after a certain
lapse of time they no longer contract tetanus. We have found that the
greater part of the tetanus toxin continues for some time in the blood
of frogs injected and kept at a low temperature. A small quantity of
this blood withdrawn almost two months after the last injection produced
fatal tetanus in a mouse. We do not know how frogs eliminate the toxin,
but it has been demonstrated that in this case it causes no production
of antitoxin. Morgenroth has confirmed this result.
[Sidenote: [349]]
Reptiles must be regarded as vertebrates exhibiting a most pronounced
natural immunity against tetanus. They show an unlimited resistance to
enormous doses of tetanus poison, and this at low, medium, or high
temperatures (30°–37° C.). Green lizards withstand considerable doses of
tetanus toxin. Although they do not contract tetanus, they get rid of
the poison exceedingly slowly. Thus, a lizard kept at a temperature of
20° C., and injected with an amount of toxin sufficient to kill 500
mice, at the end of two months still retains in its blood such an amount
of the poison that one-tenth of a c.c. will cause fatal tetanus in a
mouse. Turtles present an analogous case. The marsh turtle, _Emys
orbicularis_, tolerates very large amounts of tetanus toxin, injected
subcutaneously, and this at both low and high temperatures, at 30° C.
and beyond (36°–37° C.). The toxin passes quickly into the blood and
remains localised there for a very long time. In a turtle kept in an
aquarium at the laboratory the blood was tetanigenic for the mouse even
four months after an intraperitoneal injection of the toxin. In another
turtle which lived at incubator temperature (36°–37° C.), the blood was
still toxic two months after a subcutaneous injection of tetanus toxin
in quantity fatal for 500 mice. In turtles kept at 36° C. I observed
abundant transudations into the peritoneal cavity, and the fluid, very
poor in formed elements, was found to be very tetanigenic. It must be
accepted, therefore, that the toxin is retained in the blood plasma with
which it passes into the transudation. Every kind of cell must exhibit a
very marked negative chemiotaxis against tetanus toxin for this poison
to be retained so long in the body fluids. Under these conditions it is
not surprising that in turtles I was never able to observe the slightest
antitoxic power in the blood. Their great natural immunity must be due
to some other factor.
The alligator (_Alligator mississipiensis_) has also been found to be
quite refractory to tetanus both at low and at high temperatures.
Outwardly alligators behave exactly as do turtles, that is to say, after
the injection of various and sometimes very large doses of toxin they
exhibit no morbid symptom either general or tetanic. But the particular
changes which occur in their organism differ essentially from those met
with in the turtle. The toxin is rapidly eliminated from the blood of
the alligator, even when it is kept at a relatively low temperature (20°
C.). Under these conditions of temperature, however, the blood does not
become antitoxic although it has lost its tetanigenic property. When,
however, the alligators are kept at a higher temperature (32°–37° C.),
antitoxic power is developed in their blood, often with very great
rapidity. Quite young alligators (weighing about 500 grammes) are
capable of producing antitoxin, though somewhat slowly. A month after
the first injection of the tetanus toxin their blood is incapable of
causing tetanus in mice, but is not yet antitoxic. A month later,
however, it never fails to prevent an attack of tetanus when mixed with
fatal doses of the toxin and injected into mice.
Older alligators develop antitoxic power much more rapidly, and on
several occasions we have found, to our great astonishment, that, as
early as 24 hours after injection of the toxin, their blood was
distinctly antitetanic. The blood of the same alligators, tested before
the injection of the toxin, like the blood of normal alligators
generally, exhibited no antitoxic property.
In several experiments we took the rectal temperature of our animals and
were never able to observe the slightest rise corresponding to the
temperature of the water in which the alligators lived.
[Sidenote: [350]]
It cannot be doubted then, that, in spite of the facility with which
these reptiles produce tetanus antitoxin, their immunity does not depend
on this antitoxic property. Thus, young alligators which have resisted a
single dose of toxin sufficient to kill 6000 mice must owe their
immunity to some other cause than the antitoxic power of the body
fluids, for their blood does not begin to exhibit this property until
two months after injection.
These same reptiles are also very refractory against cholera toxin, even
in large doses; they react to the injection by the development of the
corresponding antitoxin. On the other hand they are very susceptible to
diphtheria toxin, small quantities of which are quite sufficient to
bring about a fatal intoxication.
Snakes, like other reptiles, are refractory against tetanus toxin. In
the study of their natural immunity, however, we are confronted by the
difficulty that their blood is naturally toxic for laboratory animals.
This toxin, analogous to the ichthyotoxin of eel’s serum, has been
compared with snake venom against which the snakes themselves enjoy a
very marked immunity.
[Sidenote: [351]]
Not venomous snakes only exhibit immunity against their own poison. Long
ago Fontana[505] observed that non-venomous snakes resist the bite of
the viper and even subcutaneous inoculation of its venom. Phisalix and
Bertrand[506] confirmed these observations and were able to show that a
non-venomous snake (_Tropidonotus_) will withstand a dose of venom
capable of killing from 15 to 20 guinea-pigs. Seeking for the cause of
this natural immunity, these observers came to the conclusion that it is
due to the presence in the blood of toxic substances analogous to those
of the venom of the viper. These same substances are found also in the
labial glands of the upper jaw of the _Tropidonotus_ and can from
thence, according to the view of Phisalix and Bertrand, pass into the
blood as an internal secretion. Calmette[507] has shown that the blood
of snakes, injected in a non-toxic dose, vaccinates certain mammals
against snake venom, and Phisalix and Bertrand have even obtained an
antitoxic effect by injecting a mixture of snake’s blood, heated to 58°
C., with lethal doses of venom. There is, then, in this example
something analogous to what we have described in scorpions, with this
difference, however, that the blood of these Arachnids is already
antitoxic, to a certain degree, whilst that of snakes only becomes so
after it has been modified by heat.
The classic example of immunity against a bacterial toxin amongst Birds
is that of the fowl, which is highly refractory against the tetanus
toxin. In the very earliest researches on this poison injections were
made into vertebrates of very different kinds, and a very striking
feature was the facility with which fowls resist very large quantities
of tetanus toxin. However, as is almost always the case, this immunity
has been found not to be absolute. By means of enormous doses, injected
subcutaneously or into the muscular tissue, tetanus of the most typical
kind, ending in death, has been induced in fowls, and in fowls weakened
by cold, tetanic intoxication, even with smaller doses, has been set up.
By injecting the toxin directly into the brain, according to Roux and
Borrel’s method, the fowl may be still more easily tetanised. Thus, von
Behring[508] observed that by injecting one milligramme of the toxin
into the brain of a fowl, weighing one kilo, tetanus may infallibly be
produced.
After the brilliant and fruitful discovery of the antitoxic property of
the blood, made by von Behring in collaboration with Kitasato, we were
justified in concluding that immunity against toxins and, amongst
others, natural immunity, might depend on the power of the body fluids
to neutralise the toxins. This hypothesis has been formulated at various
times, but it was for the first time subjected to experimental control
by Vaillard[509], and specially in connection with tetanus in the fowl.
The blood or blood serum of these birds, when mixed in varying doses,
small, medium, and large, with tetanus toxin, was never found to be
capable of preventing susceptible animals (mice, guinea-pigs, rabbits)
from contracting tetanus: these animals so treated behaved just as did
the controls inoculated with toxin only.
[Sidenote: [352]]
The great resistance of the fowl against tetanus,—one of the most
typical examples of natural immunity against a microbial poison,—cannot,
therefore, be explained by the presence in the body fluids of an
antitoxin capable of neutralising and rendering innocuous the tetanus
toxin. On the other hand, we are not justified in attributing it simply
to the absence of corresponding receptors in the sensitive nerve cells.
Since the fowl readily contracts tetanus when the toxin is injected
directly into the brain or when the fowl is weakened by cold, it is
evident that the sensitive elements never fail to absorb and fix any
poison that is presented to them. In ordinary cases, however, when the
fowl exhibits its remarkable resisting power against the toxin injected
in very large quantity, subcutaneously, into the muscles or into the
peritoneal cavity, the poison does not reach the sensitive cells, being
arrested and rendered innocuous whilst circulating in the tissues of the
organism.
Von Behring[510] is of opinion that in examples of natural immunity,
such as the one just examined, the principal cause of the refractory
condition depends upon the impermeability to the toxin of the capillary
wall of the vessels. It is, however, difficult to maintain this thesis
in regard to tetanus in the fowl, when it is remembered how readily
tetanus toxin passes through filters and membranes, and especially in
view of the fact that weakening of the fowl by means of cold renders it
susceptible to doses of toxin which are tolerated without inconvenience
by normal fowls.
We are, therefore, compelled to place the natural immunity of the fowl
against tetanus toxin in the category of cell immunities. This toxin, as
we have said, must be arrested _en route_ before it reaches the cells of
the nerve centres. But where and how does this beneficent arrest take
place? Ten years ago Vaillard demonstrated that the blood of fowls that
have received an injection of tetanus toxin causes typical tetanus in
susceptible animals. This tetanigenic property of the blood persists for
a certain number of days. When it is measured by the quantitative
method, it is found that all or almost all the tetanus toxin injected
into the peritoneal cavity of the fowl passes into the blood and remains
there intact for a variable number of days. From a morphological point
of view the blood, immediately after the injection of the toxin,
exhibits a hyperleucocytosis of greater or less duration.
[Sidenote: [353]]
When the fowls are killed at the stage when their blood becomes
tetanigenic (as the result of the injection of the toxin into the
peritoneal cavity), it can be demonstrated that their viscera are not
capable of producing tetanus in susceptible animals except in so far as
they contain blood. It is only the vascular organs, rich in blood, such
as the spleen, liver, kidneys, thyroid gland and bone-marrow, that
impart tetanus and then only in so far as they have not been freed from
blood. Of the various organs only the genital glands, ovaries or testes,
absorb a certain amount of the injected toxin. Very young testes or the
smallest ovarian ova containing as yet no trace of yellow yolk, when
injected into mice, produce a fatal tetanus.
In fowls, insusceptible to tetanus toxin, this toxin is found, then, in
the sexual glands and in the blood. When, in order to ascertain the
exact localisation of this toxin, we measure the tetanigenic power of
the whole blood as compared with that of the aseptic exudations induced
by the injection of gluten-casein, and necessarily much richer in
leucocytes, we get the result that the exudations contain more tetanus
toxin than does the blood. We are led, therefore, to the conclusion that
this poison is absorbed, at least in part, by the leucocytes, and it is
in these elements and in the genital cells that we must look for the
factors which arrest the toxin and prevent its reaching the nerve
centres.
Cellular or histogenic immunity is often contrasted with chemical
immunity without taking into consideration the real analogies and
differences to be found between them. It is evident that in both groups
the organism of the animal modifies the introduced toxins and that this
modification is a chemical process. In cellular immunity, however, this
act is preceded by certain biological phenomena, such as the reaction of
the formed elements and the absorption of the noxious substance.
Immunity in these cases is more complex than in the example where the
toxin is neutralised by a direct action of the body fluids, but
ultimately it always resolves itself into a chemical or perhaps
physico-chemical action of the substances of the organism of the animal
on the toxic substances of the poisons.
[Sidenote: [354]]
In Mammals examples of natural immunity against certain poisons are not
rare. Almost a century ago Oken made the observation that a person who
tried to poison a hedgehog with opium, hydrocyanic acid, arsenic or
mercury bichloride usually failed in his attempts because of the great
resisting power of this animal. Harnack demonstrated that the hedgehog
will withstand a dose of potassium cyanide six times as great as that
necessary to kill a cat in a few minutes (0·01 grm.). In Lewin’s[511]
experiments the hedgehog was found to resist the injection of powdered
cantharides in a quantity seven times as great as that which infallibly
kills a dog and greater also than the lethal dose for man. The same
observer also confirms the observation that a much larger dose of
alcohol must be used in order to intoxicate a hedgehog than is required
to obtain the same effect in the rabbit or even in the dog. Horvath[512]
fed hedgehogs for a fairly long period with living cantharides. These
Insectivora devour their venomous prey without showing any sign of
illness except a certain degree of emaciation. When Lewin tried to
ascertain the cause of this natural immunity of the hedgehog he examined
the blood of this animal for a substance antitoxic to cantharidine. His
experiments were all negative; but it is difficult to come to any
definite conclusion in this matter from the fact that the blood and
blood serum of the normal hedgehog are toxic for the small laboratory
animals. A similar objection had already been brought forward by
Phisalix and Bertrand in connection with their experiments, analogous to
those of Lewin, on the immunity of the hedgehog against the venom of the
viper.
[Sidenote: [355]]
It has long been known that hedgehogs have a liking for certain reptiles
and wage an implacable war on snakes in general and on the viper in
particular. In its attack the hedgehog tries to avoid being bitten, but
when, as often happens, it fails to evade a bite the inoculation of the
viper’s venom appears to be well borne. This observation has been
confirmed experimentally. Phisalix and Bertrand[513] have shown that the
resistance of the hedgehog to the viper’s venom is about forty times as
great as that of the guinea-pig, that is to say the hedgehog, though far
from possessing an absolute immunity, nevertheless exhibits a much
greater resistance than do most animals. Lewin[514] convinced himself of
this fact as regards adult hedgehogs, though young animals, according to
him, are much more susceptible. Thus, he has seen a young hedgehog that
had been bitten by a viper die after nine days’ illness. This
observation speaks in favour of the conclusion that the immunity of the
hedgehog might be naturally acquired rather than a really natural
immunity. The hedgehog, hunting all kinds of small animals, might often
be bitten by vipers and in this way acquire its immunity against the
venom. Under these conditions we can readily conceive that the blood of
this “insectivoran” might be placed in a position to develop a specific
antitoxic property.
When Lewin tried to satisfy himself of the existence of this property by
direct experiment he could only show that the blood of the hedgehog was
powerless to prevent the lethal effect of the viper’s venom on small
animals. But here, as in his researches on cantharidine, he did not take
into account the inherent toxicity of the blood of the hedgehog.
Phisalix and Bertrand[515], who have also studied this question, have
obtained results at variance with those of Lewin. They demonstrated
first of all that the blood of normal hedgehogs was capable of
intoxicating and even of killing laboratory animals such as the
guinea-pig. It is quite natural, therefore, that the mixture of this
fluid with viper’s venom could not be tolerated. It was, however,
sufficient to heat the blood of the hedgehog to 58° C. for it to become
not only innocuous of itself, but even for it to exhibit an antitoxic
action against snake venom. Thus, guinea-pigs which had received 8 c.c.
of heated hedgehog’s serum into the peritoneal cavity, were at once in a
condition to resist double the lethal dose of viper’s venom. Phisalix
and Bertrand conclude, therefore, that “the natural immunity of the
hedgehog against the viper’s venom is due to the presence in its blood
of an immunising substance.” The same observers[516] satisfied
themselves that horse’s serum and even that of the guinea-pig exercise
an undoubted antivenomous action; yet these animals are anything but
insusceptible to snake venom. Moreover, the necessity to heat the blood
to 58° C., as a preliminary measure, deprives this conclusion of the
degree of certainty one would like to have in such a matter. On the
other hand, the greater susceptibility of young hedgehogs prevents us
from putting the immunity of the adult in the category of natural
immunity properly so called.
[Sidenote: [356]]
Analogous considerations apply in the case of the mongoose (_Herpestes
ichneumon_), carefully studied by Calmette[517], according to whose
researches the Antilles mongoose is not very susceptible to snake venom;
it readily withstands doses very large relatively to its size, but its
immunity is not absolute. It owes much of its mastery in its fights with
venomous snakes to its extraordinary agility. The blood of the mongoose,
mixed with venom, exhibits an undoubted antitoxic power, though this is
not sufficient to prevent the death of susceptible animals. We have no
data to enable us to explain the origin of this antitoxic property, but
it is probable that here again we have an example of relative immunity,
acquired during life. Calmette points out, however, that his ichneumons
came from Guadeloupe, where no venomous snakes are found. We may, of
course, suppose that the feebly antitoxic power of the blood of these
mammals might be due to other snakes or to species of animals whose
blood possesses a certain venomous property[518].
[Sidenote: [357]]
We have far more exact data on the natural immunity of certain mammals
against toxins of microbial origin. The example most thoroughly studied,
one which has become, one might say, classic, is that of the rat against
diphtheria toxin. Since the discovery of this toxin, the first
well-studied bacterial poison, a discovery made by Roux in collaboration
with Yersin, it has been recognised that mice and rats tolerate large
quantities of diphtheria cultures or of their filtered products. A rat
resists a dose of the diphtheria poison capable of killing several
rabbits. To explain this great natural immunity it was suggested that
the antitoxic property of the body fluids could be called in. It was
supposed that the rat’s blood was, by its very nature, endowed with the
power of neutralising the toxin of diphtheria. But, as in the tetanus of
fowls, it was not long before facts rendered this hypothesis untenable.
Kuprianow[519] studied this question under the direction of Loeffler and
gave an account of the results of his experiments, which proved that the
blood of the sewer rat, which is very refractory against diphtheria,
contains no substance that will neutralise the morbific action of
diphtheria toxin on susceptible animals, especially the guinea-pig.
It was necessary to seek some other explanation, and the idea that the
immunity of the rat depends on the insusceptibility of its living cells
to the diphtheria poison was seized upon. The experiments carried out by
Roux and Borrel[520] demonstrated the incorrectness of this hypothesis.
The immunity of rats to subcutaneous or intraperitoneal injection of
diphtheria toxin is very marked. But a very small dose (0·1 c.c.) of
this poison, introduced directly into the cerebral substance of the rat,
produces a complete paralysis, which lasts for several days, and ends in
the death of the animal. Roux and Borrel conclude from this “that the
brain of the rat is specially sensitive to the action of the diphtheria
poison, and that as this animal does not die as the result of the
injection of large quantities of toxin into the subcutaneous tissue, it
is because the toxin does not reach the brain.” These authors have
pointed out analogous facts in connection with other examples of natural
immunity. The rabbit, which withstands a hypodermic injection of 30
centigrammes of chlorhydrate of morphia, is killed by 1 milligramme only
of this salt, introduced directly into the brain. Here, again, neither
the cellular insusceptibility nor the antitoxic property of the blood
(no “antialkaloidal” power could ever be demonstrated) can explain the
immunity, which appears to be due rather to the factor which arrests the
poison on its way to the nerve centres.
[Sidenote: [358]]
In spite of the insufficiency of our knowledge as regards natural
immunity against soluble poisons we are quite justified in affirming
that this category of phenomena comes mainly into the domain of the
cells. The body fluids of animals which exhibit this immunity have been
found to be antitoxic in a few species only (scorpion, snake, hedgehog,
mongoose). And for the majority of these it is possible to invoke
special causes, such as the internal secretion of snake and scorpion
venoms by the glands which manufacture them, or the acquisition of an
antitoxic power during life resulting from wounds or from the absorption
of venomous food. The theory of the insusceptibility of the cells of
animals naturally refractory to toxins must also be rejected; it is
incompatible with well-established facts. Nothing remains, then, but to
assume that the formed elements are the principal factors in this
natural immunity, and that they interpose to prevent the passage of the
poisons towards the very susceptible nerve cells.
CHAPTER XII
ARTIFICIAL IMMUNITY AGAINST TOXINS
Adaptation to poisons.—Artificial immunity against bacterial and
vegetable toxins and against snake venom.—Principal
methods of immunisation.—Immunisation by toxins and
toxoids.—Inoculation against diphtheria toxin.—Phenomena produced
in the course of vaccination against toxins.—Rise of
temperature.—Leucocytosis.—Development of antitoxic
power.—Properties of antitoxins.—Mode of action of
antitoxins.—Action of antitoxins _in vitro_.—Their action in the
organism.—Influence of living elements on the combination of
antitoxin with toxin.—Antitoxic action of non-specific serums, of
normal serums and of broth.—Immunity against toxins is not in
direct ratio to the amount of antitoxins in the body
fluids.—Hypersensitiveness of an animal treated with
toxin.—Diminution of the susceptibility of the organism immunised
against toxins.
Hypotheses as to the nature and origin of antitoxins.—Hypothesis of
the transformation of toxins into antitoxins.—Hypothesis
of receptors detached from cells as the source of
antitoxins.—Hypothesis of the nervous origin of tetanus
antitoxin.—Fixation of tetanus toxin by the substance of
the nerve centres.—The relations between saponin and
cholesterin.—Anti-arsenic serum.—Part played by phagocytes in the
struggle of the animal against poisons.—Probable part played by
phagocytes in the production of antitoxins.
[Sidenote: [359]]
Although scientific men succeeded only a little more than ten years ago
in vaccinating against poisons by artificial methods, savage races and
ancient peoples at a very remote period undoubtedly possessed methods of
counteracting the effects of certain venomous substances. The frequent
observation of cases in which doses of poisons, insufficient to cause
death, brought about a more or less durable resistant condition, must
result in the elaboration of artificial means of preventing the
intoxications.
Von Behring[521] points out that analogous facts must have been known to
the physicians of ancient times; and it is in such knowledge that we
must look for the source of the dogma put forward by Hippocrates, that
the factor which produces a disease is also capable of curing it.
[Sidenote: [360]]
To Pliny we are indebted for the now well-known story, that Mithridates
of Pontus possessed the means of protecting himself against various
poisons by a process of adaptation, and, amongst others, by the use of
the blood of Pontine ducks to which he had given poisons by the mouth.
The adaptation of horses and of the highlanders of Styria to arsenic, as
well as that of the many morphinomaniacs to morphia, is known to
everybody. A man, habituated to morphia, is able to consume daily a dose
several times the fatal one; indeed, cases have been known of people
acquiring the power of consuming two, and even three, grammes of morphia
per diem. Man may acquire an adaptation to toxic substances of the most
diverse character, such as arsenic, alcohol, morphia, nicotine, etc.
[Sidenote: [361]]
Even when we had obtained much information concerning acquired immunity
against micro-organisms we still knew nothing of the mechanism of such
adaptation, or as to the possibility of acquiring a special immunity
against bacterial poisons. Charrin and Gamaleia’s discovery that animals
vaccinated against a micro-organism are just as susceptible to its toxic
products as normal animals, led Bouchard[522], in whose laboratory it
was made, to say that the idea of the adaptation of cells to bacterial
poisons must be dropped. He developed this thesis at the International
Congress at Berlin in 1890, and formulated it as follows: “When we
inject a healthy animal and a vaccinated one with the soluble products
of the micro-organism which has been used for the vaccination, the dose
required to kill each animal is exactly the same. Let us not speak,
then, of the training of the leucocytes, and of the adaptation of the
nerve cells to bacterial poisons: it is pure rhetoric.” At this time we
had only just commenced to acquire exact knowledge concerning the toxins
of micro-organisms. For a considerable period they were sought for
amongst the ptomains, very stable substances allied to the alkaloids;
here, however, we were working in a wrong direction. It was not until
the classic researches of Roux and Yersin[523] on diphtheria toxin,
published in 1888 and 1889, that the true nature of bacterial poisons
was revealed. It was found that we were not dealing with ptomains, but
with soluble ferments, substances of indeterminate chemical composition,
allied to the albuminoids, and, like them, unstable. The methods adopted
by Roux and Yersin in their study of diphtheria toxin enabled other
investigators to discover the analogous toxins of several other
bacteria. Knud Faber[524] and Brieger and Fränkel[525] soon succeeded in
separating the toxin from the tetanus bacillus, a toxin capable of
producing in animals tetanic contractions as typical as those obtained
with cultures of the tetanus bacillus.
These investigations inaugurated a new era in microbiology and enabled
us to attack the problem of acquired immunity against bacterial toxins
scientifically. Within a few months of the declaration made by Bouchard
at the Berlin Congress, there appeared, almost simultaneously, the
earliest publications on the possibility of vaccinating laboratory
animals against the toxins of diphtheria and tetanus by artificial
methods. Immediately after the discovery of these poisons, the attempt
was made to immunise various species of animals against them, but here
very great difficulties were met with; the animals, after receiving
increasing doses of toxin, became thin and ultimately died. It occurred
to Fränkel[526] that the toxic action of the diphtheria poison might be
weakened by subjecting it to a temperature of 60° C. Independently, von
Behring and Kitasato[527] used chemical substances, especially iodine
trichloride, to attenuate the action of the tetanus and diphtheria
toxins. The animals which resisted these modified poisons were found to
be capable of tolerating gradually increasing doses of unaltered and
very active toxins. By the use of these methods it was found possible to
obtain a definite and lasting immunity against these microbial products.
[Sidenote: [362]]
The discovery of the possibility of vaccinating against bacterial toxins
was soon followed by the demonstration of the antitoxic power of the
blood of animals that had acquired such artificial immunity against
these poisons. Everyone knows of and appreciates von Behring and
Kitasato’s great discovery. It opened up a new and fruitful field of
research from most diverse points of view. Ehrlich[528] was able to
apply it to the vaccination of animals against the vegetable poisons
ricin, abrin and robin, and thus to establish rigorous methods of
immunisation and to obtain very important results concerning immunity
against toxins in general. He also succeeded in demonstrating that
animals vaccinated against these vegetable poisons, which, by their
nature, approximate to the microbial toxins, develop in their blood a
most marked antitoxic property.
Some years later, the discovery of antitoxins was extended to snake
venoms, poisons of animal origin which, like the vegetable poisons
studied by Ehrlich, present a chemical composition analogous to that of
the microbial toxins. Phisalix and Bertrand[529] and Calmette[530],
working independently, discovered methods of vaccination against snake
venom and were able to demonstrate the existence of an antitoxic power
of the blood in immunised animals.
The works above briefly referred to gave us the fundamental basis of our
present knowledge on acquired immunity against toxins.
It would be very interesting to be able to determine whether the lower
animals can be vaccinated against the toxic substances to which they are
susceptible. Unfortunately in the study of this problem we encounter
very great difficulties. Making use of various methods I have often
tried to solve it. The crayfish is susceptible to snake venom and to the
ichthyotoxin of eel’s serum, and I have tried at various times to
vaccinate it against these poisons. The results, however, were so
inconstant and even contradictory that I was unable to draw any definite
conclusion from them.
[Sidenote: [363]]
It is, indeed, very difficult to vaccinate the lower vertebrata against
poisons. Several attempts have been made in my laboratory to immunise
frogs against tetanus toxin, but without success. Calmette and
Deléarde[531] obtained the best results with abrin. They succeeded in
vaccinating frogs—which are not very susceptible to this vegetable
toxin, though they are far from presenting a real natural
immunity—against doses which are absolutely fatal for the control
animals. These observers, however, had to proceed very cautiously, and
they allowed a very long interval between each injection of abrin. The
blood of their vaccinated frogs not only did not prove to be antitoxic
against abrin, when injected into mice, but for long retained sufficient
of this toxin to poison normal mice. This experiment certainly tells
against the hypothesis that the acquired immunity of frogs is due to the
development of a specific antitoxic power in their body fluids, but it
does not settle the question definitely since it may be objected that
the blood, whilst toxic for mice, might, still, be antitoxic for the
frog. The antitoxin of this blood might merely be incapable of
neutralising all the abrin present. Fresh investigations, then, are
necessary.
Even in the higher vertebrata, it is often very difficult to obtain a
real vaccination against the various toxins. In the small mammals, which
exhibit a great susceptibility to these poisons, it is specially
difficult to obtain an artificial immunity. As Vaillard and von Behring
have demonstrated, it is possible to vaccinate such animals by means of
gradually increasing doses of unmodified toxins, but this method demands
much time, is often dangerous, and hence is not very practical. Poisons
that act through the alimentary canal are the most serviceable for
vaccination, as has been demonstrated by Ehrlich. This investigator had
to abandon the vaccination of mice by means of subcutaneous injections
of ricin on account of the sloughing set up at the point of inoculation.
He then had recourse to vaccination by way of the mouth, which gave very
good results, not only with ricin but also with abrin. This mode of
vaccination, however, is applicable to a small number of poisons only.
We can also vaccinate mammals, even laboratory rodents, such as rabbits
and guinea-pigs, by means of unmodified snake venom, but this method is
a very delicate one and must be carefully watched. It is necessary to
begin with very small doses of venom, continue them for some time, and
increase the amount of venom injected very slowly. Calmette[532]
modified this method by inserting, below the skin and leaving it there,
a piece of chalk impregnated with small quantities of venom and
surrounded by collodion through which the venom diffuses very slowly and
continuously.
[Sidenote: [364]]
Large mammals, sheep, oxen and horses, can be more easily vaccinated by
means of unmodified toxins, but they also require to be treated with
very special precaution. Salomonsen and Madsen[533] have given the
history of their horse, immunised with diphtheria toxin. Into a mare
weighing 665 kilos they were able to inject at the commencement only 1
c.c. of this toxin, and the dose had to be increased very carefully.
In the presence of all these difficulties in the use of unmodified
toxins for vaccination, a different method is now generally adopted in
the immunisation of animals, small or large, for the purpose of
scientific research or for the preparation of toxins on a commercial
scale. Vaccination is commenced with toxins modified by heat or by
chemical substances. The diphtheria and tetanus toxins, those most
employed in the serotherapeutic industry, are subjected to various
degrees of heat. Fränkel[534] was the first to make use of this method
for vaccination against diphtheria, and Vaillard[535] for vaccination
against tetanus. It consists in introducing large doses of filtered
cultures, heated to progressively lower degrees of temperature, 60°,
55°, 50° C., and then giving gradually increasing quantities of filtered
cultures whose toxicity is unaltered. This method is very convenient for
small animals, but for large mammals it is greatly simplified by
injecting for a certain period toxins heated to 60° C., and, later,
replacing these by unmodified toxin.
[Sidenote: [365]]
Phisalix and Bertrand[536] applied an analogous method to the
vaccination of the guinea-pig against the venom of the viper. This
poison, which resists much higher temperatures than do the tetanus and
diphtheria toxins, received a preliminary heating to 80° C. in order
that it might be inoculated without danger into small animals. Under
these conditions it confers a certain immunity, but even when heated to
80° C. it, in many cases, still remains sufficiently active to produce
fatal results. For this reason, in the vaccination of animals for the
preparation of antivenomous serum on a large scale, Calmette had
recourse to another method, that of attenuating the venom by means of
chemical substances.
Von Behring and Kitasato[537] were the first to make use of iodine
trichloride in the vaccination of animals against the toxins of tetanus
and diphtheria. In their early experiments this substance was injected
before the toxins were introduced. Later, the mixture was made _in
vitro_ and then injected into the animals. Roux devised another method
which had the advantage of being simple, certain, and easily employed,
for which reason it was soon introduced into commercial and scientific
practice. It consists in the injection of mixtures of the tetanus or
diphtheria toxins with Lugol’s iodo-ioduretted solution. The iodine, in
small doses, instantly neutralises or modifies these poisons and is
itself borne well, even by small animals. By employing progressively
increasing doses of these mixtures, in which the amount of iodised
solution becomes smaller and smaller compared with that of the toxin, we
are able, without difficulty, to vaccinate the most susceptible animals
and enable them to withstand considerable doses of the pure toxin. By
this method it is possible to immunise guinea-pigs against the most
active tetanus toxin. The method serves equally well for the preparation
of horses for injections of unmodified toxins. For a longer or shorter
time (according to the susceptibility of the horse) toxins which are
mixed with Lugol’s iodised water are injected. Having made sure of the
resistance of the horse, larger and larger quantities of pure,
unmodified toxin may be introduced with impunity.
For the immunisation of mammals of all sizes (guinea-pigs, rabbits,
dogs, horses) against snake venom, Calmette, in his work at Lille, also
makes use of venom modified by chemical substances, but his method
differs from those we have just described. During several weeks he
injects increasing quantities of venom, mixed with decreasing quantities
of a solution of 1:60 of hypochlorite of lime. After this treatment the
animals become capable of tolerating fatal doses of unmodified venom and
can be injected with larger and larger doses.
[Sidenote: [366]]
In recent years a method of vaccinating horses against certain microbial
toxins, and especially against the diphtheria toxin, by means of
mixtures of toxin and antitoxic serum, or with these two products
successively, has been introduced. Babes[538] was the first to extol
this method as the best for obtaining a high and durable immunisation.
Afterwards, several other observers, amongst whom I may cite Pawlowsky
and Maksutow[539], Palmirsky, and especially Nikanoroff[540], took up
this question, and communicated very encouraging results. Von
Behring[541] also found it very useful in certain cases. Thus, for the
vaccination of guinea-pigs against tetanus toxin, he recommends the
injection of a mixture containing antitoxin and an unneutralised excess
of toxin. Under these conditions he easily succeeds in immunising these
small animals in cases where all other methods fail. As a general method
of vaccination against toxins, however, this method has not fulfilled
its promise, and Roux, who tried it several times, was not at all
satisfied with it.
[Sidenote: [367]]
This method of immunisation by mixtures of toxin and antitoxin is often
spoken of as the method of vaccination by _toxones_. This name,
“toxone,” was first applied by Ehrlich[542] to a product developed by
the diphtheria bacillus in culture media, a product less and differently
toxic than is the true diphtheria toxin, yet capable of neutralising
antitoxin. The idea of toxones presented itself to Ehrlich in connection
with a fundamental fact noted by him, namely, that when to a non-toxic
mixture of diphtheria toxin and antitoxin there is added one and even
several lethal doses of the former, the animal is not affected. To make
it succumb to intoxication it is sometimes necessary to add more than 20
lethal doses of toxin. To explain this paradoxical result, Ehrlich
formulated the hypothesis that, in the soluble products of the
diphtheria bacillus there exist two poisons: (1) the true toxin which
exhibits a very strong affinity for antitoxin, and (2) the toxone which
possesses less avidity for this antibody. When to an inactive mixture of
the products of diphtheria bacilli and of antitoxin, there is added a
fresh quantity of these same products, the added toxin, owing to its
greater affinity, replaces the toxone of the previous combination. In
the mixture to which is added one or several lethal doses of diphtheria
poison, the toxone only is found free, all the toxin being combined with
the antitoxin, and, as the toxone is only feebly toxic, the animal
resists without suffering any serious illness.
Madsen[543] adopted the theory of the diphtheria toxone, and affirmed
that this substance poisons but slowly, produces neither early nervous
symptoms nor loss of hair, but excites slight oedema at the point of
inoculation and late paralyses. Susceptible animals may die from
toxones, but very much later than as the result of poisoning by the
toxins.
Ehrlich’s pupils have extended the theory of toxones to other bacterial
poisons. Thus Madsen[544] has described a similar toxone in tetanus
poison—the tetanolysin of Ehrlich—which dissolves the red blood
corpuscles, and Neisser and Wechsberg[545] refer to a toxone in the
poison produced by the staphylococcus.
Ehrlich also describes _toxoids_ as occurring in diphtheria poison. The
toxone, he maintains, is a product of the diphtheria bacillus itself,
but the toxoids (protoxoids and syntoxoids) represent the toxin modified
without further aid from the bacillus. The toxoids, though not toxic,
retain all their avidity for antitoxin. According to Ehrlich’s
conception, the molecule of toxin, under the influence of various
factors, readily loses its toxic or _toxophore_ group, capable of
poisoning the animal, whilst still retaining its _haptophore_ group, the
group that combines with the antitoxin. The toxoids then would represent
this haptophore group of the diphtheria toxin. Without being injurious
to animals, the toxoids are capable of neutralising the antitoxin and of
setting up in the animal the formation of this antibody. In the
experiments carried out by the method of Babes and of the Russian
authors we have just mentioned, there would be, according to the view
held by Ehrlich and his school, an immunisation by the toxoids.
[Sidenote: [368]]
The toxones, however, are also capable of vaccinating against the toxin
and the toxone and of giving rise to the production of a diphtheria
antitoxin, active against these two poisons. This is what is affirmed by
Madsen[546] and by Dreyer[547], according to a communication made by the
latter to the International Congress of Medicine held at Paris.
[Sidenote: [369]]
By means of the various methods briefly described above, is obtained a
real acquired immunity against the various bacterial and vegetable
poisons and the venoms. On the other hand, with the methods of
vaccination mentioned in the eighth chapter, which confer a substantial
immunity against micro-organisms, we cannot demonstrate, in the
vaccinated animals, a resistance against the corresponding toxins
greater than in the unvaccinated control animals. The animals, so
thoroughly vaccinated against certain micro-organisms that they
withstood enormous doses of culture, did not become capable of resisting
the minimal lethal dose of the poison. We are led to conclude,
therefore, that immunity can only be obtained against certain of the
toxins. For this reason we must regard the attempt made by von Behring
to obtain a real immunisation against the toxin of cholera as an
important forward step. Before von Behring’s attempt, various species of
animals had been frequently and very substantially vaccinated against
the cholera vibrio, but these animals, even when most thoroughly
vaccinated, were completely non-resistant to the cholera toxin. Von
Behring suggested to his pupil Ransom[548] the idea of immunising
guinea-pigs, not with microbial cultures living or dead, as had usually
been done previously, but exclusively with the fluids of the cultures,
deprived of the vibrios by filtration. In order, however, to attain the
desired object, it was necessary to prepare fluids sufficiently active
to poison the unvaccinated control guinea-pigs with certainty. The
results of these investigations confirmed his anticipation, and Ransom
soon found himself in possession of guinea-pigs well vaccinated against
the cholera poison. He was mistaken, however, in supposing that, in all
cases of immunity acquired against Koch’s vibrio, we have to do, in the
main, with a purely antitoxic immunity. An investigation carried out in
the Pasteur Institute[549], whilst confirming the facts discovered by
Ransom, lead to different results as regards their interpretation. It
was demonstrated that the immunity against the vibrio is in no way
founded on a resistance against its toxin and that we have to do with
two very different acquired immunities. The vaccination obtained with
the bodies of the micro-organisms induced a refractory condition against
infection by the living vibrio, but not the slightest resistance against
the toxin. The immunity, on the other hand, which is conferred by the
injection of soluble products, deprived of the micro-organisms, is
effective not only against the toxin of cholera, but also against
infection by the vibrio. When an animal is vaccinated with cultures, or
even with the bodies only of the vibrios, cholera toxin is introduced,
but the toxin, under these conditions, is incapable of setting up
antitoxic immunity. It would appear that the presence of the vibrios may
constitute some obstacle to the production of this immunity.
Soon afterwards, Wassermann[550] pointed out that the same rule applies
in the case of the _Bacillus pyocyaneus_. With whole cultures of this
bacillus he obtained in guinea-pigs an immunity exclusively against
infection, whilst with cultures in a fluid medium, deprived of the
bacilli, he was able to vaccinate his animals both against the pyocyanic
toxin and against the infective peritonitis produced by the living
micro-organism. The same double immunity could also be obtained in
laboratory animals against the typhoid bacillus and several other
bacteria.
When animals were subjected to different methods of vaccination against
toxins, the manifestation of certain phenomena more or less constant was
observed; amongst these must be pointed out especially the rise of
temperature, a local reaction and certain modifications in the body
fluids.
Fever is a very general symptom in the course of the vaccination of
mammals. A rise of temperature is almost always observed as a result of
the injection of toxins. It is very variable, both as regards duration
and intensity, and cannot serve as an indicator of the result of the
vaccination. In this respect, such great differences have been observed
that the attempt to establish any general laws has had to be abandoned.
[Sidenote: [370]]
[Sidenote: [371]]
Local reaction is also a phenomenon which is very frequently observed
during vaccination; to this von Behring[551] paid great attention. He
and his collaborators found that normal horses when injected
subcutaneously with small or large doses of tetanus toxin did not
present any exudation at the seat of inoculation. The horses which died
as the result of a tetanus intoxication and those which got better
behaved from this point of view in much the same fashion. In horses,
however, which are being vaccinated and which are periodically subjected
to gradually increasing doses of toxin, tumefaction at the seat of
injection is never absent. Von Behring attributes this difference to the
primordial insusceptibility of the living elements which govern
exudation in the subcutaneous tissue to tetanus poison. It is only
during the process of vaccination that these cells become susceptible
and capable of manifesting a visible reaction. I consider that this
difference is due more probably to a change in the chemiotaxis of the
various elements which contribute to the inflammatory exudation
reaction, from a negative to positive type. The cells do not react at
the commencement, not because they are not susceptible to the toxin, but
rather because their susceptibility is too great. During the course of
vaccination they become sufficiently adapted to the poison to be able to
manifest their normal inflammatory reaction. This explanation certainly
harmonises with the fact that during the period of vaccinations in
general and of vaccination against toxins in particular, the blood
usually presents a more or less distinct hyperleucocytosis. Now, as is
well known, this phenomenon of hyperleucocytosis is one of the most
striking manifestations of a positive chemiotaxis in white corpuscles.
It is true that, as to this reaction during the course of vaccination,
the views of observers are not unanimous. Besredka[552], as the outcome
of his work on this subject, expresses himself very distinctly. “During
the course of an immunisation against diphtheria toxin,” he writes, “one
always observes a marked reaction in the goat, either at the beginning
or at an advanced stage of the period of injections and especially in
the first few hours after injection” (p. 322). Nicolas and Courmont[553]
in their first memoir maintain that hyperleucocytosis “is not necessary
for immunisation.” Nevertheless, in the description of their
experiments, which were performed on horses vaccinated against
diphtheria, it is clear that the number of white corpuscles is often
markedly increased. Further, in several cases they describe the
formation of tumours at the point of inoculation, some of which end in
suppuration. Under these conditions, it is not possible to deny a
vaccinal reaction on the part of the leucocytes. Later, Nicolas,
Courmont and Prat[554] published a second memoir on the same subject, in
which they seek to confirm their view of the uselessness of
hyperleucocytosis in vaccination against the poison of diphtheria. They
give details of experiments on several species of animals and insist
specially on the conditions in which they have not observed
hyperleucocytosis. “The doses from the first have always been extremely
weak and with the addition of Lugol’s solution to attenuate them; only
very gradually have we reached stronger doses, as _that is one of the
indispensable conditions for the avoidance of leucocytic variations_,
whilst obtaining a good and rapid immunisation” (p. 974). These special
precautions to avoid hyperleucocytosis demonstrate clearly that this
phenomenon is usually produced during the course of vaccination. It is
quite natural that we should, by proceeding very slowly and with small
doses of toxin, succeed in diminishing or even suppressing the afflux of
leucocytes; but this fact cannot in any way minimise the importance of
the leucocytic reaction in vaccination. In these particular cases, this
reaction may take place without the number of leucocytes in the blood
being noticeably increased. In reading the details of the experiments
made by the Lyons observers, it will be seen that, in spite of all their
precautions, they were unable to prevent the production of
hyperleucocytosis. In all their cases, where they took the precaution to
count the leucocytes several times a day, there was an undoubted
increase of these cells. We may here recall Salomonsen and Madsen’s
account of the immunisation of a horse against diphtheria toxin, in
which they point out the frequency of tumefactions and even of
abscesses. In most cases the pus was sterile, which renders it probable
that the white corpuscles had accumulated at the seat of inoculation as
the result of some influence exerted by the diphtheria toxin.
[Sidenote: [372]]
By far the most important and remarkable change met with in animals
vaccinated against toxins and venoms, consists in the appearance of
antitoxic power in their blood and fluids in general. This fact was, as
already mentioned, first demonstrated by von Behring and Kitasato[555]
in the blood of rabbits immunised against tetanus. The blood itself, or
the blood serum, mixed with a quantity of tetanus toxin more than
sufficient to cause fatal poisoning, sets up no disease when injected
into animals. In their earliest researches, von Behring and Kitasato
kept the mixtures in contact _in vitro_ for 24 hours, before injecting
them into test animals. Later, they found that this prolonged contact
outside the body was unnecessary and that they could obtain successful
results by injecting the serum of vaccinated animals and the toxin
simultaneously, even at different points of the body. This discovery was
immediately afterwards applied by its authors to diphtheria and, in the
case of both intoxications, confirmed by numerous observers.
[Sidenote: [373]]
For some time we were satisfied with vaccinating small laboratory
animals and establishing the antitoxic power of their blood serum;
later, the vaccination of large animals, especially horses, was
commenced with the object of obtaining large quantities of antitetanus
and antidiphtheria serum for medical use. During the course of these
experiments the principal characters of the antitoxic fluids were
established. It was deemed desirable to isolate the antitoxic substance
from the blood serum in order to get rid of every unnecessary and
inactive admixture, so that the antitoxin might be used in as pure a
form as possible. This idea of isolating the antitoxic substance had,
however, soon to be abandoned as impossible of realisation. Antitoxin is
a non-crystallisable substance, of unknown chemical composition, which
adheres firmly to the albuminoid substances of the serum. It is usually
regarded as belonging to the same albuminoid group of substances, though
it is not possible to prove this satisfactorily. Von Behring[556],
however, who studied this question in collaboration with Knorr, denies
the albuminoid nature of tetanus antitoxin. After demonstrating that
this antitoxin, when the antitetanus serum is submitted to dialysis,
passes through the dialysing membrane, these observers found that they
could not obtain the characteristic reactions of albuminoids in the
dialysed fluid. It must be admitted, however, that this negative result
is not sufficient to justify a denial of the albuminoid nature of
antitoxin. When Nencki and Mme Sieber[557] sought to produce the
reactions of albuminoid substances with the digestive juice of
_Nepenthes_ (the well-known insectivorous plant) they got no result; but
after the concentration of the juice _in vacuo_, it at once gave the
characteristic reaction with nitric acid, and also with acetic acid,
potassium ferrocyanide and Millon’s reagent.
The antitoxins may be precipitated along with the globulins and are
distinguished, in general, by a fairly great resistance against physical
and chemical influences. In this respect they are allied to the
agglutinins, the fixatives and the precipitins, considered elsewhere,
and are sharply distinguished from the cytases. The antitoxins resist
temperatures which destroy the cytases and remain unaltered to beyond
60°–65° C. They are more stable than the delicate toxins of tetanus and
diphtheria, but they are more easily altered than the toxins of cholera,
of _Bacillus pyocyaneus_ and the venoms. When stored in a dry state in
the residue of evaporated serums and protected from light and air, the
antitoxins will keep for a very long time without showing any notable
attenuation. This property is very important in practice.
[Sidenote: [374]]
The antitoxins, in this respect also resembling the fixatives and the
agglutinins, are humoral substances in the strictest sense of the term.
They are found not only in prepared serums but abound also in the plasma
of the circulating blood, and in the plasmas of the lymph and of
exudations. Vaillard and Roux[558] have shown that the clear acellular
serous fluid of the oedema produced by the slowing of the circulation in
rabbits vaccinated against tetanus toxin, is as antitoxic as the blood
itself. Even the aqueous humour of a strongly immunised animal is
antitoxic, though to a less degree. On the other hand, the saliva and
urine exhibit very little antitoxic power, even when they are derived
from animals hyperimmunised against tetanus toxin. Milk, as first
demonstrated by Ehrlich[559], is fairly rich in antitoxin, although much
less so than the blood. According to the estimation of Ehrlich and
Wassermann[560], in the same immunised animal, milk contains
one-fifteenth to one-thirtieth of the amount of diphtheria or tetanus
antitoxin contained in the blood. Pus is always less antitoxic than
blood or blood serum. According to Roux and Vaillard (_l. c._, p. 82),
the pus of their rabbits vaccinated against tetanus toxin was only
one-sixth or one-eighth as antitoxic as the serum of the blood. In
Salomonsen and Madsen’s[561] antidiphtheritic horse the cellular
sediment of the pus was about one-half as antitoxic as the blood.
For the development of the antitoxic property in the fluids of the body,
it is not essential that animals should belong to species susceptible to
the corresponding toxin. Animals naturally most refractory against the
poisons of diphtheria and tetanus are also capable of producing
antitoxins. Vaillard[562] demonstrated this fact in the fowl. This bird,
which is naturally refractory against tetanus, usually acquires a very
marked antitetanic power in its blood after one or more injections of
tetanus toxin. He observed, however, that, in fowls thus treated, at a
stage when the fluids of the body are antitoxic, the albumen of the egg
is not so. The antitoxin, therefore, does not pass into this nutritive
secretion, as it does into the milk of mammals. On the other hand, as
has been demonstrated by F. Klemperer[563], the vitellus of the eggs of
fowls treated with tetanus toxin in time acquires an antitoxic property
of the most marked character.
[Sidenote: [375]]
[Sidenote: [376]]
The antitoxins, found especially in the fluids of the body but only
scantily in the cells, exert some action on the toxins. What is the
nature of this action? This question, much studied and discussed, is one
of very great importance in connection with the general problem of
acquired immunity against toxins. In his first memoir, written in
collaboration with Kitasato, von Behring (_Deutsche med. Wchnschr._,
Leipzig, 1890, S. 1113) formulates his first thesis as follows: “the
blood of a rabbit immunised against tetanus possesses the property of
destroying tetanus toxin.” This idea of destruction, which would remove
all toxic power from the poison, would naturally present itself to the
mind and was at once accepted by a great many observers, but the
numerous facts now accumulated on the subject will not allow us to
accept a real destruction of toxins by antitoxins. Tizzoni[564] was one
of the first to point out certain contradictions between the theory of
destruction and the phenomena produced in animals injected with tetanus
toxin and antitoxin. Buchner[565] also brought forward new facts which
led him to conclude that antitoxin, instead of acting directly on the
toxin, exerts its influence exclusively on the living elements, thus
protecting the animal against intoxication. Amongst the arguments
advanced by the Munich observer, the principal one is drawn from the
different action of mixtures of tetanus toxin and antitetanus serum on
various species of animals. It has been clearly shown that the
guinea-pig is more susceptible to tetanus than is the mouse. In
poisoning with tetanus toxin it requires an absolutely larger quantity
of toxin to kill the guinea-pig than to kill the mouse. But if we take
into account the weight of these animals, the conditions change
completely. Thus, to cause a fatal tetanus in a guinea-pig, which weighs
twenty times more than a mouse, we need only inject into the former a
dose at most ten times greater than that necessary to produce fatal
intoxication in the mouse. Buchner prepared a mixture of tetanus toxin
and antitetanus serum which, in the mouse, produces no tetanic
phenomenon or only sets up feeble and transient symptoms. According to
the theory of direct action, we must assume that in this mixture the
toxin is completely or almost completely neutralised by the antitoxin of
the serum. But when Buchner injected the same quantity of mixture into
guinea-pigs, without increasing it in proportion to the greater weight
of these animals, he produced a tetanus of the most marked character.
There has, consequently, remained in the mixture a sufficient amount of
free toxin, whose tetanigenic action is manifested in the guinea-pig, an
animal, as we have seen, more susceptible than the mouse. Buchner’s
experiment has been verified by several observers. Roux and
Vaillard[566] carried out others which afford similar evidence. The same
mixture of tetanus toxin and specific serum which is borne without the
least difficulty by normal guinea-pigs, causes typical tetanus in other
guinea-pigs of the same weight, and apparently in the best of health,
but which have been immunised some time before against the Massowah
vibrio. In another series of experiments, Roux and Vaillard injected
into guinea-pigs a very large amount of antitetanus serum “capable of
immunising them thousands of times,” and, shortly afterwards, a lethal
dose of tetanus toxin. The normal guinea-pigs were thoroughly resistant
to this test, whilst several guinea-pigs into which were also injected
the products of other micro-organisms, acquired tetanus. Analogous
results were obtained with mixtures of diphtheria toxin and
antidiphtheria serum. Roux concludes from these facts “that the
antitoxins act on the cells.” Against the theory of the destruction of
toxins by antitoxins, he invokes the influence of heat on mixtures of
these two substances. Calmette[567], under Roux’s inspiration and in his
laboratory, carried out various experiments on antivenomous serum. A
mixture of this with snake venom, in such proportion that the poison
became inactive, regained its toxicity after being heated for five
minutes at 68° C. A normal animal, injected with this mixture, succumbed
as if it had received pure venom. On being heated at 68° C. the serum
lost all its antitoxic power over the venom, and the latter, which only
becomes modified at a much higher temperature, remained intact. Later, a
similar result was obtained by Wassermann[568] in his experiments with
pyocyanic toxin. This poison is resistant at even higher temperatures
than is snake venom, whilst the antitoxin of the serum is destroyed
under the same conditions as are the other antitoxins. Taking advantage
of these peculiarities, Wassermann boiled the mixture of pyocyanic toxin
and antitoxin serum, being careful to dilute it with two volumes of
distilled water before doing so. This mixture which, before it was
heated, was quite innocuous for guinea-pigs, again became a fatal poison
after the destruction of the antitoxin.
[Sidenote: [377]]
These experiments prove clearly that, in the action of the antitoxin on
the toxin, there can no longer be any question of an actual destruction
of the latter, a view which has been accepted by both von Behring and
Ehrlich. But, as pointed out by Roux at the International Congress at
Budapest in 1894, the manifestation of the toxic action of the venom
after it has been heated along with antitoxin, may be reconciled with
the view that the combination between the two substances, if such take
place, must be very unstable. This same remark may be applied to
Wassermann’s experiment. Therefore the great majority of observers, if
not all, admit that the antitoxin combines with the toxin to form an
innocuous and unstable substance which can be decomposed by heat and by
other agents. The researches on the action of antitoxins _in vitro_ have
had a powerful influence in determining this view.
[Sidenote: [378]]
We have already in Denys and van de Velde’s[569] experiments an
indication of the direct action of certain antitoxins. These observers
showed that the serum of animals vaccinated against a Staphylococcus is
capable of neutralising _in vitro_ a particular toxin to which van de
Velde gave the name of _leucocidin_. When it was added to a drop of the
exudation from a rabbit, this leucocidin in a very short time destroyed
the white corpuscles, by dissolving the cell content but leaving the
nucleus untouched. When Denys and van de Velde prepared mixtures of
leucocytes, leucocidin and antileucocidic serum _in vitro_, the white
corpuscles retained their normal condition for a very long time. The
leucocidin was, therefore, rendered inactive by the direct influence of
the corresponding antitoxin. These facts have been confirmed by
Bail[570] and other observers and even extended to certain other
microbial toxins. Thus, the _Bacillus pyocyaneus_ produces a leucocidin
which kills the white corpuscles and dissolves their contents[571]. With
the object of facilitating experiments with these leucocytic poisons and
the corresponding antitoxic serums, Neisser and Wechsberg[572], of the
Institute of Experimental Therapeutics at Frankfort, invented a method
which allows us to observe the phenomena of the destruction of the
leucocytes and of the antitoxic power in test tubes, without having
recourse to a microscopical examination. They applied the fact,
discovered by Ehrlich, that living formed elements reduce methylene blue
and, depriving it of its oxygen, decolorise it. Leucocytes from aseptic
exudations are introduced into tubes and a weak solution (2%) of
methylene blue is poured on them. To prevent the re-oxidation of this
colouringmatter by the oxygen of the air, the surface of the fluid is
covered with a layer of liquid paraffin. If the leucocytes are living,
the lower blue layer becomes decolorised in a short time (in about two
hours); when the corpuscles are dead, decoloration does not take place.
By adding to the mixture of leucocytes and colouring matter some
leucocidin, alone or along with antileucocidic serum, it is possible not
only to observe with the naked eye the phenomena which take place in
these cases, but also to estimate to some extent the proportions of
poison and counterpoison.
All these researches make it clear that the antitoxin acts directly on
the leucocidin. Similar facts have been noted as regards certain other
organic poisons and their antitoxins. Shortly after the discovery of
antileucocidin by Denys and van de Velde, Kanthack made a communication
to the Physiological Society in 1896[573], exhibiting tubes in which the
coagulating action of Cobra venom on the blood had been prevented by the
addition of antivenomous serum. Of all the experiments, however, made to
prove the direct action of antitoxin on toxin, Ehrlich’s[574] have
played the most important part in the study of this question. Ehrlich
directed his attention to ricin which, as Kobert demonstrated, has the
property of agglutinating the red corpuscles of defibrinated blood. This
phenomenon can be easily observed _in vitro_. In tubes containing red
blood corpuscles, the addition of ricin causes these corpuscles to
agglutinate into clumps and to fall to the bottom of the tube, leaving a
clear supernatant fluid. After adding progressively increasing
quantities of antiricic serum to the tubes containing fluid blood and
ricin, Ehrlich was able to demonstrate that small quantities of
antiricin merely retarded the precipitation of the red corpuscles,
whilst larger doses completely prevented it. Having studied the
proportions of ricin and its antidote, necessary to retard and prevent
the fatal poisoning of animals, Ehrlich was struck by the parallelism
which is exhibited between the action of the antitoxin in the living
animal and that in the test tubes.
[Sidenote: [379]]
The study of anticytotoxins, discussed in the fifth chapter, has
furnished another opportunity of observing the action of antitoxins _in
vitro_. Camus and Gley and H. Kossel were the first to observe the
action _in vitro_ of antitoxic serum against the ichthyotoxin of eel’s
serum. Since this observation, this phenomenon has been repeatedly
studied in the antihaemolysins and antispermotoxins. The antidiastatic
serums also act _in vitro_ and, as their effect can be demonstrated on
soluble ferments placed in contact with unorganised bodies, such as
gelatine and casein, the purely chemical character of the reaction is
all the more strikingly shown. We are indebted to von Dungern, Briot and
Morgenroth for accurate observations on this subject.
Martin and Cherry[575] made use of a different method to demonstrate the
direct action of antitoxins on toxins which exhibit their toxic power on
the animal organism. They chose snake venom mixed with antivenomous
serum. The mixtures were filtered under great pressure [50 atmospheres]
through a film of gelatine, under the idea that, if the venom and
antitoxin were not chemically combined, the former alone, owing to its
much smaller molecules as compared with those of the antivenom, would
pass into the filtered fluid. This fluid should, under these conditions,
possess a toxic power for animals, when the mixture, used for
filtration, was deprived of the larger molecules. Martin and Cherry left
the venom and the antitoxic serum in contact for periods of varying
length, before filtering the mixtures. As the result of a series of such
experiments carried out according to this scheme, they found that the
product of the filtration made after some minutes’ contact between the
two substances, was distinctly toxic; whilst the filtrate obtained after
a contact of half-an-hour was absolutely innocuous. From their
observations these authors conclude that the antitoxin enters into
chemical combination with the venom, but that the combination does not
take place instantaneously, a certain amount of time being necessary for
its accomplishment.
[Sidenote: [380]]
In addition to the time factor others have an influence on the
combination between toxins and antitoxins, as is seen from
Ehrlich’s[576] and Knorr’s[577] investigations. Both observers have
shown that antitoxin neutralises the toxin more slowly in dilute
solutions than in more concentrated form. For this reason, when animals
are injected with very weak solutions, the toxin may manifest its action
before it can be neutralised by the antitoxin; this may lead to
erroneous conclusions. On the other hand, according to data furnished by
these authors, temperature also exerts an influence on the combination.
Lowering the temperature retards, whilst raising it accelerates the
neutralisation of the toxins by the antitoxins. Insisting on the purely
chemical character of the combination between these two substances,
Ehrlich and Knorr adduce the fact that this combination, in cases where
we have a complete neutralisation of the toxin, follows, most
rigorously, the law of multiple doses, that is to say, in order to
render innocuous a hundred doses of toxin we have only to take a hundred
times the quantity of antitoxin.
The series of facts summarised above demonstrate distinctly that
antitoxins act directly on toxins. But how can this result be reconciled
with the observations given above according to which must be admitted
the no less real influence of the organism of the living animal on
intoxication by mixtures of antitoxin with toxin? Knorr[578] sought at
first to minimise the importance of the facts brought forward by Buchner
and Roux. He failed to corroborate Buchner’s results and found that the
injection of mixtures, made with very large doses of tetanus toxin
(20,000 times the minimal lethal dose) and corresponding quantities of
antitetanus serum, brought about the same effect in guinea-pigs and
mice. By modifying the quantity of antitoxin, he rendered the mixture
equally innocuous or equally toxic for these two species. But the data
given by Knorr are quite sufficient to prevent us from accepting his
conclusion. In his experiments, as in those of Buchner, the guinea-pigs
manifested a greater susceptibility and died from mixtures which, in
mice, caused merely a tetanus of medium intensity.
[Sidenote: [381]]
Some have sought to explain Buchner’s experiment by assuming that the
mixtures, lethal for the guinea-pig and innocuous for the mouse, owed
their toxic action to the presence of the _tetanus toxone_ and not of
the true tetanus poison, the _tetanospasmin_. This hypothesis of
toxones, as stated above, was put forward by Ehrlich as the outcome of
his ingenious researches on the constitution of the diphtheria poison.
As, however, the toxones must act differently from the toxins, we can
only attribute to their action the results in those cases where the
guinea-pigs die without presenting typical symptoms of true tetanus,
that is to say without spasms. Now, in Buchner’s experiments, a much
larger proportion of these animals, injected with the same mixtures as
the mice, succumbed and exhibited the characteristic tetanic
convulsions. Even in those cases, however, where the death of the
guinea-pigs might be attributable to an intoxication by the toxone, the
general result could not be altered. The toxones are, according to
Ehrlich, manufactured by the micro-organisms in the culture media and
form an integral part of the natural microbial poisons. Again, they are
neutralised by antitoxic serums. If, therefore, in spite of there being
the same quantity of toxones and of antitoxin in the mixtures, these
mixtures become more toxic for the guinea-pig than for the mouse, we
have an indication that some special change must take place in the
animal to upset the conditions of toxicity.
Weigert[579] accepts the accuracy of Buchner’s experiment, which,
indeed, can no longer be denied, but explains it on the hypothesis that
there is some substance in the animal possessing a very great affinity
for the toxin. This substance is supposed to be capable of decomposing
the innocuous combination of the antitoxin with the toxin, just as heat
does in Calmette’s and Wassermann’s experiments, described above. In
both cases the toxin would be set free to exert its noxious action. Such
a hypothesis is very probable, because it agrees with direct
observation, but it compels us to accept some new phenomenon which is
produced not _in vitro_, but in the living animal, and which carries on
its work in a very different fashion in the guinea-pig and in the mouse.
In the present imperfect state of our knowledge it is very difficult to
form any idea of the precise conditions which must intervene in the
organism of the guinea-pig to cause the tetanus toxin to act in a
mixture with antitoxin which is much more innocuous for the mouse. In
order, however, to satisfy those who seek to understand these complex
phenomena, it may be useful to cite another example of antitoxic action
in which certain factors are distinguished by their simplicity.
[Sidenote: [382]]
Lang, Heymans and Masoin[580] have demonstrated that hyposulphite of
soda prevents poisoning by prussic acid. This terrible poison becomes
innocuous if we take care to introduce into the animal by any channel
whatever (subcutaneously, intravenously, or by the stomach) a sufficient
quantity of hyposulphite of soda. Under these conditions the sulphite is
substituted for the hydrogen of the prussic acid, transforming the
poison into sulphocyanic acid, which has no action on the organism. The
hyposulphite of soda, then, acts as the antitoxin of the prussic acid,
thanks to a chemical reaction of substitution between bodies of simple
composition. We have never yet succeeded in reproducing this reaction
_in vitro_, whilst in the animal body it is effected with very great
ease. Consequently, we are quite justified in invoking special
conditions in the body of the living animal; this, however, does not
preclude the possibility of a transformation of the toxic substance into
an innocuous substance through a chemical reaction. It is probable that
analogous phenomena may also be met with in the action of true
antitoxins on the microbial toxins or allied substances (venoms,
vegetable toxalbumins).
[Sidenote: [383]]
The case of the destruction of micro-organisms, which is now more easily
studied because it is possible to observe with the eye the fate of these
organisms in the animal, is a further source of valuable information.
The direct action of cytases on certain bacteria, such as the cholera
vibrio, can be just as easily demonstrated _in vitro_ as can the action
of antiricin on ricin. If we proceeded to argue from this, a perfectly
accurate observation, that the living animal plays no part in the
destruction of the micro-organisms and that this destruction takes place
always in a fashion analogous to Pfeiffer’s phenomenon _in vitro_, we
should undoubtedly arrive at an erroneous conclusion. We know already,
as has been indicated in previous chapters, that the granular
transformation of vibrios is only part of a whole series of phenomena of
destruction of micro-organisms, the great majority of which phenomena
require more or less active intervention of the animal organism. In
reality, matters usually go on in a very complicated fashion, in which
direct and indirect actions are blended in varied proportions. In the
examples described elsewhere, we see, alongside the granular
transformation, an agglutination into clumps and immobilisation, and an
ingestion and intracellular destruction of micro-organisms. The final
phase, no doubt, is always a chemical or physico-chemical action,
exerted against the micro-organism, but how varied are the means used to
bring about this result! We may surely be allowed to suppose that
analogous phenomena may take place in the action of antitoxins on the
toxins.
[Sidenote: [384]]
Just as, in the analysis of the influence of serums on the
micro-organisms, it was found useful to study the action of certain
fluids less complicated than the anti-infective specific serums, so we
may utilise information furnished by the antitoxic action of fluids
other than the true antitoxins. Cases are by no means rare in which
normal serums exert a certain influence on toxins. Thus, Pfeiffer[581]
noted that the normal blood serum of the goat has the power to prevent
fatal poisoning by the cholera toxin. Freund, Grosz and Jelinek[582]
observed an analogous action of solutions of nucleohiston on diphtheria
intoxication and Kondratieff[583] demonstrated the same action of an
extract of the spleen on the tetanus poison. Calmette[584], in
collaboration with Deléarde, studied the influence of a whole series of
fluids on abrin intoxication. Whilst physiological saline solution was
absolutely incapable of preventing the death of animals, fresh broth
exerted an undoubted antitoxic power. Amongst normal serums, ox serum
exhibited a certain antirabic property. More, however, than the serums
of normal animals, have those of animals immunised against various
toxins other than abrin (antitetanus, antidiphtheria, antivenomous
serums, &c.) been found to possess the power of preventing intoxication
by abrin. These facts are connected with others of analogous nature,
previously demonstrated by Calmette[585], of which I may cite the
following: the serum of animals vaccinated against tetanus toxin is
active, though to a less degree, against snake venom; the serum of
rabbits vaccinated against rabies, a serum powerless to protect against
this disease, is, however, very markedly effective against the same
venom; the serum of animals immunised against snake venom is also
antitoxic against scorpion venom (I have myself had the opportunity of
confirming this fact on several occasions). In all these examples, the
serums have proved to be less efficacious against poisons other than the
toxin with which the animals that furnished the blood had been treated.
Ehrlich[586], too, has demonstrated that animals vaccinated against
robin (toxalbumin of _Robinia pseudacacia_) produce a serum, antitoxic
not only against this poison but also against ricin. It need scarcely be
added that in all these cases of non-specific action of serums derived
from vaccinated animals, no question of any antitoxic effect of normal
serums can enter. In all the experiments just summarised, the serums of
normal animals, used as controls, were found to be inefficacious.
[Sidenote: [385]]
If, in the case of the non-specific action of serums, it were allowable
to advance the hypothesis of a direct influence of these fluids on the
toxins, it would still be impossible to sustain this view where broth
fulfils the antitoxic _rôle_. This fluid, much simpler in composition
than any serum, is an excellent culture medium for micro-organisms and
one in which the toxins develop well and can be kept for a fairly long
period. There is, therefore, not the slightest ground for assigning to
it any direct antitoxic action, on the contrary, everything leads us to
regard it as an indirect agent, which acts by stimulating the reaction
of the animal organism. Here, then, the case would be quite analogous to
that of the action of broth as a protective agent against certain
bacterial injections, a subject already discussed in the tenth chapter.
In this same category of indirect influences also, must be ranked the
example of the antitoxic action of the blood of the crayfish against
scorpion venom. I have demonstrated in a series of experiments that the
fresh blood of the crayfish has the power to prevent fatal intoxication
of mice by scorpion venom. Injected in a dose of from 1 to 1·25 c.c.,
several minutes or an hour before the injection of the rapidly fatal
dose of scorpion venom, the crayfish’s blood exerts a very distinct
preventive action. It might be supposed from this that the crayfish
belongs to the group of animals insusceptible to scorpion venom. This,
however, is not the case. The crayfish is very susceptible to this
poison and succumbs to a quarter the dose necessary to kill a mouse. The
blood of the crayfish is, therefore, completely ineffective as a
protective to the crayfish itself, and only exerts its action when
introduced into the body of the mouse. It might be concluded that it is
only after it has been drawn from the crayfish that the blood acquires
its antitoxic power. Experiment contradicts this supposition. Crayfish
blood, when injected into another crayfish, in equal or greater amount
than is necessary to protect a mouse, is incapable of preventing fatal
intoxication by scorpion venom, although, here again, the crayfish
received only one-quarter of the dose of venom used for the mice.
[Sidenote: [386]]
We are, therefore, compelled to believe that the crayfish’s blood is
antitoxic for the mouse, not in virtue of its direct neutralising action
on the venom, but owing to some indirect influence on the organism of
the mouse. It is impossible to define, exactly, the mechanism of this
action. We may suppose that the blood of the crayfish contains some
substance which, by itself, is insufficient to prevent the intoxication,
but which becomes active in the presence of some other substance, also
inefficacious by itself, met with in the organism of the mouse. Here we
should have something analogous to what is met with in immunity against
micro-organisms where both fixatives and cytases intervene to bring
about the destruction of micro-organisms. By making researches _in
vitro_ on the action of the fluids on bacteria, we may easily observe
certain phenomena which appear to indicate their direct influence. Take
the case of the fluid of an oedema from an animal vaccinated against the
cholera vibrio which renders this micro-organism motionless and
agglutinates it _in vitro_; the oedema of an unvaccinated animal
produces no such effect. If, however, we were to conclude from this fact
that, in the oedema of the living animal or in its subcutaneous tissue,
everything goes on as in the test-tube and that no other phenomenon of
reaction against the vibrios is produced, we should fall into a grave
error. It is extremely probable that, in the resistance of the living
animal against the toxins, the phenomena are more complicated than are
those observed _in vitro_. The example of the blood of the crayfish
which prevents the poisoning of the mouse, without having any influence
on that of the crayfish itself, may here serve as a guide to us. It is
possible that, as in the struggle against the micro-organisms, we have
here a co-operation of two substances, each one of which, by itself, is
inactive. One of these substances would be found pre-existent in the
blood of the crayfish, the other forming part of the organism of the
mouse. Perhaps the action of this blood is even more complicated and
only becomes active through the mediation of some constituent of the
living cell.
Our study of immunity against toxins long ago revealed cases in which
this resistance cannot be attributed simply to the antitoxic action of
the body fluids. Animals vaccinated against living micro-organisms may
succumb to infection in spite of the presence of a strong anti-infective
power of the body fluids; similarly animals immunised against toxins may
die from intoxication in spite of the antitoxins contained in their
fluids. Facts of this order are not rare. Roux and Vaillard[587] on
several occasions observed animals which died from tetanus although they
had a large supply of antitoxin in their blood. Von Behring[588] and his
collaborators, Knorr, Ransom, and Kitashima, also collected a large
number of analogous facts. They showed that horses that have been
treated for a long time with tetanus toxin and whose blood serum is very
antitoxic, still experience marked illness after fresh injections of
toxin and may even succumb, in spite of the presence of a large amount
of antitoxin in their blood. In these cases the morbid phenomena are
undoubtedly different from those typical of tetanus. Instead of the
muscular contractions which characterise this disease, the above
observers noted disturbance in the regulation of the body temperature,
exudative inflammation around the point of inoculation, impairment of
appetite and fall of body weight. Sometimes they observed muscular
tremors and marked feebleness in the movements. These symptoms differing
from those of typical tetanus, it may be asked whether this poisoning is
not due to special substances other than tetanus toxin in the fluids
injected. Von Behring does not think that this is the case, for he found
that by adding antitetanus serum the formation of exudations at the seat
of inoculation was suppressed. These exudations, then, must be
attributed to the tetanus toxin.
[Sidenote: [387]]
In the cases where animals immunised against diphtheria toxin fall ill
and even die as the result of fresh injections of toxin, in spite of the
presence of a large quantity of antitoxin in their blood, we might also
cast doubts on the diphtheritic character of the poisoning, because the
clinical picture of this poisoning is not a very typical one. At the
Pasteur Institute, where a large supply of antidiphtheria serum is
prepared, we see, from time to time, horses, which have long been
undergoing the process of immunisation and are furnishing a very good
serum, suddenly fall ill and die from intoxication, without presenting
any symptom of infective disease. On one occasion, there was actually
quite a small epidemic of fatal poisonings as the result of the
injection of a quantity of diphtheria toxin not exceeding the doses
which had been well borne previously. Amongst the horses, inoculated
with the same toxin, five of the best furnishers of serum died. The
others, some of which were producing only a weak serum, remained
unaffected.
Von Behring and Kitashima[589] have given a detailed history of a young
horse which had become very susceptible as the result of vaccination
with diphtheria toxin. It finally succumbed to the intoxication in spite
of the presence of diphtheria antitoxin in its blood.
If, in these examples, we have any reason to doubt the specific nature
of the intoxication, all doubt must give way before the case described
by Brieger[590]. One of his goats, well immunised with tetanus toxin,
which, for months, had furnished a good serum and even an antitetanus
milk, after an injection, stronger than the preceding ones, was seized
with tetanic contractions. These, becoming general, brought about the
death of the animal with the symptoms of classic tetanus. The blood,
drawn off after death, exhibited strong antitoxic power.
[Sidenote: [388]]
As the result of these observations von Behring formulated the theory of
a hypersusceptibility acquired during immunisation. “Paradoxical as it
may appear,” he writes[591], “there can no longer exist any doubt that
horses which have acquired a high immunity as the result of treatment
with tetanus toxin, present a histogenic hypersusceptibility of the
organs which react against the tetanus toxin.” In support of this thesis
von Behring compares the effect produced by this toxin on horses
immunised with this same poison and on normal horses treated with
antitoxic serum from other horses. The former, in spite of the fact that
they contain in their blood 1,500 times more antitoxin than do the
latter, are, nevertheless, less refractory to tetanus toxin. This feeble
resistance is due, in von Behring’s opinion, to the much greater
susceptibility of the living elements in the horses treated with
repeated doses of the poison.
Von Behring’s theory of this form of acquired specific
hypersusceptibility has been confirmed by several well-observed facts.
These show that, in the animal subjected to treatment by toxins,
phenomena of very diverse order are evolved simultaneously: on the one
hand, cell reactions which bring about the production of antitoxins; on
the other, an increase in the susceptibility of some of the living
elements to the specific poison. We are, however, justified in asking if
the great difference between the immunity of animals treated with toxin,
and that of others treated with antitoxic serum, can be altogether
attributed to this hypersusceptibility?
Let us examine in a little more detail some examples of this
hypersusceptibility. We know that the guinea-pig is characterised by its
great natural susceptibility to the toxins of tetanus and diphtheria.
Small doses of these poisons are quite sufficient to produce in it a
fatal intoxication. But it is possible to diminish greatly this feeble
resistance of the guinea-pig by frequent injections of very small
quantities of toxin. Knorr[592] increased their susceptibility to
tetanus toxin by daily injections of one-tenth of a minimal lethal dose.
The animals died before they had received the ten tenths of this dose.
The hypersusceptibility produced under these conditions might be so
great that one-fiftieth of the minimal lethal dose was capable of
causing death. From these facts we can understand the great difficulty
experienced in the earlier attempts to vaccinate guinea-pigs by means of
unmodified toxin.
Von Behring and Kitashima[593] made analogous researches on the
susceptibility of guinea-pigs to diphtheria toxin. By frequent
injections of very small doses of this poison they succeeded in killing
these animals with ¹⁄₄₀₀ of the minimal lethal dose _distributed over
several injections_. They never succeeded in vaccinating guinea-pigs
with increasing doses of pure diphtheria toxin. Their animals died even
when they commenced with one-millionth of the minimal lethal dose.
[Sidenote: [389]]
Here, then, we have examples of the greatest hypersusceptibility that it
is possible to observe. When we compare it with the changes in the
antitoxic power of the blood, we find that these are even more marked.
Thus, Salomonsen and Madsen’s horse, to which we have already referred,
presented extraordinary oscillations in this power. After receiving,
during the course of immunisation, a fresh dose of diphtheria toxin, the
antitoxic value of its blood suddenly fell more than one-third (35%). In
order to neutralise, completely, this dose of toxin, when injected into
a normal animal mixed with antitoxic serum from this same horse, a very
small quantity of the blood of the latter would have been sufficient.
The injection into the immunised horse should have passed unperceived,
as this animal contained in its body more than 50 litres of strongly
antitoxic blood. Nevertheless the antitoxic power of this blood fell
12,000 times more than it ought to have fallen according to the
calculation made upon the data just indicated. This fall is incomparably
greater than the increase of susceptibility to toxin in the most
significant examples reproduced above.
[Sidenote: [390]]
As the fact above cited is not at all unique, it is probable that the
phenomena which appear in the animal subjected to vaccination by toxins,
must be much more complicated than is usually supposed. If the fresh
injections of these poisons bring about a specific hypersensitiveness on
the one hand, and on the other a great fall in antitoxic power, followed
by its still more notable augmentation, it is evident that the
introduction of toxins must give rise to a great perturbation in the
cell functions. The general analogy between acquired immunity against
micro-organisms and against toxins probably rests on similar bases.
Kretz[594] has already advanced the hypothesis that, in antitoxic
action, two factors, comparable to the cytases and fixatives in the
antimicrobial action, co-operate. In the absence of one of these
elements we can understand that the one which remains may be incapable
of bringing about the neutralisation of the toxin. For this reason the
antitoxic serum may act very differently in the organism of the animal
which produces it and in that of a normal animal which receives it. An
explanation which is adequate for the antitoxic action of the blood of
the crayfish injected into mice serves equally well in the case of the
antitoxic influence of the serums of animals which themselves succumb to
intoxication.
Wassermann’s[595] experiments on the anticytase serums might appear to
supply an argument against the hypothesis we are defending. Having shown
that animals injected with antityphoid serum die of intoxication when
serum which prevents the action of the cytases is introduced
simultaneously, Wassermann put the question: May not the action of the
antitoxins be prevented by this same anticytase serum? To solve this
point he injected into guinea-pigs a mixture of antidiphtheria serum
with toxin in excess and a fairly strong dose (3 c.c.) of anticytase
serum, upon which we have already spoken (see Chapter VII). The animals,
so treated, behaved exactly as did the animals used for control which
received the same quantities of antitoxin and toxin but without the
addition of anticytase serum. Wassermann concludes from these
experiments that the exclusion of the cytase, contrary to what takes
place with antimicrobial serums, in no way impedes the action of the
antitoxins. This conclusion, which appears at first sight to be
justified, cannot, however, be accepted, as the two examples chosen by
Wassermann, typhoid infection and diphtheria intoxication, differ very
profoundly from each other. In the former, we have an experimental
typhoid peritonitis which kills the control animals in less than 24
hours, whilst the second is diphtheria in which the controls do not
succumb until the sixth day after injection. The effect of the
anticytase serum being only very transitory, it is quite natural that
this should manifest itself in an infection of short duration and should
not do so in a slow intoxication. Besides, Wassermann himself has shown
that in several other cases of immunity against micro-organisms (the
bacilli of influenza and of leprosy) the injection of his anticytase
serums does not interfere with the perfect resistance of the animals.
But even were it demonstrated that the cytases really play no part in
immunity against toxins, the intervention of some other similar factor
could always be evoked.
[Sidenote: [391]]
The analogy between immunity against micro-organisms and that against
toxins may facilitate the study of the relations between the latter and
the antitoxic power of the body fluids. In the preceding chapters we
have described examples in which animals possess a protective power in
their blood but are not refractory to the corresponding infection; on
the other hand, we have cited cases in which acquired antimicrobial
immunity exists without the blood presenting any appreciable protective
power. The idea of measuring acquired immunity against micro-organisms
by the measurement of the protective or agglutinative power of the blood
must therefore be abandoned, and it is impossible to regard immunity
against toxins as a function of the antitoxic property of the body
fluids. As we have seen, animals completely refractory to tetanus, such
as the cayman, whose immunity does not depend on the antitetanic power
of the blood, develop antitoxin after the injection of toxin. A similar
state of affairs, but less pronounced, has been demonstrated by Vaillard
as occurring in the fowl. The fowl, in spite of its very marked natural
immunity against tetanus, produces antitetanin as the result of the
introduction into its body of tetanus toxin; the rabbit, on the other
hand, a susceptible animal, may acquire a real immunity without the
development of any antitoxic power in its fluids. An additional fact was
noted by Vaillard[596]. He showed that the repeated inoculation of
tetanus spores along with a small quantity of lactic acid, made below
the skin of the tail of rabbits procured for them an immunity against
tetanus toxin, although no antitoxic property appeared in their blood.
In his experiments, one hundred volumes of blood serum were found to be
incapable of neutralising a single minimal lethal dose of the toxin. The
rabbit, however, still remains quite capable of developing antitetanic
power in its fluids. All that is necessary is to inject into it some
tetanus toxin heated to 60° C. or treated with Lugol’s iodo-ioduretted
solution. As the outcome of his researches Vaillard concludes that the
antitoxic property of the body fluids “is not sufficient ... for the
general interpretation of acquired immunity, as it cannot be
demonstrated in all animals which have become refractory.”
[Sidenote: [392]]
[Sidenote: [393]]
The facts I have just mentioned were demonstrated early in our study of
the antitoxic power of the animal organism. Since then a large number of
analogous data have been collected. Recently, von Behring and
Kitashima[597] have had to abandon the immunisation of monkeys against
diphtheria toxin because of the poor yield in antitoxin which they
obtained. The blood of one of their monkeys that had acquired a
resisting power against very large doses of diphtheria toxin showed only
a very moderate antitoxic power. In establishments where antitoxic
serums are prepared on a large scale the workers have become convinced
that the yield of antitoxin has no direct constant ratio to the immunity
of the animal. This has been demonstrated repeatedly at the stables of
the Pasteur Institute. Thus, of two horses, treated at the same time and
in exactly the same way with diphtheria toxin, one furnished a very good
antitoxic serum which was maintained at 200 units Ehrlich, rising up to
400 units, whilst the other never reached 150 units[598]. And yet both
these animals possessed the same immunity against diphtheria toxin. They
tolerate considerable doses of toxin and react merely by a slight or
insignificant rise in temperature. In another series of horses, which
have been immunised for nearly seven years, one remained capable of
yielding a large quantity of antitoxin, seeing that the value of its
serum oscillated between 200 and 300 units. After five years of this
state of things the antitoxic power began to fall considerably, without,
however, any corresponding loss of immunity. Indeed, an injection of 250
c.c. of toxin (of which 0·002 c.c. was sufficient to kill a guinea-pig)
began, at the commencement of the present year, to be borne without the
least febrile reaction. An attempt was made to raise the antitoxic power
of the blood by making intravenous injections of toxin and of diphtheria
culture, but in vain. The yield of antitoxin continued to fall and it
became necessary to employ this horse for another purpose than the
preparation of antidiphtheria serum. This is by no means an isolated
example. Of a large number of treated horses it frequently happens that
certain individuals, without being particularly susceptible to a given
toxin, are found to be incapable of producing any corresponding
antitoxin[599].
In presence of the fact that animals very resistant to toxins may
possess no, or only an insignificant antitoxic power in their fluids,
and that, on the other hand, animals in which this property is highly
developed may succumb to intoxication, it may be readily understood that
immunity against toxins and the antitoxic power of the body fluids may
be two distinct conditions. Von Behring has clearly demonstrated the
fact of the cellular hypersensitiveness of the animal immunised against
the corresponding toxin and has laid great stress upon this fact. He
came[600] to the conclusion that “the immunity of the tissues and the
production of antitoxin follow a parallel course in their development so
slightly that, in spite of an abundant accumulation of antitoxin, the
susceptibility of the elements of the tissues may increase in an
extraordinary fashion.” If, during the course of immunisation, this
susceptibility can increase so greatly, it is probable _à priori_ that
under certain circumstances it might also diminish notably. After
demonstrating “that in time the antitoxin disappears from the blood of
animals immunised with toxins without any consequent disappearance of
immunity,” von Behring formulated the conclusion that in these animals
“the living elements of the animal, which were previously susceptible to
the poisons, have acquired an insusceptibility towards the same
substances.” This result fully accords with the facts of the change of
the negative chemiotaxis of phagocytes into positive chemiotaxis for
micro-organisms during the acquisition of anti-infective immunity.
[Sidenote: [394]]
Later, von Behring[601] changed his opinion. Whilst still accepting the
change of cellular susceptibility in the direction of hypersensitiveness
in animals immunised against toxins, he refused to admit the change in
the opposite direction. The cells, according to him, never lose any of
their susceptibility, so that acquired immunity against toxins cannot be
obtained otherwise than by means of antitoxins capable of neutralising
the poison in a susceptible or hypersusceptible animal. This new theory
von Behring upheld in several papers and it is met with in his most
recent publications. Nevertheless, certain well-established facts compel
us to accept an immunity against toxins as coming about as the result of
a diminution of the susceptibility of the vaccinated animal. Parallel
with his researches on the increase of the susceptibility of guinea-pigs
to tetanus toxin, researches discussed above, Knorr[602] describes
analogous experiments on rabbits. When these animals are injected with
fractions of the minimal lethal dose, frequently repeated, the rabbit
not only does not become hypersusceptible to tetanus but exhibits a
greater and greater insusceptibility. Whilst guinea-pigs, treated
according to this method, die from tetanus before they have reached the
minimal lethal dose, rabbits, as the result of frequent injections of
small quantities of tetanus toxin, become capable of resisting five
times the lethal dose (for normal rabbits) without exhibiting the
slightest symptom of illness. Against the attribution of this result to
the acquired insusceptibility of the living animals it might be objected
that the immunity, in this case, may depend on the antitoxic power of
the fluids of the body, developed with great rapidity. Such an objection
cannot be raised in the case of horses which become insusceptible to
toxins after a long period of vaccination. The horse whose history was
given above, when discussing the diminution of antitoxic power, may
serve as an example. At the commencement of its vaccinal period, in
1894, it reacted to the injection of 10 c.c. of diphtheria toxin by a
rise of temperature of 1° C. Four years later, when its blood had become
very antitoxic (350 units per c.c.), it was necessary to inject 350 c.c.
of toxin to obtain the same rise of temperature. Quite recently, having
now lost the greater part of its humoral antitoxic power, this horse
exhibited no rise of temperature after an injection of 250 c.c. of
strong diphtheria toxin. The diminution of the specific susceptibility
is produced in this case in a most marked fashion; it is not therefore
to the antitoxic property of the body fluids that this case of immunity
must be attributed.
[Sidenote: [395]]
The insusceptibility acquired against poisons of different kinds is
observed also in cases where the adaptation is not accompanied by the
production of humoral antitoxic properties, as in the immunity of frogs
against abrin. This form of immunity may be traced through the organic
series down to such lowly developed organisms as the plasmodium of the
Myxomycetes, which as we have seen readily becomes adapted to different
poisons (see Chapter II).
It can be clearly seen, then, that immunity against toxic substances is
a very complex phenomenon which it is impossible to reduce simply to an
antitoxic function of the fluids of the body. For this reason we cannot
accept a theory which would confine this kind of immunity within the
narrow limits of a simple reaction between two substances, a reaction
quite comparable to that observed in a test-tube. Attempts have been
made to determine with almost mathematical precision the conditions
under which it is possible to communicate to the animal a resistance
against microbial toxins and formulae have been constructed to define
these conditions. But the application of these formulae has been found
to be a much more difficult matter. In Prussia, with the sanction of the
Government, regulations have been enacted as to the procedure to be
followed in the testing of antitoxic serums, and a paragraph has been
added which requires a post-mortem examination of the guinea-pigs
employed for this purpose in the case of diphtheria antitoxin. “The dead
animals,” says this instruction, “must be submitted to a post-mortem
examination, and special attention must be directed to the presence of
any pre-existing diseases (tuberculosis, pseudotuberculosis, pneumonia)
which may have induced hypersusceptibility in the animals under
experiment.” Do we not see in this a proof of the important intervention
of the organism of the living animal which may modify the results of
calculations based upon too rigorous formulae? It must not be forgotten,
too, that in addition to the three diseases named in the instructions,
we have a number of other factors which may influence the receptivity
and the resistance of animals. We have already cited Roux and Vaillard’s
experiments which demonstrated that even animals which have been
previously subjected to vaccinal inoculations against certain
micro-organisms, exhibit a hypersusceptibility to mixtures of toxins
with antitoxins.
In view, then, of this complexity of the phenomena of acquired immunity
against toxins, it would be very important could we learn something of
the nature and origin of antitoxins. Unfortunately, as we shall see,
these questions are, as yet, far from having received a satisfactory
solution.
[Sidenote: [396]]
Struck by the fact that antitoxins exert a specific action on the toxin
which has been employed in the treatment of the animals that produce the
serum, certain observers have sought an explanation on the hypothesis of
a transformation of toxin into antitoxin. We have already seen that
antitoxic action is not always absolutely specific; we have serums which
prevent intoxication by various kinds of poisons, e.g. antitetanus
serum, which is active against both tetanus toxin and snake venom. There
is, however, a great quantitative difference between the influence of
the antitoxin on the toxin with which the animals have been prepared and
on a different poison. Thus, in the example just cited, in order to
neutralise snake venom it is necessary to use a much larger quantity of
antitetanus serum than against the toxin of tetanus. The classical
example of the specific influence of antitoxins is the absolute
inactivity of antidiphtheria serum against tetanus and the same
non-effect of antitetanus serum against diphtheria intoxication. The
most simple explanation of this specificity of action appeared to be the
supposition that each antitoxin contains a part of the corresponding
toxin, modified by the organism of the animal. H. Buchner[603] advocates
this hypothesis. I myself[604] said “that it is probable that
antitoxins, at least in great part, represent a modification of the
toxins prepared by certain cells in the animal body; this product is
then poured into the blood.” This view was stated as a “probability” and
consequently contains no affirmation in the least definitive. I was,
therefore, quite prepared to give it up under the weight of the crushing
criticism formulated by several very distinguished observers. It was
objected; first, that antitoxin is produced by animals in very great
disproportion to the quantity of toxin they have received; secondly,
that the animals which receive an injection of antitoxin eliminate it
from their body much more rapidly than do those which prepare it in
their own body; thirdly, that antitoxins are sometimes found in the
blood of healthy animals, who have had no attack of the disease nor any
injection of the specific toxin. Let us examine these objections more
closely, objections all based on well-established facts.
[Sidenote: [397]]
It has been shown that the antitoxin produced by the animal is
sufficient to neutralise a quantity of toxin much greater than that
which was injected into the animals supplying the antitoxic serum.
Knorr[605], from his experiments, calculated that a horse reacts to one
unit of toxin by the production of 100,000 units of antitoxin. This
statement certainly does not allow us to affirm that all the antitoxin
corresponds to toxin, but it does not eliminate the possibility that
toxin, subjected to the influence of the cells of the animal body, may
be found, in a modified form, in the product of these elements. This
hypothesis would be quite sufficient to explain the very remarkable
specificity of antitoxins.
[Sidenote: [398]]
If the toxin, in order to be modified by the living cells, must be
subjected to some special action on the part of the latter, we can
readily understand that this process must demand a greater or less
length of time; this would lead to a much slower elimination of the
antitoxin than in the case where it had been injected, ready prepared,
into a normal animal. From the commencement of his researches on
immunity against poisons, Ehrlich[606] distinguishes two kinds of this
immunity, an _active immunity_ which is obtained as the result of the
introduction of toxins into the animal, and a _passive immunity_,
another form of the refractory condition which is set up by the
injection of antitoxic serum formed in the actively immunised animal.
Von Behring[607] applies the term _isopathic immunity_ to active
immunity, and to passive immunity that of _antitoxic immunity_. It is
generally admitted that the first kind of immunity is more slowly
acquired, but that it persists for a much longer period than the second
(passive or antitoxic immunity) which is acquired immediately after the
introduction of the antitoxin, but which, on the other hand, lasts for a
short time only. This view is supported by numerous observations on the
very rapid disappearance of the refractory condition. According to von
Behring the great difference in the duration of the isopathic and
antitoxic immunities is only an apparent one. It is due to the fact that
antitoxins are usually introduced along with the serum of different
species which sets up a strong reaction and is rapidly eliminated from
the animal. Thus the antitoxic serum of the horse is usually injected
into small animals such as guinea-pigs, rabbits, and mice. When,
however, von Behring injected horses with antitoxic serums from other
horses, the antitoxic immunity lasted almost as long as in animals
vaccinated with toxins. Ransom[608] has developed this thesis in a work
carried out in von Behring’s Institute at Marburg, and supports it by
comparative researches which demonstrate the more rapid disappearance of
the antitoxin when introduced with the serum of a different species than
when introduced with that of the same species.
Even should we accept the current view on the greater duration of the
antitoxic power of the blood in isopathic immunity, the hypothesis of
the transformation of toxin by the cells of the animal is not
necessarily invalidated. If a part of the toxin introduced into the
animal remains stored for some time in an organ it is evident that only
gradually can it be subjected to the transforming action of the cells.
It is impossible, in the present state of our knowledge, to demonstrate
this proposition, but we may invoke in its favour the prolonged
persistence of red blood corpuscles when introduced into the body of a
different species of animal (see Chapter IV). These corpuscles are in
the end always completely digested but the process is of long duration.
[Sidenote: [399]]
The same hypothesis will also explain a fact, first demonstrated by Roux
and Vaillard[609]. They have shown that after repeated bleedings of
rabbits immunised against tetanus, the antitoxic property of the blood
was soon raised to almost the same value as before. Salomonsen and
Madsen[610] have confirmed the fact of the regeneration of antitoxin
after the bleeding of their animals (horses and goats) immunised against
diphtheria. Those authors who do not accept the possibility of the
transformation of toxins in the production of antitoxins, regard these
facts as absolutely incompatible with the hypothesis which they attack.
Thus, Weigert[611] considers that the regeneration of antitoxin after
bleeding can only be understood by accepting that antitoxin, like the
blood, may be reproduced in the actively immunised animal without any
fresh introduction of toxin. It is, however, just as simple, we think,
to explain the fact in question by the hypothesis of a provision of
toxin stored up in certain cells. This also is sufficient explanation of
another observation made by Salomonsen and Madsen[612], who showed that
pilocarpin is capable of augmenting the production of antitoxin. Since
it is the living cells which transform the toxin and excrete the
antitoxin, it is quite natural to suppose that every factor which
stimulates cell function may be capable of causing an increase of the
product transformed by the cells.
The third argument invoked against the possibility of the transformation
of toxins into antitoxins is based on the fact that the serum of normal
horses has sometimes a certain degree of antitoxic power against
diphtheria toxin. The horses have never suffered from diphtheria,
therefore the antidiphtherin of their blood has nothing to do with
diphtheria toxin. It is not known why the blood serum of certain
untreated horses is from the first active against diphtheria toxin,
whilst that of others exerts absolutely no action on the same poison. We
know only that this property is far from being constant in the equine
species. Perhaps it is acquired as the result of the penetration into
the animal of some pseudo-diphtheria bacillus, whose frequency and
number are very great. In order that the microbial products may give
rise to the formation of antibodies, it is not at all necessary that the
micro-organisms should produce an evident disease. Thus, to cite one
example only, Foerster[613] observed a considerable agglutinative power
against the typhoid cocco-bacillus in the serum of a child which was
found living among a family of typhoid patients but which, itself,
presented no morbid symptom.
The criticism, directed against the hypothesis that modified toxin
enters into the production of antitoxin, may not be sufficient to show
the incorrectness of this view; it does not follow, however, that the
view is right. In the present state of our knowledge it is impossible to
solve the problem definitely, and as the hypothesis of transformation
gives us the best idea of the specificity of the action of antitoxins,
it has a right to be taken into consideration as much as any other.
[Sidenote: [400]]
Ehrlich[614] has formulated another hypothesis to explain not only this
specificity but the origin of antitoxins in general. This is the
ingenious hypothesis of side-chains or of receptors, which has already
been considered in other chapters of this work. It is now for the first
time brought forward in relation to the antitoxins properly so-called,
that is to say substances capable of preventing intoxication by
microbial toxins. In order to make his hypothesis as clear as possible
Ehrlich begins by explaining its bearing on the concrete example of
tetanus antitoxin. “When we introduce into an animal a small quantity of
tetanus toxin, it is easy to obtain exact proof that it is quickly fixed
by the central nervous system, probably by the motor cells of the
ganglia; that the central nervous system more than any other organ
attracts the tetanus toxin and retains its toxic molecules very firmly.”
There we have the side-chains of the protoplasm fulfilling this rôle and
subjecting the living protoplasm to the prolonged action of the poison.
Once it is combined, the side-chain becomes incapable of fulfilling its
normal function, and there is induced on the part of the living elements
the production of new chains of a similar character. Following the law
that the reaction is stronger than the action, there is an
over-production of these side-chains which finally so embarrass the cell
which has developed them that they are excreted by it into the blood
plasma. Once expelled into this plasma, they continue to manifest their
affinity for the tetanus toxin, an affinity which must be even greater
in the case where the chains are found in the blood than when they were
connected with the cell. Owing to this affinity, these chains, now in
the blood, fix the tetanus poison introduced into the animal and prevent
it from reaching the susceptible nerve elements. Antitoxins, according
to this hypothesis are, therefore, nothing but overplus side-chains
poured into the body fluids. Ehrlich extends his theory to a whole
series of bodies capable of causing the formation of antitoxins and
antidiastases. “It is probable,” he says, “that all analogous bodies can
only become toxic to the animal on condition that the animal is capable
of fixing their toxophore groups in certain of the organs that are
important for its life” (p. 17).
[Sidenote: [401]]
According to this theory tetanus antitoxin must pre-exist in the central
nervous system of the normal animal. In the immunised animal, the
side-chains must be reproduced in very great quantity in the nerve cells
and pass thence into the circulation. Indeed, Wassermann, a supporter of
this theory, made a search for tetanus antitoxin in the nerve centres of
normal animals. In collaboration with Takaki[615] he made the important
discovery that the brain and spinal cord of small mammals (guinea-pigs
and rabbits) when triturated with tetanus toxin prevent the
manifestation of its toxic action in animals most susceptible to
tetanus. The brain was always found to be more active than the spinal
cord. The property of neutralising the toxin of tetanus belongs to the
solid parts of the nerve centres; the fluid of the cerebral emulsion is
incapable of exercising this action.
This discovery was soon confirmed. Ransom[616] demonstrated it almost at
the same time, and independently of Wassermann and Takaki; and the fact
is indisputable. It remains to be seen whether the “antitoxin” of the
nerve centres of normal animals is really the same as that which is
found in the fluids of animals immunised against tetanus toxin, as is
accepted by Wassermann and the other partisans of the side-chain theory.
The former is characterised by a very local reaction; it is incapable of
being dissolved and distributed through the body of the animal. This is
shown by Marie’s[617] experiments, and my own[618], all carried out in
my laboratory. All that is necessary is to introduce, beneath the dorsal
surface of the thigh of a guinea-pig, a quantity of the cerebral
substance sufficient to neutralise several times the lethal dose of
toxin, and below the skin of the ventral aspect of the same thigh, a
lethal dose of this toxin, when it will be found that the guinea-pig
contracts a fatal tetanus. The antitoxic action of the nerve substance
extends, therefore, for a short distance only; it is strictly local.
[Sidenote: [402]]
The view that the action of the substance of the pounded nerve centres
is different from the neutralisation of the toxin by the antitoxin of
the body fluids is further confirmed by the fact that the fixation of
the tetanus poison by the cerebral substance is very transient. We have
shown that a mixture of toxin and pounded cerebral substance, that does
not produce any tetanic symptom when injected into the peritoneal cavity
of guinea-pigs, sets up a grave tetanus when it is injected
subcutaneously into the thigh. In the latter case the toxin becomes
separated from the particles of the cerebral substance that had fixed
it. Danysz[619] convinced himself that the mixture of pounded brain with
tetanus toxin when it is left in physiological saline solution, in
distilled water, or in a 10% solution of sea salt, allows the tetanus
toxin to pass into the macerating fluid. The fixation of the toxin to
the cerebral substance is, therefore, more comparable to the mordanting
of colouring matters by the tissues than to a real combination.
Observers who have repeated the experiments of Wassermann and Takaki
have been greatly struck by the difference between the action of the
pounded cerebral substance and that of the living brain upon the tetanus
toxin. Whereas the former, taken from the guinea-pig, an animal very
susceptible to tetanus, prevented intoxication when employed in minimal
dose, the living brain of the same species was found to be incapable of
neutralising the most minute quantities of toxin. On the other hand,
Roux and Borrel[620] have shown that the brain of rabbits, whether
untreated or vaccinated against tetanus, was very susceptible to the
action of the tetanus toxin. This toxin, injected directly into the
brain, set up in both groups of rabbits a special and characteristic
cerebral tetanus. On the other hand, when a little of the cerebral
substance of the rabbits, mixed _in vitro_ with tetanus toxin, was
injected into other susceptible animals, these remained unaffected.
This great difference between the antitoxic action of the living brain
and that of the pounded cerebral matter, on the one hand, and the
rigorous localisation of the antitetanic influence of this cerebral
substance, on the other, have suggested to several observers the idea
that the brain cannot be regarded as the organ of formation of the true
antitoxin, such as is found in the fluids of immunised animals. This
view has been expressed by Roux and Borrel, Marie and ourselves.
Knorr[621] also shares this view, being struck by the fact that rabbits
attacked by tetanus remain for weeks with contractions, but are
incapable of producing in their nerve-cells sufficient antitoxin to
disintoxicate them, although their blood is already loaded with
dissolved antitoxin.
[Sidenote: [403]]
At this period it was generally supposed that, in accordance with
Ehrlich’s theory, the hypothetical side-chains were capable, under
certain conditions, not only of fixing the tetanus toxin, but also of
neutralising it. It was said, therefore, that these chains, reproduced
in large quantities in the cerebral cells, must exercise their
neutralising action in the brain itself. Consequently, when it was seen
that, in Roux and Borrel’s experiments on vaccinated rabbits, this organ
was itself affected, it was concluded that the brain must not be
regarded as the producer of the antitoxin.
Later, Ehrlich and his supporters, amongst whom I will name especially
Weigert, have developed the theory of side-chains in a much more
detailed fashion, leading to a different interpretation of several facts
previously established. Ehrlich distinguishes in the toxin molecule a
_haptophore group_ which combines with the side-chain or the
corresponding receptor of the living elements, and a _toxophore group_
which produces the poisoning of the protoplasm. The side-chains,
inactive for the toxophore group, neutralise only the haptophore group.
Consequently, when these side-chains are numerous in the nerve elements
which produce them, they may be a source of great danger to this living
element, by attracting the toxic molecules. In this case, these chains,
or receptors, serve to attract the poison, just as the badly adjusted
lightning-conductor attracts lightning. For this reason rabbits
vaccinated against tetanus become tetanic when the toxin is injected
directly into the brain. It is only at a distance from the nerve centres
that the receptors, excreted into the body fluids, fulfil their rôle of
true antitoxins. There they combine with the haptophore group of the
toxic molecule, leaving the toxophore group intact; this latter group,
however, diverted from the nerve-cells, is incapable of exercising an
injurious action.
[Sidenote: [404]]
From this point of view not only the cerebral tetanus of vaccinated
rabbits, but also the hypersusceptibility of immunised animals, upon
which von Behring has so strongly insisted, may be explained. The
argument, drawn from these facts, against the nervous origin of tetanus
antitoxin, loses, therefore, much of its weight. If we confront this
hypothesis with the other data collected on the question, the solution
of the problem becomes beset with great difficulties. Previous to the
discovery made by Wassermann and Takaki, I attempted to solve the
problem by removing from fowls portions of the brain and spinal cord,
proposing to take advantage of the fact that birds, which are capable of
producing antitoxins, withstand these operations fairly well. My hopes
were not fulfilled; I could never keep my fowls alive long enough to
complete the experiment. We must, therefore, for the present, be content
with indirect arguments. If the nerve centres do really produce the
tetanus antitoxin and excrete it into the blood, we ought at a given
moment to find in these organs a greater quantity of this substance than
in the blood and the other organs. The reader will recall the researches
of Pfeiffer and Marx, and of Deutsch, who demonstrated the possession of
a greater richness in protective substance by the phagocytic organs of
animals, treated with micro-organisms, than by the blood serum. The same
result might be obtained by a comparative investigation of the tetanus
antitoxin in the nerve centres and the blood of animals immunised
against tetanus. My experiments directed to this point have not been
favourable to the hypothesis of the nervous origin of tetanus antitoxin.
In fowls, killed as soon as tetanus antitoxin began to appear in the
blood, the brain and spinal cord did not exhibit the slightest antitoxic
power[622]. We might be tempted to explain this result as due to an
accumulation of toxin in the nerve centres which would prevent the
manifestation of the antitoxin. For this reason, in my later
researches[623], I made use of animals that had been long immunised, but
whose blood was still antitoxic. I killed a fowl which had not received
any toxin for about eight months, and a guinea-pig into which the last
toxic injection had been made almost two years before the date of this
experiment. After removing a portion of the brain the blood of these two
animals was found to be more antitoxic than before the operation, which
indicated that the source of the antitoxin was as yet uninjured. To
ascertain whether this source was to be found in the nerve centres I
made a comparative determination of the antitoxic power of the brain, of
the spinal cord and also of several other organs, of the blood and of
the exudations. The result was still negative. The nerve centres were
found to be less antitoxic than the blood and other fluids of the body,
and even less active than such organs as the liver and kidneys.
[Sidenote: [405]]
[Sidenote: [406]]
In support of the hypothesis of the nervous origin of tetanus antitoxin
there remains, then, only the fact of the retarding action of the
cerebral substance upon tetanus. In the absence of other arguments this
assumes a preponderating importance. We have seen that this action is
based on a fleeting and not very firm fixation of the toxin by certain
parts of the brain and the cord. Are we justified in regarding this as
comparable to the more stable fixation observed in living animals
susceptible to tetanus intoxication? Soon after Wassermann and Takaki’s
discovery I pointed out that the pounded brain of frogs mixed with
tetanus toxin does not prevent animals, into which this mixture is
injected, from contracting fatal tetanus. This observation was confirmed
by Courmont and Doyon[624], in several series of experiments carried out
under various conditions. They found that “the brain of the frog, heated
or unheated, when mixed with tetanus toxin even for several hours, at
the temperature of the laboratory or at 38° C., even in considerable
doses, does not possess any neutralising property.” This fact would not
be in any way wonderful if we had to do with an animal insusceptible to
tetanus; but in the frog, as we have said in the preceding chapter, this
is far from being the case. In the cold it does not readily become
tetanic, but above 25°–30° C. it becomes very susceptible. The tortoise,
which is very refractory to this intoxication, has a brain which, when
pounded and mixed with tetanus toxin, exerts a certain preventive power
over susceptible animals. Nevertheless, the brain of the living frog, as
demonstrated by Morgenroth, absorbs this toxin. There is, therefore, a
difference between the absorption of the tetanus poison by the living
elements and by the pounded cerebral substance. A similar result is
obtained with several other toxins. Diphtheria poison is very toxic when
injected directly into the brain of the guinea-pig or rabbit. Even the
rat, as demonstrated by Roux and Borrel[625], is readily affected by
this toxin under these conditions. Doses which when inoculated
subcutaneously are well borne by the rat, when introduced into the brain
set up a fatal intoxication in this animal. And yet the brain, when
pounded and mixed with diphtheria toxin, can never protect susceptible
animals from intoxication. Numerous attempts to reproduce Wassermann and
Takaki’s experiment with the diphtheria poison have always been
unsuccessful. Attempts to obtain the same result with snake venom have
also given negative results. Calmette[626] made several experiments with
emulsions of rabbit’s brain and snake venom with the object of
ascertaining whether the elements of the nervous system possess against
venom the same properties as against tetanus toxin. “None of these
emulsions”—concludes Calmette—“exhibited either the slightest protective
or antitoxic power _in vitro_. There is, therefore, no analogy of action
between what takes place in the nerve elements against tetanus toxin and
against venom.” Nevertheless venom, like diphtheria toxin and tetanus
toxin in the frog, exerts an undoubted action on the nerve centres.
Again, the protective fixation of poisons to the cerebral substance is
not the exclusive privilege of tetanus toxin. Kempner and
Schepilewsky[627] obtained the same result with the toxin of botulism
(produced by van Ermenghem’s anaerobic micro-organism which sets up
intoxication of intestinal origin in certain cases of poisoning by
food). The brain and spinal cord of the guinea-pig, when triturated with
physiological salt solution and mixed with botulinic toxin, prevents
intoxication in susceptible animals, exactly as in Wassermann and
Takaki’s experiments with tetanus.
When Kempner and Schepilewsky wished to obtain some idea as to the
substance or substances in the nerve centres which fix the toxin of
botulism and thus prevent poisoning, they found that lecithin and
cholesterin, mixed with this toxin or injected separately and
simultaneously, protected mice just as completely as did the cerebral
substance. On the other hand, they found a difference as regards the two
substances when injected before the toxin was introduced; they were then
unable to prevent poisoning, though the cerebral substance exerted an
undoubted protective influence. Kempner and Schepilewsky also showed
that heating altered the preventive action of lecithin and cholesterin
less than it did that of cerebral emulsion.
[Sidenote: [407]]
These observers extended their researches to the protective action of
fats and demonstrated that olive oil when emulsified and neutralised
with soda and mixed with twice and even four times the lethal dose of
botulinic toxin, prevented the contraction of a fatal poisoning by mice.
Tyrosin also protected mice against this intoxication, not only when
injected simultaneously with the poison, but even when introduced into
the animal 24 hours before the poison was administered. Kempner and
Schepilewsky conclude “that not only with the substance of the nerve
centres, but also with various other substances, they were able to
obtain a certain protective effect against the toxin of botulism” (p.
221). Their experiments with cholesterin and tyrosin were suggested to
them by the previous researches of Phisalix[628] who demonstrated that
the bile salts, as well as the two substances I have just mentioned,
would protect animals against the venom of the viper.
Bearing all these facts in mind, it appears to be probable that in the
above cases it is principally the fatty matters of the nerve centres
that temporarily fix these toxins, and allow the animal organism to
divert the poisons from their morbific action. From this point of view,
it is interesting to note that the toxic action of the tetanus poison
can also be prevented by other substances than the emulsion of the nerve
centres. Thus Stoudensky[629] demonstrated, in an investigation carried
out in Roux’s laboratory, that carmine fixes the tetanus toxin and
prevents its action on the guinea-pig. As in the case of the cerebral
substance, this fixation by carmine is very unstable. When the carmine
that has fixed the tetanotoxin is macerated in distilled water it gives
up the poison to the water which is then capable of producing tetanus.
Such fixation does not end, any more than in the case of the cerebral
substance, in the destruction or disappearance of the toxin. Carmine if
first dissolved or macerated in water (especially if heated) loses its
fixative power and can no longer prevent tetanus poisoning.
Sterilisation, at 120°, 100° and even at 60° C., of the carmine,
suspended in physiological salt solution, caused it to lose its
protective action, although dry heat applied to it in closed tubes did
not destroy this power.
[Sidenote: [408]]
In many respects carmine, which is derived especially from the adipose
body of the cochineal insect, exerts an antitoxic influence analogous to
that of maceration with the nerve centres. If fats play a special part
in this action, we can readily understand how a brain, such as that of
the frog, poor in fatty matters, cannot fix the tetanus toxin and
prevent its morbific action. In any case the fact that certain
substances of diverse nature, acting on toxins, exert an influence
similar to that of the pounded mass of the nerve centres, does not allow
us to accept Wassermann and Takaki’s experiment as proving the nervous
origin of tetanus antitoxin. The analogy with the facts bearing on the
anticytotoxins, collected and described in the fifth chapter, also tells
against this hypothesis. We would here remind the reader that the two
constituent parts of the antispermotoxin, the anticytase and the
antispermofixative, develop in castrated animals and are consequently
produced outside the spermatozoa, elements susceptible to the
spermotoxin. The facts collected concerning the antihaemotoxins indicate
also that these substances have some other origin than the red blood
corpuscles.
[Sidenote: [409]]
[Sidenote: [410]]
This latter supposition appears to be in contradiction to Ransom’s[630]
very interesting researches on the haemolytic action of saponin, carried
out in Meyer’s laboratory at Marburg. This glucoside, owing to its
property of fixing itself on the stroma of these corpuscles dissolves
the red corpuscles of many vertebrates. The cholesterin of this stroma
combines with the saponin, as the result of which the red corpuscles
become altered and allow the haemoglobin to diffuse. But this same
substance, cholesterin, which causes the poison to penetrate into the
red blood corpuscles, prevents the solution of these elements when they
are bathed in blood-serum. This fluid, in fact, acts as the antitoxin to
saponin and does so just because it contains cholesterin. The
cholesterin of the serum, fixing the saponin, prevents it from affecting
the red corpuscles, thus fulfilling the function of a well fitted
lightning conductor. On the other hand, when the cholesterin of the
stroma of these corpuscles is linked on to the saponin, it renders them
the disservice of a defective lightning conductor. The accord between
these facts and the postulates of Ehrlich’s theory led Ransom to suppose
that in the haemolysins and antihaemolysins, cholesterin perhaps played
a similar part. His experiments convinced him that this was not the
case. As it is generally accepted, after Calmette’s[631] experiments and
according to Ehrlich’s view, that the alkaloids and the glucosides in
general are incapable of setting up the formation of antitoxins, we
might regard the attempts to find an antisaponin and to settle whether
it is identical with cholesterin as useless. But in regard to these
delicate questions we must be careful not to give too great weight to _a
priori_ arguments. It was believed until quite recently that substances
with very complex molecules, such as the albuminoids, toxins and soluble
ferments, must always give rise to the production of antibodies in the
animal; whilst the simpler substances whose chemical nature was better
defined could never lead to this. Facts acquired in recent years have
led to a modification of this view. In our fifth chapter we have already
spoken of the fruitless attempts of Ehrlich and Morgenroth to obtain
certain antifixatives. And yet the fixatives, as is shown by the results
of the researches of Bordet and myself, belong to the category of
substances which are quite capable of setting up the formation of
antibodies. Again, certain mineral poisons, quite unexpectedly, gave
rise to the development of the counterpoison in the animal body. This
fact forced itself upon Besredka[632] in his researches on the
adaptation to arsenic made in my laboratory. His experiments were
undertaken for the purpose of studying the mechanism of the refractory
condition against a poison, apart from any antitoxic action whatever,
which, according to previous investigations, seemed excluded. This
action, however, was exhibited in such a degree that it could not be
ignored. The serum of animals immunised against arsenious acid was found
to possess both protective and antitoxic properties against a dose of
this poison killing a rabbit in 48 hours. It is true that
Morishima[633], in a research carried out in Heyman’s laboratory at
Ghent, has thrown doubt upon these results. His objections, however,
cannot refute the statements of Besredka which rest on very precise and
numerous experiments which I witnessed. Morishima left out of account
several important circumstances and carried out his experiments without
any continuous check by means of control animals. It must be said also
that the resistance of the rabbit against arsenic depends on many
different factors and that, at certain seasons, it is much more
difficult to adapt them to the poison than at others. It is only by
numerous researches extending over a very long period that we can arrive
at precise and conclusive results.
From these observations there is every inducement for us to attempt to
ascertain whether, by subjecting animals to repeated injections of
saponin, it is possible to augment the antisaponic power of their
blood-serum and whether, if this takes place, the antitoxic action is
due to a rise in the amount of cholesterin in this serum. I therefore
requested Besredka to carry out some experiments bearing on this point.
Guinea-pigs, injected with progressive doses of saponin for more than
two months, at the end of this period showed no increase in the
antisaponic power of their serum. They followed the rule established by
Ehrlich; they developed no antitoxin against a glucoside. Moreover, they
gave us no new information as to the origin of these antibodies.
[Sidenote: [411]]
In his first memoir in which the theory of side-chains is treated,
Ehrlich insists on the nervous origin of antitetanin as an example of
the production of antitoxins by animals susceptible to poisons. Now,
however, that he has come to distinguish haptophore and toxophore groups
in the toxic molecule, it is to the side-chain, which fixes the first
group, that Ehrlich attributes prime importance. “The formation of
antitoxins”—he says[634] in the opening address at his Institute at
Frankfort—“would, therefore, be absolutely independent of the action of
the toxophore elements.” In other words, for a cell to be capable of
producing antitoxin, it is not at all necessary that it should be
susceptible to the toxic influence of the poison; it is only necessary
that it should possess receptors, or side-chains, capable of combining
with the haptophore group of the toxin. Thus it is possible, as we have
described above, to produce antitoxins, with modified toxins whose toxic
action is _nil_ or almost so, but which have retained their power of
combining with antitoxic substances. According to Ehrlich, these
modified toxins are _toxoids_, in which the toxophore group is
completely destroyed; “whilst the haptophore group, the producer of
immunising substances, is retained in its integrity.” It is evident then
that, under such conditions, the tetanus antitoxin might be developed
elsewhere than in the nerve centres. For that it would be sufficient
that outside the nerve cells there should be other living elements
capable of fixing the tetanus toxin, or, to use Ehrlich’s phraseology,
elements, possessing side-chains, having an affinity for the haptophore
group of the tetanus poison.
Dönitz[635] has already expressed the view that in the rabbit the
tetanus toxin may be fixed not only by the nerve elements but also by
the various other cells.
[Sidenote: [412]]
[Sidenote: [413]]
The existence of such cells, outside the nervous system, is not merely
hypothetical. It is shown very clearly in Roux and Borrel’s experiments
on cerebral tetanus. In order to produce this disease in the rabbit, it
is sufficient to introduce a very small dose of toxin directly into the
brain. When inoculated subcutaneously with much larger quantities of the
same tetanus poison, the rabbit remains in good health or exhibits
merely a slight and transient tetanus. “The resistance of the rabbit
against the tetanus toxin, injected under the usual conditions”—conclude
Roux and Borrel[636]—“is not due, then, to a relative insusceptibility
of the nerve centres, but to the fact that much of the poison introduced
does not reach the nerve cells and is destroyed in some part of the
animal.” In the guinea-pig, as shown by the same investigators, the
difference of the dose of tetanus poison, necessary to produce fatal
tetanus by intracerebral or by subcutaneous injection, is minimal or
nil, from which it may be argued that in this very susceptible animal
there is no destruction of toxin outside the nerve centres and that the
whole of the poison introduced makes its way without hindrance as far as
these organs. Ehrlich, in his report to the International Congress of
Medicine in Paris (August, 1900), accepted these results, as seen from
his tenth and eleventh propositions: “The receptors exist, sometimes in
certain tissues only, sometimes in the majority of the organs (action of
tetanus poison in the guinea-pig and in the rabbit),” “... the presence
of numerous receptors in the organs of less vital importance may bring
about—thanks to a kind of diversion of the toxin molecules—a diminution
in the susceptibility of the animal to this toxin[637].” We must here
recall the differences between the susceptibility of the guinea-pig and
that of the rabbit to small doses of tetanus toxin frequently repeated
as in Knorr’s experiments already referred to. The guinea-pig, subjected
to these injections, dies in a tetanic condition long before it has
received the minimal lethal dose for this species when injected in a
single dose. The rabbit, on the other hand, is very tolerant of repeated
doses and even rapidly acquires an immunity against five minimal lethal
doses for the rabbit (injected at once). Knorr explained this difference
as due to the hypersusceptibility of the nerve centres in the guinea-pig
and to their acquired insusceptibility in the rabbit. The experiments of
Roux and Borrel on the cerebral tetanus of rabbits vaccinated against
tetanus, have demonstrated that this insusceptibility is not produced in
these animals. We must, therefore, seek some other explanation. In
rabbits subjected to small repeated doses, the poison is more and more
prevented by certain living elements from reaching the nerve centres.
Further, it is neutralised by the antitoxin which is rapidly produced.
We find from Knorr’s[638] researches that in rabbits antitoxin appears
in the blood in cases where, affected with a transitory tetanus, their
limbs remain contracted for weeks. In guinea-pigs, affected with the
same form of tetanus, antitoxin in appreciable quantity is never found,
even after complete recovery. All these facts accord with the hypothesis
that there exist, outside the nervous system, certain living cells which
absorb the tetanus toxin and produce antitoxin. Rabbits and fowls
possess this property in a much greater degree than do guinea-pigs. The
fowl, according to Knorr, develops “a large quantity of antitoxin,
whilst the tetanic symptoms are still augmenting.” In this animal, as we
have been able to show[639], a portion of the tetanus toxin is absorbed
by the leucocytes. By exciting aseptic exudations in fowls into which I
had previously injected this toxin, I was able to convince myself that
these exudations, much richer in leucocytes than was the blood, were
also much more tetanigenic than was the blood. I observed also a more or
less pronounced leucocytosis after the injection of non-lethal doses of
tetanus toxin into fowls. It is possible that the leucocytes were actual
agents in protecting the animal against the penetration of this poison
to the susceptible nerve centres.
The great susceptibility of leucocytes to microbial toxins serves to
indicate that these cells are of some importance in the struggle of the
animal against these poisons. Their injection usually sets up a marked
hyperleucocytosis of the blood. On this point Chatenay[640], working in
my laboratory, has carried out a series of experiments on animals
poisoned by bacterial (tetanus and diphtheria), phanerogamic (ricin and
abrin) and animal (snake venom) toxins. He was able to demonstrate a
striking analogy between them and the phenomena which occur in bacterial
infections. When death takes place at the end of a very short period,
the number of leucocytes markedly diminishes; if the animal lives beyond
24 hours or resists completely, a hyperleucocytosis, often of very
marked character, is produced. In the guinea-pig, which is so
susceptible to tetanus, the leucocytosis observed occurs even after
injections of quantities of tetanus toxin equal to several lethal doses,
and it is only after the introduction of an amount equal to one hundred
times the lethal dose that the number of leucocytes remains stationary
or shows a diminution. Here we have something analogous to what takes
place against the anthrax bacillus in the same animal. The penetration
of this deadly organism sets up a marked leucocytosis, but the
accumulated leucocytes are incapable of seizing the bacilli or of
preventing their noxious action. In other species of animals, such as
the rabbit and the fowl, the intervention of the leucocytes against the
anthrax bacillus, as well as against the tetanus toxin, is more
effective.
[Sidenote: [414]]
If this toxin, instead of being injected in solution, be introduced
along with the bodies of the micro-organisms which contain it, the
struggle on the part of the animal takes place under more favourable
conditions and even very susceptible animals may afford evidence that
they offer a high resistance. Vaillard and Vincent[641] have shown that
if we inject living tetanus bacilli, or the spores of these bacilli,
deprived of free toxin, into guinea-pigs a great accumulation of
leucocytes, which prevent the production of infection and intoxication
by devouring the bacilli and their spores, takes place. The toxin
contained in the ingested bacilli remains innocuous; this affording
evidence of the protective part played by the leucocytes against the
toxin. The same interpretation may be offered to explain the survival of
animals very susceptible to tetanus, when the tetanus poison, mixed with
pounded cerebral substance or with carmine powder, is injected. In these
mixtures the toxin, as mentioned above, becomes attached to certain
substances of the triturated brain or to the grains of carmine. This
fixation is very unstable, the toxin is readily set free; but, when
introduced into the body of the animal, the mixture induces a great
accumulation of leucocytes which seize the cerebral particles and the
grains of carmine and along with them take possession of the toxin.
Wassermann and Takaki’s experiments and those of Stoudensky are easily
explained if we assume two protective acts: the first of these consists
in fixing the toxin, thus preventing it from diffusing and rapidly
reaching the living nerve cells; the second is the absorption of the
toxin fixed by the leucocytes,—cells endowed with receptors for the
haptophore group of the toxin, but insusceptible to its toxophore group.
When one of the two factors is absent, tetanus cannot be prevented. It
is for this reason that in Courmont and Doyon’s experiments with
emulsion of the frog’s brain, mixed with tetanus toxin, the inoculated
animals died from tetanus in spite of an accumulation of leucocytes.
This fact affords additional proof that, under these conditions, the
toxin does not become anchored to the particles of the pounded cerebral
substance, this anchoring being a condition necessary for the effective
reaction of the leucocytes.
[Sidenote: [415]]
[Sidenote: [416]]
The absorption of the tetanus toxin becomes evident when we study in
detail the phenomena produced in the experiments carried out according
to Vaillard’s methods with tetanus spores and those of Wassermann and
Takaki with poison to which cerebral emulsion has been added, or
according to Stoudensky’s method with grains of carmine. When, however,
it is desired to bring forward rigorous proof of the presence of the
tetanus toxin inside the leucocytes charged with spores, with granules
of cerebral substance or with grains of carmine, very great difficulties
are encountered. How, indeed, is it possible to demonstrate this poison
fixed upon these various bodies, a poison, the presence of which cannot
be demonstrated except by its injection into the animal? For this, in
the study of the reaction of the organism of the animal against the
poisons, it is very important to have recourse to substances, whose
presence can be demonstrated more easily than can the microbial toxins.
We must first have recourse to the alkaloids, especially atropin, which,
in this respect, present numerous advantages. We know that rabbits
resist considerable doses of sulphate of atropin, even when this poison
is injected directly into the blood. On the other hand, when it is
introduced into the brain, according to Roux and Borrel’s method, even
small quantities are quite sufficient, as demonstrated by Calmette[642],
to produce a fatal poisoning. The intracerebral injection of the
one-hundredth part of a dose which, when introduced into the circulation
of the rabbit, produces no disturbance, in the same animal at the end of
a few minutes sets up an enormous pupillary dilatation with symptoms of
very lively excitation, increase of the reflexes, and general
anaesthesia. These phenomena are succeeded by paralysis and death, which
supervenes three or four hours after the injection. The natural immunity
of the rabbit against atropin falls therefore into the same category as
that against morphin. It is not due to the innate insusceptibility of
the nerve cells, but to something which prevents the alkaloid from
reaching these living elements. With the object of ascertaining the
mechanism of this immunity, Calmette injected into the veins of rabbits
a fairly large quantity of sulphate of atropin (0·2), he then bled these
animals and collected from their blood the plasma and the white
corpuscles, separating them by centrifugalisation. When injected into
the brain of other rabbits, these constituents of the blood did not act
in the same way. Whilst large doses of plasma set up merely a short
period of excitation and a very transitory pupillary dilatation,
corresponding quantities of leucocytes caused grave disturbances,
sometimes followed by death in from seven to twelve hours. Calmette
concludes from his researches that the atropin does not remain in the
fluid part of the blood, since mere traces of it are found in the serum,
but that it is seized and absorbed almost immediately by the
leucocytes[643]. This result has been confirmed by Lombard[644] by
another series of experiments. After injecting very large quantities of
sulphate of atropin into rabbits and guinea-pigs, he bled these animals
and separated out the elements of their blood. Instead of introducing
these elements into the brain of rabbits, he injected them into cats,
animals very sensitive to atropin. The cats which received the red
corpuscles and the plasma exhibited very insignificant symptoms of
poisoning. Those, on the other hand, which were injected with a
corresponding quantity of leucocytes, had much graver symptoms of
intoxication, such as photophobia with maximal pupillary dilatation,
dysphagia and persistent diarrhoea.
It is, therefore, to the absorption of the atropin by the leucocytes
that naturally refractory animals owe their immunity, an immunity which
is very marked in spite of the susceptibility of the nervous elements of
these animals. We have been able to obtain this result thanks to the
delicate physiological reactions obtained with certain alkaloids. As
regards arsenic the demonstration could be pushed even further, for the
absorption of this mineral poison by the leucocytes has been established
by chemical analysis.
[Sidenote: [417]]
When engaged in my researches on the leucocytic phenomena in
intoxications I succeeded[645] in showing that in rabbits subjected to
rapidly fatal doses of arsenious acid, there is a marked diminution in
the number of white corpuscles in the blood. On the other hand, in
rabbits habituated to arsenic, the same doses which brought about
hypoleucocytosis and death of the control rabbits, induced a
considerable rise in the number of leucocytes. Later, Besredka[646] made
continuous and detailed researches upon this subject and obtained most
interesting results. In order to simplify the conditions of experiment,
he studied the reaction of the organism of the animal after the
introduction of a red trisulphide of arsenic[647], a not very soluble
salt, easily recognisable by its colour and markedly toxic. When
non-lethal doses of this salt were injected into the peritoneal cavity
of the guinea-pig, there was, first a transitory fall in the number of
the white corpuscles in the peritoneal fluid, followed by a
hyperleucocytosis of the most marked character. Of the leucocytes
accumulated in the exudation the macrophages almost exclusively seized
the yellowish-red granules of the trisulphide of arsenic. Very shortly,
the whole of the salt injected was found within the peritoneal
leucocytes, and the animals in which this marked phagocytosis occurred
remained in good health. The ingested granules could be observed for
several days in the macrophages; but in course of time, these arsenical
particles were broken up into very small granules and ultimately
disappeared. Here, then, we have an intraphagocytic solution of the
trisulphide of arsenic and very probably a transformation of this salt
into some other arsenical combination, innocuous to the animal. This
soluble substance escapes from the macrophages and is finally excreted
by the urinary passages.
[Sidenote: [418]]
Since the phagocytes ingest the trisulphide of arsenic and render it
innocuous, it was to be anticipated that the elimination of these
protective cells would lead to a fatal poisoning by doses which, under
normal conditions, are readily withstood by guinea-pigs. When Besredka
used sacs of reed pith containing non-fatal quantities of the red
trisulphide and introduced them into the peritoneal cavity of
guinea-pigs these animals were not long in exhibiting symptoms of
poisoning and died at the end of a longer or shorter period, this
varying with the amount of poison introduced. Even when the phagocytic
reaction had been impaired as the result of a previous injection of
carmine powder, the guinea-pigs died after doses of trisulphide of
arsenic which, under ordinary conditions, did not kill them. The
phagocytes in this experiment devoured numerous grains of carmine and
were rendered incapable of ingesting enough of the trisulphide of
arsenic to save the animal. On the other hand, when Besredka set up a
previous accumulation of macrophages in the peritoneal cavity of his
guinea-pigs, he succeeded in rendering these animals resistant to doses
of trisulphide of arsenic that, under normal conditions, were fatal. The
whole of these facts converge to show that the phagocytes, thanks to
their power of seizing the trisulphide of arsenic and of modifying it
within them, exercise a beneficent and immunising action on the organism
of the animal. The analogy of the main facts concerning this protective
influence with that observed in the immunity against infective
micro-organisms is indeed very considerable.
Having determined the part played by the macrophages in the resistance
of the organism of the animal against a not very soluble salt of
arsenic, Besredka proceeded to study the leucocytic phenomena in
poisoning by soluble arsenical compounds. In his experiments he made use
of potassium arsenite and he found that when lethal doses were injected
the guinea-pigs showed a diminution of leucocytes in the blood in less
than 24 hours, whilst with non-lethal doses, he produced a marked
hyperleucocytosis. When he injected lethal doses into rabbits accustomed
to arsenic, these animals manifested an increase of white corpuscles,
just as in animals injected with non-lethal doses. These oscillations in
the number of leucocytes, like those which have been observed after
poisoning by trisulphide of arsenic, certainly indicate that the
organism and its defensive cells behave in the same way to both slightly
soluble and very soluble salts of arsenic. In the first case it was easy
to demonstrate that the accumulation of leucocytes in the blood and in
the peritoneal exudation terminated in the ingestion of the granules of
trisulphide. With potassium arsenite, it was not so easy to prove the
point; a chemical analysis of the elements of the blood, however, has
given a decisive answer. After injecting the lethal dose of this soluble
salt into rabbits accustomed to arsenic, Besredka bled them in order to
separate the plasma, leucocytes and red corpuscles. Several experiments
made on these rabbits gave a concordant result which this observer sums
up thus: “Although the bulk of plasma and of red corpuscles was much
greater than that of the leucocytes, it was in the latter only that
arsenic was found” by chemical analysis. It was only in those cases
where the animals survived, and manifested hyperleucocytosis, that
Besredka succeeded in demonstrating the presence of arsenic in the white
corpuscles.
[Sidenote: [419]]
These experiments, excluding any doubt as to the protective part played
by the leucocytes against arsenical intoxication, of course suggested
the idea of investigating whether the nerve elements, submitted to the
direct influence of potassium arsenite, exhibit any real susceptibility
to this poison. The injection of solutions of this salt into the brain
demonstrated that the one-hundredth part of an ordinary lethal
subcutaneous dose was sufficient to cause fatal poisoning. This fact,
then, falls into line with other facts, already numerous, as to the
susceptibility of the nerve centres to microbial toxins, alkaloids and
other poisons. But in the case of potassium arsenite, it was even more
easily demonstrated than in the other cases that immunity natural or
acquired, is connected with the absorption of the poison by the
leucocytes. These cells, themselves much less susceptible to the toxic
action than are the nerve elements, protect them from contact with the
poison.
It is manifest that arsenic is not the only mineral substance capable of
being absorbed by the phagocytes, and there are already on record well
established facts in support of this thesis. Some time previous to the
researches on arsenical poisoning just summarised, Kobert, then in
Dorpat, set his pupils, Stender, Samoïloff, Lipsky and others[648] to
make systematic researches on the fate of iron in the animal organism.
For this purpose these observers made use of a very soluble preparation
of iron—or better expressed, as soluble as possible—Dr Hornemann’s
_ferrum oxydatum saccharatum solubile_, which does not precipitate in
alkaline media. They proved that a small quantity of the iron introduced
into the animal is eliminated by the kidneys and the wall of the
intestine, but that the greater part of the metal is arrested in the
organs, especially the liver, spleen and bone marrow. The iron is there
absorbed by the leucocytes which hold it for some time and then throw it
into the intestine.
[Sidenote: [420]]
I have had the opportunity of observing this circulation of Dr
Hornemann’s soluble salt in the organism of several species of
vertebrates. Some time after its introduction into the organism by the
blood vessels, peritoneally or subcutaneously, the iron may be found (by
means of the microchemical reaction with potassium ferrocyanide)
accumulated in the various phagocytes, especially the leucocytes, the
stellate Kupffer’s cells of the liver and the macrophages of the splenic
pulp. The non-phagocytic cells, as, for example, Ehrlich’s basophile
leucocytes, so abundant in the lymph of rats, take up very little of
this iron, although the macrophages and microphages are full of it[649].
Against these facts Weigert[650] has advanced the objection that the
leucocytes absorb only the iron precipitated in the form of granules,
but my own researches allow of no doubt that not only granular but
dissolved iron is absorbed. This discussion, however, loses much of its
importance in view of the results obtained with potassium arsenite.
According to Samoïloff[651], soluble salts of silver in the animal
organism undergo a fate similar to that of Hornemann’s soluble iron salt
and are absorbed by the phagocytic elements. It must be noted, further,
that according to the experiments of Arnozan and Montel[652], the
leucocytes absorb such drugs as calomel and salicylate of soda.
[Sidenote: [421]]
These observations all clearly show that the phagocytes must not be
looked upon as cells capable of seizing merely the dead bodies of
micro-organisms and of animal cells, always fearing and avoiding poisons
and only able to come forward when protected by some other antitoxic
function. The phagocytes no doubt often exhibit a negative
susceptibility for many poisons, when these are introduced into the
animal organism in too large a quantity. But these cells are most
resistant to toxic substances and protect the higher elements from the
poison. Under these conditions, it is quite natural to assign to the
phagocytes the rôle of the fighting agents of the animal organism
against poisons and we may even enquire whether these elements do not
produce the antitoxins. It has been pointed out that it is very
difficult to attribute this function to the cells susceptible to the
toxic action,—the spermatozoa in the production of antispermotoxin, the
red blood corpuscles in the development of antihaemotoxin, or the nerve
cells in the production of tetanus antitoxin. Moreover since, according
to Ehrlich’s theory, it is only the haptophore group which excites the
formation of antitoxins on the part of the elements which possess the
corresponding receptors, it is quite possible that the phagocytes,
thanks to the facility with which they absorb the poisons, occupy an
important place as producers of antitoxins. I have already formulated
this hypothesis, and several investigators, amongst whom may be cited
Gautier[653] and Courmont[654], have received it favourably, though in
the imperfect state of our knowledge, it cannot, as yet, be fully
demonstrated. It might perhaps be objected against this hypothesis that
in many instances, after the injection of micro-organisms living or
dead, in spite of a vigorous leucocytic reaction the organism of the
animal does not produce any antitoxin. In such cases, there is clearly a
development of antibodies, such as the fixatives, whose phagocytic
origin may reasonably be claimed, but no true antitoxins. It must not be
forgotten, however, that the various kinds of phagocytes present,
amongst themselves, great differences, and that perhaps certain only of
these elements are capable of producing antitoxins. When
micro-organisms, living or dead, are introduced into an animal it is
found that antitoxins do not as a rule appear in the fluids; in these
cases the reaction is set up mainly by the microphages. The macrophages
represent the principal source of antitoxins. In cases where these
phagocytes ingest the micro-organism the blood exhibits an undoubted
antitoxic power. Such is the case with bubonic plague in the human
subject, where the micro-organism is readily ingested by the
macrophages. Here we obtain antitoxic serums even after the introduction
of living or dead organisms into the animal, a fact observed by Roux and
his collaborators. Another fact in favour of the hypothesis I am
defending is furnished to us by the cayman. As noted above, this
reptile, of all known animals, supplies antitoxins most quickly and
easily. In the cayman the leucocytic system is composed of eosinophile
microphages filled with granules, and of macrophages. As the eosinophile
cells are only very weakly phagocytic, it is the macrophages almost
exclusively which intervene in the reaction against the micro-organisms.
It is probable, then, that in the cayman and in animals inoculated with
the plague bacillus the exclusion of the microphages from the struggle
constitutes a factor favourable to the production of antitoxins and at
the same time favourable to the manifestation of the activity of the
macrophages.
[Sidenote: [422]]
If these latter phagocytes play the primary rôle in the excretion of
antitoxins in the fluids of the body we should expect to find this
function exercised not only by the motile macrophages of the blood and
lymph, but also by the fixed macrophages, so widely diffused through
almost all the organs.
I advance this hypothesis for what it is worth, simply as a guiding idea
for new researches in this field, of which so much is still
unknown[655]. The brief account of the actual state of the question of
artificial immunity against toxins, has indicated to us that this is a
problem far more difficult of solution than is that of acquired immunity
against micro-organisms. The mere fact that these latter can still be
found some hours or even days after their entry into the refractory
animal, affords a great advantage in these researches as compared with
those on toxins which are lost, often almost immediately, after their
injection. Consequently our knowledge of antimicrobial immunity is more
advanced than is that on immunity against the soluble products of
micro-organisms.
The facts narrated in this chapter support the thesis I have defended on
the subject of immunity against micro-organisms—that antimicrobial
immunity in no way depends on a previous resistance against the toxins.
As a general rule the immunity against micro-organisms is developed more
readily than the immunity against their toxic products and at an earlier
stage.
Although much still remains to be done in the elucidation of the
mechanism of antitoxic immunity, the principal data acquired on the
subject of this immunity have undoubtedly led to applications of the
highest importance, as will be set forth in one of the following
chapters.
CHAPTER XIII
IMMUNITY OF THE SKIN AND MUCOUS MEMBRANES
Protective function of the skin.—Exfoliation of the epidermis as a
means of ridding the animal of micro-organisms.—Localisation and
arrest of micro-organisms in the dermis.—Intervention of
phagocytes in the defence of the skin.
Elimination of micro-organisms by the conjunctiva.—Microbicidal
function of the tears.—Absorption of toxins by the
conjunctiva.—Protection of the cornea.—Elimination of
micro-organisms by the nasal mucosa.—Protection by the respiratory
channels.—Dust cells.—Absorption of poisons by the respiratory
channels.
Alleged microbicidal property of the saliva.—Part played by microbial
products in the protection of the buccal cavity.—Antitoxic
function of the saliva.
Antiseptic action of the gastric juice.—Antitoxic function of pepsin.
Protective function of the alimentary canal.—Absence of microbicidal
power from the intestinal ferments.—Protective function of the
bile.—Antitoxic rôle of the digestive ferments.—Favouring and
retarding functions of the intestinal micro-organisms.—Destruction
of toxins by these micro-organisms.
Defensive rôle of the liver. Protective function of the lymphoid
organs of the alimentary canal.
Protective function of the mucous membrane of the genital
organs.—Autopurification of the vagina.
[Sidenote: [423]]
In the preceding chapters the phenomena of immunity which are exhibited
within the animal body in which the portals were open for the
penetration of the micro-organisms and their poisons have been studied.
We had to deal almost exclusively with experimental immunity, the study
of which constitutes the basis of our present knowledge concerning the
general problem of immunity. In natural immunity, however, things do not
follow the same course. The micro-organisms and their toxins are not
introduced directly into the tissues and blood by means of a syringe or
other instrument. The micro-organisms have to make their own way through
the skin and the mucosae, tissues which offer a resistance more or less
serious and effective; or they may have to take up their abode in the
cavities of the animal organism, in order that they may be able to
inundate it with their poisons. We must here review briefly these
natural barriers to microbial invasion.
[Sidenote: [424]]
The skin constitutes a protective covering of great importance in
connection with the preservation against microbial invasion of the
delicate parts of an animal. In many of the lower and higher animals,
and even in man himself, the skin becomes the seat of a microbial flora,
often very abundant, in which may be found, in addition to certain
inoffensive organisms, other minute parasites more or less harmful. The
pyogenic cocci, staphylococci and streptococci, are constantly found on
the human skin, most frequently hidden in the depths of the canals of
the hair follicles. These micro-organisms seize every favourable
opportunity to attack the organism, producing such local lesions of the
skin as acne, pimples, boils, and erysipelas, or even becoming
generalised in the blood and tissues, as in the septicaemias and
pyaemias. To the skin, therefore, must be assigned a very important
function in the prevention of the invasion of micro-organisms which are
found constantly on the surface of the body or which, along with all
kinds of dirt, are brought there accidentally.
[Sidenote: [425]]
The skin is able to fulfil this protective function from the fact that,
in most animals, it is covered with a not very permeable layer of some
considerable thickness. In the majority of the Invertebrata, of all
classes, the surface of the body is clothed with a chitinous layer,
sometimes very thin and capable of folding and following all the
movements of the body; or again it may be impregnated with calcareous
salts and very hard, as in the case of the integument of Insects and
Crustacea, and the shell of the Mollusca. In all cases this cutaneous
sheath constitutes a formidable obstacle to the entry of
micro-organisms. Even in animals of very small size the thin cuticle is
effective in preventing any invasion by these parasites. Thus the
_Saprolegniae_, fungi so fatal to many aquatic animals, are often quite
unable to pass through this cuticular layer. In order to pass this
obstacle their germs must take advantage of some fissure or wound,
produced by other causes. _Daphniae_, too, may often be observed to
succeed in ridding themselves of the _Monospora_ with its needle-like
spores by means of a mechanism which we have already described in
chapter VI. The white corpuscles of the blood surround the spores of
this parasite and transform them into an innocuous detritus. Sometimes,
however, a number of these fine spores manage to perforate the cutaneous
investment of the small crustacean; quite a small opening is made in the
chitinous wall, which in itself is a source of no danger. As soon,
however, as a spore of the _Saprolegnia_ approaches this opening, it
immediately begins to thrust a process through the small lesion, and
from that moment the fate of the _Daphnia_ is sealed. Incapable of
opposing the slightest phagocytic resistance to the filaments of the
fungus, it is invaded throughout by the mycelium and soon dies.
The integrity of the skin being so important for the preservation of
life, a fairly perfect mechanism has been elaborated for the maintenance
of this integrity. All animals, no matter what their position in the
animal scale, are liable to lesions and wounds of the surface of their
bodies. In the _Daphniae_ I have often[656] observed wounds produced by
the bites of other aquatic animals. The surface of these wounds soon
becomes covered with a rich microbial vegetation. The leucocytes are
brought up to the injured point and there produce a protective layer;
but, at the same time, a rapid proliferation of the neighbouring cells
of the epidermis takes place; this closes the wound and separates the
skin, so reconstituted, from the micro-organisms. Everything resumes its
original order and the leucocytes soon disperse, regaining the blood
stream.
These phenomena, which can be readily observed under the microscope in
such small and transparent animals as the _Daphniae_, may serve as the
prototype of those of a number of analogous processes in the animal
kingdom. The thicker and more solid the cuticular investment, the more
fully it guarantees the animal against the penetration of
micro-organisms. Cuénot[657] made the observation that Crustacea,
furnished with such a hard envelope as is the carapace of the Decapods,
are completely defenceless from the moment parasitic micro-organisms
make their way into their bodies. These intruders quietly instal
themselves in the tissues, without causing the slightest phagocytic
reaction, and thus bring about the inevitable death of the host. The
protection of the animal in this case is, so to speak, associated with
the resistance offered by the carapace.
[Sidenote: [426]]
Again, in many of the Vertebrata, the skin has a hard, thick sheath,
e.g., the scales of fishes and of reptiles. Man, with his supple and not
very thick skin, is less well endowed; this, however, does not prevent
him from defending himself against the entry of micro-organisms by the
cutaneous path. Sabouraud[658], a well-known dermatologist, has given a
very concise and at the same time very complete sketch of the part
played by the skin in the protection of the body against
micro-organisms; from this author the following data are borrowed.
The epidermic layer sets up a defence by the production and expulsion of
corneal cells. In the normal course of the life of the epidermis, the
cells of the deeper layers, coming to the surface, become exfoliated and
are thrown off. “There is thus produced, a continual exfoliation of the
dead layers, and a continual eviction of such micro-organisms as are
living on them. The epidermis is dense and its cells have a hard
envelope; the micro-organism is not endowed with motion, or at least not
with sufficient to be of service in effecting an entrance. It can only
penetrate the epidermis by multiplication _in situ_, a micro-organism
originates alongside another, another in front of it, and in front of
this again others. In this way they burrow between the apposed cells
just as a root penetrates into the ground; so great is the resistance of
the horny cells that we never find any micro-organisms within them, but
between them only” (p. 734). The epidermic cells, containing
micro-organisms, exfoliate, and the skin is thus ridded of them.
Frequently the process, as it goes on constantly and slowly, is
invisible; but often, on the other hand, it becomes exaggerated and
manifests itself in the form of a cuticular desquamation which leads to
the elimination of a large number of micro-organisms. The patient may
retain “such pellicles for ten years, and even longer, without
presenting anything else but these, and there are many other chronic
squamous infections in which the course is uncomplicated by even an
erosion or the slightest wound.”
The connective tissue of the human skin is also fully able to defend
itself; it is extremely vigorous and represents a real obstructing and
resisting tissue. The penetration of parasites sets up in it a
thickening of the fibrous tissue; this effects a localisation of the
microbial focus. To appreciate the effectiveness of this dermic defence,
we have only to compare the slow growth of lupus, a form of cutaneous
tuberculosis, with that of tuberculosis of the lungs and other viscera,
or the slow evolution of farcy, or cutaneous glanders, with that of the
visceral form of the disease.
[Sidenote: [427]]
If we examine more closely the process by which the dermis surrounds the
intruders with a fibrous capsule, we readily recognise in it a reaction
of the macrophages of the skin. In lupus these phagocytes seize the
tubercle bacilli, combining to form giant cells and giving rise to an
exaggerated development of the connective tissue fibres. Moreover, when
the skin is menaced with a microbial invasion, not only the local
macrophages but the leucocytes are mobilised. The migratory white
corpuscles travel through the epidermis and the connective tissue layer.
In spite of the absence of a lymphatic circulation in the epidermis, the
leucocytes penetrate into this layer “and, in a section through the
normal epidermis, it is very rare not to find here and there some
deformed and flattened leucocyte, surprised just as it was creeping
between the cells of the _rete mucosa_ or of the _stratum granulosum_.”
Immediately that the epidermis or the dermis finds itself menaced with a
microbial invasion, an accumulation of leucocytes of all kinds is
produced at once; this may remain microscopic or it may assume
proportions visible to the naked eye. Frequently the subjacent
epithelium throws off epidermic scales which are filled with leucocytes;
often also the leucocytic foci in the dermis become emptied, the
micro-organisms being expelled along with their enemies the phagocytes.
The tissues of the skin proper defend themselves against micro-organisms
as well as they are able; but so soon as the danger becomes serious
there is sent to their succour a whole army of mobile phagocytes. This
example of the defence made by the cutaneous investment may serve as a
prototype of that of every other region of the body. Alongside a local
action, there is always an intervention of mobile phagocytes; but when
this action becomes insufficient, a much more abundant accumulation of
leucocytes than is found in ordinary cases is immediately produced.
Like the skin, the mucous membranes are invested with an epithelial
layer, which serves as a barrier to the entry of micro-organisms. But
whilst the surface of the normal skin is dry or barely moistened by the
secretory products of the cutaneous glands, the mucous membranes are
always humid, a condition favourable to the multiplication of
micro-organisms. Hence the mucous membranes which are most exposed to
contact with the air and with external objects, always contain a larger
or smaller number of organisms, amongst which the pathogenic species,
notably staphylococci, pneumococci and streptococci, are the most
common. The part played by the animal organism in getting rid of these
micro-organisms becomes more complicated than in the case of the defence
made by the skin.
[Sidenote: [428]]
The first of the mucous membranes to be exposed to contamination by
micro-organisms is the conjunctiva of the eye. At the moment of birth it
is in contact with the vaginal mucous membrane and acquires from it some
of its micro-organisms, both innocuous and pathogenic. Tears fulfil the
function of averting the danger resulting from this proximity and from
the presence of micro-organisms in the conjunctival sac in general.
Ophthalmologists have shown that these tears transport the organisms
into the nasal cavity by means of the lachrymal canal. To determine this
point Bach[659] introduced a number of Kiel water bacilli along with
pyogenic staphylococci into the conjunctival sac of various individuals.
Seedings made with the tears showed a very rapid disappearance of the
two organisms, which passed into the nose where their presence could be
demonstrated by making plate cultures of the nasal mucus. Enormous
numbers of the Kiel bacilli, introduced into the conjunctival sac, were
all transferred to the nasal cavity, on the average, by the end of
half-an-hour. The pyogenic staphylococci persisted on the surface of the
conjunctiva for a longer period, but they also passed in large numbers
through the lachrymal canal into the nose.
[Sidenote: [429]]
Certain observers, notably Bernheim[660], thought that the tears, in
addition to their purely mechanical defensive action, were capable of
destroying the micro-organisms by their microbicidal power. Bach[661]
submitted this question to a minute examination and came to the
conclusion that several species of bacteria, introduced _in vitro_ into
the tears of healthy persons or of those who were suffering from
conjunctivitis or certain other ocular diseases, disappeared somewhat
rapidly. Comparative experiments with tears previously heated to 58° and
even to 70° C., in most cases gave the same results, that is to say,
they caused a rapid disappearance of the organisms introduced. From
these facts the author concluded that it is probably to the salts
contained in the tears that their bactericidal action is due. Control
experiments made with physiological saline solution and with various
mixtures of mineral salts met with in the tears have been found by Bach
to cause a like disappearance of the same species of organisms. Well
water, and even distilled water, gave the same result. In all these
cases it is evident that, in the tears, there is no bactericidal cytase
comparable with that found in the serums and other body fluids which may
contain this phagocytic diastase. The experiments with heated tears
demonstrate this clearly. On the other hand, these same experiments lead
one to suppose that the diminution and even the disappearance of the
micro-organisms in the tears, is due to a large extent, and perhaps
completely, to an agglutinative action of the salts, a fact which has
been demonstrated by several observers.
In all these cases it is indisputable that the mechanical part played by
the tears is the most important of the defences offered by the
conjunctiva of the eye against the microbial invasion. That this defence
is not always sufficient is proved by the frequency of conjunctivitis,
as well as by the ease with which certain micro-organisms, inoculated
into the conjunctival sac, set up a general infection. This is specially
the case with the coccobacillus of human plague. When it is introduced
into the conjunctival sac of susceptible animals (rat, guinea-pig, &c.),
it passes thence into the nasal cavity and soon produces a generalised
and fatal infection. The conjunctival membrane, even when perfectly
intact, readily absorbs certain poisons. Everyone knows the rapidity
with which atropin, when introduced into the conjunctival sac, causes
dilatation of the pupil. But the mucous membrane may serve also as the
port of entry for toxins of microbial origin. Several observers, and
especially Morax and Elmassian[662], have demonstrated that the
diphtheria poison placed upon an unbroken conjunctival membrane, where
the epithelial layer is uninjured, sets up local lesions which progress
very slowly but which terminate in the formation of actual false
membranes. Nevertheless, it must be admitted that the intact epithelial
layer of the conjunctiva exerts a certain defensive action against the
penetration of toxins, although a very slight lesion of this layer will
allow of the ready absorption of the diphtheria poison and the formation
of false membranes.
The cornea likewise, so long as it is intact, exhibits a marked
resistance against the penetration of micro-organisms and of toxins.
When it becomes injured in any way its epithelium is repaired with great
rapidity, as has been well demonstrated by Ranvier[663], who has shown
that the walls of the wound close by a process of epithelial “soldering”
in a purely mechanical fashion, without the intervention of any
preliminary proliferation of the epithelial elements. Thanks to this
very rapid obliteration the micro-organisms are prevented from
penetrating not only into the interior of the cornea, but into the
anterior chamber of the eye.
[Sidenote: [430]]
It has already been pointed out that the ocular conjunctiva gets rid of
the introduced micro-organisms chiefly by removing them mechanically and
sending them through the lachrymal duct into the nasal cavity. This, in
turn, defends itself by making use of a similar method. In his
experiments on the Kiel red bacillus, inoculated into the conjunctival
sac of man, Bach demonstrated that in a very short time these
micro-organisms are carried into the nasal cavity. He showed also that
they do not remain long in the latter position and that their number
decreases hourly.
Twenty-four hours after the introduction of these bacilli into the
conjunctiva none, as a general rule, are to be found in the nasal mucus.
This expulsion of the micro-organisms likewise takes place by mechanical
means, aided by the movements of the vibratile cilia. It is evidently to
this process that the mucous membrane owes its relative freedom from
micro-organisms. Frequently, when examining the nasal mucus or when
making cultures therefrom, one is astonished at the small number of
micro-organisms found in the nasal cavities of persons in good health.
Thomson and Hewlett[664] have certainly gone too far when they affirm
that the upper regions [i.e. the Schneiderian membrane] of the nasal
cavity are, in almost 80% of cases, free from micro-organisms. But it is
certain that in these regions we do find a small number only of the
bacteria which exist in greater abundance in the lower (cutaneous)
passages of the nose.
[Sidenote: [431]]
To explain this paucity of micro-organisms in the nasal cavity, Wurtz
and Lermoyez[665] have assumed the existence of a bactericidal property
in the nasal mucus. They affirm that the anthrax bacillus, after contact
with this mucus for several hours, loses its virulence for the most
susceptible animals, and that several other micro-organisms—the
staphylococci, the streptococci, and the _Bacillus coli_—become
attenuated under the same conditions. Others who have studied this
question have come to a different conclusion. Thomson and Hewlett found
that the nasal mucus is not bactericidal, although it prevents the
multiplication of micro-organisms. F. Klemperer,[666] denies the
bactericidal property of the nasal mucus. He could never demonstrate the
destruction of micro-organisms by the mucus, and he also observed that
bacteria do not multiply at all readily in this medium. These results
confirm the hypothesis that the defensive action of the nasal mucous
membrane against microbial invasion is mainly effected by the mechanical
elimination of the numerous micro-organisms which continually reach it.
Amongst these organisms are some which are conspicuous for the ease with
which they multiply in the body, taking the nasal cavity as a starting
point, e.g. the micro-organisms of influenza, the plague bacillus,
which, according to several observers, is very virulent when introduced
by the nostrils[667], and the leprosy bacillus. This last, according to
Goldschmidt[668], Sticker[669], and Jeanselme[670] often enters the
human body by way of the nose.
It is certain that the olfactory apparatus deprives the inspired air of
a large number of the micro-organisms which it carries. These organisms
deposited on the mucous membrane are expelled with the nasal mucus. A
number of the foreign organisms, carried by the air, may, however,
surmount this first barrier and penetrate further into the trachea and
bronchi, whence, helped by the movements of the vibratile cilia, they
are usually expelled along with the mucus.
[Sidenote: [432]]
[Sidenote: [433]]
In spite of this double defence it has been recognised that very minute
corpuscles and, amongst others, micro-organisms may overcome every one
of these obstacles and reach the pulmonary alveoli. Here, under the name
of “dust-cells” (“cellules à poussière”)—“Staubzellen” of the German
writers—located in the alveoli, are described certain large
mononucleated elements which contain granules of foreign origin, usually
deposits of soot, of a deep black. This permeability of the normal lung
tissue for dust particles and pigmented corpuscles has been closely
studied and clearly demonstrated by J. Arnold[671] and his pupils.
Several observers have tried to determine whether micro-organisms,
introduced by the respiratory channels, behave like other bodies.
Animals were made to inhale, or there were introduced into the trachea,
cultures of bacteria pathogenic for the animals experimented upon. The
results so obtained have been very contradictory. Morse[672],
Wyssokowitch[673], and Hildebrandt[674], never succeeded in inducing
anthrax by the introduction of anthrax bacilli into the lungs of normal
animals. They concluded, therefore, that the uninjured pulmonary tissue
was impermeable by virulent micro-organisms. H. Buchner[675] with his
collaborators and pupils maintaining the opposite view, declare that
rabbits that have inhaled anthrax bacilli or their spores always succumb
to a fatal attack of anthrax. These contradictory results were
attributed to differences in the methods employed, and an attempt was
made to perfect the methods of research, especially to prevent the
penetration of the anthrax bacilli by lesions of the trachea or by any
channel other than that of the pulmonary tissue. Gramatschikoff[676],
under Baumgarten’s direction, undertook a series of experiments in order
to determine whether it was possible for the anthrax bacillus to
traverse the pulmonary tissue. He introduced through the trachea of
rabbits and guinea-pigs an anthrax culture, afterwards washing the
respiratory passages with a large quantity of broth or of physiological
saline solution. Several of the animals so treated did not succumb to
the inoculation, and Gramatschikoff concluded that it was impossible for
the anthrax bacillus to make its way through the wall of the normal
pulmonary tissue. He was satisfied that some of the injected organisms
were destroyed in the lung, although he was unable to see how this
bactericidal action was determined. In these experiments a large
quantity of fluid was introduced after the bacilli; this might wash away
the bacilli and convey them to situations where they could exert no
morbific action; moreover the anthrax bacilli used were of doubtful
virulence (the injections made to control the virulence in the
subcutaneous tissue were in nearly every instance made with quantities
of fluid greater than those introduced by the trachea), and
Gramatschikoff’s results could not be accepted as deciding the question.
On the other hand, H. Buchner’s inhalation experiments made with spores,
and the study of the organs of animals so treated, leave no doubt as to
the possibility of the invasion of an animal by the respiratory channels
by the anthrax bacillus. Furthermore, the “rag-picker’s disease” and the
“wool-sorter’s disease,” or pulmonary anthrax, developed in man as a
result of the inhalation of dust charged with anthrax spores,
demonstrate most clearly that it is possible for the anthrax bacillus to
enter the body by the respiratory channels. The pulmonary mycoses,
produced by the penetration of the _Aspergillus fumigatus_ in the human
subject, offer confirmatory evidence.
[Sidenote: [434]]
In spite of the fact that the pulmonary tissue is not impermeable to
pathogenic micro-organisms, it is none the less true that it exhibits a
very marked resistance to infection by this channel. It is, however,
neither the thickness of the wall, as in the case of the skin and the
mucous membranes, nor the mechanical elimination with the help of the
vibratile cilia or of the secretions, that constitute the means of
defence in the respiratory alveoli. Here the cell elements are charged
with the duty of ridding the lungs as much as possible of the
micro-organisms which enter. Ribbert[677] and his Bonn pupils,
Fleck[678] and Laehr[679], observed this fact long ago. They showed that
the spores of _Aspergillus flavescens_ and the staphylococci, injected
into the veins or into the trachea, penetrate into the pulmonary
alveoli, where they are soon seized by the “epithelial cells” and the
leucocytes. Laehr observed that this phenomenon is produced at the end
of a few hours, and that the ingested cocci within the phagocytes
undergo a progressive degeneration and at last disappear.
Tchistovitch[680], working in my laboratory, studied micro-organisms
pathogenic for the rabbit—the anthrax bacillus, the coccobacillus of
fowl cholera, and the bacillus of swine erysipelas—ingested by the
“dust-cells” of the alveoli. He has added the important observation
(already referred to in chapter IV) that these phagocytic elements are
not epithelial cells at all, but are really macrophages of lymphatic
origin. They are not found in the alveoli of new-born animals, but soon
appear there and instal themselves in such a manner that for long one
was led to regard them as true epithelial cells of the pulmonary tissue.
This tissue, invested with an extremely delicate covering, is incapable
of defending itself against the invasion of micro-organisms, but the
animal organism comes to its aid by sending a permanent army of
macrophages which evict from the alveoli, so far as is possible, both
micro-organisms and other foreign bodies. Under these conditions, we can
readily understand that similar cells which fulfil the same protective
function, are also found in the neighbouring bronchial glands. It has
long been recognised that the macrophages of these glands are often
crammed with various kinds of granules of foreign origin, which have
made their way into the lungs with the inspired air.
Toxic substances can be absorbed by the mucous membrane of the
respiratory channels. Roger and Bayeux[681] have shown that no lesion is
required in order that diphtheria poison may invade the mucous membrane
of the trachea, and so produce typical false membranes. The lung, we
know, is accessible to gaseous toxic substances; moreover, its surface
readily absorbs fluid poisons.
The protection of the digestive system is more complex than that of the
respiratory passages; this is not remarkable, when we consider the
greater complexity of the organs of digestion and the varied conditions
which they present with regard to microbial invasion.
[Sidenote: [435]]
The buccal cavity, so exposed to the entry of extraneous micro-organisms
along with the food and the external air, has a very rich microbial
flora, in which Miller[682], the author of our most complete work on
this subject, has recognised in man more than thirty species. Several
representatives of this flora, e.g. the _Leptothrix_ and the
_Spirochaeta_ are constantly present, and are very characteristic of the
buccal cavity of man. With them are frequently found pneumococci,
staphylococci, and streptococci, whose pathogenic power is undoubted.
Virulent diphtheria bacilli are also met with in a certain number of
quite healthy persons. It is astonishing that, in spite of this state of
things, wounds in the mouth heal very rapidly, and operations on the
buccal cavity done with insufficient or no aseptic precaution do not, in
the great majority of cases, set up infective complications of the
slightest importance. After certain buccal operations we are often
confronted with a complicated and open fissure; nevertheless the wound
thus left exposed is not ordinarily the seat of any infection either
local or generalised.
It is often asked, how under these conditions does the mouth defend
itself against the vast number of formidable micro-organisms. When the
theory of the bactericidal power of the body fluids was dominant, and
appeared to explain several important points in the general problem of
immunity, the saliva was studied from this “bactericidal” point of view.
Sanarelli[683], as the outcome of patient and laborious researches, came
to the conclusion that the human saliva acted as an antiseptic and
destroyed a large number of micro-organisms. It is true that he
recognised its efficacy only when few bacteria were subjected to its
action; but even when the saliva was incapable of killing a large number
of micro-organisms, it did not allow them to develop—it was a bad
culture medium; moreover, it had the power of attenuating the virulence
of certain pathogenic bacteria, such as the pneumococcus, so frequently
found in the mouth.
The conclusions of the Italian observer did not, however, meet with
general acceptance. Miller[684] did not believe that the saliva exerted
any bactericidal action, raising the objection that the absence of
nutritive value in the human saliva for bacteria is explained by the
fact that in his experiments Sanarelli employed filtered saliva, which
consequently had been deprived of much of its nutritive
substances,—epithelial débris, mucus, etc. Hugenschmidt[685], working in
my laboratory, carried out a special research on the influence of the
human saliva on micro-organisms, and arrived at conclusions quite at
variance with those reached by Sanarelli. In spite of the variety of
micro-organisms made use of, he could never satisfy himself that the
saliva had any bactericidal property.
[Sidenote: [436]]
He sometimes saw, no doubt, a certain slowness of growth or even the
destruction of certain of the micro-organisms sown at the commencement
of the experiment, but this was very slight and rather exceptional. In
most cases the micro-organisms, introduced into the saliva, grew
rapidly, so that their number, in a very short time, became very
considerable. Where the saliva brought about any diminution in the
number of micro-organisms, this semblance of bactericidal action could
be noted not only in the normal saliva, but; also, as in the lachrymal
secretion above described, in saliva heated to 60° C. Against certain
micro-organisms—the torulae and the staphylococci—the heated saliva
acted more vigorously than did the unaltered saliva. It is consequently
impossible to draw any parallel between the action of the saliva and
that of the cytases.
Since the saliva often contains (according to certain authors even
constantly) small quantities of potassium sulphocyanide, it seemed to be
worth while to ascertain whether this salt is capable of destroying
micro-organisms. The experiments carried out by Hugenschmidt, in order
to settle this point, demonstrated that when given in doses comparable
to those met with in the saliva, the potassium sulphocyanide exerts no
bactericidal action.
Powerless as an antiseptic, the saliva fulfils an important function in
ridding the mouth of micro-organisms in a mechanical way. The parotid
secretion and that of the other salivary glands dilutes the bacteria and
carries them from the pharyngeal cavity into the stomach. Hence, in
diseases where the salivary secretion is much diminished, the mouth
becomes the most important portal of entry for micro-organisms capable
of setting up secondary infections. The saliva is further useful in
diluting the alimentary detritus and preventing its stagnation and
decomposition in the buccal cavity.
[Sidenote: [437]]
In addition to the direct mechanical part played by the saliva, it
performs a very important indirect function. This fluid contains
microbial products and diastases, and is capable of exciting in the
leucocytes a positive chemiotactic activity. Hugenschmidt demonstrated
the fact by introducing into animals small capillary glass tubes
containing saliva. A certain time after being placed in position, these
tubes became filled with considerable masses of immigrated leucocytes.
The same result was obtained with guinea-pig’s saliva, enclosed in
capillary tubes and introduced into the peritoneal cavity of the same
species. Here, again, the leucocytes assembled in the tubes and ingested
the micro-organisms found in the saliva. The influence of the saliva on
the afflux of the leucocytes must be regarded as an act important for
the protection of the buccal cavity, and it is probably due to this
attraction of leucocytes that lesions of this region heal so quickly.
The leucocytes are very numerous in the glands of the mouth and the
tonsils always supply large quantities of them.
We must not lose sight of the fact that the epithelial covering of the
bucco-pharyngeal cavity also constitutes an important protective factor.
Just as on the surface of the skin, the corneal cells are in a permanent
state of desquamation, so the cells in the mouth are being constantly
renewed. This desquamation increases especially during mastication, when
enormous numbers of cells are thrown off; after every meal there is a
partial renewal of the surface of the lining of the buccal cavity. Being
covered on their surface, and in their interstices charged with
innumerable micro-organisms, the epithelial cells carry away with them
all this population from the mouth.
The numerous micro-organisms which persist in the mouth, in spite of all
these means for getting rid of them, must also play a certain part in
the defence against infections. It is very probable that many of these
saprophytes impede the multiplication of certain pathogenic bacteria;
but at present it is impossible to define more exactly these phenomena
of microbial competition. It is only because we have analogies in other
regions of the body that we are able to defend this position.
[Sidenote: [438]]
The saliva, incapable of destroying the micro-organisms themselves, is
able to act on their soluble products, as on certain other poisons. In
this relation the action of the saliva on snake venom is most familiar.
Wehrmann[686], who has made researches on this subject in Calmette’s
laboratory at Lille, has shown that the amylase (ptyalin) of human
saliva, mixed with very rapidly fatal doses of venom, quite prevents its
toxic action. Von Behring[687] reminds us on this point that the ancient
Psylli (a race of northern Africa), at the beginning of our era,
employed their saliva as an antidote against snake bites.
[Sidenote: [439]]
Powerless to kill the micro-organisms, the saliva carries them off
mechanically to the exterior or, more frequently, into the stomach. The
acid medium of this great reservoir exerts a very marked effect on these
microscopic organisms. It has long been recognised that the gastric
juice prevents putrefaction and can arrest it even when it has become
very advanced. From this observation an antiseptic action of this juice
was inferred. Bacteriological researches, undertaken to determine the
nature of this action, have demonstrated that several species of
micro-organisms die very shortly after being placed in contact with the
gastric juice _in vitro_. Straus and Wurz[688] found that even anthrax
spores and the tubercle bacillus could be destroyed by gastric juice,
after a prolonged sojourn in a sufficient quantity of this fluid.
Comparative researches, made with aqueous solutions of hydrochloric
acid, have demonstrated that the bactericidal action of the gastric
juice depends solely on the amount of this acid that it contains, that
is to say, the pepsin plays no part in the process. This juice exerts no
true digestive action on the micro-organisms, but it destroys a certain
number of them by its hydrochloric acid. This antiseptic action may also
be inferred from a series of demonstrations on the exaggerated microbial
multiplication in cases where the gastric juice has been poor in
hydrochloric acid. Several observers have confirmed this bactericidal
action of the gastric juice which is exerted specially against certain
species capable of causing grave infective diseases. On the other hand,
certain bacteria and other lower fungi are quite resistant to the
antiseptic action of this fluid; they adapt themselves very readily to
an existence in the stomach. Consequently there exists in this organ,
even in animals such as the dog, whose gastric juice contains most
hydrochloric acid, a special flora, whose most characteristic feature is
the relative insensibility to the acidity of this medium. The
Blastomycetes, along with the yeasts and the Torulae, constitute the
most frequent representatives of this flora; alongside these may be
grouped the Sarcinae and certain acidophile bacilli. Miller[689] has
isolated several of these micro-organisms from the contents of the
stomach, and has observed that, mixed with the food, they resist the
action of the gastric juice, even that of the dog, whose hydrochloric
acid content is greater than in man and many of the other mammals[690].
But these acidophile micro-organisms have no pathogenic power and
consequently are not much to be feared. It is very doubtful whether even
the infective bacteria which are easily killed by the gastric juice _in
vitro_, are often destroyed in the stomach. The typhoid coccobacillus,
which has shown itself to be so sensitive to the destructive action of
the gastric juice of man, of the dog, and of the sheep, is, from the
experiments of Straus and Wurz, quite capable of passing through the
stomach without being affected. Stern[691], as the result of his own
researches, as well as of those of his pupils, came to the conclusion
that this micro-organism is not in the least affected by the gastric
juice of a healthy man, containing the normal amount of hydrochloric
acid. It was only in cases of hypersecretion and of hyperacidity that
the micro-organisms of typhoid fever were destroyed before they reached
the small intestine.
The cholera vibrio also can pass through the stomach and its acid juice.
After Koch’s demonstration of the great susceptibility of this organism
to acids _in vitro_, it was generally concluded that it must perish in
the normal content of the stomach. Many cases have since been recorded
in which the cholera vibrio was found, in times of cholera epidemics, in
the faeces of healthy persons. In order to get into the large intestine
it had to pass through the normal stomach. In experimental cholera in
young suckling rabbits, a large number of vibrios were also found in the
distinctly acid contents of the stomach, and they were seen to pass into
the small intestine without any neutralisation of the acidity of the
stomach taking place. This example proves, once again, that the
phenomena that occur within the living body cannot be identified with
those that go on in the test-tube, _in vitro_.
[Sidenote: [440]]
Whilst the acidity of the gastric juice exerts a certain influence on
micro-organisms, the pepsin which it contains acts unfavourably on their
toxins. There are many poisons which are readily absorbed, without being
modified, by the mucous membrane of the stomach. Even the venom of
snakes can, under certain conditions, produce its toxic effect as it is
absorbed through the stomach. Thus, according to the experiments of
Wehrmann[692], pepsin exerts a very feeble action on this poison. On the
other hand, this diastase has a marked action on certain bacterial
toxins. Gamaleia[693] pointed out that pepsin destroys the diphtheria
toxin. Charrin and Lefèvre[694] have shown that it also weakens other
microbial toxins. According to Nencki and Mmes Sieber and
Schoumow-Simanowski[695], the gastric juice of the dog destroys
relatively small quantities of the diphtheria poison. A gramme of the
juice is capable of rendering innocuous 50 lethal doses of this toxin,
but, in order that this action may be produced, a prolonged contact of
the two substances is required. Since the neutralised gastric juice acts
in the same way, this effect must be attributed not to the acidity of
the gastric juice, but rather to the amount of pepsin it contains. This
diastase acts much more powerfully on the tetanus toxin, 1 gramme of
gastric juice neutralising 10,000 doses lethal for the guinea-pig. On
the other hand, abrin is not modified by the gastric juice according to
the researches of Répin[696], carried out in Roux’s laboratory.
Nevertheless, its action when administered by the stomach is feeble, and
Ehrlich[697] has been enabled to vaccinate small animals against this
vegetable poison by availing himself of his knowledge of this fact.
Répin explains this result as due to the very slight absorption of abrin
by the gastrointestinal mucous membrane. This same factor, Répin thinks,
may contribute also to the failure of various toxins when ingested. This
rule, however, is not an absolute one. Thus, the toxin of the botulinic
bacillus of van Ermengem[698] is not destroyed by the digestive
diastases, and it is certainly absorbed by the mucous membrane of the
alimentary canal. For this reason, when it is introduced by way of the
stomach, it exhibits a very violent toxic activity.
[Sidenote: [441]]
The stomach, though capable, through its acid, of preventing the
multiplication of certain micro-organisms, protects, very feebly, the
rest of the digestive apparatus. As soon as, in the duodenum, the
acidity is weakened or neutralised, the various micro-organisms commence
to multiply and soon develop very abundantly.
In the animal series the intestine proper presents a very great
variability, and even, in closely allied species, exhibits considerable
differences. From the particular point of view which interests us these
differences are very marked. Alongside insects, such as the silkworm,
the larvae of cockchafers and others, whose intestinal canal contains a
very rich bacterial vegetation, we have others which contain exceedingly
few micro-organisms or, indeed, none at all. This last condition is
represented by the caterpillars of small Lepidoptera, and notably by
those of several species of clothes-moths. These differences correspond
to the variety of the juices and digestive ferments met with in these
Invertebrata. As the physiology of digestion in these animals is as yet
little understood, it is at present impossible to define clearly the
conditions which regulate these phenomena. In any case, it is very
probable that the soluble digestive ferments destroy the micro-organisms
and prevent them from growing in the intestinal content. Otherwise it is
difficult to explain why the larvae of clothes-moths, which live in old
dusty textile fabrics, where the germs of bacteria are not wanting,
present a digestive canal from which micro-organisms are entirely
absent. The digestive juices, adapted to digest wool and even wax, are
evidently capable also of digesting the bodies of micro-organisms. In
other insects, which feed on vegetables and on substances less difficult
to digest, micro-organisms develop in the intestinal content, as in many
of the higher animals. Insects often have their intestine lined by a
very delicate chitinous membrane which offers no obstacle to the
absorption of the products of digestion, but prevents the
micro-organisms from reaching the epithelial layer. We have here a
defensive apparatus against microbial invasion, which must be the more
useful because this membrane is thrown off and renewed at each moult,
thus enabling the insect to rid itself at one swoop of a large number of
its microscopic inhabitants.
[Sidenote: [442]]
In the Vertebrata the canal of the pancreas and that of the small
intestine are always populated by a greater or smaller number of
micro-organisms, amongst which bacilli predominate. We know the great
difficulty experienced every time we wish to make experiments on the
pancreatic digestion outside the animal body. The digestive fluid,
alkaline and containing many bacteria, is soon transformed into a
microbial _purée_. We are then obliged to have recourse to antiseptics
to arrest this development and to bring into prominence the digestive
rôle played by the soluble ferments of the pancreas. This well-known
fact may be used as an argument against the existence of any kind of
bactericidal power in the small intestine of higher vertebrates. Even in
those animals which are distinguished by the remarkable poorness of
their intestinal flora, we fail to reveal the presence of bactericidal
substances. The Crustacea, e.g. the crayfish, and certain worms, such as
the _Ascaris_, contain few micro-organisms in their intestine. The
former feed on putrescent substances, the latter inhabit the small
intestine of man and animals, populated by myriads of bacteria. It might
be supposed that, under these conditions, the intestinal content must
contain a mass of micro-organisms or, if that be not the case, that it
must contain some substance which is powerfully bactericidal. In
reality, neither one nor the other of these suppositions receives any
confirmation. The intestines of the two Invertebrata I have named are
very poor in micro-organisms and their contents do not exhibit the
slightest bactericidal power. When a little of their contents is placed
in tubes and kept at a suitable temperature it is not long before it
becomes filled by a great number of bacteria of various kinds.
To explain this poverty of the microbian flora of the intestines in
these examples we must postulate some kind of mechanical purification,
facilitated by the peristaltic movements of the digestive canal.
[Sidenote: [443]]
Even in animals which have an abundance of micro-organisms in the small
intestine, there must be produced some phenomenon which brings about the
disappearance of a certain number of them. In mammals the small
intestine always contains far fewer micro-organisms than does the large
intestine; in birds, the coecum is much richer in bacteria than is the
rest of the digestive canal. Schütz[699] has attempted to demonstrate
the disinfecting power of the small intestine in the dog by feeding it
on substances to which he had added a large number of Gamaleia’s vibrio
(_Vibrio metchnikovi_). After convincing himself that micro-organisms
perish in the digestive canal and are never found in the excrementa,
Schütz introduced into his dogs a cannula, one branch of which passed
into the pylorus, the other into the duodenum. By means of a small
apparatus he could readily interrupt the communication between the
stomach and the intestine. The vibrios, mixed with biscuit, and softened
with water, introduced directly into the duodenum (whilst the stomach
was kept completely isolated), penetrated into the large intestine in
small numbers only. The lower part of the colon, the rectum and the
excrements gave no cultures of vibrios and did not give rise to any
growth except that of the _Bacillus coli_. In this case the disinfection
of the intestine took place without any help from the gastric juice.
Further, when Schütz killed dogs, after giving them food in which
vibrios were mixed, these organisms were found in the intestine only.
The gastric acidity, therefore, is not capable of killing these
organisms, or of preventing them from passing into the small intestine,
in which alone they were killed. It was only with the aid of purgatives,
such as castor-oil or calomel, that Schütz succeeded in preserving the
vibrios in the intestines and in finding them in the dejecta. This
observer did not carry his investigations further and did not make out
the mechanism by which the small intestine destroyed such large numbers
of vibrios. He supposes that alongside a mechanical factor, such as the
very active peristaltic movement, there exist others, perhaps chemical
processes, capable of killing these micro-organisms.
[Sidenote: [444]]
This question of the defensive action in the small intestine is,
consequently, far from being settled. The data collected indicate merely
that the problem is a very complex one. It has been shown, however, that
very virulent bacteria may pass through the digestive canal not only
without injuring the animal but even meeting their own death in this
organ. The anthrax bacillus, so fatal to mice and guinea-pigs, may be
swallowed by these animals without the slightest danger to them. It may
then be found in the small intestine, but not in the large intestine,
this proving that the gastric acidity is incapable of destroying them
outright. To produce generalised anthrax by way of the intestine, it was
necessary that the animals should swallow the spores of anthrax along
with spiny plants, as in the experiments of Pasteur and his
collaborators[700], or along with sand or powdered glass. In these cases
the intestinal lesions served as the port of entry for the bacillus, the
intact mucous membrane of the intestine preventing their penetration.
Mitchell, in an unpublished work, undertaken in my laboratory, succeeded
in giving fatal anthrax to guinea-pigs, even when he fed them with
spores mixed with the “crumb” of bread soaked in milk. During the whole
period of the experiment the animals took no food capable of producing
lesions of the wall of the intestine. But examples of infection under
these conditions are altogether exceptional. In the great majority of
instances the animals were not attacked. The same rule applies also to
many other micro-organisms, which can be ingested with impunity although
their inoculation into the blood and tissues sets up infections which
are inevitably fatal. Many animals may, without running the least risk,
swallow large numbers of bacteria which in man produce grave intestinal
disease. Thus, it has never been possible to produce typhoid fever
regularly and with certainty in any of the species of animals to which
masses of typhoid coccobacilli were given by ingestion. We may recall
the difficulties which so many investigators have met with in inducing
intestinal cholera in laboratory animals, which are so refractory to
Koch’s vibrio. Only very young animals, especially unweaned rabbits, are
capable of contracting fatal intestinal cholera, but these animals may
contract it not only from the true cholera vibrio but also from
Gamaleia’s vibrio. As soon as rabbits begin to feed on vegetables they
acquire an immunity which is insuperable.
It is most assuredly not the digestive ferments of the intestine that
protect the animal against infection through the intestine. The contents
of every part of the small intestine of the Vertebrata permit an
abundant development of all sorts of bacteria, and in solutions of
trypsin not only do pathogenic and resistant micro-organisms grow
luxuriantly, but also saprophytes and the most inoffensive bacteria.
Weigert[701] influenced by this fact even saw in it an objection to the
theory that the destruction of micro-organisms in the animal, notably
that which is effected by the phagocytes, is to be regarded as an act of
digestion. It is a remarkable fact that whilst trypsin is so powerless
against micro-organisms the intracellular ferments, and especially
microcytase, whose kinship with the group of trypsins is undeniable, are
able to bring about their digestion so completely.
[Sidenote: [445]]
It was thought that among the digestive fluids the bile more especially
exerts a definite antiseptic power. It is undeniable that this fluid is
not indifferent for certain bacteria. Talma affirms that it is
bactericidal for several micro-organisms, especially the diphtheria
bacillus. In many of his experiments, however, the bile proved to be
incapable of killing micro-organisms introduced directly into the
gall-bladder. According to the researches of Gilbert and Dominici[702]
the bile does not prevent the abundant development of micro-organisms
capable of setting up diseases of the biliary passages, such as the
_Bacillus coli_. I have tried to prevent the multiplication of the
cholera vibrio by the addition of bile, but my results were entirely
negative. If the bile in an undiluted state has such a slight action
upon so many kinds of bacteria, it is evident that we cannot count upon
its antiseptic action when it passes into the small intestine, where it
is mixed with all sorts of other substances.
[Sidenote: [446]]
The digestive fluids of the small intestine, either those that are
non-bactericidal, the pancreatic juice, or those that are not very
active, the bile, are, nevertheless, capable of producing a marked
influence on certain poisons, and amongst others on certain microbial
toxins. According to the experiments of Nencki and of Mmes Sieber and
Schoumow-Simanowski (_l.c._), trypsin is much more antitoxic against the
diphtheria poison than is pepsin. Thus, the pancreatic juice of both the
rabbit and the guinea-pig destroys this toxin much more actively than
does the gastric juice. The pancreatic juice of the dog exerts a very
powerful action on the same toxin. A gramme of this fluid neutralises
ten thousand lethal doses of the toxin. Wehrmann, also, found that
trypsin inhibits the poisonous action of snake venom. Bile also exerts
an action upon certain poisons. Mixed with diphtheria and tetanus toxins
it prevents their pathogenic effect. It also neutralises the venom of
snakes, as has been observed by Fraser[703], Phisalix[704] and
Calmette[705]. All the venoms, when placed in contact with fresh bile
for 24 hours, induce no injurious effect when the mixture is injected
into normal animals. Bile, heated to 100° C., and even to 120° C., is
still, though more feebly, active. To obtain these results, however, it
is indispensable to prepare, beforehand, a mixture of the two fluids.
When injected separately, whether at the same time as, before, or after,
the venom, the bile does not prevent poisoning. The venom when injected
directly into the gall-bladder of the rabbit sets up fatal intoxication
to the same degree as does the same dose of venom introduced
subcutaneously. Calmette, who made this experiment, explains this
negative result as due to the too rapid absorption of the venom, which
has not had time to be affected by the destructive action of the bile.
A protective action of the bile has been determined with regard to two
viruses, the micro-organisms producing which are not, as yet, known.
Koch[706] succeeded in vaccinating Bovidae with the bile of animals that
had died from rinderpest, and Frantzius[707] prevented animals from
contracting rabies when he inoculated into them rabic virus mixed with
the bile of rabbits that had succumbed to that disease. Vallée[708]
points out, however, that the bile of the normal rabbit produces exactly
the same effect. Here, then, we have to do with a preventive action of
the bile, as such, against the rabic virus. In the present state of our
knowledge it is impossible to say whether this influence of the bile is
directed against the toxin or against the unknown micro-organism.
Analogy would lead us to accept the former of these two suppositions.
The bile, active against certain poisons, does not, however, prevent
poisoning by cholera toxin nor by that of botulism, two most typical
intestinal intoxications.
[Sidenote: [447]]
Since diastases and the digestive juices are incapable of affecting
micro-organisms and since certain of these latter perish in the
intestines we must seek some other cause for their destruction. It is
probable that the vital competition among the micro-organisms, whose
rôle could be foreseen in the buccal cavity, is of still greater
importance in relation to the phenomena of pathogenic action or of the
innocuousness of infective bacteria in the intestinal canal[709]. This
complex and difficult chapter, up to the present, has been studied in a
very imperfect fashion. In our observations on cholera we have remarked
that under certain conditions the cholera vibrios do not develop on
gelatine plates, except in the neighbourhood of certain adjuvant
micro-organisms such as the Torulae and the Sarcinae. Guided by this
fact we have succeeded in producing intestinal cholera in suckling
rabbits, with races of vibrios which, when ingested alone by these
animals, remain innocuous or set up the disease only occasionally. We
have convinced ourselves of the helpful action of certain
representatives of the gastro-intestinal flora upon true cholera[710].
Following on these observations, it was quite natural to suppose that
this flora might also contain micro-organisms capable of hindering the
development and toxic action of the cholera vibrio. We have even
advanced the hypothesis that these “hindering” micro-organisms in the
flora of the digestive canal may explain the immunity of animals, of
many human individuals, and even of the population of unattacked towns,
to intestinal cholera. We should have, then, in the intestinal contents,
inhabited by a number of micro-organisms and deprived of bactericidal
juices, an important factor which in many cases guarantees a refractory
condition. It must be stated, however, that prolonged studies, carried
out with the object of demonstrating in suckling rabbits the precise
part played by these micro-organisms which prevent cholera, have not
given any satisfactory results. This we attribute to our very imperfect
knowledge of the microbial population of the digestive organs.
[Sidenote: [448]]
If the destruction by representatives of the normal intestinal flora of
the micro-organisms which penetrate into the intestines has not as yet
been satisfactorily demonstrated, the power of these latter to destroy
microbial toxins cannot be doubted. We[711] have shown that a great
number of micro-organisms develop well in broth cultures of the tetanus
bacillus which contain a quantity of specific toxin. This toxin is
destroyed under the influence of this microbial vegetation, but the
production of antitoxin never results. Charrin and Mangin[712] have
observed similar facts.
As the destruction of bacterial toxins by micro-organisms takes place
with great constancy and rapidity, it is quite natural to suppose that
the same phenomenon occurs also in the intestinal canal of living
animals in which pathogenic micro-organisms have succeeded in secreting
their toxic products.
The liver having long been recognised as the purifying organ of the
products resulting from digestion, it has been asked if it might not
also play a part in the destruction of microbial poisons. Certain facts
point to its inhibiting influence on the action of nicotine, atropin,
and of certain other alkaloids, and we have other facts which
demonstrate the power of the liver to transform into urea the ammoniacal
substances arising from the activity of the digestive glands. When
Nencki, Pawloff, and their collaborators[713] succeeded in making the
portal vein communicate with the vena cava, and thus were able to
suppress the purifying function of the liver, they found that their dogs
became poisoned in consequence of the accumulation of ammonia in the
animal organism.
Guided by these data as to the protective rôle played by the liver an
attempt was made to apply them to the action of this organ on bacterial
toxins such as the diphtheria poison. The numerous attempts undertaken
in this direction have given negative results: the liver was not found
to be capable of destroying this toxin. Bouchard, Charrin and Ruffer
have studied the action of the liver on the pyocyanic toxin. They
thought that they could make out a certain antitoxic action of this
organ, but, later, Charrin[714] convinced himself that the bacterial
secretions are only “moderately modified” under these conditions, and
that it is more especially the parts soluble in alcohol which undergo
modification in the liver. Now, the true bacterial toxins, as is well
known, are distinguished by their insolubility in alcohol. Moreover in
the numerous experiments made by Roux and Vaillard and so many other
observers on the tetanus and diphtheria toxins there has never been any
evidence of any kind of antitoxic action of the liver.
[Sidenote: [449]]
The digestive organs are furnished throughout with a defensive apparatus
against micro-organisms; this consists in an accumulation of lymphoid
tissue in the form of patches or groups of solitary glands:—the tonsils,
Peyer’s patches, and the solitary glands of the intestine. These organs
produce a large number of phagocytes which are able to come into close
contact with the micro-organisms. Ribbert[715] and Bizzozero[716] have,
independently or almost simultaneously, described glandular masses in
the coecum of the rabbit in which they recognise the presence of many
micro-organisms derived from the intestinal content. They noted that the
greater number of these bacteria were within cells, and regarded this as
an example of phagocytic reaction. Manfredi[717] was able to confirm
this interpretation by the demonstration that the ingested
micro-organisms were dead. Later, Ruffer[718] studied this question in
my laboratory. He observed intestinal phagocytosis in Peyer’s patches in
several species of animals, and showed that the lymphoid tissue
contained large macrophages filled with bacteria and microphages in
process of intracellular digestion. Amongst these latter he recognised
leucocytes, which in turn contained micro-organisms. The accumulation of
phagocytes in the lymphoid organs of the digestive canal constitutes, so
to speak, the last act of a struggle which is spread over a very wide
field.
Some years ago Stöhr demonstrated[719] that the wall of the intestine,
and especially the tonsils and other lymphoid organs, are traversed by
an enormous number of leucocytes which execute a kind of migration
towards the cavities containing micro-organisms. This continual and
normal condition is often termed Stöhr’s phenomenon. It is evident that
we have here a process of phagocytic defence in which the leucocytes,
disseminated through the digestive canal, give chase to the
micro-organisms that are nearest to the living portions of this organ.
When we remove a particle of mucus from the surface of the tonsils of a
person in good health we always find that it contains leucocytes,
especially microphages, filled with micro-organisms of all kinds.
[Sidenote: [450]]
The protection of the digestive mucous membrane is a more complicated
process than that of other mucous membranes, and many of the points
concerned therein are still obscure and need to be elucidated by further
research. It might be thought that the phenomena, associated with the
defence of the mucous membrane of the genital organs, being much more
simple and yet of similar nature, should be much more easily made out,
and that these would throw light on several aspects of the problem of
the general defence of the animal. Obstetricians and gynaecologists have
certainly given much attention to this question as regards the female
genital organs, but we are still far from possessing a satisfactory
knowledge of this subject. There already exists quite a literature on
the question, dominated by the work in two volumes published by Menge
and Krönig[720], but a satisfactory solution has still to be obtained.
At birth the vulva and the vagina are free from micro-organisms, but
they soon become inhabited and a fairly abundant flora, in which may be
recognised certain predominant species, such as the bacillus of
Doederlein, is developed. Micro-organisms, therefore, can exist in the
vulva and the vagina, and yet, when we introduce into these organs
cultures of various bacteria, saprophytic or pathogenic, they soon
disappear. We have the phenomenon to which Menge has given the name of
“autopurification” of the female genital organs. He himself, as well as
his predecessors, Doederlein and Stroganoff, tried to make out the
mechanism of this purification. In the new-born female child the
phenomenon is less complicated than in the adult. According to Menge the
acidity of the vaginal secretion in these infants at first prevents the
development of a large number of bacteria. Associated with this factor
is a marked emigration of leucocytes, which destroy the bacteria by an
act of phagocytosis, or perhaps by their products that have escaped into
the vaginal mucus. As a third element to which much importance is
attributed, we must accept the intervention of acidophile bacteria which
grow well in acid secretions but which hinder the development of other
micro-organisms. Doederlein concludes that it is more especially to the
bacillus which bears his name that the vagina owes its protection
against infective germs. Menge, however, attributes this action to a
whole series of bacteria.
[Sidenote: [451]]
After introducing a quantity of the _Staphylococcus pyogenes_ into the
vagina of new-born females, Menge found that they grew for a certain
length of time. Their presence excited a great accumulation of
leucocytes in the vaginal mucus, this being followed by a very marked
ingestion of the micro-organisms, but it was only from the moment when
the vagina became peopled with the bacteria which constitute its normal
flora that the staphylococci began to disappear. This process of
autopurification only ceased three days after the introduction of these
bacteria. Menge asked himself whether some purely mechanical element did
not contribute to rid the vagina of the micro-organisms which had
entered it. To settle this point he introduced into this cavity grains
of vermilion, and as these latter remained there for a longer period
than did the micro-organisms, he concluded that the vagina was incapable
of purifying itself by mechanical means. We must, however, in these
experiments take into account the fact that the micro-organisms which
Menge introduced into the vagina excited considerable reaction,
accompanied by a marked leucocytosis. Under these conditions there
should be produced a greater quantity of the mucous secretions which
could much more readily carry off with them the micro-organisms that had
come into the vagina than the smaller quantity could deal with the
vermilion. It is very probable, therefore, that, just as in the case of
the other mucous membranes, that of the female genital organs is capable
of mechanically expelling fine particles, and especially
micro-organisms.
[Sidenote: [452]]
With the object of throwing further light on the problem of the
autopurification of the vagina, Cahanescu[721], working in my
laboratory, undertook experiments on the females of several species of
mammals. The mare, as producing the greatest amount of vaginal mucus,
was selected by this observer as suitable for the settling of this
question of the bactericidal power of this secretion. The result was
absolutely negative, even when such an inoffensive saprophyte as the
_Coccobacillus prodigiosus_ was used. The autopurification of the vagina
of the female dog, rabbit and guinea-pig, was found to be neither very
marked nor very active. The micro-organisms introduced into the vagina
usually remained there for some time. Of all the factors in the
microbial destruction which Cahanescu was able to make out that of the
accumulation of leucocytes was the most active. Sometimes he observed an
extraordinary amount of phagocytosis, whilst in other experiments this
was slight or even absent. Many of the leucocytes being killed in the
vaginal mucus, it is possible that in some cases a certain bactericidal
action of the cytases which have escaped from these dead leucocytes is
set up. It is true that the vaginal secretion of the mare did not
exhibit this antimicrobial property _in vitro_, but in the other animals
experimented upon it was found impossible to make similar experiments,
the quantity of mucus being too small. In woman the acidity of the
surface of the mucous membrane of the vulva and of the vagina, so
frequently present, may play a certain part in the protective action
against those bacteria which cannot tolerate the acid medium, but the
animals studied by Cahanescu, even female dogs, do not possess this
advantage, their mucous membranes usually having an alkaline reaction.
In the urinary channels this acid reaction also plays a part, as one of
the defensive agencies against the penetration of bacteria. This may
also be effective in man and other animals that have an acid urine. In
many other animals, however, where the urine is alkaline micro-organisms
do not pass into the deeper parts of the urinary organ under normal
conditions. Here it is to the outflow of the urine that the bladder owes
its immunity against pathogenic micro-organisms and saprophytes. When we
connect two flasks containing sterilised broth in such a way that the
fluid flows slowly from one of them into the other, the former never
becomes contaminated by the micro-organisms which are present in the
latter, in which latter the broth is soon transformed into a _purée_ of
bacteria, whilst in the former the broth remains unaffected and aseptic.
This purely mechanical factor has been well brought out by
Preobrajensky[722] as the result of work carried out in Duclaux’s
laboratory. The sterility of the normal urinary bladder must be
attributed to a similar cause. When the urine begins to stagnate in the
bladder it very readily becomes contaminated.
[Sidenote: [453]]
Since the acceptance of the view that the suprarenal capsules serve to
neutralise the effect of certain toxic substances elaborated in the
body, there has been an inclination to assume that these organs might
also fulfil an antitoxic rôle against microbial poisons. The hypothesis
was advanced that this function might be shared by the suprarenal
capsules with the thyroid gland and with certain other problematical
organs. We have already stated (Chapter V) that the suprarenal capsules,
in some experiments where spermotoxin was injected into rabbits,
exhibited a certain antispermotoxic power. But, up to the present, no
exact fact has been observed that would favour the idea of an antitoxic
action of the above-mentioned organs against bacterial toxins. Roux and
Vaillard[723], in their great work on tetanus, have made experiments in
this direction, but their results did not justify them in giving a
positive answer to the question.
Nature does not make use of antiseptics to protect the skin and the
mucous membrane. The fluids which moisten the surface of the mouth and
of other mucous membranes are not microbicidal, or are so to a very
slight degree, and then rather of an exceptional nature. Nature rids the
mucous membranes and the skin of a large number of micro-organisms,
eliminating them by epithelial desquamation, and expelling them along
with fluid secretions and excretions. Nature, like the doctors of the
present day who replace antisepsis of the mouth, intestine, and other
organs by washing with pure physiological saline solution, has chosen
this mechanical method. She avails herself of the help of inoffensive
micro-organisms to prevent pathogenic micro-organisms from taking up
their abode in these positions, and she is constantly sending to all the
mucous membranes and the skin an army of mobile phagocytes which explore
the ground and rid it of micro-organisms. When these begin to get more
numerous the phagocytic reaction becomes more intense. A struggle takes
place between the two living elements—phagocytes and micro-organisms. In
those cases where the animal remains unaffected the former gain the
upper hand.
CHAPTER XIV
IMMUNITY ACQUIRED BY NATURAL MEANS
Immunity acquired after recovery from infective diseases.—Immunity
acquired in malaria.—Humoral properties of convalescents from
typhoid fever.—Preventive power of the blood of persons who have
recovered from Asiatic cholera.—Antitoxic power of the blood of
persons who have recovered from diphtheria.
Immunity acquired by heredity.—Absence of hereditary immunity properly
so-called.—Immunity conferred by the maternal blood and by the
yolk.
Immunity conferred by the milk of the mother.
[Sidenote: [454]]
It has long been known that an attack of one of many of the infective
diseases brings about a refractory condition of the organism against
that disease, a condition which persists for many years, and may even
endure for life. Even before the microbiological era of medical science
had arrived it had been fully established that a person who had
recovered from small-pox might come in contact with and nurse small-pox
patients without risk of contracting a second attack of the disease. The
same thing has been observed purely empirically in several other
infective diseases, such as whooping-cough, typhoid fever, scarlatina,
mumps, etc. On the other hand it has been shown that certain infective
diseases, such as fibrinous pneumonia, erysipelas, recurrent fever, and
influenza, do not leave behind them the slightest trace of an immunity.
It has often been observed, indeed, that after a first attack of any of
these diseases there is a marked susceptibility to a second attack.
Between these two extremes come the infections which are followed merely
by a refractory condition of shorter duration than that which follows
the diseases of the first group. The first of this intermediate group is
measles, which gives rise to a relatively long immunity, then come in
order bubonic plague, anthrax, cholera, etc.
[Sidenote: [455]]
It should be stated that the first attack of any of the infective
diseases causes modifications more or less permanent in the organism,
and is always followed by immunity. Even in erysipelas, a disease where
the relapses are so frequent that certain individuals are, so to speak,
predestined to re-acquire it at short intervals, an immunity is
produced, but a very transient one. Since the discovery of the
streptococcus of erysipelas by Fehleisen[724], this observer, and
several other investigators, have inoculated it into persons affected
with malignant tumours. In the course of a series of experimental cases
of treatment it was noted on several occasions that after a first
inoculation, followed by typical erysipelas, a period of immunity was
developed, during which the introduction of the streptococcus produced
no result. It has also been observed that recurrent fever, when
inoculated into monkeys, sets up a very transient but real refractory
condition. In fibrinous pneumonia, also, the relapses are generally
separated by periods of immunity, of longer or shorter duration.
[Sidenote: [456]]
It was generally supposed that an attack of malarial fever was not only
not followed by any immunity, but that a first attack predisposed the
organism to a second. Facts of this kind have often been observed and
cannot now be questioned. Nevertheless, an acquired immunity against
malaria is developed under certain conditions. During his travels in New
Guinea, Koch[725] found that in certain regions whilst most children
below ten years of age are attacked by malaria, and Laveran’s parasite
can be demonstrated in their blood, older children and adults are
completely immune from this infection. Koch is convinced that in this
instance we have an example of immunity acquired by natural means as the
result of an attack of malaria at the younger age. This great observer
bases his conclusion on the fact that unattacked adults, coming from
districts where the children contain the parasite, do not contract
malaria when they migrate to other malarial regions, whilst natives
coming into these same regions from districts where malaria does not
exist are soon attacked. Max Glogner[726] has attempted to explain these
facts established by Koch, on the assumption that the unaffected adults
simply benefit by their natural immunity and that we have here a kind of
selection: the adults who are susceptible to malaria die as the result
of this disease, whilst others, naturally refractory, resist and show
themselves incapable of contracting the disease even in other malarial
regions. Glogner in support of his view cites the case of the children
of the orphanage at Samarang (Java), who for many years are subject to
relapses and to malarial re-infections and are incapable of acquiring
the slightest immunity. According to Koch, Glogner’s example cannot be
compared with that of the children of New Guinea. In the former case,
the natural course of the disease is interrupted by treatment with
quinine, which must prevent immunity being set up; whilst, in the
latter, the children are abandoned to their fate, and, receiving no
treatment, slowly acquire a true immunity. It is evident that this
acquired immunity in malaria is a complex phenomenon on which fresh
researches must be made; but it cannot be questioned that, under certain
conditions, it comes under the general rule and can be naturally
acquired.
This general rule is that, in infective diseases, immunity is usually
developed after a first attack. The acquired refractory condition is of
very long duration in certain cases, but very transitory in others. To
the discovery of the vaccination by attenuated micro-organisms, made by
Pasteur and his collaborators, the objection was often made that many
diseases, such as anthrax, might relapse. This is undoubtedly the case;
the anthrax bacillus may attack the same individual several times;
nevertheless the acquired immunity against this disease is very real,
though the refractory condition lasts for one or a few years only,
instead of persisting for a very much longer period, as in the case of
typhoid fever, mumps, and small-pox. Bearing in mind the possibility of
a relapse in the case of these infective maladies, attempts at
artificial vaccination should never be relinquished.
[Sidenote: [457]]
Among the examples of immunity acquired by natural means must be cited
that of syphilis, a very special case. It has long been known and
demonstrated by numerous experiments on man, that individuals who have
presented the primary symptoms of syphilis contract a marked immunity
against a new infection. The syphilitic chancre does not relapse, and
yet this very manifest and persistent immunity does not prevent the
individual, immune against re-infection, from continuing to be ill and
of being the field for the later syphilitic phenomena. This special
refractory condition has done great service in establishing the etiology
of certain diseases which we were justified in suspecting to be of
syphilitic origin. Many clinical observers have accepted this origin for
general progressive paralysis. Others deny any causal relation between
the two diseases. Krafft-Ebing[727] has resolved this question by the
application of the law of acquired syphilitic immunity. The inoculation
of the syphilitic virus into ten persons attacked by general paralysis
was followed by no chancre at the seat of inoculation and by no other
primary or secondary symptom of syphilis. The patients with general
paralysis present a real immunity against these symptoms; consequently
general paralysis is a tardy manifestation of syphilis.
The acquired immunity against re-inoculation by the syphilitic virus is
established immediately after the end of the period of incubation of the
first infection, and is of lifelong duration[728]. Besides this very
special and, so to speak, partial immunity, there exists in syphilis a
second form of acquired immunity which is of a more general nature.
According to the law known as the law of Baumès-Colles, the mother who
suckles her infant, hereditarily infected with syphilis through the
father only, enjoys a real anti-syphilitic immunity.
[Sidenote: [458]]
In tuberculosis the few facts of acquired immunity that have been
observed present a certain analogy with those bearing on immunity in
syphilis. A large number of well-observed facts demonstrate that a
person who has suffered from scrofula or has manifest symptoms of
tuberculosis properly so called, cannot count upon an immunity against
pulmonary phthisis. It might, then, be supposed that no acquired
refractory condition exists in connection with this disease. Koch[729]
has clearly demonstrated, however, that tuberculous guinea-pigs, into
which the bacilli of tuberculosis have been introduced subcutaneously,
react against these bacilli in a very special manner. The presence of
these micro-organisms immediately sets up an active inflammatory process
at the point of inoculation; this brings about the expulsion of the
bacilli with the exudation; a voluminous slough is developed, which,
when shed, carries with it a large number of bacilli, a process followed
neither by the formation of a permanent ulcer nor by hypertrophy of the
neighbouring glands. As in syphilis, the animal has acquired immunity
against re-infection by the tuberculous virus, which, however, in no way
prevents the first inoculation from becoming generalised and setting up
a fatal tuberculosis of almost all the organs. Koch’s observations,
which have served as the basis of his researches on tuberculin, have
been confirmed by other investigators. The reaction of the tuberculous
organism against re-infection has received the name of “Koch’s
phenomenon.”
Clinical medicine has afforded many data of the highest importance
bearing on the establishment of an acquired immunity in many infective
diseases; but a scientific study of the mechanism of this immunity could
only be founded on the result of microbiological researches obtained
during the recent period of scientific activity. The general conclusion
to be drawn from these researches is that the immunity, acquired by
natural means, is very analogous to that which is obtained artificially
by vaccination by the various methods already mentioned. The phenomena
observed in animals inoculated with the various known vaccines present a
great resemblance to those that obtain during recovery from a disease
contracted under natural conditions. To support this thesis it would be
necessary for us to survey the mechanism of healing, which would carry
us too far afield, the subject being far too vast to be summarised here.
We must, then, content ourselves with a few remarks inserted for the
instruction of the reader on this subject.
Those diseases against which no remedy exists are most suitable for
furnishing us with important information on immunity acquired by natural
means. We have already seen in the case of malaria to what point
therapeutic treatment can modify the natural course of the phenomena.
For this reason it will be useful to consider first the immunity
acquired as the result of a first attack of typhoid fever. The immunity
which develops in this example is both marked and persistent; the
therapeutic intervention which might disturb the natural phenomena is
_nil_.
[Sidenote: [459]]
As yet we do not know the mechanism of healing in typhoid fever. This
disease affecting the human species exclusively (the experimental
peritonitis of animals, set up by the typhoid coccobacillus, is
distinguished by very marked differences), it is very difficult to find
a means of studying it at all satisfactorily during the phase of
recovery. Even in default of this knowledge, however, it is possible to
gather some idea as to the changes which the blood plasma undergoes, not
only during the course of an attack of typhoid fever, but also during
and after convalescence.
Some time ago Chantemesse and Widal[730] observed that the blood serum
of persons attacked by typhoid fever acquires the property of inhibiting
the experimental peritonitis set up by the typhoid coccobacillus in
laboratory animals. The blood of the patient becomes “preventive.”
Against this conclusion the objection has been raised that in the large
doses of serum employed by the above observers a protective effect can
be obtained, even when using the blood of normal men, i.e. neither
suffering from typhoid fever, nor having recovered from this disease.
Later researches, however, have confirmed the observation made by
Chantemesse and Widal. It is no doubt true that the injection of half a
cubic centimetre of normal human serum into the peritoneal cavity of an
untreated guinea-pig is often sufficient to render it refractory to a
dose of typhoid coccobacilli fatal to the control animal. We have an
ordinary protective action, such as described in Chapter X. The blood of
typhoid patients is, however, capable of protecting normal animals, in
doses which exhibit not the slightest protective action if normal blood
be used.
[Sidenote: [460]]
The protective power of the blood serum of convalescents has been
studied very carefully by Pfeiffer and Kolle[731]. In certain
individuals very small quantities (0·001 c.c.) of this fluid were quite
sufficient to confer on guinea-pigs an immunity against fatal typhoid
peritonitis. This power was at its maximum only during the first weeks
of convalescence. In one case, in which these observers were able to
study the properties of the blood on two separate occasions, they found
that two months after the first examination there had been a marked
falling off in the acquired protective power. In a second case, where
the blood was examined a year after the patient had recovered from a
grave attack of typhoid fever, they found only feeble indications of
this specific protective property. “Everything seems to indicate,”
conclude Pfeiffer and Kolle, “that the protective typhoid substances
were rapidly eliminated by the blood stream. If further researches
should confirm these results, as yet few in number, we might conclude
therefrom that the immunity which, after an attack of typhoid fever,
persists for years, frequently even for the rest of life, would be
independent of the amount of ready-prepared protective substances in the
blood” (_l.c._ p. 218). The facts upon which this conclusion is based
confirm the general thesis that even acquired immunity is in no way the
function of any humoral property.
We know that in the protective serums there is constantly found the
specific fixative (the sensibilising substance of Bordet, the
intermediary body or amboceptor of Ehrlich). It was, therefore, quite
natural that this substance should be sought in the blood of patients
who were suffering, or had recovered, from typhoid fever. Bordet and
Gengou[732] easily demonstrated, by the method described in Chapter ix,
the existence of typhofixative in the blood serum of two individuals
convalescing from this disease.
Widal and Le Sourd[733] extended this discovery to the blood taken
during the course of the disease from typhoid fever patients. The ten
cases studied by them all gave a positive result, whilst all the samples
of blood from persons suffering from various other diseases possessed no
typhofixative. As yet we do not know whether this substance persists for
any length of time after recovery or not. In this respect we have much
more information concerning another humoral property of typhoid
patients,—specific agglutination. Guided by the fact that, even during
the course of the disease, the blood of persons suffering from typhoid
fever acquires protective properties, Widal sought to find out whether
the agglutinative power of the fluids of the body appears equally early.
We know that his studies gave a positive answer, and that the blood of
typhoid patients may have agglutinative properties from the first day of
the disease. This fact was made use of by Widal to establish the serum
diagnosis of typhoid fever, a method now generally used in clinical
medicine. The question which most interests us at this moment is whether
this acquired agglutinative property persists for any length of time
after the recovery of the patient, and whether it can be employed as the
measure of immunity obtained.
[Sidenote: [461]]
In certain cases the serum was found to be fairly strongly agglutinative
for a considerable period after recovery had taken place. But these
cases are rare, and the agglutinative power, like the protective power
of the blood, usually begins to decrease very soon after recovery.
Bensaude[734] observed that the former disappeared between the 10th and
95th day of apyrexia. Widal and Sicard[735] have noted in certain of
their cases the complete disappearance of the agglutinative power of the
blood, which took place in one case on the 18th, in another on the 24th
day of defervescence. In many convalescents, fifteen to thirty days
after the commencement of apyrexia, the agglutinative power begins to be
attenuated.
Previous to these researches on the protective and agglutinative
properties, Stern[736] had already put the question: May we not draw
some general conclusion as to the bactericidal power of the blood serum
of convalescents from typhoid fever? He found that the typhoid
coccobacilli did not thrive so well in the blood serum of persons in
good health as in that of convalescents, in which they give abundant
cultures. Widal and Sicard (_l.c._) subjected this question to a fresh
examination, and showed that in this respect there exists no constant or
marked difference. Thus, in ten samples of serums from individuals who
had never been under the influence of the typhoid infection, four were
found to be bactericidal for the typhoid coccobacillus. In twelve other
samples, drawn from convalescents from typhoid fever, five exhibited a
bactericidal power against the same micro-organism.
All the researches made on acquired immunity after recovery from typhoid
fever demonstrate clearly that, in this case, it is impossible to
attribute it to humoral modifications, which are usually more transitory
than the immunity.
[Sidenote: [462]]
The immunity which follows an attack of cholera is far from being either
as powerful or as prolonged as that which follows typhoid fever. Certain
individuals have contracted cholera twice during the same epidemic, but
such cases are exceptional, whilst acquired immunity, temporary at
least, may be looked upon as the general rule. Many points in the
pathogenesis of intestinal cholera are still obscure; nevertheless we
are justified in affirming that this disease is a real intoxication by
the cholera poison manufactured, in the small intestine of man, by
Koch’s vibrios. The action of the vibrionic toxin is sufficient to set
up a grave and often fatal attack of cholera; but in the majority of
cases a secondary infection by the vibrio which penetrates into the
intestinal wall, denuded of its epithelial layer, is associated with the
action of the poison. Sometimes this micro-organism becomes generalised
in the animal, and is found in the blood and in many of the organs.
The facts I have here briefly summarised may be utilised to explain
certain characters which are found in the fluids of individuals who have
recovered from an attack of cholera. Soon after the discovery of the
tetanus and diphtheria antitoxins, and almost immediately after the
demonstration of the protective power of the blood, taking advantage of
the epidemic of Asiatic cholera, which developed in Europe from 1892,
the new data began to be applied to that disease. We have already
referred in a preceding chapter to the fact that the blood serum or the
blood of those in good health and who have never had Asiatic cholera, is
capable of preventing cholera peritonitis in the guinea-pig inoculated
with Koch’s vibrios. In order to obtain this protective action, the
injection of a pretty large dose, about half a c.c., is necessary. This
property is in no sense specific, for the same blood, injected in the
same doses into guinea-pigs, will protect them not only against this
vibrio, but also, and indifferently, against many other bacteria, such
as the typhoid coccobacillus, the _Bacillus coli_, etc.
The blood or blood serum, coming from those who have recovered from
Asiatic cholera, may, on the other hand, acquire a specific protective
power. It will, indeed, prevent infections by other micro-organisms;
but, to obtain this effect, it is necessary to inject the same
quantities of it as of the blood coming from normal individuals. On the
other hand, when we wish to prevent cholera peritonitis in the
guinea-pig, we need introduce minute doses only of the serum of persons
who have recovered from an attack of cholera. Lazarus[737] was the first
to make this interesting observation. In three cases of cholera studied
by him, the serum withdrawn some time after recovery exhibited an
extraordinary protective power: a decimilligramme of the blood serum of
these patients was quite sufficient to prevent the death of a guinea-pig
inoculated intraperitoneally with the cholera vibrio. Soon after, G.
Klemperer[738] made a similar observation in two other cases that had
recovered, but the blood, in his convalescents, was much less active
than was that in the cases cited by Lazarus.
[Sidenote: [463]]
Issaeff[739], working in Koch’s Institute in Berlin, examined the blood
of several persons who had recovered from cholera, and found that the
serum had always acquired a specific protective property; this property
never developed before the third week from the commencement of the
disease, and had completely disappeared as early as three months after
this period. Several examples studied by A. Wassermann[740] and
Sobernheim[741] fully corroborate this conclusion. Our own
researches[742] on twenty-four cases indicate a very great variability
in the protective power of the blood of persons who had recovered from
cholera. We were able to demonstrate its presence in rather more than 58
per cent. of these cases. Sometimes this power was almost as marked as
in the example given by Lazarus, whilst in others it was very feeble,
often even _nil_. We were unable to demonstrate any relation between the
gravity of the disease and the strength of the protective power of the
blood. Thus, in a moderately severe case of cholera, a very small
quantity of serum (0·001 c.c.) was sufficient to protect the guinea-pig
from fatal cholera peritonitis, whilst in another, an extraordinarily
grave case, even a quantity of 2 c.c. was incapable of producing the
same effect. In these two cases the blood had been withdrawn at the
corresponding period after the commencement of the disease
(seventy-third and seventy-fifth days). Sobernheim (_l.c._) found the
protective power of the serum most marked in a person who had cholera
vibrios in his normal dejecta, but who was always in good health and was
only examined because he was living with cholera patients.
[Sidenote: [464]]
All these observations point to the fact that neither recovery from, nor
immunity against, cholera can be regarded as a consequence of the
protective power of the blood. This power does not manifest itself until
some time after complete recovery has taken place, and then disappears
too soon, that is to say at a moment when acquired immunity ought still
to be maintained. On the other hand, the irregularity in the protective
power of the blood indicates that this humoral property is something
secondary. Since Asiatic cholera is an intoxication by the cholera
toxin, we can readily understand that the protective power, resulting
from the invasion of the living parts of the organism by the vibrios,
should here play a part of little importance. We know already that this
power is due to the presence of substances manufactured by phagocytic
elements, placed in contact with vibrios. In the experimental infection
of rabbits by the cholera vibrio, as demonstrated by Pfeiffer and Marx,
the cells of the spleen, of the lymphatic glands, and of the
bone-marrow, produce the protective substances. We have no idea of the
source of these substances in Asiatic cholera in man.
Asiatic cholera, being an example of intoxication of intestinal origin,
it might be supposed that the antitoxic power of the body fluids should
be specially manifested after recovery has taken place. On this point
our knowledge is as yet very imperfect, because it was not until after
the end of the last epidemic of cholera that we learnt how to prepare
the toxin. In a case of cholera (M.S.), contracted in our laboratory,
the blood serum was examined to ascertain its protective power and its
antitoxic activity. This fluid, withdrawn more than three weeks after
the commencement of the disease, was found to be protective only in a
large dose (0·5 c.c.), in which dose even the serum of normal persons is
capable of producing the same effect. It was found in an experiment with
suckling rabbits that the antitoxic property of the blood serum of M.S.
was _nil_. It did not prevent these rabbits from dying of intestinal
cholera after the absorption of the vibrios, in spite of a dose of three
c.c. of serum injected some time previously.
This experiment, unique up to the present, is, of course, insufficient
to enable us to affirm that recovery from Asiatic cholera may take place
without the development of antitoxic power in the body fluids. That this
is so is, nevertheless, probable. In other intoxications of microbial
origin, certain data have been collected which point to the same
conclusion. Thus, Knorr[743] observed that the blood of guinea-pigs
which had recovered from tetanus did not exhibit any antitetanic power.
Vincenzi[744] made a similar observation in a man who had recovered from
tetanus.
[Sidenote: [465]]
[Sidenote: [466]]
We are much better informed as to the antitoxic property of the blood of
persons who have recovered from diphtheria. Klemensiewicz and
Escherich[745] have studied two cases of diphtheria in which the
defibrinated blood withdrawn some time after recovery was found to be
protective for the guinea-pig against a lethal dose of diphtheria
bacilli. This fact has been confirmed by several other observers,
especially by Abel[746] and Orlowski[747], the latter of whom made his
researches under the direction of Escherich. In these experiments the
antitoxic power of the blood was demonstrated against diphtheria toxin
employed without bacilli. According to the data collected by the above
authors the antitoxic property of the body fluids was not exhibited
during the early days of convalescence, but was well marked in the
second week after recovery. This power was maintained for a short time
only, disappearing in a few months. Amongst the observations collected
on this subject the most interesting is that made by Escherich. In an
infant examined for the first time whilst it was still in good health,
the blood was incapable of protecting the guinea-pig. Some time after
this negative result had been obtained the child was attacked by a mild
diphtheria, which gave rise to the development of antitoxin, for its
blood when again examined exhibited a very high antitoxic power. This
proves most clearly that even a slight attack of diphtheria is capable
of producing antitoxic power in the body fluids. This observation may be
utilised to explain the frequency of the presence of this property in
the blood of persons in good health who, according to their own
statements, have never had diphtheria. This fact has been established by
the researches of A. Wassermann[748], Abel (_l.c._), and Orlowski.
According to the last observer, the blood in one-half the children in
the hospital at Gratz who had not been attacked with diphtheria was
antitoxic against the diphtheria toxin, sometimes even to a higher
degree than was that of the children who had recovered from this
disease. Wassermann has demonstrated that in adults this
antidiphtheritic power of the blood is even more frequent than in
children, and that it increases with age. Nevertheless, these persons
affirm that they have never had an attack of the disease. To explain
this very paradoxical fact, Wassermann asked himself whether the
individuals whose blood was antidiphtheritic did not owe this property
to the action of pseudo-diphtheria bacilli. Although incapable of
causing the disease, these bacilli might, perhaps, exert a certain
immunising influence and give rise to the production of an antitoxin
active against true diphtheria toxin. Researches, directed to the
clearing up of this point, have not led Wassermann to reaffirm his
suggestion. It must be observed that the varieties of these
pseudo-diphtheria bacilli are numerous, and that some of them, perhaps,
may be capable of fulfilling the function suggested by Wassermann. On
the other hand, it is proved that the specific and virulent diphtheria
bacillus may be found in the throat of persons in good health either
without inducing diphtheria, or only giving rise to a very slight form
of disease of very short duration. We must bear in mind that in persons
who have not had typhoid fever, but who live among patients suffering
from this disease, the blood may be very agglutinative (Foerster); that
in others, unattacked by cholera but containing Koch’s vibrios in the
intestine, the blood may acquire a high specific protective power
(Sobernheim). It is probable that the same rule applies also to the case
of diphtheria, and that, consequently, the blood of persons in good
health, but containing the diphtheria bacillus in their bodies, may
acquire antitoxic power.
This humoral power, once developed, may even be transmitted from the
mother to the foetus and so become hereditary. Abel (_l.c._) examined
the blood serum of four adult women, taking it from the placenta after
parturition. Each time it was found to be distinctly antitoxic against
the diphtheria toxin. Later, Fischl and Wunschheim[749], working in
Chiari’s laboratory in Prague, studied the blood of new-born children
from the same point of view. They showed that in the majority of cases
this fluid prevents the production of a fatal disease in the guinea-pig,
in spite of the injection of several lethal doses of very virulent
diphtheria cultures. The blood of new-born children is equally capable
of neutralising the diphtheria toxin, that is to say, of protecting
animals against poisoning by this toxin. The above observers do not
doubt that this antitoxic power comes directly from the maternal blood
through the placental circulation. This fact appears to throw some light
on the phenomena of immunity acquired by heredity.
[Sidenote: [467]]
Until quite recently we have had very vague notions as to the
possibility of transmitting to descendants the immunity contracted as
the result of recovery from an infective disease or after vaccination.
It has long been known that natural immunity may be transmitted
hereditarily. Certain families or certain races are characterised by a
special insusceptibility to certain infective diseases. It must even be
admitted that this innate immunity has been transmitted from generation
to generation. It is quite otherwise with acquired immunity. We know
that as a rule the characters acquired during life are not transmitted
to descendants; it is only in special cases, in the very lowest
organisms, such as the bacteria and their allies, that we may observe
the conservation of certain acquired characters through an infinity of
generations. The attenuation of bacteria or the absence of the formation
of spores, once acquired under special conditions, may thus be
transmitted to their descendants who develop and live under normal
conditions.
[Sidenote: [468]]
[Sidenote: [469]]
After the discovery of anthrax vaccine by Pasteur, Chamberland and Roux,
and an attempt had been made to vaccinate large flocks of sheep, it was
an easy matter to investigate whether immunity acquired by the parents
was transmissible to their offspring. Several observers, amongst whom I
may specially cite Chauveau[750], Rossignol and Cienkowski, got together
a certain number of data bearing on this question. These data showed
distinctly that, in certain cases, the lambs born from vaccinated sheep
presented, from birth, an undoubted resistance to the anthrax bacillus.
This fact, however, was neither constant enough nor sufficiently marked
to enable us to count upon the young animals being in a refractory
condition, and thus avoid having to submit them to vaccination by the
two Pasteur vaccines. This necessity threw into the background the
researches on the hereditary transmission of acquired immunity. It was
only much later that this question was again taken up on a purely
theoretical basis. Ehrlich[751], to whom science is indebted for so many
works of the highest importance upon immunity, again took the initiative
in exact and minute researches upon the heredity of immunity, acquired
as the result of vaccination against toxins. In this relation he studied
the immunity of the descendants of animals immunised against
phanerogamic toxins, such as ricin, abrin and robin, and later, in
collaboration with Hübener[752], that of the offspring of animals
vaccinated against tetanus toxin. Ehrlich proved very clearly that the
antitoxic immunity acquired by the father is never transmitted to his
progeny. This fact alone is quite sufficient to show that it is not a
true immunity that is met with in young animals born of mothers who have
acquired a refractory condition; true immunity is transmitted by the
sexual elements, the spermatozoon and the ovum. Certain observers,
Tizzoni[753] and his collaborators Cattani and Centanni, thought they
could overthrow the rule established by Ehrlich. They believed that the
male rabbit, vaccinated against rabies, was capable of transmitting its
immunity to its progeny. Charrin and Gley[754] expressed the same
opinion as regards animals of the male sex vaccinated against
experimental pyocyanic disease. But the very precise experiments of
Wernicke[755], Vaillard[756] and Remlinger[757] upon a whole series of
infective diseases and intoxications, such as diphtheria, cholera
peritonitis, anthrax, experimental typhoid septicaemia, etc., showed
conclusively the correctness of Ehrlich’s results. Well-vaccinated
males, even when hypervaccinated, never transmit their immunity to their
descendants. This acquired property, like so many others, is not
hereditary in the strict sense of the word. The females, on the other
hand, with rare exceptions, transmit their acquired immunity to their
young, but this transmission can in no way be attributed to the ovum; it
is here, then, no longer a question of hereditary immunity properly so
called. According to Ehrlich the female furnishes in her blood plasma
the antitoxin which passes into the circulation of the foetus. In all
respects this is allied to the so-called passive immunity (or antitoxic
immunity of von Behring). It is due entirely to the direct introduction
of antitoxin, manufactured by the cells of the maternal organism, into
the body of the progeny. The living elements of the foetus play no part
in it, and it is for this reason that the antitoxins and immunity in the
new-born animal disappear so very rapidly,—within a few weeks after
birth. Wernicke accepts the views of Ehrlich in their entirety. He found
that the immunity of female guinea-pigs was transmitted to the new-born
animal; but this hereditary transmission was exhausted in a single
generation; it was not found in the second generation. Wernicke was able
to demonstrate that the refractory condition in guinea-pigs, born of
mothers vaccinated against diphtheria, persisted for three months.
Vaillard found that it was retained in certain cases for an even longer
period,—up to the fifth month. On one occasion he even observed the
transmission of the immunity to a second generation. A female
guinea-pig, born of a mother immunised against tetanus, gave birth to a
young one which, when tested a month after birth with a ten times lethal
dose of the toxin, contracted merely a slight tetanus.
From this fact, as well as from the fact that the immunity of the young
ones born of vaccinated mothers persists longer than does that conferred
by the injection of antitoxic serum, Vaillard concludes that there
exists a kind of hereditary immunity which is “fixed” by the cells. He
thinks that not only the antitoxins and other antibodies but also
certain living elements, especially the leucocytes, are able to pass
from the maternal blood into that of the foetus and to transmit to it
the properties acquired by the mother. At this point we may recall the
facts demonstrated by von Behring and Ransom that antitoxin persists
much longer in the blood of an animal when it is introduced with the
serum of the same species. (We have described these observations in
Chapter XII.) Now, since in hereditary transmission the antitoxin passes
over with the blood plasma of the same species, whilst in the
experiments on antitoxic immunity it is generally injected with the
serum of a different species, it is easy to understand that the former
should persist for a longer period than the latter. It is, therefore,
very probable that this immunity of the offspring from vaccinated
mothers is not in any way a case of true hereditary immunity, but is due
simply, as maintained by Ehrlich, to the passage of ready prepared
antibodies from the mother to the foetus. In the immunities against
diphtheria and tetanus we have the direct passage of antitoxins; in
transmitted immunity against infection by the vibrios of Koch and
Gamaleia, so carefully studied by Vaillard, we have, very probably, the
passage of corresponding fixatives from the mother to the foetus.
[Sidenote: [470]]
Dzierzgowsky[758] in a recent study on hereditary immunity denies the
passage of antibodies and toxins through the placenta. He thinks that
the foetus does not acquire its immunity through the blood of the
mother, but at a very much earlier period. The ovum contained in the
Graafian follicle would, according to this observer, come in contact
with a fluid very rich in antitoxin, whence it might imbibe the
necessary amount of this antibody to ensure the immunity of the new-born
animal. Dzierzgowsky bases this opinion on experiments in which
antidiphtheria serum injected into pregnant goats and dogs did not
produce any antitoxic power in the blood of the foetus. But in the
experiments on these animals the injections consisted of the serum of
the horse—a different species. This must modify, profoundly, the
conditions of the passage of the antitoxin through the placenta.
Dzierzgowsky made a single experiment upon a mare, immunised with
diphtheria toxin, and its foal. Whilst the serum of the former was
markedly antitoxic, that of the foal did not possess this property in
the slightest degree. Hence the conclusion that the antitoxin of the
mother had not passed into the blood of the foetus. But the blood of the
foal was not withdrawn until some ten months after birth. Now, as the
so-called hereditary immunity only lasts for a very short time
Dzierzgowsky’s experiment supplies no evidence against the passage of
antitoxin through the placenta.
In order to prove that the immunity against toxins may really be
acquired by the ovum, Dzierzgowsky[759] carried out a series of
experiments with the eggs of fowls immunised against diphtheria toxin.
The yolk of the egg, in accordance with the discovery made by F.
Klemperer, contained antitoxin; and this antitoxin passed into the blood
of the hatched chickens. These facts, though in themselves very
interesting, cannot be used to refute the view that antitoxins pass
through the mammalian placenta. It is true that this view is perhaps not
yet completely proved, but it accords well with all the known facts.
Thus, the frequent presence of diphtheria antitoxin in the blood of
new-born infants is explained much better on the assumption that it
passes through the placenta than that it is due to an immunisation of
the ovum surrounded, in the Graafian follicle, by antitoxic fluid. It is
difficult to conceive how this immunity could be so fully retained
during the nine months of pregnancy.
[Sidenote: [471]]
In support of his interpretation of the phenomenon of immunity
transmitted by the mother to her progeny Ehrlich invokes his beautiful
discovery of the immunity conferred by the maternal milk. A vaccinated
female is capable of communicating to her young a portion of the
antibodies manufactured in her organism, not only by the blood channels,
but also, in certain cases, by the milk with which she feeds her young.
[Sidenote: [472]]
The transmission of antitoxins by milk has been demonstrated by Ehrlich,
and this has since been confirmed by many observers (see Chapter XII).
When Ehrlich found that the immunity of the progeny is retained for a
longer time than is that which is conferred by injections of antitoxic
serum, he conceived the idea of investigating whether the cause of more
prolonged retention did not reside in the transmission of the maternal
antitoxin by the milk. With the object of verifying this he took, at the
moment when they had given birth to young, unvaccinated mice and mice
that had been vaccinated against various toxins (ricin, abrin,
tetanotoxin). He so changed the progeny that the vaccinated mothers
nourished the young born of the normal mice, whilst the normal mothers
suckled the offspring of the vaccinated mice. The result of these
ingenious and delicate experiments fully confirmed his anticipations.
The vaccinated mice transmitted their immunity not only to the young
ones to which they had given birth but also to those they had merely
nourished with their milk. This observation proved, to demonstration,
that the antitoxins are absorbed by the alimentary canal, a very
important fact from several points of view. Later researches have shown
that only very young mice are capable of assimilating antitoxin through
the intestinal wall. Adult mice, fed by Ehrlich with quantities of
antitoxic milk, acquired neither immunity nor any antitoxic property of
the blood. Later, Vaillard (_l.c._) was able to show that even the young
of other species of animals such as the guinea-pig and the rabbit are
incapable of appropriating the antitoxins from milk by the alimentary
canal. He repeated Ehrlich’s experiments with new-born guinea-pigs and
rabbits which he caused to be suckled by mothers vaccinated against
tetanus. These young rodents, so treated, were found to possess no
immunity whatever; they were not able, therefore, to absorb the
antitoxin found in the milk of their nurses. Remlinger (_l.c._) made
similar experiments with young guinea-pigs and rabbits suckled by foster
mothers which had been vaccinated against the coccobacillus of typhoid
fever. As in Vaillard’s experiments, the result was negative, the milk
of the foster mother did not communicate any refractory condition to the
nurselings. Remlinger drew the same conclusion from his researches on
the transmission of the agglutinative property of the body fluids. When
female rabbits and guinea-pigs are vaccinated during gestation the young
ones acquire, along with the immunity against the typhoid coccobacillus,
a certain agglutinative power of the blood serum. When, however, these
vaccinated females suckle the progeny of non-vaccinated mothers the
agglutinative power of the milk of the foster mother never passes into
the blood of the nurselings. Some years before this, Widal and
Sicard[760] had demonstrated the same fact that young rabbits and
new-born kittens, when fed with agglutinative milk, acquired no power of
agglutinating the typhoid coccobacillus. They agreed with Ehrlich,
however, that the blood serum of young mice fed with agglutinative milk
acquired the power of agglutinating the typhoid micro-organism.
[Sidenote: [473]]
As it was important to determine whether the human subject was capable
of acquiring a certain immunity by absorbing antibodies contained in the
milk, the study of this question was taken up, especially from the point
of view of agglutinative power. Although the relations of this
agglutinative power with immunity are very problematical it would be
interesting, bearing in mind the analogy between the agglutinative,
antitoxic, and protective properties, to ascertain whether the ingestion
of agglutinative milk can confer any agglutinative property on the blood
serum. Numerous researches in this direction were carried out in
connection with typhoid fever. Widal and Sicard (_l.c._) caused a person
to drink daily (for a period of three weeks) half a litre of milk coming
from an immunised goat, a milk which powerfully agglutinated the typhoid
coccobacillus. The blood, examined on several occasions, never showed
the slightest agglutinative power. This experiment goes to prove that,
in the adult human subject, the agglutinin does not pass from the
alimentary canal into the circulation. May it not perhaps be otherwise
in infants which are fed on milk only? An observation by Landouzy and
Griffon[761] seemed to confirm this supposition. They first demonstrated
the agglutinative power of the blood serum in a woman who had contracted
typhoid fever three months after her lying-in. Being a mild attack the
woman continued to suckle her child during the whole course of the
fever. On examination of the blood of the infant it was found that the
serum agglutinated the micro-organism of typhoid fever. These observers
did not measure the agglutinative power of the blood, either in the
infant or in the mother. This omission deprives their observation of
value since it is now recognised that normal human blood fairly
frequently exhibits some power of agglutinating the typhoid
coccobacillus. For diagnostic purposes it is necessary, therefore,
always to measure this power in order to be sure that it is higher than
that of the normal blood.
It is all the more difficult to draw any positive conclusion from the
observations of Landouzy and Griffon because in several similar cases
the result has been entirely different. Thus Achard and Bensaude[762]
have shown that the blood of an infant, suckled by a nurse attacked by
typhoid fever and whose serum became distinctly agglutinative, was
incapable of bringing about clumping of the typhoid coccobacilli.
Schumacher[763], working in Fraenkel’s laboratory in Halle, studied a
case with very great care. A woman gave birth at full term to an infant
whose blood serum exhibited a certain agglutinative power. The mother
suckled the infant from its birth. Although her milk manifested a very
considerable agglutinative property, the blood of the child exhibited
not only no increase in agglutinative power but a marked diminution. The
agglutinin of the maternal blood had not passed into the fluids of the
child.
[Sidenote: [474]]
From the point of view of the impossibility of acquiring immunity by
suckling, therefore, the human subject may be grouped with the
guinea-pig, rabbit and cat. Up to the present the mouse is the only
exception. It would be very important, with the object of finding a
means of communicating immunity by way of the intestine, to study the
precise conditions which govern this phenomenon. In hereditary immunity,
or rather in what appears to be such, those cases where the new-born
animal exhibits a resisting power induced by the vaccination to which it
has been subjected in the womb of the mother must be borne in mind. We
have already cited the example given by Remlinger of rabbits and
guinea-pigs born refractory against the typhoid coccobacillus, which had
been injected into the mother animals. In those cases where the
vaccination of the mothers has been carried out during the period of
gestation the immunity of the young ones is more permanent than when it
was completed before that period. Into this same group come those cases
where women, vaccinated during the course of pregnancy, give birth to
infants refractory to vaccine. Similar facts have been reported by
veterinary surgeons with regard to sheep-pox; Arloing, Cornevin, and
Thomas[764] have offered similar demonstrations with regard to
symptomatic anthrax.
These results may be more or less closely associated with those where
the child attacked by an infective disease immunises the mother. Such
facts are rare. We know that a healthy mother may give birth to a
syphilitic child; the affected father introducing the virus with the
sperm, the contaminated foetus contracts the disease and the new-born
infant is syphilitic. According to Ehrlich and Hubener (_l.c._ p. 54),
the foetus instead of infecting the mother sets up in her a refractory
condition. It must be confessed that as yet we do not understand the
mechanism of this immunity; but in any case we have here to do with an
example of immunity naturally acquired under very special conditions.
Here again must be recognised another form of immunisation:—where the
child born of a syphilitic mother remains healthy and contracts syphilis
neither by the breast nor through the kisses of the mother. Here,
undoubtedly, we have an immunity against syphilis acquired in the womb
of the mother, who may, however, readily communicate her disease to
other persons by means which are without effect on her own infant. This
example comes under the law of Profetta. Here again the mechanism of the
acquired immunity is absolutely unknown.
[Sidenote: [475]]
It must be admitted that, generally, we are still very imperfectly
informed concerning immunity as acquired by natural paths. In cases
where this immunity is developed as the result of an attack of an
infective disease the phenomena observed closely resemble those that
have been observed after vaccination by living, fully active, or
attenuated viruses, by micro-organisms which have been killed, or by the
products of these micro-organisms. These vaccinations which bring about
isopathic (von Behring) or active (Ehrlich) immunity give rise to
transient and mild diseases and are confined almost completely to the
diseases contracted by natural means which terminate in recovery and
give rise to a refractory condition. The immunisation of the foetus
comes into the same series.
On the other hand, the immunity which was believed to be hereditary and
which results merely from the direct passage of the antibodies of the
blood or of the milk of the mother to the foetus and to the child come
into a group of cases characterised by what Ehrlich has termed a
condition of passive immunity. We have already discussed (Chapter X) the
thesis that this term “passive” is applicable only in rare cases. Most
frequently it is necessary that the living cells of the organism which
receives the antibodies—antitoxin, fixatives or others—should contribute
their quota in order to ensure the refractory condition. This rule is
undoubtedly applicable to the examples of immunity acquired by the
new-born progeny of unaffected mothers.
CHAPTER XV
PROTECTIVE VACCINATIONS
Vaccinations against I. Small-pox.—II. Sheep-pox.—III. Rabies.—IV.
Rinderpest.—V. Anthrax.—VI. Symptomatic Anthrax.—VII. Swine
Erysipelas.—VIII. Pleuropneumonia in the Bovidae.—IX. Typhoid
Fever.—X. Plague.—XI. Tetanus.—XII. Diphtheria.
[Sidenote: [476]]
In the preceding chapters I have attempted to present to the reader a
general view of the phenomena of immunity against infective
micro-organisms and against their toxic products. I shall now attempt to
give a review of the facts acquired in connection with the prevention of
the infective diseases of man and of the chief domestic animals by means
of vaccination. Vaccinations as we know can be carried out either with
viruses the constituents of which have not as yet been recognised, with
micro-organisms grown on various nutrient media, with virulent or
attenuated micro-organisms, or with microbial products deprived of the
micro-organisms by which they have been built up. In addition to these
methods we may vaccinate with protective or antitoxic serum and other
body fluids, with normal serum, or with a whole series of fluids not
excepting water.
[Sidenote: [477]]
I. _Vaccination against small-pox._—We naturally commence the series
with vaccination against small-pox, which is one of the oldest and one
of the best known, having been practised in every country in Europe for
more than 100 years. Small-pox, a very contagious and fatal malady, was
very rife in the 18th century. Large cities like London and Paris were
severely affected. One-tenth of the total mortality was due to this
disease. According to statistical information, very exact for that
epoch, the deaths from small-pox in London during the course of the
second half of the century (1751–1800) numbered more than 100,000
(102,112) persons. During the first half of the same century this
disease caused great ravages in France, especially in Paris, where,
according to certain statistics (Haeser), about 14,000 persons died in
1716.
[Sidenote: [478]]
Variolisation or “inoculation” coming to Europe from the East, had come
into extensive use when, at the end of the 18th century, the discovery
was made that cow-pox, the varioliform disease of the Bovidae, produced
in persons who milked cows suffering from this eruption an immunity
against small-pox. This idea, popular in origin, was known to breeders
in England, France, Germany, and Holland; we have thus an indication
that this knowledge must date from a fairly distant period. Jenner gave
the question a scientific and experimental basis, and it was only after
his intervention that vaccination by the contents of the pustules of
cow-pox began to spread more generally. During the 19th century an
immense amount of material bearing on this question was collected; we
have thus been enabled to attain absolutely exact results, and that in
spite of the very imperfect state of our knowledge on the etiology of
small-pox and of cow-pox. Long ago Chauveau[765] demonstrated that the
virus of these diseases must be organised, because that of the vaccine
would not pass through a filter. This organism has been carefully
sought, but sought in vain in spite of all improvements in
microbiological methods. It was thought that the cocci so often found in
the contents of the vaccinal pustule was the specific micro-organism of
cow-pox. Such was the opinion of the illustrious botanist Cohn[766]. It
was soon shown, however, that this was not the case. The cocci,
principally staphylococci, are “secondary” micro-organisms which may be
absent from the vaccine without its losing anything of its action. A
search was then made for the micro-organism of the vaccine among the
protozoan organisms. L. Pfeiffer[767] announced the discovery of a
species of vaccinal _Amoeba_. Guarnieri[768] has even described various
stages in the reproduction of this hypothetical parasite; but
Salmon[769] demonstrated, in a work carried out in the Pasteur
Institute, that we had here to deal merely with leucocytes which had
entered epithelial cells and had there undergone marked degeneration.
Funck[770] thought that he was able to confirm the discovery of the
sporozoon of vaccinia, but his error was easily demonstrated
(Podwyssozki and Mankowski)[771]. Up to the present, then, we have no
knowledge of either the micro-organism of small-pox or of that of
vaccinia. We still employ, as formerly, the virus taken from the
vaccinal pustule. Even the relations which exist between the two viruses
and the two diseases which they have set up have not yet been settled.
Several authors believe that the bovine disease is only a modified and
attenuated form of human small-pox; whilst others maintain that we have
two very different exanthemata, one of which—cow-pox—is capable of
setting up immunity not only against itself but also against small-pox.
[Sidenote: [479]]
For a long time, in order to vaccinate against small-pox, the contents
of the vaccinal pustules which formed on the human subject after an
original inoculation of the virus of cow-pox were employed. But a number
of cases of infection by syphilitic virus and certain other accidents
caused this method to be abandoned. A number of years ago, however,
there spread throughout Europe and into several countries of other
continents another method, which consists in vaccination by “animal
lymph,” that is to say, by the contents of pustules developed on the
skin of the calf. This method was first carried out at Brussels in 1868,
under the direction of Warlomont, at the Institute founded by the
Belgian Government for the preparation of vaccine. The original virus
came from a genuine case of cow-pox and has since been kept up by
uninterrupted passage from calf to calf. The virus is introduced into
the shaved skin of the region between the groin and the udder as far
forward as the umbilicus. It is inoculated superficially into the
epidermis by cuts one centimetre long. At the points of inoculation
characteristic pustules develop; from these the vaccinal content is
withdrawn, on the fifth day in summer or the sixth in winter. The
contents are removed by pressure and by scraping the pustules. The
scrapings are mixed with water and glycerine. The vaccine thus prepared
is put into small glass tubes which are sealed at both ends. This
method, with slight modifications, has extended to many other countries,
and is carried out either in private establishments or in State
institutions as in Germany. For the purpose of purifying the vaccine it
is diluted and then allowed to sediment or it may be subjected to
centrifugalisation. The object of these measures is to rid the “lymph”
of the micro-organisms which accompany it. This object is, however, only
imperfectly attained and is moreover accompanied by an attenuation of
the vaccinal action. On the other hand, precautions are taken to ensure
all possible cleanliness during the operation of inoculation and whilst
the calves are under treatment. Thus, great care is taken to disinfect
the area of inoculation with alcohol or some other antiseptic and to
dress the pustules during the course of their development. Similarly the
arms of the patient to be vaccinated are well washed; following in this
the rules of asepsis rather than of antisepsis for fear that the
vaccinal virus might be destroyed by antiseptic substances. Various
instruments are made use of for vaccination and care is taken to
sterilise these before they are used. Sometimes the lancet is used,
sometimes “plumes à vaccin” or vaccinostyles, or a bistoury of
iridio-platinum (Lindenborn) etc.
When the vaccine is of good quality and the operation of vaccination is
well done, there is no doubt as to the protective result obtained
against small-pox. The observations that have been collected for a great
number of years past, in many countries, place this beyond doubt. There
are, indeed, statistics from which it is impossible to draw any precise
conclusions because they are founded upon too scanty figures or deal
with conditions that are too complex. This is the case with the Swiss
vaccinations. Certain cantons (such as Zug and Uri) have made
vaccination obligatory, whilst others (Bern, Zurich, Lucerne, etc.) some
years ago abolished the law which compels the vaccination of all
children in infancy. It happened that for some years small-pox had more
victims in the cantons of the first group than in those of the second.
The opponents of antivariolic vaccination attempted to use this as an
argument against the utility of this method. But a more detailed study
of the facts clearly shows that it is impossible to draw from it any
conclusion whatever. Even in those cantons where vaccination is supposed
to be compulsory this law is not carried out rigorously, and the number
of persons vaccinated often does not exceed that in the cantons where it
is not obligatory.
[Sidenote: [480]]
In order to gain some idea of the utility of vaccinations we must
collect statistics on a much larger scale than are those obtainable from
the Swiss cantons. Germany furnishes such statistics. Compulsory
vaccination was introduced there more than a quarter of a century ago
(1874), and statistical information has been collected with great care.
With the exception of a slight increase during the period from 1879 to
1885 small-pox has diminished progressively since the proclamation of
the new law, and has become so rare that in 1897 there were only 5 fatal
cases in the whole German Empire. In the space of 13 years (1886–1898),
in a population which embraces two-fifths of the total inhabitants of
the German Empire, there were altogether five fatal cases of small-pox
occurring in persons who had been successfully revaccinated. Moreover,
the majority of the cases of small-pox occurred in the maritime towns or
in the vicinity of the frontier of the Russian Empire.
Specially favourable results have been obtained in the German army, in
which, even before the law of 1874, vaccination was compulsory. In 25
years there occurred in the Prussian army only two cases of death from
small-pox. In summing up the statistical data on vaccination
Kübler[772], from whom we have borrowed the above statements, expresses
himself as follows: “The history of small-pox must in all cases register
the fact that this dreaded disease has, as the result of general
vaccination, not only become rare in the German Empire but that it has
almost completely disappeared” (p. 365). The example of Germany
encouraged several other countries to introduce compulsory vaccination,
and Roumania, Hungary, and Italy have in turn promulgated similar laws.
Here also it was not long before satisfactory results were obtained. In
Italy especially the mortality from small-pox has largely decreased in
recent years.
[Sidenote: [481]]
In England, where compulsory vaccination was introduced some time ago,
it was abolished in 1898. As the opposition of the people became more
manifest, the law, although it continued to exist formally, was carried
out very imperfectly. The number of unvaccinated children had gradually
increased in such a fashion that in London itself in 1897–1898 it
attained the proportion of 24·9%, whilst in certain provincial districts
it has oscillated between 78·4 and 86·4%. Under these conditions, the
abolition of the law of compulsory vaccination was only the legal
sanction of an accomplished fact. According to the details which have
been supplied to me by the Jenner Institute in London (which has taken
in hand the distribution of vaccine), vaccinations since they are no
longer compulsory have become more frequent in England, and the quantity
of vaccine distributed has increased considerably. This quantity,
however, is not adequate because small-pox has again made its appearance
in London in the form of a pretty serious epidemic[773].
In France a law is being framed which will render infant vaccination
compulsory. Up to the present this has not been the case, and small-pox
from time to time causes considerable ravages, as we may see at this
moment in Paris. During recent years the mortality from small-pox in
France has been from 90 to 100 times greater than in Germany. It is
greater amongst the female population than amongst males; this
constitutes a fresh argument in favour of vaccination. Although not
compulsory for the whole of the French population, it is so for soldiers
and for children who carry on their studies in schools, and it is for
this reason that small-pox is rarer amongst males. The most complete
demonstration of this is found in the incidence of small-pox in the
French army. In spite of a less numerous contingent of troops
(451,941–457,677) the mortality from small-pox was greater during the
period when vaccination was not yet carried out generally (1885–1887)
than during the period (1889–1896) when it was rigorously enforced on a
much larger number of soldiers (524,733–564,643). From 13·6 fatal cases
per year in the first period the annual figure fell to 6.
[Sidenote: [482]]
It follows, when we take into consideration the whole of the very
numerous data at our disposal, that the usefulness of vaccination
followed by revaccination after some (5–7) years cannot be seriously
called in question. As to the inconveniences that may be caused, they
are observed in very rare cases, and then most frequently when impure
vaccines are used, or when the vaccinated skin becomes contaminated.
According to the German statistics there were registered in the space of
13 years (1885–1897), in 32 millions of vaccinations, 113 fatal cases as
the result of infection of the wounds. In forty-six of these it was
proved that the small wound had been contaminated by impurities
introduced by those attending on them. The remaining 67 fatal cases
could be ascribed to the vaccines themselves. We must, however, still
regard these cases as too numerous and as being readily avoidable by the
adoption of rigorous asepsis. To sum up, the anti-variolous vaccination
by the virus of cow-pox constitutes a method of very great value in the
prevention of one of the most dreaded of infective diseases, but it is
evident that improvement can still be made in this branch of practice.
If science should succeed some day, as we may be permitted to hope it
will, in finding the micro-organism of vaccinia and of small-pox, and it
should succeed in growing it in pure media, it might react very
beneficially on the practical application of vaccination. The more
simple the methods, the less chance will there be of the occurrence of
those unsuccessful cases which, even now, are rare exceptions.
II. _Vaccinations against sheep-pox_ (_la clavelée_).—Sheep-pox, being a
disease very similar to human small-pox and very serious from an
economic point of view, the idea was conceived of fighting it by methods
similar to those used against small-pox. Since the 18th century there
has been practised on a large scale the artificial immunisation of sheep
by the inoculation of the virus of the sheep-pox (clavelisation) just as
the variolisation of man was practised before the discovery of cow-pox.
For this purpose it was necessary to have a considerable quantity of
virus; this was obtained by inoculating sheep-pox into the skin of
sheep. This inoculation was effected either with a lancet or, according
to Soulié’s method[774], by means of a Pravaz syringe. The pustules,
developed under these conditions, were generally of large size and
capable of furnishing a considerable quantity of the virulent lymph
(_claveau_) used for immunisation. This fluid, when gathered pure, and
kept in a closed vessel protected from light and heat, retains its
virulence for a long time: unlike what is observed in the case of
vaccine, the addition of glycerine destroys the virulence of the lymph
pretty quickly. For use, the lymph is diluted with ten times its volume
of 2% borated water; the fluid thus obtained is inoculated into the
extremity of the tail or of the ear; usually a pustule, which remains
single, is formed at the point of inoculation. Clavelisation rarely sets
up a generalised eruption which is always serious and sometimes fatal.
[Sidenote: [483]]
In France the law ordains the clavelisation of flocks in which sheep-pox
appears; but it interdicts its practice in unattacked flocks;—it is easy
to understand the reason for this; in infected flocks all, or almost
all, the sheep, gradually become ill and the illness lasts for some
time; clavelisation diminishes both the duration and the gravity of the
disease; the mortality that it causes, although sometimes very great,
the French sheep being very susceptible to sheep-pox, is always much
less than that due to a natural contagion;—on the other hand, the
clavelisation of a healthy flock, beyond the fact that it may cause
considerable losses, is attended by the special danger that it creates
centres from which the contagion may invade all the flocks of the
district.
But there are countries in which protective and general clavelisation
does not present these inconveniences—the countries where the disease is
endemic and where the sheep are very resistant to the action of its
virus. This is the case in Algeria; sheep-pox exists there permanently
without doing much damage; but the Algerian sheep, which take sheep-pox
without suffering any apparent illness, communicate to French sheep
amongst which they are introduced a very malignant sheep-pox which
sometimes kills as many as 50 per cent. of the flock. This explains and
justifies the measures recently taken by the Minister of Agriculture,
forbidding the importation of Algerian sheep into France unless they
have been vaccinated at least a month previously.[775]
In many other countries clavelisation is likewise enacted, being
authorised in cases where it may be very useful and interdicted in other
cases. In certain countries, _e.g._ Germany, Holland, and Denmark,
clavelisation can be put into force by the Government, which alone has
the right to authorise it under certain circumstances.
[Sidenote: [484]]
III. _Antirabic vaccinations._ Vaccination against rabies has this point
in common with those against small-pox and sheep-pox, that it is
effected with a virus whose micro-organism is as yet unknown. On the
other hand, it is distinguished by its efficacy during the incubation
period. When persons are vaccinated during the incubation period of
small-pox, or sheep during the same period of sheep-pox, the
vaccinations by vaccine and _claveau_ are incapable of arresting the
disease and the infections continue to follow their normal course. When,
on the other hand, we vaccinate men or animals that have been bitten by
mad animals or inoculated with the rabic virus by other means, the
antirabic vaccination, with rare exceptions, prevents the development of
rabies. This vaccination, taking advantage of the length of the
incubation period of rabies, constitutes, therefore, a special type,
intermediate between protective vaccination, properly so called, and a
therapeutic method of treatment.
It is to Pasteur that science and humanity owe the invention of this
method. Aided by his collaborators, especially by Roux, he established
in the first place a whole series of important facts on the subject of
the rabic virus and of experimental rabies. He then set himself to
elaborate a practical method capable of preventing the manifestation of
the disease in dogs inoculated with rabic virus and in men bitten by mad
animals. He succeeded in solving this problem in 1885.
Pasteur’s antirabic vaccines are prepared from the spinal cords of
rabbits that have died of experimental rabies as the result of the
inoculation of the virus bearing the name of “fixed virus.” Prepared in
the laboratory, this virus presents the characteristic feature that when
inoculated under the dura mater of rabbits it sets up in them the first
rabic manifestations after an incubation period of six or seven days.
The disease soon assumes the typical paralytic form which lasts several
days. Whilst the period of incubation presents only very limited
variation, the time of death is subject to much greater variation,
especially according to the season of the year. Sometimes the rabbits
will die on the eighth day after the inoculation of the virus: but death
may be delayed one or two days, rarely more.
It is necessary to wait for the natural death of the mad rabbits before
the spinal cord is extracted, and not to kill them before this term, for
it is only during the final moments of life that the rabic virus is
abundant and is distributed uniformly through the whole substance of the
organ. After removal from the vertebral canal the cord is suspended in
glass vessels containing solid potassium hydrate at the bottom. A whole
series of cords so prepared are then kept in a dark chamber heated to
23° C. or thereabouts. The progressive desiccation which the cords
undergo under these conditions diminishes their virulence. At the end of
several days of this treatment the desiccated cord, instead of producing
rabies in 6–7 days in rabbits inoculated under the dura mater by
trepanning, induces it after longer periods of incubation. Finally, the
cords do not produce even the slightest symptoms of the disease.
[Sidenote: [485]]
The fundamental basis of the Pasteurian method consists in the fact that
the desiccated cord, inoculated as an emulsion below the skin of
animals, produces in them a complete and permanent immunity against
inoculation of the most powerful rabic virus beneath the dura mater.
This experiment, frequently repeated on rabbits and dogs, justified
Pasteur in 1885 in attempting the first vaccinations of persons bitten
by rabid animals, especially dogs. The encouraging results of these
early attempts led to the foundation of the Pasteur Institute in Paris,
devoted, in part, to antirabic vaccinations. Shortly afterwards,
antirabic Institutes were founded in many other European towns, and
later in North and South America, in Indo-China, the East Indies, and in
Africa. At present there are in France six such Institutes (Paris,
Lille, Marseilles, Montpellier, Lyons, Bordeaux), in Russia 9, in Italy
6, etc. The last of these institutions founded in Europe is that of
Berlin, where it forms a branch of the Institute for Infective Diseases
carried on under the direction of Robert Koch. The foundation of an
antirabic institute in Berlin had a very important significance from
several points of view. In the first place, it indicates the definite
acceptance of the Pasteurian method, a method which has been discussed
so long and so keenly. Secondly, it proves that even in a State where
there is a highly organised sanitary police, antirabic vaccinations may
still be of great service.
Seeing that it was in the Pasteur Institute of Paris that the method of
antirabic vaccinations was first elaborated and that it has undergone a
very prolonged ordeal, the method there used serves as a model for the
practice of almost all other institutes. Although in some of them
methods which differ more or less from the original may have been
introduced, the fundamental principle upon which they are based remains
the same.
[Sidenote: [486]]
According to the Pasteurian method properly so called the vaccinal
inoculations are commenced with cords that have been dried for 14 days
and have thus lost their virulence. A piece five millimetres long is
pounded up with very weak veal broth. Up to 3 c.c. of the emulsion thus
prepared is injected below the skin of the flank. The same day a second
injection of the same quantity of an emulsion of a cord which has been
drying for 13 days is made at the corresponding position on the opposite
side. Each day an advance is made by injecting emulsions of cord which
are increasingly fresh and the treatment is concluded by the
introduction of virulent cords, which have been kept at 23° C. for 3
days only. The ordinary medium treatment lasts for 15 days. On the first
5 days two vaccine injections a day are made. On the last 10 days, when
gradually fresher and more virulent cords are employed, only a single
injection is made each day. The injections are made with syringes of the
Pravaz type and are carried out under conditions of rigorous
cleanliness.
If the bites are numerous, or if they are situated on exposed parts, the
treatment is prolonged for 18 days and is further distinguished in that
the cords of 4 and of 3 days are injected much more frequently.
In especially grave cases, when the bites are on the face and head, the
treatment extends over 3 weeks. A more rapid progress is made by making
four injections instead of two during the two first days; in this way a
greater quantity of the virulent cords is injected than in the first two
types of treatment.
The effect of the antirabic vaccinations is usually very good. During
the early years of their application the results were fully discussed
from all points of view, and no efforts were neglected of seeking out
objections of every kind. For the purpose of obtaining rigorously
accurate statistics a separate division was made, at the Pasteur
Institute, for the cases of persons treated after bites inflicted by
dogs whose rabic condition had been demonstrated experimentally (by the
injection of an emulsion of the bulb below the dura mater or into the
anterior chamber of the eye of the rabbit or guinea-pig). A second and
special set of statistics was drawn up of cases where the bites had been
inflicted by animals whose rabic condition had been recognised by
veterinary examination. Individuals bitten by animals that were simply
suspected to suffer from rabies were kept separate.
[Sidenote: [487]]
Thanks to this systematic classification we were able, at the Pasteur
Institute of Paris, to establish the fact that the antirabic
vaccinations performed on persons bitten by animals that were
undoubtedly mad resulted in an extremely low mortality from rabies.
Finding it impossible to attack these results, demonstrated with the
precision of a laboratory experiment, the adversaries of the Pasteurian
method alleged that, quite apart from any vaccination, the percentage of
cases of rabies in persons bitten by mad animals is not greater than
amongst the vaccinated. A hitch in the application of the new vaccinal
method soon demonstrated how entirely unfounded was this objection. At
the Bacteriological Institute of Odessa, founded in 1886, that is to say
almost immediately after the Paris Institute, the first attempts at
vaccination were followed by a mortality from rabies of 5·88 per cent.,
a figure incomparably higher than that of the Paris Institute. Analysing
the probable causes of this want of success it was found that the
Russian rabbits, being much smaller than the French ones, furnished far
too small an amount of vaccinal matter. This being the case, the
introduction of a more intensive treatment was sufficient to cause the
mortality to drop suddenly to 0·8 per cent. This fact, added to so many
other proofs, finally convinced the most sceptical and brought about a
general acceptance of the Pasteurian method.
In course of time the number of cases observed has become very
considerable and the experience gained in the manipulation of this
method very wide. The improvements made in the details of the vaccinal
practice have brought about a progressive diminution in the mortality
amongst the persons treated. From 0·94 per cent. in 1886 the mortality
(counted from the 16th day after the completion of the vaccinations)
fell in 1897 to 0·39 per cent., in 1900 to 0·28 per cent. In the space
of 15 years (1886–1900) there have been treated in Paris 24,665 persons,
of whom 107 died from rabies, giving an average of 0·43 per cent.[776].
The greatest mortality was registered during the early years of the
application of the method, and the rate of the later year’s (1896–1900)
oscillated between 0·39 per cent. and 0·20 per cent.
The results obtained in the majority of the other antirabic institutes
corroborate those of the Pasteur Institute of Paris. Thus, according to
the latest statistics of the St Petersburg Institute[777], the
mortality, in 1899, among persons who had completed their vaccinations,
was about 0·5 per cent. At Berlin[778] there were treated during the
same period 384 persons, of whom 2 died from rabies during treatment,
whilst a third succumbed on the 14th day after the close of the
vaccinations. Only this latter case ought, according to the principles
generally accepted, to be counted as an unsuccessful case, this would
give a mortality of 0·26 per cent.
Quite recently, the antirabic treatment has been so reinforced that the
treatment terminates with the injection of cords desiccated for two days
or even one day only. The results of this intensive treatment have not
yet been reported upon.
[Sidenote: [488]]
According to the statistics of the Berlin Institute rabies is far from
being so rare in Germany as was, at one time, generally supposed. During
the year 1899 its presence was demonstrated, by the experimental method,
in 206 dogs coming from various districts. It is in Silesia, Western
Prussia, and Posen that rabies in dogs has been observed most
frequently.
Antirabic vaccinations have also been performed on herbivorous animals
(sheep, goats, cattle, and horses) which are immunised by means of
injections of the rabic virus into the veins, according to the method
suggested by Nocard and Roux[779], as the result of experiments made by
Galtier[780].
[Sidenote: [489]]
IV. _Vaccinations against rinderpest._ For some time attempts were made
to find a means of immunising the Bovidae and other ruminants,
susceptible to rinderpest, against this terrible disease, which causes
great ravages in regions where it is endemic and greater still in those
regions where it only appears in epidemic form. The good results
obtained from “clavelisation” suggested the idea of immunising against
rinderpest by the inoculation of the rinderpest virus, but all such
attempts gave unsatisfactory results, the inoculation setting up a
rinderpest as grave, and often as fatal as the natural disease. Only in
recent years have we succeeded in elaborating methods of vaccination
really capable of coping effectively with rinderpest. Koch[781] went to
Cape Colony, where this disease had recently appeared and had caused
enormous losses, with the intention of finding a practical method of
arresting the scourge. In spite of his technique and incomparable skill
he was as unsuccessful in finding the parasite of rinderpest as had been
other investigators. The micro-organism of this disease remains unknown.
It was necessary, however, to seek a remedy against it. Koch, studying
the properties of the bile of animals that had died from rinderpest,
recognised that the injection of this bile into normal animals conferred
upon them a fairly certain immunity, and this fact served as the basis
on which to work out a practical method of combating rinderpest on a
large scale. At first this method was received with much enthusiasm, but
experience soon demonstrated the inconveniences it often presented.
Kolle and Turner[782], who continued the researches on rinderpest in
Cape Colony, extolled Koch’s method at the commencement of the epidemic
with the object of establishing around the original disease centre an
unaffected zone which would interfere with the propagation of the
disease. They recognised, however, that this method could not be
employed generally, for the reason that it does not set up immunity
until the end of eight days, during which period the animals may
contract the disease. Further, it demands the sacrifice of a large
number of animals in order to provide the vaccinal bile required for the
vaccinations; finally, it confers an immunity of short duration only
(four to six months).
[Sidenote: [490]]
It was necessary, therefore, to find some method that was more generally
applicable. With this object Koch himself began to study the blood serum
of animals that had recovered spontaneously from rinderpest. He was able
to assure not only himself, but several other observers, that this serum
was capable of rendering normal animals into which it is injected
refractory. Bordet and Danysz, who studied rinderpest in the Transvaal
in 1897, made many experiments in this direction and devised a method
which gave good results in practice. But it was left to Kolle and Turner
to work out a method at once simple and easily applied, one which soon
came into general use. This method is known by the name of “simultaneous
vaccinations.” It consists in the injection of a protective serum
simultaneously with the virulent blood. To prepare the former the
authors just mentioned made use of animals that had recovered
spontaneously from rinderpest or of Bovidae that had been immunised by
bile or by some other method. It was recognised that the protective
power of the serum of animals that have recovered is very small and
cannot confer immunity on normal animals, except when injected in large
doses. Kolle and Turner showed that if Bovidae that have recovered
spontaneously are injected with very large quantities of virulent blood
coming from animals fatally attacked, the protective power of the serum
of the former is markedly increased and a serum is obtained which is
active in small doses and which gives good results in practice. This
serum may be kept for a long time by the addition of a small quantity of
carbolic acid. The immunity conferred by this serum upon normal animals
is immediate, but of short duration; it is completed by making a
simultaneous injection of virulent blood; we thus obtain a double
immunity, one part immediate, the other permanent; to get this result,
however, the serum must not be mixed with the virulent blood, for when
this is done the immunity conferred is trifling or _nil_. On the other
hand, it is complete and persists for several months when the protective
serum is injected separately on one side of the body and the virulent
blood on the other.
Kolle and Turner had to defend their method against many ill-founded
objections and attacks, but they succeeded in getting it accepted, not
only in Cape Colony but also in many other parts of Africa, and in many
countries in Europe and in Asia. In 1898 it was decided at a conference
which met in Cape Town to use the method of simultaneous vaccinations to
the exclusion of all others. This method has since been applied on a
very large scale and it was not long before favourable results were
obtained. The same method has proved to be very successful with Nicolle
and Adil Bey[783] of Constantinople, who now prepare large quantities of
the antirinderpest serum, and combat this disease with great success in
the Ottoman empire. Yersin[784] adopted the same method to fight the
cattle plague in Indo-China, where it causes great ravages, especially
among buffaloes. His Institute at Nha-Trang has become a centre for the
preparation of the specific serum, which he distributes over a vast
territory. In the East Indies the simultaneous method has been applied
by Rogers[785]. In Russia, where rinderpest is endemic in many regions,
the Institute of Experimental Medicine at St Petersburg furnishes the
serum destined to prevent the propagation of this epizootic
disease[786].
In a few years this method of simultaneous vaccination has been extended
to all the countries ravaged by rinderpest and has already rendered
immense services to agriculture.
[Sidenote: [491]]
V. _Anti-anthrax vaccinations._ In the first four sections of this
Chapter we have brought together the methods which have as their basis
the vaccination by viruses whose nature is as yet unknown. Since we
cannot obtain them by artificial culture, we have to introduce them with
animal fluids:—either the contents of vaccinal or clavelar pustules, or
matter from rabic nervous centres, or again the blood of animals
attacked by rinderpest. In the case last mentioned, in order to prevent
the too serious effect of the injection of the virus, it is combined
with a simultaneous injection of protective serum.
[Sidenote: [492]]
In the case of the vaccinations against anthrax we pass to the group of
viruses whose organised nature is well known and which can be injected
in pure culture grown on artificially prepared media. This method
constitutes one of Pasteur’s most brilliant discoveries, made in
collaboration with Chamberland and Roux. Before they had found a
satisfactory method of vaccinating against anthrax these observers had
to solve the problem in connection with a less complicated and less
difficult case. From the first, in his studies on pathogenic
micro-organisms, Pasteur had devoted his attention to finding a means of
communicating immunity against these parasites. With the aid of
Chamberland and Roux he was not long in discovering a method by which it
was possible to attenuate the virulence of the micro-organism of fowl
cholera and to vaccinate fowls against this terrible disease by
inoculating them with this attenuated micro-organism. Guided by these
results Pasteur, Chamberland and Roux set to work to find the vaccine
against anthrax; they were soon confronted by a serious obstacle in the
formation of spores which prevented the attenuation of the bacilli. This
obstacle they overcame by submitting cultures of the bacillus to a
temperature of 42°·5 C. Under this condition spores do not develop, and
the bacilli become attenuated at the end of a longer or shorter period.
Although in possession of these attenuated viruses, it still needed very
laborious investigations to adapt them to the vaccination of various
species of animals susceptible to anthrax, especially sheep. In this
they were also successful, and in 1881, over 20 years ago, Pasteur and
his collaborators demonstrated the efficacy of their method on a large
number of animals. This demonstration was made at Pouilly-le-Fort before
a large commission. We may affirm that this celebrated experiment opened
a new path to science and to the practice of vaccination. It was
performed on 50 sheep, half of which were vaccinated twice with twelve
days’ interval, the other 25 sheep serving as control animals. Fourteen
days after the vaccination by the second vaccine all the 50 sheep were
subjected to a test inoculation of a very strong anthrax virus. Two days
later the vaccinated animals remained unaffected, whilst the control
animals had all succumbed to anthrax.
Similar experiments, undertaken in France, Hungary, Germany, Russia and
elsewhere, confirmed the efficacy of anthrax vaccinations and led to
their extension into all the countries where bacterial anthrax was rife.
From the year 1881 the method came into regular use, and before the end
of that year there had been vaccinated, in France alone, 62,000 sheep
and 6,000 Bovidae. Since these first attempts, made on a large scale,
gave such good results, the anti-anthrax practice was not long in
spreading through France, then into Hungary and several other European
countries. Later, it extended into other continents, especially into
South America (Argentina)[787] and Australia. Vaccinations against
anthrax were also applied to horses with the same good results[788].
In France the anti-anthrax vaccines are prepared at and sent out from
the Pasteur Institute of Paris. These vaccines consist of broth cultures
of attenuated bacilli, of which the weakest, the first vaccine, is fatal
to the mouse and small guinea-pigs. The bacilli of the second vaccine
are less attenuated, and are capable of killing not only adult
guinea-pigs but even a certain number of rabbits, when inoculated
subcutaneously. The two vaccines are races of the anthrax bacillus,
capable of producing spores which present the same degree of virulence
as the filamentous bacilli which gave them birth.
The anti-anthrax vaccines are sent out in tubes containing the quantity
necessary for the vaccination of a large number of animals. The
vaccinations are made especially in spring in order that the animals may
be protected during the hot season, which is usually more favourable to
the development of anthrax epidemics.
[Sidenote: [493]]
In the sheep the vaccines are injected below the skin on the inner
aspect of the thigh. One-eighth of a c.c. of the first vaccine is
injected with a somewhat modified Pravaz syringe. Twelve or fifteen days
later a similar injection is made on the opposite side with the second
vaccine. In the Bovidae the vaccines are injected behind the shoulders,
where the skin is thinnest. In the horse the injections must be made on
the sides of the neck and shoulders. In large mammals double the amount
(¼th of a c.c.) of each vaccine is injected.
The tubes of vaccine, once opened, should not be employed a second time.
Care must be taken to use the whole of their contents at one series of
vaccinations.
The vaccinal injections produce tumefaction at the point of inoculation
and are followed by a slight rise of temperature. But these symptoms are
of little importance and soon disappear. Serious complications and fatal
results from the vaccinations are very rare. The loss due to these
accidents is estimated at one-half per cent. in sheep and a quarter per
cent. in the Bovidae.
The refractory condition resulting from the vaccination requires for its
development a period of about a fortnight. The immunity is then very
substantial and lasts for a fairly long time. According to Chamberland
60% of the sheep retain their immunity a year after they have been
vaccinated. But as a great number of animals then become susceptible, it
is usual to revaccinate annually.
According to the statistics furnished by the vaccine department of the
Pasteur Institute there have been vaccinated, up to the 1st of January
1900, a total of 4,971,494 sheep, and 708,980 cattle. Abroad the
corresponding figures are 3,831,948 and 1,869,445. Altogether, the
number of animals vaccinated amounted to 11,381,867, of which 3,626,206
have been treated with the vaccine furnished by the Budapest Laboratory.
The results of the anti-anthrax vaccinations were found to be so
favourable that it was unnecessary to introduce any improvements in
technique. Attempts have certainly been made to prepare anti-anthrax
serums, and these have been successful, but up to the present such
serums have not been introduced into practice.
[Sidenote: [494]]
VI. _Vaccinations against symptomatic anthrax._ Symptomatic anthrax,
which is often confounded with true anthrax, is set up, as demonstrated
by Arloing, Cornevin, and Thomas, by a specific anaerobic micro-organism
to which has been given the name of _Bacillus chauvaei_. Immediately
after the discovery of the attenuation of viruses and of vaccines
against fowl cholera, the three observers above mentioned tried to apply
it to symptomatic anthrax. Finally they devised a method which was soon
adopted in practice, and which, for nearly twenty years, has been used
in the vaccination of the Bovidae in countries where symptomatic anthrax
is most prevalent. This is especially the case in mountainous districts,
such as Switzerland, the Bavarian Alps, the Dauphiné, L’Auvergne, etc.
Arloing, Cornevin, and Thomas[789] prepare two vaccines against
symptomatic anthrax by a method very different from that used in the
preparation of the Pasteurian anti-anthrax vaccines. They take the virus
from the muscles invaded by the micro-organism; they triturate a piece
of the tumefied muscle in a mortar, adding to it a few drops of water.
The mixture is filtered through muslin and the fluid dried at 37° C.; a
virulent brown powder is thus obtained. In the preparation of the
vaccines a portion of this powder is mixed with water and subjected to a
temperature of 100°–104° C. for seven hours. Another portion is heated
during the same number of hours to 90°–94° C. only. This latter forms
the second vaccine whilst the first portion constitutes the first.
In practice the two vaccinal powders are dissolved in cooled boiled
water and are introduced into the subcutaneous tissue of the animals
that it is wished to immunise. The second vaccine should be injected 8
to 12 days after the first. The vaccines are usually tolerated very well
by the Bovidae and confer upon them a definite and permanent immunity.
In spite of certain drawbacks this method, known as the “Lyons method,”
has proved to be a very serviceable one and is retained as the best
devised up to the present. Its efficacy is proved by the fact that in
the period from 1884 to 1895 in 400,000 vaccinated animals the mortality
has only been 1 per 1,000. Arloing, Cornevin, and Thomas thought that
raising the virus to a high temperature brought about a real
attenuation.
[Sidenote: [495]]
Leclainche and Vallée[790], who have recently returned to the study of
this question, have shown that this view cannot be maintained. In
reality the spores, after being heated to 90°–104° C., gave rise to
bacilli endowed with their normal and complete virulence. But the
heating in the preparation of the Lyons vaccines destroys the toxin
manufactured by the _Bacillus chauvaei_, with the result, that the
spores now become the prey of phagocytes: it is for this reason and for
this reason alone that the inoculation of these vaccines is so well
tolerated. All the spores of the vaccinal powder are not eaten by the
phagocytes: those which are found in the centre of solid particles of
the powder offer a prolonged resistance to the action of the cells, and
some of them germinating produce bacilli and give rise to a mild disease
capable of conferring immunity. The germination of these spores is
further facilitated by the presence of foreign micro-organisms in the
vaccinal powders; these organisms help to interfere with the
phagocytosis of the spores of symptomatic anthrax.
In the course of their researches, Leclainche and Vallée demonstrated
that it is easy to vaccinate animals susceptible to anthrax and to
confer on them a substantial immunity by means of a single protective
injection of a pure culture of _Bacillus chauvaei_. For this purpose
they use cultures grown in broth made from the pig’s stomach (“bouillon
de panse” or Martin’s broth) which they heat for 2 hours at 70° C. The
cultures, so treated and injected in quantities of 1 to 2 c.c. into
Bovidae, induce in them an immediate immunity. These authors are
persuaded that the vaccination by this method might be used on a large
scale with certain advantages over the method at present in use. A
single injection, instead of two, involves a great economy, and the
injection of pure vaccinal cultures obviates the accidents caused by the
foreign organisms which are found mixed with the Lyons vaccine.
On the other hand, Leclainche and Vallée think that vaccination by
serums has no future in the fight against symptomatic anthrax and should
only be used in exceptional cases.
It is evident that the Lyons method is capable of being improved and
some day may be replaced by another. Still it must be remembered that it
has already preserved a very great number of animals from certain death
by symptomatic anthrax.
[Sidenote: [496]]
VII. _Vaccinations against swine erysipelas._ Swine erysipelas is a
disease widely distributed in nearly all countries where the breeding of
pigs is carried on on a large scale. It is a very fatal disease, and it
is estimated that in France alone at least 100,000 pigs of the value of
more than five million francs succumb to it annually. Unfortunately
swine erysipelas is often confounded by breeders with other epizootic
diseases, especially pneumo-enteritis of the pig. This confusion has
often resulted in large losses to agriculture.
Soon after the vaccinations against anthrax became a part of veterinary
practice, Pasteur[791], assisted by Thuillier, took up the study of
swine erysipelas which was causing great ravages in the department of
Vaucluse. They were not long in discovering that the true cause of the
disease was a very small bacillus capable of growing in pure culture in
nutrient broth. Guided by his former investigations, Pasteur with his
collaborator undertook minute researches into the reinforcement and
attenuation of the virulence of the bacillus of swine erysipelas which
led them to the elaboration of a method of vaccination capable of
conferring on pigs a high degree of protection against the disease.
Following the line of the anthrax vaccinations, Pasteur and Thuillier
prepared two vaccines against the erysipelas, the first more attenuated
than the second. The bacilli of these two vaccines were cultivated in
broth and sent out in tubes similar to those employed in the
distribution of the anthrax vaccines.
The vaccines are in themselves innocuous and are capable of
communicating to the inoculated pig an immunity sufficiently durable to
be of real service. Young pigs being less susceptible to the erysipelas
than are the adults, it is generally preferred to vaccinate young pigs
of from two to four months. The vaccination is done at two separate
times. The first vaccine, in a dose of one-eighth of a cubic centimetre,
is inoculated subcutaneously on the inner aspect of the right thigh; the
second vaccine is inoculated in the same way, 12 or 15 days later, into
the left thigh. The immunity that follows these vaccinations is not
fully established until the end of the second week.
[Sidenote: [497]]
In spite of the many advantages of the Pasteurian method the
vaccinations against swine erysipelas have not spread so much as one
might have expected; and they have found a general application abroad
rather than in France. It is only necessary to cast a glance at the
statistics to be convinced of this. From the date of the introduction of
the Pasteurian vaccinations in 1884 up to the 1st January, 1900, there
had been vaccinated in France in all 428,746 pigs, whilst abroad, where
the vaccinations were introduced some years later, the number of pigs
vaccinated was 4,819,387. Of this number the great majority (4,194,191)
had been treated in Hungary. The losses amongst the vaccinated animals
were insignificant (1·68%) when compared with an average mortality of
20% amongst unvaccinated pigs.
This limited extension of the vaccination of pigs in France arises from
various causes. In many countries the breeding is on too small a scale
to allow of the intervention of the veterinarian and of the expenses
which the vaccinations involve. On the other hand, it cannot be denied
that the Pasteurian method presents certain drawbacks in practice. The
living, although attenuated, bacilli introduced may sometimes serve as
centres of infection, especially in cases, rare no doubt, where the
vaccinated animal contracts a chronic form of the disease. The
Pasteurian vaccines must, therefore, be avoided in districts where the
erysipelas has not yet appeared. Their application in countries already
infected presents the further drawback that the immunity requires for
its establishment a fairly long time, sufficiently long to permit the
micro-organism to kill a large number of pigs before the vaccines have
conferred any immunity upon them.
[Sidenote: [498]]
It is natural that, under such conditions, an attempt has been made to
replace the Pasteurian method by some other method less risky. Hence,
since the discovery of the principle of sero-therapy several
investigators have sought to apply it to swine erysipelas. Emmerich and
Mastbaum[792] were the first to demonstrate that the blood of rabbits,
immunised with the bacilli of this disease, acquire a very marked
protective power. They have even attempted to construct from the results
of their researches methods which might be applied practically. It is
especially however to Lorenz[793], a Darmstadt veterinarian, that we owe
the first practical application of this method. He prepared protective
serums by injecting erysipelas bacilli into rabbits and pigs, and
demonstrated that the inoculation of these serums, when combined with
that of the living bacilli, conferred upon pigs a sufficient immunity
and one that was set up immediately after the introduction of the serum.
According to Lorenz’s method it is first necessary to give a protective
injection of serum; some days (3–5) afterwards this is followed by an
inoculation of living bacilli coming from the attenuated erysipelas
known in Germany under the name of “Backsteinblattern.” About two weeks
later a further injection of the same bacilli, but in double quantity,
is given. This method, therefore, involves three vaccinal injections as
against two in the Pasteurian method. It is consequently dearer than the
latter, but, as it presents certain undeniable advantages, an attempt
was made to introduce it into veterinary practice. But being much more
complicated endeavours were made to simplify it. Voges and Schütz, by
methods which have remained secret, soon obtained a more active serum,
and finally Leclainche[794] of Toulouse, after demonstrating that the
horse is the best animal for the production of a very active serum,
succeeded in devising a method of vaccination as simple as it was
effective. He gave to it the name of “serum-vaccinations.” The first
inoculation is made with a mixture of specific serum and a culture of
living and virulent bacilli. This inoculation is well borne by all pigs
and may be made without any regard to the age of the animal. The
immunity is set up immediately after the injection of the mixture, but
it is not sufficiently durable for the requirements of practice. For
this reason Leclainche followed up the first injection by a second,
which is made ten to twelve days later and consists of an inoculation of
half a cubic centimetre of pure virus. This new method had the special
advantage of arresting, almost immediately, the mortality in an infected
piggery and of eliminating the chronic cases that are sometimes observed
after the Pasteurian vaccinations.
Leclainche[795] has already applied his method of serum-vaccinations to
more than five million pigs of all ages. “It has been found to be
constant in its effect and absolutely innocuous,” and “not a single case
of erysipelas has been met with in pigs that had received the two
vaccines,” and Leclainche hopes that his method will soon come into
general practice, and that it will be utilised in all cases where the
Pasteurian method is found to be insufficient.
[Sidenote: [499]]
As the basis of all the new methods for vaccinating pigs against
erysipelas is the preparation of serums capable of preventing the
pathogenic effect of the bacilli, the question of the determination of
the protective power of these serums comes to be one of considerable
importance. At first one was satisfied with certain approximate
estimations, but later the necessity was felt of having a more exact
measurement. Leclainche is persuaded that of all the laboratory animals
capable of being used for these experiments the pigeon is the only one
that can usefully fulfil this rôle; very susceptible to the passage
virus, it is killed by the bacillus after a regular incubation and
invasion period, and the chronic form of the erysipelas, so troublesome
in the rabbit and even in the pig, is met with in the pigeon in very
exceptional cases only. Leclainche commenced his experiments by
inoculating into the pectoral muscles of the pigeon mixtures of serum
and virulent cultures. The pigeon received 1 c.c. of a culture of a
passage virus mixed with variable quantities of serum. The serum is
ready for use in the vaccination of pigs when the pigeons resist the
injection of a mixture of ½ a c.c. of serum with 1 c.c. of a virus which
kills the control pigeons in 60 to 72 hours.
At the Frankfort Institute of Experimental Therapeutics another method
of testing devised by Marx[796] is used. In it injections, below the
skin of a series of grey mice, are made of progressively increasing
doses of the serum the strength of which it is desired to determine.
Twenty-four hours later a virulent culture of the bacillus of swine
erysipelas is introduced into the peritoneal cavity of the same mice.
The virus is so chosen that the control mice die in about 72 hours. Marx
finds that this method gives results which are much more constant and
exact than any other; this opinion is confirmed at Höchst, the largest
factory of serums in Germany.
VIII. _Vaccinations against bovine pleuropneumonia._ This infective
disease is one of the most dreaded scourges of bovine animals. Very
contagious, it has spread from central Europe not only into all the
other countries of the European continent, but into Africa, America, and
almost every quarter of the globe. The virus of this disease was
discovered in the serous exudation of hepatised lungs long before the
microbiological period of the Medical Sciences had begun.
[Sidenote: [500]]
Dr Willems of Harselt, who made an experimental investigation,
remarkable for the time at which it was carried out (more than half a
century ago), demonstrated at once the great virulence of the pulmonary
serous fluid; he found also that the effects of the inoculation of the
virus varied much according to the seat of inoculation. When made into
the trunk, the neck, or the shoulders, the inoculations are usually
fatal; at the periphery, the lower part of the limbs, at the extremity
of the ears or of the tail, the inoculation ordinarily produces merely
an inflammatory tumefaction of small extent, which is absorbed in a few
weeks; after this the animal is refractory to the natural disease.
Willems concluded from this that we may vaccinate against
pleuropneumonia by inoculating the virulent serous fluid of the lung
into the tail. Willems’ method of inoculation became a part of current
practice 50 years ago.
For the carrying out of a large number of vaccinations it is necessary
to have at one’s disposal an adequate quantity of virus; it was
therefore to meet this requirement that researches were first carried
out. The serous fluid was withdrawn from the hepatised lungs of animals
that had succumbed to the disease and was inoculated into normal Bovidae
as soon as possible, so as to avoid contamination of the fluid. In fact
this pulmonary serous fluid often contains foreign germs capable of
multiplying rapidly so that it putrefies very quickly. Pasteur showed
that it was possible to remedy these drawbacks by a very simple method
by which he could obtain a large quantity of rigorously pure virus. All
that is necessary is to inoculate a little of the pleuropneumonic virus
below the skin of a weaned calf, behind the shoulder. At the seat of
inoculation there is an abundant exudation of virulent serous fluid into
the cellular tissue, from which we are enabled to collect large
quantities of pure virus.
In some countries, as in Germany and in Australia, institutions have
been founded for the production by this method of the virulent serous
fluid necessary for these inoculations.
The virus should be inoculated into the tip of the tail of animals that
it is desired to immunise, because the temperature in this situation is
relatively low and the connective tissue is dense and not very abundant.
The inoculation is made with a lancet or a Pravaz syringe. The
vaccination is generally borne well, in spite of the reaction phenomena
which are manifested about two weeks after the introduction of the
virus. At that time a febrile condition is set up and a swelling
manifests itself at the point of inoculation, which, however, soon
retrogresses and then disappears.
[Sidenote: [501]]
The immunity conferred by Willems’ method is substantial and lasting
(for one or two years and even longer); this explains its great success
in the hands of breeders and veterinarians. Accidents following its use
are rare, and the mortality does not exceed 1 per cent.
In spite of all these advantages a new method was still desirable, a
method which would allow of the preparation of large quantities of virus
of a suitable and uniform activity under conditions of irreproachable
purity. Thanks to the discovery of the micro-organism of pleuropneumonia
which we owe to Nocard and Roux[797] this object has been achieved. With
the collaboration of Borrel, Salimbeni, and Dujardin-Beaumetz, they
succeeded in demonstrating and isolating this micro-organism, the
smallest of all known living organisms. The first steps in these
researches were very laborious, but later the organism of
pleuropneumonia was cultivated on fluid and solid media: Martin’s broth
(prepared with pigs’ stomachs) or agar with the addition of a certain
quantity (about 5%) of fresh ox serum. The serum-broth, sown with pure
pneumonic serous fluid, gives only a moderate growth, which becomes only
slightly turbid and contains micro-organisms so small that it is
impossible to distinguish them individually. They can be made out only
when massed together in irregular clumps. The minuteness of this
micro-organism is evidenced by the ease with which it passes through a
Berkefeld filter, and even through certain Chamberland candles (F). This
feature enables us to obtain the pure virus easily, a fact very
important in connection with the isolation of the micro-organism.
[Sidenote: [502]]
Once in possession of pure cultures of the micro-organism of
pleuropneumonia, Nocard and Roux attempted to make use of it in
practical vaccination. They showed that the organism separated by them
is capable of producing typical pleuropneumonia when it is inoculated
into the appropriate regions of the body of bovine animals. But when
inoculated subcutaneously or into the skin of the tail, it produces
merely a mild and transient disease which confers an immunity quite as
effectual as that set up by the inoculation of the virulent serous
fluid. It may be readily understood that, under these conditions, pure
cultures may be much more serviceably employed in the practice of
vaccination than can Willems’ virus from the fact that it is easy to
obtain large quantities of absolutely pure cultures. It is easy to
predict that the new method will soon replace the old one, very great as
are the services the latter has rendered to agriculture. Up to the
present, vaccinations with pure cultures have been made in several
districts in France with very favourable results. The Pasteur Institute
and the Veterinary School at Alfort have already distributed to
veterinary surgeons more than 5,000 vaccinal doses of culture; the
protective action of these inoculations has been at least equal to that
of the inoculations by Willems’ method and the resulting accidents have
been reduced in the proportion of 20 to 1[798].
The serum of animals hyperimmunised against pleuropneumonia possesses a
very distinct _protective_ action, but too little marked and of too
short duration to be of any use in practice; it has also a _curative_
action arresting the invading march of a pleuropneumonic congestion; but
here it is necessary to intervene early, before the appearance of fever,
and to inject large quantities of serum.
The inoculation of a mixture of virus and serum produces no congestion;
but it does not confer any immunity; the animal remains just as
susceptible as the control to the inoculation of the pure virus.
[Sidenote: [503]]
IX. _Vaccinations against typhoid fever._ In the preceding sections I
have treated more especially of the vaccination of domestic animals
against several infective diseases. The information collected on this
subject is marked by its great exactness, as it is easy to apply to
animals the most rigorous experimental method. In the case of the human
subject this is not such an easy matter. As it is impossible to submit
him to experimental proof we are obliged to be satisfied with
observation, controlled by statistical data. The experience of more than
100 years has, however, been sufficient to demonstrate the great utility
of vaccinations against small-pox with the virus of cow-pox which is
innocuous for the human subject. In the case of antirabic vaccinations
we have to deal with injections into the human subject, first of
weakened viruses and then of virulent viruses. Here, however, it is a
question of the preservation of the already infected human organism,
which, very often, only comes under treatment during the incubation
stage of rabies. One can readily understand the hesitation to inoculate
even weakened viruses into the human subject, especially when we are not
dealing with altogether exceptional cases such as we have in the
protection against rabies. We have, therefore, but few examples in which
the methods of vaccination by micro-organisms have been applied to man.
Such injections were first tried by Ferran[799] against Asiatic cholera.
Having succeeded in vaccinating guinea-pigs against experimental cholera
septicaemia, the Spanish investigator attempted to inoculate cholera
vibrios into the subcutaneous tissue of man, hoping thus to vaccinate
him against true cholera. In this way he was able to demonstrate that
the subcutaneous injection of living vibrios never sets up symptoms of
cholera. The injection is followed by a general reaction in the form of
fever, pains in the back and inflammation at the point of inoculation,
in a word, transient phenomena of little gravity. Encouraged by these
initial results Ferran, profiting by the outbreak of cholera in the
province of Valentia, injected into more than 20,000 persons living
cultures of Koch’s vibrio. The results published by him did not,
however, furnish any real proof of the possibility of conferring
immunity against intestinal cholera by means of subcutaneous injections.
Later Haffkine[800] modified Ferran’s primitive method somewhat, and
instead of living vibrios he injected vibrionic cultures killed by heat
or by antiseptics. During the cholera epidemic of 1892 and 1893 he tried
the inoculation of these killed vibrios into man, with the object of
vaccinating against Asiatic cholera. Later he went to Calcutta in order
to try his method on a large scale. He was there enabled to inoculate a
great number of persons, and the statistics which he collected appeared
to him to be favourable.
[Sidenote: [504]]
But studies on the pathogenesis of Asiatic cholera shook the foundations
of Ferran’s method. The injections of vibrios, living or killed, were
found quite capable of vaccinating animals against vibrionic peritonitis
and septicaemia, but they appear to exert no influence whatever against
poisoning by the cholera toxin. When it had been learnt how to set up
true intestinal cholera in young rabbits Ferran’s and other similar
methods of vaccination were used in vain to prevent the incidence of
this disease, which is very similar to Asiatic cholera of man. An
experiment[801] made at the Pasteur Institute in Paris upon two persons
vaccinated by Haffkine, showed that they were not protected against the
choleriform diarrhoea set up by the ingestion of the cholera vibrios. A
third person, who had never been “vaccinated” and who served as
“control,” after the ingestion of the same cholera culture, behaved
exactly as did the other two.
From all these data the conclusion was drawn that in order to prevent
intestinal cholera it is necessary to use not cultures of vibrios,
living or dead, but antitoxic serums. In fact, the majority of young
rabbits vaccinated with these serums and afterwards submitted to
infection by the cholera virus through the mouth were found to be
vaccinated against intestinal cholera. It has not been possible, as yet,
to apply this method to man, hence we are unable to give a decided
opinion. Moreover, as the methods based on Ferran’s principle have now
been abandoned I have not deemed it necessary to devote a special
section to anticholera vaccinations. I could not, however, pass it by in
silence, since the attempts to vaccinate man against cholera have led to
the trial of a similar method against typhoid fever.
Pfeiffer and Kolle[802] were the first to inoculate man with typhoid
coccobacilli sterilised by heat. They observed that these injections
caused fever, pretty violent pains in the back accompanied by vertigo,
shivering and pain at the point of inoculation, without, however, being
in any way serious to health. At the same time they found that the blood
serum of inoculated persons acquired a very marked protective power (for
guinea-pigs injected into the peritoneal cavity with lethal doses of
typhoid cultures) quite comparable to the properties discovered by them
in the serum of persons who had recovered from typhoid fever. Pfeiffer
and Kolle believed that they thus had a proof of the refractory
condition of the individuals whom they had submitted to these
injections.
[Sidenote: [505]]
These experiments were continued by Wright, Professor of Pathology at
Netley, and it is owing to his unwearied efforts that science finds
herself in possession of very important evidence on the subject of
protective inoculations against typhoid fever in man. According to a
verbal communication made to me by Wright, he has up to the present
distributed more than 300,000 doses of his antityphoid vaccine. This
vaccine he prepared in the following way[803]. The typhoid coccobacillus
is sown in carefully neutralised broth containing 1% of peptone. The
flasks of culture are kept in the incubator at about 37° C. for two or
three weeks, after which their contents are transferred to large flasks
in order to be submitted to a temperature of 60° C. This temperature is
quite sufficient to kill all the coccobacilli, but for greater surety
Wright added to his cultures one-tenth of their volume of a 5% solution
of carbolic acid or of lysol. The vaccine, thus prepared, is examined as
to its toxicity for the guinea-pig by means of subcutaneous injections.
Wright injects into man a dose of vaccine which is sufficient to kill
100 grammes of guinea-pig (of the weight of 250 to 300 grammes). This
dose often amounts to half a cubic centimetre, but it may have to be
increased to 1 c.c. and even 1·5 c.c.
The inoculations are made below the skin of the flank or in the
shoulder. They are followed by a rise of temperature which commences as
early as two or three hours after the injection. This fever is
accompanied by pains in the back, nausea, and want of appetite. There
may even be collapse; this led Wright to keep his patient in bed for
some time after the vaccinal injection. Besides this reaction, there
occurs, at the seat of inoculation, a swelling and redness, accompanied
by pain; as a rule all these symptoms have disappeared by the end of 48
hours.
[Sidenote: [506]]
Wright convinced himself that the blood serum of individuals treated by
his vaccine, at the end of a certain time acquires the property of
agglutinating typhoid coccobacilli in a variable, but usually very
marked degree. He even thought that this property might up to a certain
point serve as the measure of the immunity acquired against typhoid
fever. His own researches, however, showed him that this supposition
could not be maintained, and that the agglutinative power, varying
greatly in strength, might sometimes be absent where the immunity could
not be denied. On the other hand, he clearly showed, especially by the
experiments with serum collected at the period which precedes the
relapses, that the agglutinative property might be highly developed, in
spite of the absence of immunity. Wright then set himself to study the
bactericidal property of the serum of individuals who had been injected
with his vaccine. He devised a very ingenious method of gaining with a
minimum loss of time some idea of the fluctuations of this power of the
body fluids to kill the typhoid coccobacillus. In the first place he
demonstrated that the bactericidal property is not at all parallel to
the agglutinative power, and this has further confirmed him in his
opinion that there may be no direct relation between it and acquired
immunity. He has found further that the power of the blood serum to
destroy the typhoid coccobacillus is very variable in persons vaccinated
by his method. After injections of large quantities of these killed
bacilli this power may even be diminished for a very long period. On the
other hand, medium or small doses of the vaccine first set up a negative
stage, during which the bactericidal property is very feeble, and later
they bring about an increase of this property, often very marked. Wright
does not think that the bactericidal power can serve as the measure of
the immunity acquired by the vaccinated individuals, but he hopes that
some day a method may be found suitable for the examination of the blood
which will give us information as to the degree of immunity conferred by
the antityphoid vaccination. For the present the only basis upon which
we can form any opinion on this subject is furnished by statistics. Now
we know that it is often very difficult to collect data that are
sufficiently exact. Hence during the war in South Africa, where
one-fifth of the English troops, that is to say about 50,000 persons,
were submitted to vaccinations by Wright’s method, it is only in certain
cases that the statistical information can be utilised. Many of the
patients attacked by slight fevers are omitted from the statistics,
because from the absence of a precise diagnosis it is not known whether
they should come under the category of typhoid patients or not. In other
cases the secondary complications divert the attention of the doctors
and prevent the registration of a proper diagnosis.
[Sidenote: [507]]
Of the data collected amongst the English troops in South Africa, Wright
considers that those which were collected during the siege of Ladysmith
were the most exact, on account of the facility with which it was
possible to study and register all the cases of typhoid fever under
these conditions of complete isolation. Now it has been recognised that,
amongst the vaccinated soldiers and officers, there occurred scarcely
one-eighth as many cases of typhoid fever as occurred amongst the
unvaccinated (1,499 cases in 10,529 unvaccinated, and 35 cases in 1,705
vaccinated). The mortality amongst the vaccinated was also very much
lower. The difference to the credit of the vaccinations should in
reality be even greater, for amongst the unvaccinated are counted many
persons who having already had an attack of typhoid fever were not
submitted to vaccination.
The testimony of the majority of the medical men who followed the
results of Wright’s method closely is also favourable to the
vaccinations. Thus Henry Cayley[804] reports that the staff of a Scotch
Hospital of the Red Cross, almost all of whom (57 persons out of 61) had
received two vaccinal inoculations, escaped typhoid fever, in spite of
the numerous opportunities afforded for the contraction of the disease.
This very favourable example is also instructive in that it testifies to
the value of two consecutive vaccinations. In many other cases where one
has had to be satisfied with a single protective inoculation the results
were less brilliant. According to Howard Tooth, who made his
observations at Bloemfontein, the vaccinations according to Wright’s
method must be regarded as very useful.
Outside South Africa this method has been employed on a fairly large
number of persons in British India, in Egypt, and in Cyprus. According
to the earlier statements from India the incidence amongst the
vaccinated persons was one-third that of the unvaccinated. The most
recent statistics[805] show still more favourable results. Thus at
Meerut the incidence amongst vaccinated persons from Oct. 1899 to Oct.
1900 was one-eleventh that of the unvaccinated (2 cases of typhoid fever
in 360 vaccinated, and 11 cases of the same disease in 179
unvaccinated): the mortality (one case amongst the former, six amongst
the latter) was less than one-twelfth that of the unvaccinated.
In Egypt and in Cyprus according to the statistics communicated to Dr
Wright[806] by Col. Fawcett these vaccinations have given even better
results. In 2,669 unvaccinated persons there occurred 68 cases of
typhoid fever with 10 deaths, whilst amongst the 720 vaccinated there
was only a single case of this disease, this single case succumbing.
Here, however, we have to do with a patient who must have received the
vaccinal inoculation during the period of incubation, the disease
breaking out soon after the vaccination. This would represent in all the
cases a morbidity only one-seventeenth as intense amongst the
vaccinated.
[Sidenote: [508]]
A few isolated voices only have not pronounced in favour of the
antityphoid vaccinations and their opinion is formulated in a very
undecided fashion. Amongst the most important of these adversaries, if
indeed we may term them such, must be cited Washbourn[807], on account
of his experience in microbiology. Attached as a doctor to the Yeomanry
Hospital at Deelfontein in South Africa, he witnessed many cases of
typhoid fever and was greatly struck by the death of two persons amongst
the vaccinated patients. But he himself confesses that it is as yet
premature to judge Wright’s method, and in support of his sceptical
attitude does not offer any other satisfactory observation.
Outside the English colonies vaccinations against typhoid fever have
been tried in Russia by Wyssokowitch[808]. He inoculated 235 soldiers of
a regiment encamped at Kiew, amongst whom an epidemic of typhoid fever
had broken out. The vaccinations were carried out by means of cultures
killed with carbolic acid. We are unable to judge of the efficacy of the
method because the number of persons vaccinated was too small and the
epidemic too limited. It may be noted, however, that amongst these
individuals not one took typhoid fever, whilst amongst the unvaccinated
three cases of the disease were registered.
The antityphoid vaccinations have as yet only a very short history, and
it is, perhaps, premature to express any decided opinion on the matter.
We may, however, consider the results already obtained as offering
encouragement to continue our experiments. Everything, indeed, tends to
a recognition of the utility of vaccinations by means of killed typhoid
cultures. The statistics are as a rule good; the danger from the
protective inoculation is _nil_ or quite trifling. With the exception of
the discomfort of which we have spoken and which is transitory, no
untoward result has ever been observed.
[Sidenote: [509]]
To all this must be added the fact that from the point of view of the
pathogenesis of typhoid fever, all the probabilities point in favour of
the vaccinations. Whilst in Asiatic cholera we have to deal with an
intoxication, from the alimentary canal, an intoxication set up by
vibrionic products, against which the subcutaneous inoculation of
micro-organisms can not be effective, in typhoid fever we have to do
with a real infection. The micro-organism, although developed at first
in the small intestine, becomes generalised throughout the system.
Thanks to improved methods it can always, or almost always, be found in
the blood of the patient, and its constant localisation in the spleen
furnishes a real evidence of this. Under these conditions it is quite
natural to suppose that everything which is able to prevent the
penetration of the typhoid coccobacillus into the blood and the internal
organs ought at the same time to contribute to the protection of the
individual.
We are fully aware that science has not yet said its final word upon
this question. We are coming more and more to the conclusion that it is
necessary to make two injections instead of one. It is possible that we
may have recourse to certain improvements of the method by combining
with it the injections of antityphoid serums as a protective measure.
The near future will doubtless bring us the solution of these very
important questions.
X. _Vaccinations against human plague._ Plague, which for so long was
looked upon as the greatest scourge of humanity, has until recently
remained almost unknown from the scientific point of view. But from the
moment that it became possible to apply to its study the immense
advances realised by microbiology the thick veil which had hidden its
nature fell at a single stroke and science found itself in possession of
effective means of fighting against it. Amongst these means one of the
most important is protective vaccination.
[Sidenote: [510]]
When the last pandemic of plague broke out in Bombay and in the East
Indies in general, Haffkine was there engaged in applying his method of
vaccination against Asiatic cholera of which we have spoken in the
preceding section. Well acquainted with the results of the
bacteriological researches made on bubonic plague by Kitasato, and
especially by Yersin, he, in 1896, began to study this disease. After
the discovery made by Yersin, Borrel, and Calmette[809], who showed that
animals susceptible to human plague could be easily vaccinated against
the micro-organism which gives rise to it, Haffkine[810] endeavoured to
find a practical method for the vaccination of man. He set up a
laboratory at Bombay and, after some preliminary experiments on rabbits,
he commenced to inject human beings with pure cultures of the plague
coccobacillus. From 1897 up to the present he was able to vaccinate a
very large number of individuals, and the results obtained have
encouraged him to continue the application of his method. The principle
of this method is that which had guided him in the preparation of
anticholera vaccines and which is used for the vaccines against typhoid
fever. It consists in the employment of pure cultures of the specific
organism killed by heat. The cultures are grown in large flasks
containing peptonised broth and sown with a small quantity of the plague
coccobacilli. A little sterile butter or cocoanut oil is poured on the
surface of the fluid. Under these conditions the organism grows
abundantly and produces growths which hang down into the fluid,
reminding us of the stalactites in a grotto. This mode of development
forms one of the most typical characters of the micro-organism of human
plague. The culture flasks are kept at a temperature of about 30° C. for
five to six weeks, at the end of which period a large number of the
bodies of the micro-organisms have fallen to the bottom of the flask,
allowing much of their toxic contents to escape. The fatty layer on the
surface favours a surface development of the coccobacilli, the number of
micro-organisms in a flask being thus greatly increased.
After growing for 35 to 42 days under these conditions the cultures are
heated at 65°–70° C. for from one to three hours with the object of
killing all the micro-organisms and so rendering their injection
innocuous. To make sure of the effectiveness of this heating care is
taken to remove a small portion of the fluid and to sow it in a suitable
medium. Should this medium remain sterile the vaccine may be used. Into
adult men it is injected in a dose of 3 c.c., whilst women, children,
and adolescents receive 2–2·5 c.c., into the subcutaneous tissue.
[Sidenote: [511]]
Some hours after the injection of the vaccine the temperature rises
above normal, reaching 38°·5 to 39° C., and sometimes even 40°–40°·5 C.
This febrile condition lasts from 15 to 48 hours. It is soon accompanied
by pain, redness, and swelling at the point of inoculation. These
symptoms persist for from three to five days. The _malaise_ which
follows the vaccinations is sometimes very uncomfortable or even
painful, but never serious. Only in exceptional cases is the formation
of abscesses observed, and this is due, undoubtedly, to contamination of
the vaccines by foreign micro-organisms. The English Commission sent to
India to study plague found other micro-organisms than the plague
coccobacilli fairly frequently in the vaccine culture flasks, but, with
very rare exceptions, these micro-organisms were found to be innocuous.
By rigorously following the rules to be observed in making pure cultures
it should not be difficult to avoid this complication.
Haffkine used every effort to induce his patients to be vaccinated a
second time, being justly persuaded that two injections are capable of
ensuring a more certain and more stable immunity than is a single
injection.
[Sidenote: [512]]
From what moment immunity may be considered to be acquired has been a
matter for great discussion. From very numerous experiments upon animals
of various species, as well as many observations on man, it is now
agreed that a period of several days (5–8) from the injection of the
vaccine is required before immunity is manifested. It is for this reason
that cases of plague which have broken out before this period has
elapsed cannot be looked upon as contraindicating the efficacy of the
method.
A large amount of evidence, coming from persons who have made their
observations on the spot, is almost unanimous in endorsing the fact that
Haffkine’s vaccination protects man against plague. It is often
difficult to compile exact statistics in surroundings where so many
factors contribute to deceive even the careful observer. In spite of
this a certain amount of evidence has been collected which may be
accepted as affording us fairly satisfactory information. One of the
best groups of statistics was that collected at Damaun, a Portuguese
possession in India, into which plague was imported from Bombay in 1897,
and where a large number of vaccinations were carried out. From the
report of Haffkine and Lyons[811], in a population of 8230 persons,
rather more than one-fourth (2197) were vaccinated, the greater majority
(6033) remaining uninoculated. Amongst the former only 36 died from
plague, which corresponds to 1·6 per cent.; whilst amongst the
unvaccinated persons the disease carried off 1482 persons or 24·6 per
cent. Vaccination, therefore, according to these statistics, must have
brought down the mortality to one-fifteenth. The German Commission[812],
two members of which, Koch and Gaffky, went to Damaun to be present at
the vaccinations and to observe their efficacy, pronounced in favour of
Haffkine’s method. The English Commission[813] made reservations and
criticised the statistics of Haffkine and Lyons (who amongst others
attribute all the cases of deaths that occurred amongst the unvaccinated
to plague), but in the end this Commission also recognised the utility
of the vaccinations at Damaun.
The data collected with regard to the vaccinations at Undhera, Hubli,
and several other places in British India confirm the results obtained
at Damaun. The statistics collected at these localities are certainly
open to criticism, but the result as a whole is none the less
encouraging as regards this method of vaccination. According to the
conclusions of the English Commission the “inoculations had a
considerable effect in warding off plague attacks from the
inoculated.... The protection afforded by inoculation seems, however,
never to be absolute[814].” We do not, as yet, know the duration of the
immunity produced by Haffkine’s vaccinations; it cannot be very long to
judge from the experiments on animals, but it may last for several
weeks, probably even for months.
The vaccinations by killed cultures may be especially useful when it is
a question of limiting the extension of an epidemic that is already
established. The ease with which the vaccine can be prepared renders it
possible to obtain very large quantities of it in a short time, with
which it is possible to immunise the entire population of towns or
districts. But, as the immunity by this method requires several days for
its development and as the injections of micro-organisms, even when
killed, may be very injurious during the incubation period of plague or
immediately before the infection, it is necessary to limit the
vaccinations to persons who are not in intimate contact with the sick,
or who are, from the beginning, exposed to infection[815].
[Sidenote: [513]]
Lustig and Galeotti[816] have described another method of preparing
antiplague vaccine which can be utilised where it is of importance to
obtain a large quantity of vaccine in a very short time. Instead of
allowing the cultures to grow for five or six weeks as required by
Haffkine’s method, the Italian observers make use of cultures on agar
which have grown for two days only. The micro-organisms, removed from
the surface of the agar, are treated with a weak solution of potash
(0·75%–1%) which dissolves the bodies of the coccobacilli. This
phenomenon has sometimes occurred by the end of twenty minutes, but it
often requires an hour or more. The contact of the micro-organisms with
the alkali must never exceed three hours. The viscous mass thus obtained
is then treated with acetic acid, when a precipitate is thrown down.
This precipitate, after being washed, is used for the vaccinations. When
injected in large quantities into animals, Lustig and Galeotti’s product
sets up necrosis, but a weak dose is well borne and confers immunity
against plague. In man it is sufficient to inject two or three
milligrammes of this substance diluted with water. The vaccinal nuclein
of the Italian observers has been but little employed for the
immunisation of man in India, but it is largely used in this country for
the inoculation of horses from which to obtain an antiplague serum.
[Sidenote: [514]]
The serotherapeutics against human plague were inaugurated by the
researches of Yersin, Borrel, and Calmette (_l.c._), who demonstrated
that animals susceptible to the plague bacillus can be vaccinated and
even cured of experimental plague. The preparation of antiplague serum
has since been energetically pursued under Roux’s direction at the
Pasteur Institute. After several trials, some of which were very
encouraging, others, on the contrary, somewhat unfavourable, they
succeeded in obtaining a serum which is capable of curing plague after
it has broken out and has become grave. As in this treatise we
intentionally leave aside everything connected with healing we shall
speak only of the antiplague serum as a protective agent.
Whilst vaccinations by killed plague cultures have been practised
principally in the East Indies, the immunisation with antiplague serum
has been employed in Europe, especially at the time of the epidemics of
Oporto in 1899 and of Glasgow in 1900. In all these cases use was made
of the serum from the Pasteur Institute, up to the present the most
active of all those prepared. It is a serum obtained from horses treated
for a long period with cultures of the plague bacillus and with the
toxin of the same organism. Treatment is begun by injecting plague
coccobacilli killed by heat (70° C.). These injections are made into the
veins, with the object of avoiding the local lesions which are observed
after the subcutaneous introduction of micro-organisms. When the horses
have been rendered refractory by this treatment with dead
micro-organisms, the next step is to inject (also into the veins) small
quantities of living cultures. The doses of these cultures are gradually
increased, and end by conferring upon the animal a very strong immunity,
which is strengthened by injections of products of cultures passed
through a Chamberland filter.
Calmette and Salimbeni[817] injected prophylactically more than 600
persons menaced by plague at Oporto. These comprised the doctors and the
staffs of the laboratories of hygiene and of the disinfection services,
the firemen who removed the sick persons and the dead, the families of
those who were attacked, the members of the French colony, etc. Into
each person 5 c.c. of serum was injected below the skin of the abdomen.
These vaccinations in some cases caused nettle-rash, eruptions similar
to those so often observed after the injection of the other kinds of
serums. Of the total number injected two persons contracted plague: the
unfortunate Doctor Camera Pestana and his assistant. The former
succumbed to the disease, but the second only contracted a very mild
form of it. The study of these 600 cases, as well as of experiments on
animals, demonstrated that the immunity conferred by the antiplague
serum is set up immediately after its injection but is not of long
duration. It is probable that it lasts for 8 or 10 days, or at furthest
a fortnight only.
Similar results were obtained at Glasgow. Van Ermengem[818], who has
published a report on the epidemic in this town, mentions that more than
70 persons in good health were inoculated with the serum; each one
received 10 c.c. beneath the skin of the belly. Of these 70 persons one
was attacked with a fairly mild plague 8 days after the vaccination, and
another, a housekeeper, was attacked, 9 days after the injection, with a
congestion of the cervical glands induced by the plague bacillus. Both
cases recovered. All the other vaccinated persons, in spite of constant
exposure to the plague infection, remained unaffected. Van Ermengem was
of opinion that the two persons treated with the serum were already
infected when they were vaccinated.
[Sidenote: [515]]
The Belgian observer points out, further, the frequency of secondary
accidents which were produced in the persons vaccinated at Glasgow. Van
Ermengem himself went through the ordeal after being injected with 10
c.c. of serum as a protective measure and this gave occasion to several
critics to attack the Pasteur Institute. This is how Van Ermengem
himself puts the matter. “The accidents after the immunising
injections ... were very numerous, they were observed 33 times in 72
cases. Sometimes they were even fairly serious, to the point of causing
great suffering to the patient and of disquieting those around them. We
could describe them from thorough knowledge, since we experienced them,
but they scarcely differ from those which are observed from time to time
after the injection of antidiphtheria serum, and, like them, they
disappear without leaving the least trace” (_l.c._ p. 18).
In spite of these accidents and the necessity of renewing frequently
(every ten or fifteen days) the protective injections of serum, their
use is quite advisable in certain circumstances. They may render great
service on board infected vessels or in lazarettos (as in the case which
occurred at Frioul after the arrival at Marseilles of Arab stokers
suffering from plague), in docks, warehouses, and stores where
contaminated merchandise is found. They should also be employed to
vaccinate those coming into immediate contact with plague cases in
hospitals and in private houses. In a word, vaccinations by serum, owing
to their power of conferring a very rapid immunity, should be practised
wherever there is more or less immediate and imminent danger. Under
these conditions they are of very great service in localising the
disease.
The methods of vaccination against plague that have been employed up to
the present may undoubtedly be improved. Calmette and Salimbeni (_l.c._)
have already published the results of experiments on animals undertaken
with the object of studying the effect of a combined method of
vaccination with antiplague serum and killed cultures of the plague
bacillus. But even in their present form the methods used for protecting
individuals against this disease deserve to be regarded as conferring
great benefits on humanity.
[Sidenote: [516]]
XI. _Vaccinations against tetanus._ Tetanus unlike plague is not a
contagious disease, nor is it capable of becoming epidemic. It
constitutes, however, a very formidable disease against which all
therapeutic methods have only a very limited effect. This is a further
reason for drawing the whole attention of medical and veterinary men to
the prevention of tetanus by vaccinal injections. Tetanus is a disease
in which the intoxication plays an altogether dominant part. The tetanus
bacilli do not develop, at the point where they are introduced into the
body, unless favoured by auxiliary conditions, such as the
multiplication of other micro-organisms. Even then the organism of
tetanus reproduces itself with difficulty, and without becoming
generalised throughout the body. The poison which it secretes is however
sufficient to produce a very grave intoxication, ending most frequently
in death. In certain countries tetanus, as a sequel to various wounds,
is very frequently met with in man and in certain domestic animals, such
as the horse, donkey, pig, etc.
It is only since the discovery by von Behring and Kitasato of an
effective method of immunisation against tetanus that it has been
possible to consider the practical application of antitetanus
vaccinations. These observers demonstrated that the tetanus poison, when
treated with trichloride of iodine, had its toxic action weakened and
was transformed into an effective vaccine. Roux and Vaillard found that
the addition of Lugol’s iodo-iodurated solution to the tetanus poison
renders it capable of vaccinating all kinds of susceptible animals. It
was shown later, that even with modified active tetanus toxin, we can
still obtain good results when care is taken to inject the poison with
great circumspection.
But it is not these vaccines obtained from tetanus cultures that have
come to be used in practice. The best results are obtained by the use of
antitetanus serums. After von Behring and Kitasato’s discovery of the
power of the serum of animals immunised against tetanus to neutralise
the action of the tetanus poison, very numerous experiments were made on
the same subject. It has now become possible by treating horses with
large quantities of tetanus toxin to obtain specific serums of
extraordinary activity. Thus several serums are capable of preserving
mice against a lethal dose of tetanus poison if we inject into them a
quantity of serum equal to the one-thousand-millionth of their weight.
Serums of this strength protect domestic animals against tetanus. We
know that many operations on horses, sheep, goats, pigs, and other
mammals are very often followed by a tetanus which is usually fatal.
Castration, amputation of the tail, the ablation of proud flesh or
tumours, the operation for cryptorchitis or hernias, etc. are often
complicated by tetanus. Moreover, tetanus may frequently appear in
horses that have received wounds in the foot or in the lower parts of
the limbs, “Clous de rue,” farrier’s punctures, wire-heels, blows, etc.
[Sidenote: [517]]
With the object of remedying this state of things Nocard[819]
distributed to veterinarians about 70 litres of antitetanus serum to be
employed for protective purposes. The majority of the animals treated
(horses, donkeys, mules, bulls, rams, lambs, and pigs) received two
injections of serum at an interval of 10–12 days, 20 c.c. for large
animals and 6–10 c.c. for sheep and pigs. Of 3088 animals which received
the first injection of serum immediately after the operation not a
single one contracted tetanus. Of 400 animals which received the first
injection at a later period, 1–4 days and more after the accidental
wound of which they had been the victims, one horse only, treated five
days after the accident (farrier’s puncture), was seized with mild
tetanus, but it soon recovered. In the same localities where the results
of the vaccination were so brilliant, 314 cases of grave and fatal
tetanus occurred amongst animals operated upon or injured that were not
submitted to the serum treatment.
It may be readily understood with these facts before us why the practice
of protective vaccinations of animals against tetanus should have spread
so rapidly amongst veterinarians. The demand for antitetanus serum from
the Pasteur Institute of Paris for veterinary use increases every year
at a great ratio. Thus in 1896 there were sent out only 1511 bottles of
10 c.c. each, in 1898 the number rose to 24,959 bottles, in 1900 it
exceeded 43,000.
The efficacy of the antitetanus serum employed as a protective agent can
no longer be questioned, but it must not be forgotten that its injection
does not render the treatment of the wounds unnecessary. These wounds
should receive a rigorous antiseptic cleansing. All foreign bodies
should be carefully extracted; otherwise the prolonged presence of
tetanus spores might set up a late tetanus after the disappearance of
the transient immunity due to the serum.
[Sidenote: [518]]
The protective injections of antitetanus serum into men likely to
contract tetanus are also beginning to spread. It often happens that
bicyclists, in falling, receive injuries which are contaminated by
horse-dung or other matters which may contain the spores of tetanus. In
these cases, as in many other forms of injury, vaccination with
antitetanus serum is indicated. Thus it happens from time to time at the
Pasteur Institute that injured persons come and ask for a protective
injection of serum. Several medical men and surgeons are now accustomed
to vaccinate such of their patients as have had their wounds
contaminated by earth or dung. All the cases of this treatment which
have come to our knowledge have been followed by very good results.
XII. _Vaccinations against diphtheria._ Antidiphtheria vaccinations have
been the subject of much discussion since the discovery of the
antidiphtheria serum and its introduction into routine practice. A large
number of works were published for and against the application of serum
in protective treatment against diphtheria, especially in the early
years of its use. Later the controversy has subsided somewhat, and at
present very few writers are found who continue to decry antidiphtheria
vaccinations.
The antidiphtheria serum was discovered in 1890 by von Behring working
in collaboration with Kitasato; these observers demonstrated in
laboratory animals its neutralising action upon the diphtheria toxin. A
little later von Behring began to apply it in the treatment of
diphtheria, but the early results were far from satisfactory, and von
Behring soon recognised that it was necessary to obtain much more active
serum. Along with Ehrlich of the Institute for Infective Diseases at
Berlin he set to work to study this problem. In collaboration with
several investigators, among whom I may cite Wernicke, Wassermann, and
Kossel, he succeeded in obtaining very encouraging results as regards
the antitoxic strength of the serums and their therapeutic action on
children attacked by diphtheria.
At this time, also, Roux in Paris began, assisted by Martin and
Chaillou, to study the same question. These observers prepared serums
which for that period were very active and made a very effective
application of them upon more than 300 diphtheria patients.
From the year 1894 the use of serum began to spread in all countries,
and it was then that an attempt was made to apply it to the protection
of children in good health, but who had been specially exposed to
contagion.
[Sidenote: [519]]
It was necessary to have at command large supplies of antidiphtheria
serum; this was prepared by injecting into horses repeated doses of the
toxin manufactured by the diphtheria bacillus. The serums thus obtained
were first tested as to their protective, antitoxic, and curative action
on guinea-pigs, animals very susceptible to diphtheria. The necessity of
finding some means of measuring the strength of the serum soon arose.
Von Behring and Wernicke at first standardised it on the basis of the
number of grammes of guinea-pig which could be protected by one gramme
of serum. Later, von Behring[820] introduced the principle of the
“normal serum,” that is to say, a serum of which 0·1 c.c., mixed with 10
lethal doses of diphtheria toxin, is capable of preventing every morbid
symptom in a guinea-pig weighing 300 to 400 grammes.
Ehrlich[821] perfected this method in the following way: to tubes, each
containing 10 lethal doses of a standard toxin, are added different
amounts of serum. These mixtures are brought to the same volume of 4
c.c. by the addition of physiological saline solution, and each is
immediately injected below the skin of a guinea-pig. If 0·1 c.c. of a
serum completely neutralises the 10 lethal doses of toxin, the serum
retains its name of normal serum; in the case where 0·05 c.c. is
sufficient to bring about the same result the serum is designated double
normal serum. When 0·001 c.c. gives the same results, a hundred times
normal serum, and so on. A cubic centimetre of normal serum (that is to
say a dose capable of neutralising 100 lethal doses of standard toxin)
constitutes an “immunising unit” (Immunisirungseinheit (I.E.) of
Ehrlich). As it was soon recognised that toxins, even when kept under
the best conditions, lose more or less of their toxic power, Ehrlich had
to modify his method of standardising serum. He now makes use of a
standard antidiphtheria serum, kept in a dry condition, which is much
more constant than are the toxins. Solutions of this standard serum are
prepared and compared with the serum whose strength has to be
determined. Ehrlich has given a detailed description of the method of
procedure required to obtain exact results.
[Sidenote: [520]]
At the Pasteur Institute Ehrlich’s method has been adopted, supplemented
however by another test for the estimation of the strength of
antidiphtheria serums, a method allied to von Behring’s old method.
Various doses of the serum to be examined are injected subcutaneously
into guinea-pigs, and 24 hours later these guinea-pigs receive a
quantity of a living culture of diphtheria bacilli which kills control
animals in 30 hours. The protective power of the serum in relation to
the weight of the animal is thus determined. For example, a serum which
is said to be active at 1/100,000 has the power, in a quantity equal to
1/100,000th of the weight of the inoculated guinea-pig, of preventing a
fatal result. It was thought, at first, that the protective power,
measured in this way, would be proportional to the antitoxic property
determined according to Ehrlich’s method. But as the results given by
these two methods were often widely different, it was resolved at the
Pasteur Institute to examine by both methods all the serums intended for
use in practice. This led to the conclusion formulated by Roux[822], in
his report communicated to the International Congress of Hygiene, held
at Paris in 1900, that a serum possessing a very high protective power
(against the living diphtheria bacillus) might be only feebly antitoxic,
and _vice versâ_.
[Sidenote: [521]]
This result is explained by the fact that the antidiphtheria serums are
very complex fluids, containing several superposed properties of very
variable strength. Marx[823], of the Frankfort-on-Main Institute, tried
to shake Roux’s conclusions, bringing forward his experiments made on
guinea-pigs and rabbits injected with antidiphtheria serum into the
peritoneal cavity and into the veins. He wished in this way to avoid the
introduction of the serum into the subcutaneous tissues, whence the
absorption of the antitoxin must take place in a very irregular fashion.
In Marx’s experiments, thus carried out, the protective power of the
serums was always found to run parallel with their antitoxic power, from
which he concluded that Roux’s view was incorrect. It must not be
forgotten, however, that this view was founded on experiments in which
the antitoxin had been injected into the subcutaneous tissue before or
simultaneously with the toxin or the diphtheria bacillus. Under these
conditions the protective power is often found to be altogether
disproportionate to the antitoxic power. This fact has been observed so
carefully and with such exactness that it is impossible to deny it. Now
it is undoubted that the conditions of the experiments upon which Roux
relies correspond much more closely with those that are realised in
vaccination of man against diphtheria than with the conditions met with
in Marx’s experiments. In these vaccinations antidiphtheria serum is
injected below the skin of persons whom it is wished to protect against
the action of the diphtheria bacillus.
With the object of bringing about a unification of the methods of
estimating serums used in different countries the International Congress
of Hygiene, held at Madrid in 1898, appointed a special Commission to
settle this problem. But when the Congress met again at Paris in 1900
this Commission had not completed the task allotted to it. The
representatives of the various methods had exchanged ideas, but in
applying the same method the results obtained in various places and by
various observers presented differences too great to allow of any
understanding being arrived at. It is evident that we have here a very
complicated problem. The serums are tested on living animals in which of
course nothing like the constancy of a chemical reaction can be
obtained.
Possibly the methods of breeding and the races of the same animals in
the different countries may be quite sufficient to explain the
divergencies in the results obtained. Whatever may be the reason the
unification of serum estimation has not yet been obtained, and it is
difficult to anticipate that any better result is to be arrived at.
From all this we may draw the conclusion that the possibility of
attaining a too rigorous precision in the standardisation of serum has
been exaggerated. Our object must be to obtain results as favourable as
possible in the application of the antidiphtheria serums, and for that
purpose it is necessary to inject greater quantities than those which
may be indicated by any method of estimation. This rule is applied as
far as is possible at the Pasteur Institute.
As regards vaccination against diphtheria of persons who are in good
health but are especially exposed to infection, the question must be
accepted as settled in the affirmative.
[Sidenote: [522]]
From the commencement of our attempt to cure diphtheria by means of a
specific serum, the necessity was seen of protecting children who were
in contact with the sick persons against this disease. Small quantities
of serum were injected into such children for protective purposes. The
first results communicated in 1894 by Roux to the Congress at Budapest
being very encouraging, an attempt was made to give the greatest
possible extension to the system of vaccination by antidiphtheria serum.
In the following year, 1895, fairly numerous statistics had been
collected, and Torday[824] at Budapest, Kurth[825] at Bremen, and
Rubens[826] at Gelsenkirchen were able to publish a number of favourable
statistics. Soon afterwards, however, a fatal case occurred in the
family of a well-known Berlin doctor, Langerhans[827], an accident that
started a violent controversy and stirred up an active campaign against
serum. Langerhans’s son, a boy aged 2 years, in good health, was
inoculated with a small dose (1·2 c.c. of this serum) and succumbed
about a quarter of an hour afterwards with symptoms of suffocation. The
post-mortem examination made by Strassman[828] showed the cause of death
to be suffocation in consequence of the aspiration of food into the
respiratory passages during the act of vomiting. An examination of the
serum used by Langerhans did not reveal any toxic action on animals or
any contamination by micro-organisms. All to no purpose, the serum was
held answerable for the death of the child, and an attempt was made to
demonstrate at almost any cost that its use in human practice was
extremely dangerous. Gottstein[829] joined in chorus with the
over-excited opinion and published a denunciation of vaccinations by
antidiphtheria serum. He collected from the literature of both
hemispheres four cases, in all, in which death had occurred some time
after the injection of this serum into children not suffering from
diphtheria. A perusal of the description of these cases is sufficient to
convince one that the death could in no sense be attributed to the
serum, and that it could be explained much more easily by the fatal
action of the streptococcus, the cause of the non-diphtheritic
affections of the children that died.
[Sidenote: [523]]
The ineptitude of this denunciation must have done much to calm public
opinion, and in September of the same year, 1896, C. Fränkel[830], in a
report presented to the German Association of Public Hygiene, was able
to give a review of the state of the question of vaccination against
diphtheria, summing up in favour of the use of the specific serum.
“Taking into consideration the data collected,” he remarks, “it is
scarcely possible to doubt the value of immunisation by serum, so that
we may say positively that we are now treading a path which will lead us
to great and important results.” This very favourable opinion was due in
great measure to the vaccinations carried out in the wards of Heubner’s
Clinic at Berlin[831]. At first, injection of the antidiphtheria serum
as a protective into patients who were found in the immediate vicinity
of the children attacked with diphtheria (contacts) was deemed to be
sufficient: but in consequence of the results obtained by this method it
was decided (starting from January, 1896) to inject all children who
came into the hospital. During the first period there still occurred a
few cases of diphtheria contracted in hospital, but from the moment
systematic and general vaccinations were introduced not a single new
case occurred.
The immune condition of the vaccinated children is maintained for three
to four weeks. After this lapse of time some of them contracted
diphtheria. But it was sufficient to introduce revaccination at the end
of this period to prevent the outbreak of any further case of diphtheria
in Heubner’s wards. Results quite as favourable and as convincing were
obtained in the department for children attacked by scarlet fever.
[Sidenote: [524]]
The amount of serum injected varied, but it was usually given in doses
of 1 c.c. containing from 200 to 250 I.E. (immunising units of Ehrlich).
The serum was always found to be innocuous except in certain cases where
it set up erythemata of greater or less extension. In 460 injections 20
cases of these exanthemata were produced, that is to say 4·34%. The
frequency of these complications was not proportional to the amount of
serum injected. According to the figures communicated by Löhr the
largest doses of the serum employed did not produce exanthemata more
frequently than did the smaller quantities. Thus 117 injections of 1
c.c. only were followed in five cases by these erythemata, which
corresponds to 4·27 per cent. The hope of diminishing the frequency of
the exanthemata by diminishing the amount of serum injected was
therefore not realised. This fact lends support to the conclusion above
formulated as to the exaggeration of the importance of the measurement
of serum. If it could be established that small quantities of serum rich
in antitoxin caused cutaneous eruptions less frequently than did
stronger doses there would certainly be a great advantage in using
serums containing a very large number of immunising units for
vaccination. Perhaps serums having a great antimicrobial power but of
comparatively low antitoxic potency might even render great service in
protective treatment. Future researches undertaken in this direction
alone can give us information on this subject.
In 1896 the vaccinations in Heubner’s wards were discontinued, but the
reappearance of diphtheria in 1897[832] rendered their recommencement
necessary. 500 children were vaccinated each with 200 immunising units.
Following this no case of diphtheria broke out. The eruptions were rare
and slight.
The increasing extension of the use of antidiphtheria serum for the cure
of the disease after it has broken out has led to a greater development
in its use as a preventive measure. Thus, in the countries where
diphtheria is endemic, vaccinations by serum are now practised very
extensively. In Russia, which is one of the great hotbeds of this
disease, vaccinations by antidiphtheria serum are frequently practised.
[Sidenote: [525]]
At the Congress of Russian doctors at Kasan in 1896, Vissotsky
communicated the result of 2,185 vaccinations which gave a morbidity of
1·3%, a morbidity that must be regarded as very low indeed. A well-known
Russian physician for children’s diseases, Rauchfuss[833], who cites
these figures, has collected several other facts concerning the
prophylactic injections of antidiphtheria serum followed by good
results. In the government of Woronetz, according to the statements of
Ouspensky[834], out of 738 vaccinated persons diphtheria occurred in 2·2
per cent., which again may be considered a favourable result, especially
if we take into account the great extension of diphtheria in this
country. In Podolia, out of 537 children vaccinated in 1895, only four
cases of diphtheria occurred, a morbidity of 0·74%. In the government of
Kherson, one of the great centres of diphtheria in southern Russia, the
results appear to be less favourable: out of 543 children which received
a protective inoculation, 21 contracted the disease (or 4·6 per cent.),
of which five died. If we study these statistics more closely[835] it
will be seen that these results are far from being unfavourable. The
protective inoculations were made only once and with somewhat small
doses, nevertheless many of the cases of diphtheria broke out only at a
late period, sometimes more than nine months after the injections had
been made. Now, it is proved that these injections, although very
efficacious, produce their action for a very short time only, for a few
weeks at most. Of the five fatal cases, four did not occur until 2, 4½,
6, and 9½ months respectively after the protective inoculation. It is
impossible to look upon these statistics as affording proof of the
inefficacy of the serum. The fifth case is the only one that occurred
within a short time (15 days) of the injection, and in this instance
only 150 immunising units had been injected.
A detailed study of the other examples of antidiphtheria inoculations in
the government of Kherson leaves a very favourable impression. Out of 90
children inoculated by Wecker[836] in the district of Elisabetgrad not a
single one contracted diphtheria, which is the more remarkable as at the
time of the inoculations there existed in the same families 14 cases of
diphtheria; the chances of contamination were thus great.
[Sidenote: [526]]
Recently, on the occasion of the outbreak of a great epidemic in Paris,
the question of vaccinations by serum was again raised and earnestly
discussed at the Paris Hospitals Medical Society and at the Society for
the Study of Children’s Diseases. Voisin and Guinon[837] communicated
the history of an epidemic amongst the staff at the Salpêtrière Hospital
in the wards of idiot children, “against which protective serum
treatment was remarkably effective and absolutely innocuous.” The serum
was injected, in the case of children more than 10 years of age, in 10
c.c. doses, and into the rest in 6 c.c. doses. This measure brought
about first an abatement and then cessation of the epidemic. The
immunity after a single injection lasted from two to three weeks, and
the few cases of diphtheria which broke out amongst the infected
children were distinguished by their great mildness. Erythemata and
other post-injection complications were insignificant, so that the
protective use of the serum was fully justified. Only a small minority
of the medical men who took part in the discussion spoke against the
antidiphtheria vaccinations; once, indeed, a reference was made to the
case of Langerhans’s child, although its death was certainly not due to
the serum. It is true that in families where it is possible to keep the
children under careful observation and to intervene at the appearance of
the first symptoms of diphtheria, the preventive injections may be
dispensed with, but in practice these favourable conditions are rarely
realised, and the prophylactic serum treatment is then of great service
in preventing the outbreak of the disease.
Netter[838] communicated to the Society of Pediatrics a summary of
32,484 observations on the prophylactic injection of antidiphtheria
serum. Of this number 192 cases were noted in which the diphtheria broke
out in spite of the injections, corresponding to 0·6 per cent. of those
treated. These figures, however, included all cases of the disease which
occurred up to thirty days after the injection. Now, the immunity is
often less durable than this, and it may disappear more or less
completely twenty days and sometimes even fifteen days after
vaccination.
Netter himself made great use of antidiphtheria vaccination. It was his
custom to propose to the parents either a protective inoculation at once
or a systematic precautionary bacteriological examination of the throats
of the children not yet attacked. He regards the first method as
preferable. According to the latest statistics which he was kind enough
to communicate to me, of 152 children (in 50 families), 91 of whom
received protective inoculations, not one contracted diphtheria: whilst
in 239 other families where the children had not been inoculated there
were 52 cases of diphtheria, with 10 deaths. Many practitioners in Paris
have now pronounced themselves in favour of protective injections of the
serum, and the Society of Pediatrics, at its meeting on 11th June, 1901,
concluded the discussion of this question by proposing the following
resolution: “The Society of Pediatrics, affirming that protective
inoculations present no serious danger and confer a very considerable
amount of immunity for some weeks, recommend their use when children are
gathered together in numbers, and in families where a scientific
supervision cannot be maintained.”
The large amount of evidence collected on this question leaves no doubt
as to the real efficacy of vaccinations by antidiphtheria serum.
[Sidenote: [527]]
The summary of the results obtained by vaccination in the 12 diseases of
man and of animals I have just placed before my readers cannot pretend
to serve as a detailed guide to prophylactic practice. My object has
been merely to concentrate into one chapter the principal data upon
which this very important question rests, to bear witness to the
progress which has already been realised, and at the same time to show
that the scientific study of immunity is in very intimate relation with
its practical application. It is evident that the road is far from
traversed to its terminus, for there are many infective diseases in
which vaccinations cannot be employed, but it is none the less certain
that the path which has led to so many important and useful results
should still be followed in studying problems which up to the present we
have been unable to solve.
CHAPTER XVI
HISTORICAL SKETCH OF OUR KNOWLEDGE ON IMMUNITY
Methods used by savage races for vaccination against snake venom and
against bovine pleuropneumonia.—Variolisation and vaccination
against small-pox.—Discovery of the attenuation of viruses and of
vaccinations with attenuated micro-organisms.—Theory of the
exhaustion of the medium as a cause of acquired immunity.—Theory
of substances which prevent the multiplication of micro-organisms
in the refractory body.—Local theory of immunity.—Theory of the
adaptation of the cells of the immunised organism.
Observations on the presence of micro-organisms in the white
corpuscles.—History of phagocytosis and of the theory of
phagocytes.—Numerous attacks upon this theory.—Theory of the
bactericidal property of the body fluids.—Theory of the antitoxic
power of the body fluids.—Extracellular destruction of
micro-organisms.—Analogy between bacteriolysis and
haemolysis.—Theory of side-chains.
Progress of the theory of phagocytes.—Attempts to reconcile it with
the humoral theory.—Present phase of the question of immunity.
[Sidenote: [528]]
As protection against disease is one of the most important amongst those
questions which are engrossing the attention of humanity, it is natural
that very great attention should have been devoted to it from the most
remote times. We see primitive races, the ordinary layman, medical men,
legislators and even the most subtle thinkers devoting their energies to
the solution of the problem of immunity against poisoning and against
infections. Historical science will never reveal to us the earliest
sources of our knowledge on this question, so remote are their origins.
The wide distribution of several methods for protecting man and cattle
against certain diseases clearly proves that the origin of this practice
dates from a very early period.
[Sidenote: [529]]
The frequency of venomous snakes in many countries has inspired a dread
of these reptiles, and this must have led to the search for some method
of fighting against the poisoning after the patient had been bitten.
Thus, we find that many primitive races make use of various methods of
immunising the body against the action of venom. The Portuguese colonel,
Serpa Pinto[839], in a letter addressed to d’Abbadie, describes the
method by which he was vaccinated by the Vatuas, natives of the east
coast of Africa. These savages extract the poison of snakes and prepare
from it, by the addition of vegetable substances, a very brown glutinous
paste which they introduce into incisions made in the skin. This
operation is very painful and is followed by a swelling which lasts for
a whole week. The Vatuas assert that this method confers a sure immunity
against the venom. Serpa Pinto was never bitten by a snake, but, a short
time after he had been vaccinated, he was stung, in the Seychelles
Islands, by a scorpion without experiencing any ill effects. This
experience confirms the assertion of the Vatuas, because it has been
shown that the vaccine against snake venom is also efficacious against
the bite of scorpions. The fact that after being stung by another
scorpion ten years later Serpa Pinto was so ill that for eight days he
believed that he was going to die or at least to lose an arm, shows that
he did not enjoy natural immunity, and the innocuousness of the previous
bite must therefore be attributed to a vaccination the effect of which
had disappeared at the end of ten years.
Another vaccinal method used by primitive races is that against the
pleuropneumonia of the Bovidae. De Rochebrune[840] points out that the
Moors and the Pouls of Senegambia have “a custom whose origin is lost in
the obscurity of antiquity” which consists in the inoculation into their
herds of cattle of the virus of the epizootic pleuropneumonia. “The
point of a knife of primitive form, or of a dagger, is plunged into the
lung of an animal that has died from the disease and an incision,
sufficient to allow the virus to penetrate below the skin of the healthy
animal, is made into the supranasal region. Experience has demonstrated
the success of this protective operation.”
In Europe, the vaccinations of cattle with the virus of pleuropneumonia
have certainly been known for more than a century, for, in a pamphlet
published at Berne in 1773[841], mention is made of the “inoculation” of
Bovidae as a means of preventing the disease in England and in Holland,
a disease against which it has been recognised that remedies are
powerless.
[Sidenote: [530]]
The inoculation of the variolous virus into the healthy human subject,
which comes into the same category as the inoculation of the
pleuropneumonic virus into healthy bovine animals, is also a widely
extended and very ancient method. The Chinese[842] assert that they have
known from the commencement of the 11th century the method of immunising
against small-pox. Amongst them, as amongst the Siamese, the matter from
the variolous scab is introduced into the nostrils. In Persia
variolisation is practised by surgeons and by the staffs of bathing
establishments, who introduce the powdered scabs into scratches in the
skin. The Ashantis inoculate the variolous virus into seven places on
the arms and legs. According to the account of Timoni, a Greek physician
practising in Constantinople in the first half of the 18th century, the
Circassians and Georgians, intent upon preserving the beauty of their
daughters, make punctures at various points in the skin, with needles
charged with variolous virus. Everybody is acquainted with the fact that
it was from Constantinople that Lady Mary Wortley Montague at the same
period (1721), imported into Europe “the Greek method,” which consisted
in the inoculation of the contents of small-pox pustules with the object
of producing a benign small-pox and of protecting the vaccinated person
from severe and dangerous small-pox. This practice was widespread in
Europe during the second half of the 18th century, but as it was not
unattended by serious drawbacks an attempt was made to avoid them by the
employment of all kinds of medicaments. As these, however, were found to
be entirely ineffective, the need was felt of replacing variolisation by
some more benign method.
[Sidenote: [531]]
It is asserted[843] that in Baluchistan the custom of having cows
suffering from cow-pox milked by children who had wounds on their hands
has been widespread from time immemorial. This practice conferred upon
these children an immunity against small-pox. It cannot be denied that
the idea of being able to vaccinate with cow-pox was common knowledge
amongst breeders and dairymen in several countries in Europe, especially
in England, France, and Germany. It is stated that Edward Jenner learnt
from the country people of his native county of Gloucestershire that
contact with cow-pox protected against small-pox. Being a man of great
understanding and culture, he set himself to verify this opinion
experimentally. Having demonstrated by a great number of experiments
that the inoculation of variolous virus into persons vaccinated by
cow-pox had no ill result, he became the great propagandist of the new
method. He worked at this subject for 20 years but only decided to
publish his results (in 1798) after he had completely satisfied himself
of the great utility of vaccination with the virus of cow-pox. At first
Jenner’s discovery met with great opposition, but his method was soon
verified in France and several other countries and it was not long
before it was generally practised.
[Sidenote: [532]]
When Pasteur set himself to study the infective diseases in their
relation to micro-organisms the idea of profiting by the discovery of
these pathogenic organisms and of drawing from them a weapon against
infections soon arose in his mind. He studied Jenner’s work in order to
extract from it any indications capable of putting him into the right
path. He induced his collaborators to carry out several series of
experiments with the object of immunising the animal organism against
infective micro-organisms. During this laborious and original work
chance[844] helped in the accomplishment of his task. When, at the
conclusion of the holidays in the autumn of 1879, Pasteur and his
collaborators Chamberland and Roux wished to resume their experiments on
fowl cholera, they found to their great surprise that the
micro-organisms of this disease, usually so fatal, had become innocuous.
Fowls, that received doses of cultures much more than sufficient to
cause death, did not experience any ill effect. Prepared by his previous
knowledge and by the continual direction of his thoughts to the
prevention of contagious diseases, Pasteur divined at once the great
bearing of this check in his inoculations with old cultures, and
immediately began to make precise experiments as to the vaccinating
power of these micro-organisms which had become innocuous. These
researches led him to the discovery of two great principles: that of the
attenuation of viruses, and that of the vaccinating property of
attenuated micro-organisms. Various memoirs by Pasteur[845] established
these laws in a very exact manner; moreover he gave all the information
necessary to allow of the principal results being controlled and
verified. In France, this great discovery was at once accepted by
various investigators, though others found occasion to manifest their
scepticism. Abroad this discovery met with very lively opposition and
this from the highest authorities, who would not recognise the
possibility either of attenuating the virus or of conferring immunity
upon animals. The anthrax bacillus can be grown for a very long time on
culture media, the potato, for example, without losing its pathogenic
power in the slightest degree. Therefore, it was said, this attenuation
of virus can have no actual existence. White rats that have resisted one
or more inoculations of the anthrax bacillus may die from a later
inoculation of the same micro-organism. Therefore there is no acquired
immunity, etc. The principles laid down by Pasteur are from every point
of view of such prime importance, that very numerous experiments were
carried out at once for the purpose of verifying their exactness and the
contest was not a long one. In the course of a few years it was
universally recognised that the attenuation of viruses, and also the
vaccination by attenuated micro-organisms, were realities which
henceforth cannot be denied and which must pass into the domain of
truths definitely acquired. An attempt was then made to extend these
fresh victories to the other infective diseases. Pasteur, Chamberland,
and Roux applied themselves to devising a method of vaccinating animals
against anthrax and against rabic virus; Pasteur and Thuillier extended
their researches on this subject to swine erysipelas. From several other
quarters the search for vaccines was instituted. Toussaint made various
attempts, at times crowned with success, to immunise animals against
anthrax by means of heated anthrax blood. Arloing, Cornevin, and Thomas
succeeded in vaccinating the Bovidae against symptomatic anthrax.
Loeffler was the first in Germany to demonstrate that rabbits which had
recovered from the disease set up by the bacillus of mouse septicaemia
acquired an immunity against the attacks of this organism. It is not
necessary to cite further examples, so numerous have they become and so
unanimously confirmatory.
[Sidenote: [533]]
After the first steps had been taken along this new path Pasteur and his
collaborators began to apply the knowledge they had gained to the
preparation of vaccines capable of giving practical results. The two
anti-anthrax vaccines and the two vaccines against swine erysipelas were
the fruit of these attempts. Here, again, numerous objections were
raised against these discoveries. Sheep which had received enormous
quantities of the bacillus may die from anthrax in spite of the two
Pasteurian vaccines and from that it was wished to conclude that these
vaccines should not be employed in practice to protect sheep against the
anthrax fever. The results of experiments made on a large scale in
various parts of the globe have demonstrated the inadequacy of these
objections and these questions are now regarded as definitely settled.
[Sidenote: [534]]
So large a number of investigations, in response to the most urgent and
immediate needs, was not favourable to minute researches on the
mechanism of this immunity which had been revealed in so marvellous a
fashion. In spite of this, Pasteur applied himself to the solution of
this problem so far as this was possible under the conditions in which
he carried on his investigations. He thought that acquired immunity was
the result of the impossibility of the growth of a pathogenic
micro-organism in a medium in which it had previously been cultivated.
When the micro-organism of fowl cholera sets up in certain individuals a
disease which though grave is not fatal, or when the attenuated
micro-organism produces a simple, transient discomfort, it lives in both
cases in the fluids and tissues of the animal. This existence is
possible in consequence of the absorption of certain nutrient
substances. Once these substances are consumed they are not easily
renewed, and in consequence the vaccinated organism becomes incapable of
nourishing the special micro-organism a second or a third time. To
support this brilliant hypothesis by precise facts Pasteur made
experiments on the conditions met with in the development of the
micro-organism of fowl cholera _in vitro_. He filtered a broth culture
of this micro-organism after it had grown luxuriantly for several days,
and into the fluid, which had now become clear and transparent, he sowed
afresh the same micro-organism. No growth took place and the fluid
remained quite clear. This absence of development might be explained
either by the presence in the fluid of some excremental substance thrown
off during the first culture or by the absence of some substance
indispensable for the nutrition of the micro-organism. Pasteur excluded
the first hypothesis by an experiment which demonstrated that it is
sufficient to add to the filtered fluid a small quantity of fresh
nutritive substances to enable the micro-organism again to develop
abundantly. It is therefore to the absence of some element essential to
the existence of the micro-organism that we must attribute the immunity
enjoyed by animals which have been vaccinated or which have undergone
spontaneous cure. This is how Pasteur[846] expressed himself on this
point: “the muscle which has been much affected has, even after healing
and repair, become in some way incapable of supporting the growth of the
micro-organism, as if the latter, by a previous culture, had eliminated
from the muscle some principle that life does not bring back and whose
absence prevents the development of the small organism. There is no
doubt that this explanation, to which the plainest facts at the moment
lead us, will become general and applicable to all the virulent
diseases.”
This explanation appeared to be a reasonable one to several observers,
amongst whom I may cite Chauveau[847], the distinguished author of
important works on viruses. “In all probability this seductive theory,”
says Chauveau, “based on one of the most interesting of those clear and
decisive experiments for which Pasteur is famous, applies to the
majority of cases of immunity acquired by protective inoculation.” But
Chauveau thinks that it does not explain natural immunity, especially
that of the Algerian sheep, against anthrax, an example that he had
studied on several occasions. When he inoculated into these animals
large quantities of anthrax bacilli, not going beyond certain limits,
the sheep resisted perfectly; but injections of enormous doses were
nearly always capable of overcoming this natural immunity of the
Algerian sheep and of inducing in them a fatal anthrax. Chauveau thinks
that this fact is best explained by the presence of an inhibitory
substance in the blood plasma, whose action becomes exhausted when
distributed over a very large number of bacilli. This opinion was not,
however, shared by Pasteur[848], who raises the objection that natural
immunity can really be produced and maintained without the presence of
this inhibitory substance from the fact that fowls, which exhibit such
marked resistance against anthrax, readily contract the disease when the
temperature of their bodies is lowered. Under these conditions it is
unimaginable that an inhibitory substance has disappeared under the
influence of cold.
[Sidenote: [535]]
The controversy existent from the birth of theories on immunity shows us
that from the very commencement the problem was found to be a very
complex one, and that to attack it in a satisfactory way we must as far
as possible multiply and deepen our study of the phenomena which
accompany the resistance of the animal against pathogenic
micro-organisms. Thus, Chauveau[849] was not long before he undertook
experiments having for their object the determination of the fate of
anthrax bacilli when injected into the blood vessels of Algerian sheep.
He found that these organisms disappeared from the blood at the end of a
few hours, but they were then to be found accumulated in the lung,
spleen, and certain other viscera. In these positions the bacilli become
incapable of reproducing themselves and in refractory individuals soon
disappear, being opposed by the inhibitory substances of the blood
plasma.
The two theories just sketched have this point in common, that they both
attribute the natural or acquired immunity to humoral and purely passive
properties. According to one theory it is the impoverishment of the
fluids of the animals which prevents the development of the pathogenic
organism, whilst according to the other it is the presence of some
bacterial poison which brings about the same result. To give
experimental support to his theory Pasteur brought forward his attempts
at sowing micro-organisms in culture media exhausted by a previous
development of the same organism, eliminating, so to say, the active
influence of the animal organism. It is true that, in order to explain
natural immunity, it was necessary to ascribe a rôle to the
“constitution” and to the “vital resistance,” interpreting this, as
Naegeli had already done, in the sense of a competition for the oxygen
and the nutritive substances between the parasites and the cells of the
body.
[Sidenote: [536]]
Adopting this point of view, Hans Buchner[850], a pupil of Naegeli,
attempted to gain a more precise idea of the conditions under which
acquired immunity against infective diseases is set up. He developed his
theory in various publications; this theory consists, briefly, in the
property of the animal organism to reinforce the local resistance of the
organs by means of an inflammatory reaction. The starting-point of this
local theory is the thesis that each pathogenic micro-organism can only
manifest its pathogenic action when it enters the particular organ in
which it is capable of living and maintaining itself. Thus, the
pneumonococcus can live in the lungs only, the cholera vibrio in the
intestines only, and so on. Every time that a pathogenic micro-organism
becomes localised in its special organ, an inflammatory action is set up
which results in the reinforcement of the living elements of the organ
in question. Inflammation, therefore, is regarded by Buchner as a
salutary reaction, which acts, not directly on the exciting morbific
cause, but through the mediation of the specific cells of the organs.
This theory of immunity led Buchner to propose arsenical treatment as a
remedy against microbial disease, because arsenic is, of all drugs, the
one capable of setting up the greatest inflammatory reaction.
Another German observer, Grawitz[851], proposes a theory of acquired
immunity, according to which a first attack of an infective disease sets
up “the adaptation of the cells to the power of energetic assimilation
of the fungi.” This reinforced adaptation is transmitted to the
descendants of the cells which have acquired it, and for that reason the
immunity may persist for months, and even years. Grawitz attempted to
base his views on experiments on the acquired immunity against the
fungus of the lily of the valley, but Loeffler[852] soon demonstrated
that this thesis could not be maintained, and that the immunity assumed
by Grawitz did not, in reality, exist.
It will be seen that all the theories summarised above are marked by
their vague character and want of precision; this is not at all
astonishing when we take into consideration the very imperfect knowledge
of the phenomena of immunity. It is evident that if we wish to gain a
satisfactory idea of the mechanism of the resistance of the animal body
against pathogenic micro-organisms, we must inform ourselves as to the
modifications which take place in the organs and tissues at the time of
the acquisition of the immunity, and also find out what becomes of the
micro-organisms in a refractory animal.
We have seen that Chauveau demonstrated that anthrax bacilli when
injected into the vessels of Algerian sheep disappear, but he was unable
to say anything as to the way in which this disappearance was brought
about in nature. Buchner accepted the reinforced resistance of inflamed
organs without being able to describe the phenomena which manifest
themselves during the inflammation of tissues invaded by the pathogenic
micro-organisms.
[Sidenote: [537]]
Independently of these theoretical and rather speculative views on
immunity, there has been an addition to our scientific assets of fairly
exact data on the relation of certain pathogenic organisms to the organs
and tissues of susceptible or refractory animals. When, as a result of
the labours of Davaine and Obermeyer, the attention of pathologists,
especially of those working at pathological histology, was drawn to the
part played by micro-organisms in infective diseases, a diligent search
was instituted for these organisms in sections of the organs of persons
who had died from various diseases. Masses of cocci especially were
found in the organs of individuals who had died from diphtheria,
puerperal fever, and various forms of pyaemia. In the course of these
investigations attention was drawn fairly frequently to the presence of
micro-organisms inside the white corpuscles of pus and of other morbid
products. Amongst the first to make this observation I may cite
Hayem[853] in France, and Birch-Hirschfeld[854], Klebs, Rindfleisch, von
Recklinghausen, and Waldeyer in Germany. Klebs[855] speaks of the
presence of micro-organisms in infected wounds, in the interior of
contractile white corpuscles, and attributes to these cells the
principal rôle in the transport of these parasites in the lymphatic
tissue. Waldeyer[856] cites a case of puerperal fever in which the
corpuscles of the peritoneal pus were filled with bacteria. Similar
observations were by no means rare; and they led to a general conclusion
that micro-organisms meet with such favourable conditions inside the
leucocytes that they would contribute to their dissemination through the
body. This opinion had become so general that when Koch[857], in frogs
inoculated with anthrax bacilli, made the discovery of round cells
containing large numbers of these micro-organisms he did not hesitate to
conclude that the bacilli found a favourable medium in the substance of
these elements. Now the frog, under ordinary conditions, is refractory
to anthrax.
[Sidenote: [538]]
As early as 1874, however, Panum[858] had given expression to the view,
in a vague fashion it is true, that leucocytes might assist in the
destruction of micro-organisms. In his memoir on putrefactive poisons we
find a note wherein occurs the following reflection: “For the solution
of the question as to how and in what situations the ordinary bacteria
of putrefaction disappear, an interesting communication made by
Birch-Hirschfeld seems to me to furnish an indication. According to this
observer the micrococci, introduced into the circulation, are deposited
in the lymphatic glands and in the spleen, after having, for the most
part, entered into the blood corpuscles. That the ordinary bacilli of
putrefaction really die in the body is proved, not only by the
circumstance that they remain inactive after the acute paroxysm of
putrid intoxication has been happily surmounted, but also by the
important observations made by Eberth on the innocuousness of the
inoculation of ordinary bacteria into the cornea.” These lines contain
the indication that the corpuscles of the blood (in this case
undoubtedly leucocytes) ingest the bacteria introduced in the blood
current and destroy them.
Some years later, in 1877, Grawitz[859], in connection with his
researches on the parasite of the lily of the valley, made the remark
that the fungi, when introduced into the blood of mammals, are seized by
the white corpuscles and thus “withdrawn from contact with the
assimilable fluid.” Gaule[860] who, as we know, sought to demonstrate
that the _Drepanidium_ of the frog’s blood is nothing but the fragments
of cell nuclei transformed into ‘Würmchen,’ has described the structure
of these organisms in the amoeboid cells of the spleen. “I happened on
one occasion,” he writes, “to observe an amoebocyte of the spleen of the
frog which in a short time ingested three ‘Würmchen,’ and then went away
briskly without leaving any trace of where it had been. Following its
movements I was able at the first to make out within the contents of the
amoebocyte the refractile body of the ‘Würmchen.’ But this body became
paler, and half-an-hour later it had been completely assimilated.”
Undoubtedly these “Würmchen” were nothing but parasites (_Drepanidium_),
and have no connection with the cell nuclei of frogs. Their ingestion,
followed by destruction, was, therefore, a defensive act on the part of
the body manifested by the amoeboid cells of the splenic pulp.
[Sidenote: [539]]
[Sidenote: [540]]
In the same year, 1881, in which this observation by Gaule was
published, Roser[861], assistant in surgery at Marburg, published a
small pamphlet on the lower animals. In this pamphlet the possibility of
growing certain unicellular organisms in urine and milk and the
adaptation of these organisms to saline solutions received special
mention. At the end of one of his paragraphs Roser expresses his views
on immunity, although this subject was not discussed at all in his
pamphlet. He expresses himself thus: “The immunity of animals and plants
in complete health depends in my opinion: (1) on the relative quantity
of salt contained in their fluids, and (2) on the property of their
contractile cells of ingesting the enemy which enters the animal body”
(p. 18). As these statements have been put forth without receiving any
further development, in the midst of all kinds of other speculations, it
is not astonishing that the words I have just quoted, as well as Roser’s
pamphlet itself, should not have attracted the attention of either
zoologists or medical men. In the reviews for these two sciences
(Schmidt’s _Jahrbücher_ and the _Zoologischer Jahresbericht_ of the
Zoological Station at Naples) it is not even mentioned. It appears that
not only did other biologists and medical men attach no importance to
Roser’s speculations, but that the author himself did not claim any
great value for them. I draw this conclusion from the fact that five
years after his first pamphlet he published a second on inflammation and
healing[862] in which he does not apply his theory of immunity to
explain these two phenomena. This new work is of an even more
speculative character than was the first, and instead of attempting to
show any relation between the anti-infective part played by the
leucocytes and their migration during inflammation, Roser insists on the
fundamental independence of this phenomenon of healing. For him the
inflammation, accompanied by diapedesis, must not be looked upon as a
healthy reaction of the body, but as a manifestation of disease. The
heat which is observed under these conditions must be attributed in part
at least to the production of heat by infective micro-organisms. I must
confess that Roser’s two pamphlets were unknown to me for many years,
and it was Hueppe who drew my attention to them by his mention of them
in the fourth edition of his work on bacteriological methods[863] which
appeared in 1889. I had then, independently of the Marburg surgeon and
by a totally different path, arrived at my conclusions as to the part
played by the amoeboid cells. At the commencement of my researches on
healing and immunity the passages cited above from the publications of
Panum, Gaule, and Grawitz were also unknown to me. Having long studied
the problem of the germinal layers in the animal series, I sought to
gain some idea of their origin and significance. The part played by the
ectoderm and the entoderm appeared quite clear, and the former might
quite reasonably be regarded as the cutaneous investment of primitive
multicellular animals, whilst the latter might be regarded as their
organ of digestion. The discovery of intracellular digestion in many of
the lower animals led me to regard this phenomenon as characteristic of
those ancestral animals from which might be derived all the known types
of the animal kingdom (excepting, of course, the Protozoa). The origin
and the part played by the mesoderm appeared the most obscure. Thus,
certain embryologists supposed that this layer corresponded to the
reproductive organs of primitive animals: others regarded it as the
prototype of the organs of locomotion. My embryological and
physiological studies on sponges led me to the conclusion that the
mesoderm must function in the hypothetically primitive animals as a mass
of digestive cells, in all points similar to those of the entoderm. This
hypothesis necessarily attracted my attention to the power of seizing
foreign corpuscles possessed by the mesodermic cells. This fact has long
been recognised. It was known that the white corpuscles of the
Vertebrata often contained various kinds of cells, especially red and
white blood corpuscles. It was known, also, that the amoeboid cells were
capable of ingesting granules of coloured substances. When making an
injection of indigo into the vessels of _Thetys_, Haeckel[864] in 1858
was surprised to find the blue granules inside the amoeboid blood
corpuscles of this beautiful gasteropod mollusk. This fact has since
been confirmed by many observers, and the capacity of the amoeboid cells
to take up foreign bodies became recognised as a general phenomenon.
Nevertheless this phenomenon was not regarded as being analogous to
digestion. Thus Haeckel[865] himself, in his researches on the
calcareous sponges, advocated the view that the foreign bodies
penetrated into the interior of the viscous protoplasm in a purely
passive fashion.
[Sidenote: [541]]
Observations that I made on sponges and on certain pelagic animals,
transparent and of simple organisation, convinced me that the presence
of foreign corpuscles in the amoeboid cells of the mesoderm must be
attributed to an active ingestion by these cells which, in every
respect, might be compared to the phenomena of intracellular digestion
in the epithelial cells of the digestive canal of many of the lower
animals. In order to demonstrate this fact clearly it was necessary to
bring forward exact experimental proof. I set myself, therefore, during
my stay at Messina in 1882 and 1883, to study the rôle of the amoeboid
cells of the mesoderm from the point of view of intracellular digestion.
I found it an easy matter to demonstrate that these elements seized
foreign bodies of very varied nature by means of their living processes,
and that certain of these bodies underwent a true digestion within the
amoeboid cells. My principal thesis, that is to say the idea of the
intimate relations between the entoderm and the mesoderm, was thus fully
confirmed.
Pondering over these results, which were quite new at the time, the idea
suggested itself to me that the digestive function, so profoundly rooted
in the mesodermic elements, must play a part in many of the vital
phenomena of animals. Starting from this standpoint, I succeeded in
demonstrating that, during the very complicated metamorphoses of
Echinoderms, such as the _Synaptae_, the amoeboid cells of the mesoderm
fulfil a function in the atrophy of numerous larval organs. I have never
prosecuted any medical studies; but some time before my departure for
Messina I listened to the reading of Cohnheim’s treatise on General
Pathology, and I was struck by his description of the facts and of his
theory of inflammation. The former, especially his description of the
diapedesis of the white corpuscles through the vessel wall, seemed to be
of momentous interest. His theory, on the other hand, appeared to be
extremely vague and nebulous. It occurred to me that a comparative study
of inflammation in lower animals of simple organisation would certainly
throw light on the very complex pathological phenomena in the
Vertebrata, even in the frog which had served as the starting-point for
Cohnheim’s remarkable experiments.
[Sidenote: [542]]
Since, in the atrophy of the larval organs of the _Synaptae_, the
essential rôle is accomplished by the amoeboid cells of the mesoderm
which accumulate and unite into masses, the richness of inflammatory
exudations in white corpuscles may perhaps signify that these corpuscles
have a very important function to fulfil. This reflection led me to make
the following experiment: to wound and introduce spines beneath the skin
of very transparent marine animals; if my hypothesis should be well
founded this should bring about an accumulation of amoeboid cells at the
injured spot. I selected for this purpose the large Bipinnaria larvae of
starfish, so abundant at Messina, and inserted prickles of the rose into
their bodies. Very shortly these prickles were found to be surrounded by
a mass of amoeboid cells such as we see in human exudation as the result
of the introduction of a spine or other foreign body. The whole process
took place under my eyes in a transparent animal possessing neither
blood nor other vessels, nor a nervous system. The first point was
settled. The inflammatory exudation must be considered as a reaction
against all kinds of lesions, the exudation being a more primitive and
more ancient phenomenon in inflammation than are the functions of the
nervous system or of the vessels.
I know quite well that, at the period when I made my researches (1882),
pathologists regarded inflammation as the consequence, if not always, at
least in the majority of cases, of the penetration of micro-organisms.
From this followed the conclusion that the diapedesis and accumulation
of white corpuscles in inflammatory diseases must be regarded as modes
of defence of the organism against micro-organisms, the leucocytes in
this struggle devouring and destroying the parasites. According to this
hypothesis the significance of inflammation at once became simple and
clear. With the object of verifying my hypothesis I began to make
experiments on the lower animals, so abundant in the Straits of Messina,
and to make myself acquainted with the results that had been obtained in
general pathology and in pathological histology. A perusal of Ziegler’s
treatise on Pathological Anatomy made it clear to me that in these
branches of medical science there had long been accumulated a great
number of observations fitted to facilitate the acceptation of the new
hypothesis on inflammation and healing. Numerous and well-established
facts on the absorption of extravasated blood, on the fate of the
coloured corpuscles in the body, on the presence of micro-organisms
inside leucocytes, etc., confirmed me in my view.
[Sidenote: [543]]
When I had got together certain information and a number of facts in
support of my hypothesis I communicated the results to my lamented
friend, Kleinenberg, at that time Professor in the University of
Messina. Both medical man and zoologist, he was well qualified to offer
a judgment upon the matter; this judgment was favourable. Sometime later
I had the great pleasure of meeting the celebrated Professor Virchow at
Messina. I imparted to him my ideas and he was kind enough to come with
me to examine my preparations of Bipinnaria larvae and other lower
animals in which I had set up the phenomena of inflammation without the
assistance of nervous or vascular systems. This eminent observer greatly
encouraged me to continue my investigations. When I explained to him my
view that the inflammatory reaction on the part of the amoeboid cells
could only be understood by accepting the hypothesis that the white
corpuscles gave chase to the micro-organisms and destroyed them, Virchow
replied that in pathology just the opposite was invariably taught. The
general opinion was that micro-organisms were certainly found inside the
leucocytes and that they made use of these cells as a means of transport
and of dissemination through the body.
[Sidenote: [544]]
During my stay at Messina my researches were limited to the lower
animals, but later I began to study inflammation and the phenomena of
infection in the Vertebrata. It was not until eight months after I had
commenced my researches in this direction that I decided to publish my
results. I first set them forth in an address given at Odessa before the
Congress of Naturalists and Medical Men in 1883. Later, they were
published in a special article inserted in Claus’s _Arbeiten_ at
Vienna[866], and in a small work which appeared in the _Biologisches
Centralblatt_[867]. I sought especially to develop the idea that the
intracellular digestion of unicellular organisms and of many
Invertebrata had been hereditarily transmitted to the higher animals and
retained in them by the amoeboid cells of mesodermic origin. These
cells, being capable of ingesting and digesting all kinds of
histological elements, may apply the same power to the destruction of
micro-organisms. In order to support this conclusion I introduced
various kinds of bacteria into the bodies of some of the lower animals
and I demonstrated that they were ingested and destroyed by the amoeboid
cells. It was evident, however, that this proof was not sufficient. I
then set myself to study the diseases of small Invertebrata sufficiently
transparent to be observed directly under the microscope. The
_Daphniae_, those small crustacea so numerous and so frequent in
fresh-water, furnished me with a favourable medium in which to study a
real struggle which takes place between their leucocytes and the spores
of a vegetable parasite belonging to the group of the Blastomycetes. In
many cases the amoeboid cells guarantee the integrity of the animal by
devouring a large number of these spores and transforming them into an
inert detritus. In other cases, on the contrary, the fungi get the upper
hand in the struggle; they succeed in germinating and in overcoming the
resistance of the leucocytes by reproducing themselves rapidly and by
killing these cells with their poisons. The history of this disease and
of this struggle was published in _Virchow’s Archiv_[868].
Some time afterwards I published in the same journal my work on the
anthrax bacillus[869], in which I attempted to demonstrate that in the
Vertebrata also the invasion of pathogenic micro-organisms sets up a
desperate struggle between them and the amoeboid cells.
In these four works I made use of the term “phagocytes” to designate the
amoeboid cells capable of seizing and digesting the micro-organisms and
other formed elements. To the theory based on this property of the
defensive cells I gave the name of “theory of phagocytes.”
I thought, as already mentioned above, that the observations on
absorption and leucocytes, which had been accumulating for years in
pathological histology, had sufficiently paved the way for a favourable
reception to the idea that the amoeboid cells are defensive elements of
the body capable of guaranteeing to it immunity and cure. In this I was
mistaken. It was precisely the specialists in this branch of science who
from the first manifested the most lively opposition to this theory.
However, in the Presidential Address delivered before the 66th meeting
of the British Association held at Liverpool in 1896, Lord Lister
said[870]: “If ever there was a romantic chapter in pathology, it has
surely been that of the story of phagocytosis.” These words encourage me
to put before the reader the essential features of this story.
[Sidenote: [545]]
My first two memoirs published in 1883 did not in any way attract the
attention of the medical public. These investigations had a character
that was too zoological to be noticed by pathologists. But the two
following publications, in which I treated of the _Daphnia_ disease and
especially of bacterial anthrax, immediately roused severe criticism.
Baumgarten[871], the well-known pathologist, opened the battle by the
publication of a review of my researches on phagocytosis. He attempted
to sap the basis of my theory, and not contented with _à priori_
arguments, he set his pupils to make a series of researches on the fate
of micro-organisms in the refractory animal. These researches resulted
in several theses for the doctor’s degree which sought to demolish every
point of the theory of phagocytosis.
Later, Baumgarten[872] published a long and above all admirably written
analytical article entitled: “Zur Kritik der Metschnikoff’schen
Phagocytentheorie,” in which, with much talent and wit, he attempted to
demolish the bases and conclusions of the phagocytic theory.
Baumgarten regards the precise observations which I had been
accumulating for some years as incorrect and refuted by the observations
and experiments of his pupils. The arguments that I give to justify my
theory are, according to the same critic, contrary to logic and to
truth. If the phagocytes are really elements destined to guarantee the
integrity of the animal organism how is it, asks Baumgarten, that just
at the moment of greatest danger, when the blood and the tissues are
invaded by the micro-organisms, the leucocytes are conspicuous by their
absence? The answer that there is no predestination in the phagocytosis,
and that the danger is the greater the more feeble the phagocytic
reaction—a fact which is in perfect harmony with the law of causes and
with the principles of the evolution of species according to Darwin’s
theory—did not satisfy my critic. He says: “If the interpretation which
Metschnikoff gives of the activity of the leucocytes appears to be
rather the product of a rich imagination than the result of the
objective observation of the seeker, it matters little that his account
of the development of the leucocyte in what he wishes to see in it
should be in conformity with the principles of the theory of evolution”
(p. 4).
[Sidenote: [546]]
I was able by numerous researches[873] to refute point by point the
objections based on the work of Baumgarten’s pupils, but that did not
prevent him from persisting in his negation. Only, commencing by writing
long articles, he contented himself, later, with denying the theory of
phagocytosis in small annual notes, appearing in his reviews of works on
bacteriology, which were unsupported either by argument or by any facts
mentioned in his abstracts.
Baumgarten’s example was followed by many other pathologists. Ziegler,
the well-known author of a text-book on pathological anatomy that has
certainly had a wider circulation than any other work, vigorously
attacked the theory of phagocytosis. As it was precisely from this
treatise that I had acquired my knowledge of the large number of facts
that had accumulated in pathological literature on the part played by
leucocytes in resorption, I was persuaded that Ziegler, who had
collected these statements, would be one of the first to recognise the
importance of phagocytosis in inflammation, healing, and immunity. But
this distinguished pathologist, in several of his publications[874],
expressed himself very vigorously against the phagocytic theory. The
intervention of these cells, according to him, must be purely accidental
and their rôle in the defence of the body against the micro-organisms
very insignificant. The better to demonstrate this thesis he caused his
pupils to undertake investigations on several infective diseases, and
these young observers all arrived at the same result, that phagocytosis
has nothing to do with the struggle of the animal against the anthrax
bacillus or against the bacillus of symptomatic anthrax. It is the less
necessary to enter into these details now because I have, in the
preceding chapters, given sufficient proofs of the incorrectness of the
objections advanced by Ziegler’s school. It has been demonstrated most
conclusively (by Lubarsch’s researches, as well as by many other works)
that in anthrax in man phagocytosis, denied by one of Ziegler’s pupils,
is most marked. It is likewise well known from the researches of Ruffer,
Leclainche and Vallée, as well as from my own observations, that in
symptomatic anthrax, in which the phagocytic reaction is denied by
another of Ziegler’s pupils, it is a very important and highly developed
feature.
[Sidenote: [547]]
The opposition emanating from another eminent pathologist, Weigert[875],
particularly impressed me, because this investigator is known not only
to be an observer of great accuracy but to possess a mind of great
imagination and generalising power. In several papers he put forward his
utmost ingenuity to demolish the phagocytic theory root and branch. He
would recognise neither the importance of phagocytosis in healing and
immunity, nor the defensive function of the giant cells. Weigert,
however, contented himself with formulating theoretical objections, and
no works directed specially against the doctrine of phagocytosis have
issued from his laboratory. It must be stated, however, that although
there has been such opposition on the part of certain of our most
eminent pathologists, others amongst them have, from the beginning,
expressed themselves in more favourable terms. Thus, Virchow[876], in an
introductory article in the 101st volume of his _Archiv_, continued his
friendly attitude with regard to the works on phagocytic defence and
spoke of them as opening up a new field of research. Ribbert[877], in a
series of publications, maintained the importance of the phagocytes in
the resistance offered by the animal to the aggression of
micro-organisms, and pointed out, especially in connection with the
diseases set up by the staphylococci, the frequency of the ingestion of
these parasites by the leucocytes. He insists specially on a
modification of the phagocytic reaction, which consists in the
accumulation of white corpuscles around the centre of microbial
infection. In these cases, without the occurrence of any real ingestion
of the micro-organisms into the substance of the phagocytes, these
organisms may have their morbific manifestation hindered by the
assemblage of the white corpuscles. It is needless to insist that this
act, which I referred to in my first work in 1883, constitutes the
prelude to a true phagocytosis and is closely bound up with this
defensive phenomenon. Another pathologist, Hess[878], supports the
theory of phagocytosis by confirmatory researches of great value.
[Sidenote: [548]]
The pathologists who were adversaries of the phagocytic theory combined
their efforts to demolish it, without troubling themselves to replace it
by any other theory of defence on the part of the body which might more
easily be made to accord with their principles and their statements.
Baumgarten certainly tried to prove that micro-organisms perish in cases
where immunity is produced or healing occurs, not as the result of the
phagocytic reaction or of any other manifestation on the part of the
menaced animal, but simply “of themselves” (von selbst), that is to say,
they have simply accomplished the normal cycle of their existence and
die a natural death, this bringing about healing and immunity. As may be
readily understood he was unable to bring forward the slightest evidence
of the correctness of this hypothesis, which, I believe, has never been
accepted by anyone, nor even been defended by its author. In this
respect the attacks directed against the theory of phagocytosis by
bacteriologists have been of a very different character. Not content
with overturning this hypothesis, these observers have sought to build
upon its ruins new theories capable of offering a better explanation of
the phenomena of immunity. I must here confess at the outset that these
attacks have been much more important than those coming from the
pathologists and pathological anatomists, and have led to discoveries of
the greatest value.
One of Fodor’s experiments[879], one not altogether new, served as the
point of departure for much work and for a large series of objections
directed against the phagocytic theory. The Hungarian investigator found
that the defibrinated blood of the rabbit was capable of destroying _in
vitro_ a great number of anthrax bacilli. From this it was concluded
that the fluids of the living body possessed a bactericidal power
sufficient to explain the immunity against infective micro-organisms.
The destruction of the anthrax bacillus by defibrinated blood was
confirmed by a young American investigator of great talent,
Nuttall[880], who carried out an important work on this subject in the
laboratory and under the direction of Flügge at Breslau. He was able to
follow step by step, by the observation of anthrax bacilli on the warm
stage, their degeneration under the action of the defibrinated blood.
This destruction of the bacilli took place outside the phagocytes. The
same phenomenon could be shown by the method of gelatine plate cultures.
The bacilli, subjected to the influence of the defibrinated blood of
rabbits and other vertebrates, usually died or were markedly injured.
The blood when heated to 55° C. completely lost its bactericidal power.
[Sidenote: [549]]
These observations, perfectly exact in every detail, gave Flügge[881]
and his assistant Bitter[882] the opportunity to criticise vigorously
the theory of phagocytosis. The cells were said to be incapable of
ingesting living micro-organisms; these latter must be previously
destroyed by the bactericidal action of the body fluids, and it was only
their dead bodies which were devoured by the phagocytes.
Flügge based his criticism upon considerations of a general character
and upon observations made mainly by Nuttall. “There is no necessary
point of analogy,” says the learned Breslau hygienist, “between the
ingestion of food and the struggle against infective micro-organisms,
nor between nutritive substances and living micro-organisms” (p. 225).
“From Nuttall’s results it must evidently be accepted as possible that
the phagocytes can ingest dead bacteria only and that they have not the
power of ridding the body of the living infective agents” (p. 226). The
following passage is especially significant. “When we examine, with an
open mind, a series of preparations which show the relations between the
phagocytes and the bacteria in various infective diseases, the
phagocytes sometimes present themselves as the victims of the bacteria,
which continue their triumphal march; sometimes they produce the
impression of tombstones lying in large numbers behind the line of
battle and after the end of the struggle. On the other hand, they in no
way force themselves upon our notice as instruments of slaughter which
the attacked organism makes use of to defend itself” (p. 227).
These arguments have been regarded by many investigators in all
countries as perfectly sufficient to overthrow the phagocytic theory.
The bactericidal power of the body fluids became the rallying cry of a
great number of works always directed to the same object: to replace the
rôle of phagocytosis by that of a bactericidal power of the body fluids.
It is quite unnecessary to weary the reader with a list of the very
numerous publications that have appeared on this subject in every
European language. But it is not possible to pass over in silence the
work of some of the principal partisans of the humoral theory of
immunity.
[Sidenote: [550]]
The first place amongst these works certainly belongs to von Behring’s
memoir[883] on the natural immunity of white rats against anthrax. As
already stated in Chapter VI of this work, von Behring discovered the
very remarkable power possessed by the rat’s blood of destroying anthrax
bacilli with very great rapidity. This investigator did not hesitate to
conclude therefrom that this bactericidal property of the blood must, in
the rat, bring about a great resistance against anthrax. We should have
in this case, then, an example in which the immunity did not depend in
any way upon phagocytosis, but would be bound up entirely in a purely
humoral property.
With the object of deciding whether the bactericidal property of the
blood is really the general and essential cause of natural or acquired
immunity, von Behring, in collaboration with Nissen[884], carried out a
long series of experiments, the results of which, however, did not
confirm their expectations. They found that in animals well vaccinated
against certain bacteria (notably Gamaleia’s vibrio or _V.
metschnikovi_), the blood plasma undoubtedly acquires a high specific
bactericidal power, but at the same time they satisfied themselves that
the blood, even of well immunised animals, was generally incapable of
killing the micro-organisms. The bactericidal property, then, according
to their researches, presented itself not as a general character but as
one of limited importance. These facts even led von Behring to abandon
the theory of the bactericidal power of the body fluids as an
explanation of immunity.
This theory found many warm partisans, especially at Munich. Emmerich
had already announced at the International Congress of Hygiene, held at
Vienna in 1887, that in the blood of rabbits vaccinated against the
bacillus of swine erysipelas an antiseptic substance of remarkable
activity is produced. To this, exclusively, in this instance, and not to
the phagocytes, he attributed the acquired immunity. Later,
Emmerich[885] in an investigation carried out in collaboration with di
Mattei developed this view. We may refrain from giving any account of
the contents of their memoir as well as from criticising their
conclusions, as this has already been done in Chapter IX. Let us content
ourselves with stating that our own experiments, as well as those made
later by Mesnil, have demonstrated the inaccuracy of Emmerich’s
statements.
[Sidenote: [551]]
Another Munich bacteriologist, H. Buchner, at first expressed
himself[886] very favourably on the theory of phagocytosis. He regarded
it as more capable of explaining most of the phenomena of immunity than
was his own older local theory. But little by little he declared himself
in formal opposition to the cellular theory of immunity and went over to
the camp of his sometime adversaries. He adopted[887] the humoral theory
of the bactericidal action of the body fluids, upon which subject he
carried out several important investigations. He was able without
difficulty to confirm Nuttall’s discovery of the disappearance of the
microbicidal power when the defibrinated blood was heated to 55° C., and
he added to this fundamental fact many others of great value. He
demonstrated the part played by the salts in the exercise of this
bactericidal power, and laid great stress on the fact that this power
depends on the presence of a special substance of albuminoid nature, to
which he gave the name of _alexin_. Buchner[888] combatted with success
the idea that I had expressed, according to which the bactericidal power
of the body fluids is reduced in great part to a plasmolytic action of
the blood serum upon certain micro-organisms. It cannot be denied that
my hypothesis is only very partially applicable, and that the larger
share in the bactericidal action of the body fluids belongs to the
alexins. Buchner also made the study of this action more easy by the
demonstration that the red blood corpuscles of a foreign species
undergo, under the action of the blood and of the serums, a globulicidal
action comparable to that which occurs in the case of micro-organisms.
Whilst Flügge, von Behring and many others of the old partisans of the
bactericidal theory of the body fluids abandoned it more or less
completely as an explanation of immunity, Buchner remained faithful to
it and tried, aided by the collaboration of his pupils, as far as
possible to defend it.
[Sidenote: [552]]
In France this humoral theory was adopted chiefly by Bouchard[889] and
his pupils, amongst whom I must cite more particularly Charrin and
Roger. They sought to confirm it by personal researches, the greater
part of which were carried out upon the bacillus of blue pus. These
investigators studied it especially in relation to acquired immunity. A
comparison of the mode of development of the pyocyanic bacillus in the
serum of susceptible animals and of vaccinated animals of the same
species, convinced them of the great importance of the action of the
body fluids. In cases where these fluids were found to be incapable of
killing the micro-organisms they exerted over them an injurious
influence, either by attenuating their virulence, or by producing more
or less important modifications in their forms and functions. The
essential cause of natural or acquired immunity was always attributed by
Bouchard’s school to the property of the body fluids. The phagocytes
were said to intervene only secondarily, either to carry off the dead
bodies of the micro-organisms, or to ingest the bacteria, rendered
inoffensive by the humoral action.
The humoral theory of immunity, with some slight modifications, spread
very generally into every country, and many investigators accepted it
without reserve. But certain observers ventured to run counter to the
general current and raised objections of principle against the theory of
the bactericidal power of the fluids of the body. After the principal
facts established by the partisans of this theory had been confirmed, it
was asked whether the phenomena of the destruction of micro-organisms
observed _in vitro_ are really equivalent to those produced in the
refractory animal. A glance at the data brought together with so much
zeal was sufficient to demonstrate that this parallelism does not exist.
The blood of animals susceptible to certain micro-organisms was found to
be bactericidal for these organisms, whilst that of refractory animals
was incapable of destroying them. It is useless to cite examples, so
numerous are they. On the other hand, the bactericidal power of the body
fluids, so marked for certain pathogenic organisms such as the anthrax
bacillus and especially the cholera vibrio and the typhoid
coccobacillus, is insignificant or _nil_ as regards many bacteria
against which refractory animals are not wanting.
[Sidenote: [553]]
All these facts throw doubt on the predominating part played in immunity
by the bactericidal power of the body fluids. Lubarsch[890] attacked the
humoral theory, showing by a great number of experiments that animals
whose fluids are very bactericidal _in vitro_ are very susceptible to a
much smaller quantity of bacteria of the same species introduced into
the body. Thus, the defibrinated blood and the blood serum of rabbits
destroy a large number of bacteria in a very short time, whilst the
rabbits themselves contract fatal anthrax after the introduction of a
small number of these micro-organisms into the blood vessels. This
contradiction cannot be explained except by the profound changes which
the blood must undergo outside the body. Facts of the same nature have
been shown for the anthrax of rats by Hankin, Roux, and ourselves, as
described in Chapter VI.
[Sidenote: [554]]
The International Congress of Medicine, assembled at Berlin in 1890, was
the first occasion on which I spoke publicly of the new theories of
immunity. In the addresses given at the general meetings, leaders of
medical science in several countries summed up their opinion on this
question. Koch[891], in his memorable report, declared that the new
acquisitions had destroyed the basis of the theory of phagocytes, and
that consequently it must give place to the humoral theory of immunity.
Bouchard took up a more conciliatory position, but, according to him,
the bactericidal power of the fluids of the body was the primary and
essential cause of immunity. The phagocytes only intervened later, in
order to finish the work begun without their assistance. Lord Lister
expressed himself[892], on the other hand, much more favourably on the
subject of the theory of phagocytosis. This observer, who is not only a
great surgeon, but is perhaps even more remarkable for his great powers
of generalisation, has paid special attention to the problem of
immunity. With the object of clearing up this very complicated and at
the same time important question, Lord Lister seized the occasion of the
meeting of the International Congress of Hygiene in London in 1891, to
bring about an exchange of views between the partisans of the various
theories of immunity. Under his presidency he devoted an entire sitting
of the Section of Bacteriology to the discussion of this question.
Buchner presented a report[893] drawn up exclusively from the point of
view of the humoral theory and devoted to the demonstration of the
slight importance of phagocytosis, and also to the preponderant part
played by the alexins dissolved in the body fluids and circulating in
the plasma of the blood. He attempted to harmonise the facts on the
bactericidal power of serums observed _in vitro_ with the special
conditions to be met with in the animal body. He specially insisted on
the point that, in the blood and the organs, the alexins cannot act with
the same rapidity that they can in test tubes containing serum. In this
way he recognised that between the bactericidal action _in vitro_ and
that in the body of the animal, there exists a marked difference, but he
would not consent to attribute it in the latter case to the intervention
of the phagocytes.
Roux[894] also made a report on immunity at the same sederunt, speaking
very distinctly in favour of the cellular theory. A chemist by
inclination, he was sympathetic at first to the humoral theories of
immunity. Working with Pasteur, and side by side with him, Roux, from
the beginning of the new era of medical science, had made numerous
experiments on the part played by the body fluids in immunity. But as
the results were not sufficiently precise and demonstrative they were
soon abandoned. The attachment of Roux, however, to the humoral theories
was manifested in his work, carried out in part with Chamberland[895],
on the subject of vaccination by means of microbial products. Later,
having obtained a deeper knowledge of various facts concerning natural
and acquired immunity, he rallied to the cellular conception and
developed it in his report presented to the above Congress in London.
Several microbiologists took part in the discussion, and I myself[896]
was able to communicate certain facts concerning the immunity of
guinea-pigs, acquired as the result of vaccination against Gamaleia’s
vibrio. I chose this example because it presented, according to von
Behring and Nissen, the clearest case of a bactericidal property
developed during the course of immunisation. I was able to furnish the
proof that, in the vaccinated animal, the micro-organism in question, in
spite of the great bactericidal power of the blood serum _in vitro_,
remains alive in the animal body for a long time, and that its
destruction is effected by the phagocytes, which ingested it alive. In
this example I showed that the leucocytes of the exudation, that have
ingested vibrios, may still furnish cultures of this organism if they
are taken from the body and transferred in hanging drop to the
incubator.
[Sidenote: [555]]
The fact that, even in the case which appeared most to favour the
humoral conception of acquired immunity, phagocytes play the principal
part, must to many members of the Congress have appeared sufficiently
significant. Indeed, several observers who were present at the debates,
received the impression that the phagocytic theory had not been
overturned by its adversaries. At this period the question of the
importance of antitoxins from the point of view of immunity had scarcely
been raised. The great discovery made by von Behring and Kitasato was
already accepted by everyone; but there was no ground for attributing to
it any general importance. In fact, though proved for tetanus and
diphtheria, and extended by Ehrlich’s beautiful experiments to the
vegetable toxins (ricin, abrin, and robin), the antitoxic property of
the fluids of the body presented itself rather as a special than as a
general phenomenon. It is in this sense that Roux had assigned to it its
place in the chapter of immunity. The two diseases, against which
antitoxic serums had been discovered, are certainly distinguished from
the great majority of infections by the localisation of the
micro-organisms and the abundant secretion of their toxins.
It was only after the London Congress that this question came
prominently forward. Von Behring thought that the antitoxic power of the
body fluids is generally distributed in all cases of acquired immunity,
and that micro-organisms, introduced into the animal possessing this
power, become incapable of any pathogenic manifestation. Certain facts,
brought together in Bouchard’s laboratory, tell against the hypothesis I
have just mentioned. With the object of throwing light on this question
I began, immediately after the close of the Congress, to study the
acquired immunity of rabbits against the micro-organism of the
pneumo-enteritis of pigs. I was able to demonstrate[897] that in this
case the resistance of the animal against the micro-organisms does not
depend on the acquisition of any antitoxic property by the body fluids;
such a property is completely absent. At the same time I showed that the
serum of vaccinated rabbits possesses a very marked protective power
against infection by the coccobacillus of pneumo-enteritis. It was for
the first time proved that independently of the antitoxic and
bactericidal properties of serums, there exists another special
property, the anti-infective property. This I conceived to be of the
nature of a stimulant action on the part of the phagocytes.
[Sidenote: [556]]
It has already been stated in an earlier chapter that before the
discovery of antitoxins Richet and Héricourt[898] had observed an
immunising action of the serum of animals refractory to staphylococci.
These observers were content with this demonstration and did not seek to
penetrate more deeply into the mechanism of the action of their serum.
For this reason when von Behring and Kitasato announced their discovery
of antitoxic serums it was generally thought that the antistaphylococci
serums were also antitoxic serums. The immunity against the
micro-organism of the pneumo-enteritis of pigs taught us that here we
might have to deal with quite a different matter. It was soon
demonstrated that the serum from the immunised animal might in fact,
without being antitoxic, present the same anti-infective property as in
the case of pneumo-enteritis. That was first proved in the case of the
experimental disease set up by Koch’s cholera vibrio.
[Sidenote: [557]]
The reappearance of cholera in Europe in 1892 drew the attention of
bacteriologists to this disease, and was the occasion of many new
researches on immunity against the cholera vibrio. Several important
works on this question were published by Pfeiffer[899], at this period
director of the scientific staff of the Koch Institute at Berlin. He
obtained, in animals well immunised against the cholera vibrio, a serum
endowed with a high anti-infective power but entirely without any
antitoxic property. The guinea-pigs themselves, very resistant to the
cholera peritonitis, were found, on the other hand, to be very
susceptible to the minimum lethal dose of the cholera poison. The
absence of antitoxic power in the fluids of the body taken in connection
with a well-marked phagocytic reaction in a large number of cases of
immunity, natural and acquired, has turned the scale in favour of the
cellular theory. The impossibility on the part of those who maintain the
purely bactericidal theory of the body fluids, to reply to the
objections above mentioned has accentuated this favourable movement.
Just at this moment, when the theory of phagocytes might be regarded to
have obtained the rights of citizenship, a discovery was made which
appeared to overturn it completely. I have mentioned more than once that
the attempts of the partisans of the bactericidal theory of the body
fluids have failed whenever it was necessary to give evidence of their
action in the refractory animal. Instead of a destruction of the
micro-organisms in these fluids, it was always found that they perished
inside the phagocytes. These facts have even led to the manifestation of
a desire to harmonise the humoral theory with the theory of
phagocytosis. Denys, with certain of his collaborators, and Buchner and
his pupils came to the conclusion that the alexins are merely leucocytic
products. As regards the theory of phagocytosis we have this section,
who attribute an important function in healing and immunity to the
emigration of the leucocytes towards, and their accumulation at the
menaced spot. They admit that the leucocytes really represent the
healing elements of the animal body; it is not, however, they say, their
phagocytic functions which confer upon them this rôle but their power of
secreting alexin. These bactericidal substances act outside the
phagocytes—in the plasmas of the blood and of the exudations—and
phagocytosis only intervenes at a later period and secondarily.
This new modification of the bactericidal theory of the body fluids has
often been termed by Buchner a connecting bridge between the humoral
theory and the cellular theory of immunity.
In the midst of this movement of conciliation, Pfeiffer[900] in 1894
published a work on the immunity of the guinea-pig against experimental
cholera peritonitis. He maintains that here the destruction of the
vibrios takes place without any co-operation on the part of the
phagocytes and exclusively by means of the body fluids. The vibrios,
before their complete destruction and solution in the fluids of the
body, are transformed into granules, presenting the transformation to
which we have given the name of Pfeiffer’s phenomenon.
[Sidenote: [558]]
Several of Pfeiffer’s pupils have confirmed his view in connection with
the cholera vibrio, and have extended it to several other
micro-organisms such as the typhoid coccobacillus. The destruction of
the micro-organisms in these cases is brought about, according to
Pfeiffer and his collaborators, not by the alexins of Buchner, but by a
separate substance. The protective anti-infective serum contains it in
an inactive state only; but immediately this serum is introduced into
the body of a normal animal, the bactericidal substance is acted upon by
the endothelial cells and becomes “active,” capable of destroying a
large number of vibrios. Pfeiffer has developed this theory more
especially in an article published in 1896, entitled “Ein neues
Grundgesetz der Immunität[901].” Pfeiffer’s observation and his theory
built upon it gave a new lease of life to the humoral theory and for
some time many observers believed that the theory of phagocytosis was
now finally overturned. Fränkel[902] announced, in a public address,
that science in its progressive march has “discovered the methods of
defence employed by the animal organism against its most dreaded
enemies, methods which have nothing in common with phagocytosis, which
act quite independently of the phagocytes and manifest an action so
energetic that we may calmly eliminate all other factors.” This view is
based on the discovery of antitoxins and the bactericidal substance
studied by Pfeiffer.
It will be readily understood that as soon as I learnt of the existence
of a real extracellular destruction of micro-organisms I at once began
to study it in order to find out its real importance amongst the
phenomena of immunity. First of all, I examined Pfeiffer’s phenomenon in
connection with the cholera vibrio[903], and I was able to show that it
was produced only under special conditions. The pre-existent phagocytes
must be greatly injured before the cholera vibrios can be transformed
into granules. Phagolysis (so I termed this transitory damage to the
phagocytes) is indispensable for the manifestation of Pfeiffer’s
phenomenon in the peritoneal fluid. When it is suppressed, by preparing
the phagocytes by means of injections of various fluids, we find that,
instead of Pfeiffer’s phenomenon, phagocytosis is almost instantaneously
produced. In positions where very few or no leucocytes are pre-existent,
as in the subcutaneous tissue, Pfeiffer’s phenomenon is never observed.
[Sidenote: [559]]
Even in the case of the cholera vibrio the extracellular destruction is
observed, therefore, only in special cases. Most of the other pathogenic
micro-organisms do not undergo this destructive process at all under
conditions in which the cholera vibrio exhibits Pfeiffer’s phenomenon in
a marked degree. These facts appeared to justify me in the conclusion
that the destruction of micro-organisms takes place in the animal body
by means of soluble ferments, the result of phagocytic digestion. These
ferments are found under the normal condition within these phagocytes
and escape from them when they are destroyed or receive some transient
injury. This conclusion was in flat contradiction to the theory and
statements of Pfeiffer, who attributed an important function to the
endothelial secretions. To settle this controversy I tried to obtain
Pfeiffer’s phenomenon outside the body, that is to say independently of
any co-operation from the peritoneal endothelium. It is sufficient to
add a little peritoneal lymph, rich in leucocytes, to the inactive
anti-infective serum, to obtain in hanging drops the transformation of
the cholera vibrios into granules.
Bordet[904], in my laboratory, repeated this experiment with the object
of determining its essential mechanism. He succeeded in obtaining
Pfeiffer’s phenomenon _in vitro_, not only by adding peritoneal lymph
from a normal guinea-pig to the specific serum, but also by adding to it
a drop of fresh blood serum from the same animal. The analysis of the
phenomena which take place under these conditions led Bordet to the
following hypothesis. The destruction of micro-organisms in vaccinated
animals takes place by the co-operation of two substances. One of these
is Buchner’s alexin which is found normally in the phagocytes; it sets
up bacteriolysis properly so-called when it is enclosed within the
leucocytes or after it has escaped from them at the time of phagolysis.
To attain this end, however, the alexin needs the co-operation of
another substance. This is the protective or sensibilising substance of
Bordet. It circulates in the plasmas and carries a specific character
which is absent from the alexin. I need not here insist at any length on
this theory, because it has already been sufficiently explained during
the course of this work.
[Sidenote: [560]]
The data on the restricted part played by Pfeiffer’s phenomenon and on
its mechanism, above summarised, have been attacked by Pfeiffer and by
several other observers, but they have received general confirmation, so
that their accuracy can no longer be in doubt. Objections were also
raised to Bordet’s view of the mechanism of bacteriolysis. Thus, Abel
has criticised it in the following argument[905]: “In spite of the
soundness and the boldness of the majority of Bordet’s statements on the
importance of the various factors, and especially of the leucocytes in
immunity, it cannot be doubted that later researches will modify and
correct his interpretations which we, in Germany, do not accept in their
full extension. Up to the present, the victory in the various rounds has
always been with Pfeiffer, whose researches, solid and exempt from bias,
have made him, to use a sporting expression, the ‘favourite’ with all
those who follow attentively the international contest in the arena of
the problem of immunity.” Abel is certainly a highly esteemed
bacteriologist, but he is not a good prophet, and he assumes a mistaken
attitude in looking at the subject from a “national” point of view[906].
In Germany much interest is taken in scientific movements and, very
naturally, original and new theories are there criticised and discussed.
But that does not justify one in putting forward against an opinion the
statement that it is not accepted in Germany. In this country, so rich
in scientific work, we find partisans of the most opposite views. In any
case, in the conflict between Pfeiffer on the one hand, and Bordet and
myself on the other, things have not turned out as Abel predicted. The
two substances which act in the destruction of the micro-organisms are
now accepted by the whole world. The intimate relations between the
alexins and the leucocytes are equally recognised by very many
observers. The fact that the alexins are confined within phagocytes has
been confirmed by several observers, and has received a very convincing
proof from Gengou’s experiments on the comparative action of the serum
and blood plasma against micro-organisms. The existence of phagolysis,
denied at first by some observers, has been verified by others and can
now no longer be doubted.
[Sidenote: [561]]
The relations between the sensibilising substance and the phagocytes are
less easily grasped than are those between the alexins and the
leucocytes. Nevertheless, the experiments made by Pfeiffer and
Marx[907], have led these observers to recognise that the former arise
from the spleen, the lymphatic glands, and the bone marrow, that is to
say, organs which are pre-eminently phagocytic. This result has been
confirmed by Deutsch and must be regarded as definitely settled. All the
data collected in recent years have, therefore, confirmed the view that
the destruction of micro-organisms in the refractory animal presents
itself as a special example of their absorption by formed elements. This
truth was so fully recognised in our laboratory that the analogy between
bacteriolysis and the destruction of animal cells was looked upon as
quite natural and evident. Bordet had for some years past observed that
the blood serum of certain animals presented a marked analogy in its
agglutinative property in regard to micro-organisms and in that against
red blood corpuscles. In 1898, studying the fate of the spirilla of the
goose in the peritoneal cavity of guinea-pigs (see Chapter VI), I
observed that these micro-organisms underwent the same changes both
within and outside the phagocyte; this fact appeared to me to be in
perfect harmony with the whole of our knowledge concerning the
absorption of formed elements and on intracellular digestion.
Bordet[908], prepared by his preceding researches on the agglutination
of the red blood corpuscles, set himself to study the fate of the red
corpuscles in the animal body. He easily established a close
relationship between the development of the bacteriolytic property and
the haemolytic power of the serum of animals prepared by repeated
injections of bacteria and of blood. His results were soon (January,
1899) confirmed by Ehrlich and Morgenroth[909], who supplemented them
with the important statement that Bordet’s sensibilising substance, or
intermediary substance (E. and M.), has the property of attaching or
fixing itself to the red blood corpuscles.
[Sidenote: [562]]
The works on haemolysis, carried out during the last three years by
Ehrlich and Morgenroth on the one hand, and by Bordet on the other, have
allowed us to extend our study of the mechanism of the action of the two
substances on micro-organisms and on animal cells. Ehrlich has extended
his ingenious theory of antitoxins to the bacteriolytic substances,
which he regards as side-chains detached from the cells and capable of
absorbing the toxins. In a series of remarkable investigations, most of
them carried out in collaboration with Morgenroth, Ehrlich has developed
his theory which attempts to offer an account of the essential mechanism
which presides over the destruction of micro-organisms and over the
neutralisation of their poisons. This theory is at present in full swing
of development. Some of his points contradict several of the conclusions
in Bordet’s works. Whilst the latter maintains that the sensibilising
substance becomes fixed as a mordant, Ehrlich regards it as entering
into chemical combination with the molecular group of the
micro-organisms and of the animal cells. According to Bordet, the alexin
of the same species of animal is always the same substance. Ehrlich
energetically maintains the plurality of the alexins, to which he gives
the name of complements.
This controversy has caused a most interesting exchange of views and has
led to experiments which are remarkably ingenious, but it must be
admitted that as yet all the points in dispute are not definitely
settled. It is evident that we have here a new line of research which
promises most fruitful results for science.
We have described in various chapters of this work the fundamental
elements of Ehrlich’s theory. Many think that this theory is, in
principle, antagonistic to the theory of phagocytosis, but we have
already observed that this view cannot be accepted. It is true that
Ehrlich maintains that the bacteriolytic and cytotoxic ferments which we
have called _cytases_ (alexins or complements) circulate in a state of
solution in the blood plasma, whilst, according to the theory of
phagocytosis, they are found under normal conditions inside phagocytes.
But this view has nothing to do with the basis of the theory of
receptors, or of Ehrlich’s side-chain theory, according to which the
antitoxin and certain other antibodies (intermediary substance) are
regarded as products detached from cells having an affinity for the
toxins and the microbial products.
The theory of phagocytosis seeks to establish the part played by these
cells in the destruction of micro-organisms. It maintains that the vital
manifestation of the phagocytes, irritability, mobility, and voracity,
constitutes an essential factor in ridding the animal of
micro-organisms, because the true bactericidal ferment is contained
within the phagocytes, except in cases of phagolysis. The destruction of
the micro-organisms follows the laws which govern the absorption of
formed elements in general. This absorption, finally, is the work of two
soluble digestive ferments, one of which (fixative) is readily excreted
by the phagocyte into the plasmas of the blood and exudations. The
theory of phagocytosis seeks to establish these principles with the
greatest possible exactness, but it has not yet ventured to penetrate
more deeply into the phenomena of intracellular digestion which are
confounded with the action of soluble ferments in general. This problem
is still far from being satisfactorily solved.
[Sidenote: [563]]
In spite of very numerous objections, of which the principal ones have
already been mentioned, the theory of phagocytosis, within the limits
indicated, so far from being overturned, has become more and more
consolidated, thanks to the numerous observations made since its
foundation. It is for this reason that the opposition has calmed down of
late years and that in many works the opinions expressed have become
more favourable to the rôle of phagocytosis in immunity.
Soon after the Congress of Hygiene in 1891, the Pathological Society of
London devoted several meetings to a discussion of the question of
immunity. Many eminent observers took part in these debates, which were,
in general, favourable to this theory of phagocytosis[910].
At the International Congress of Hygiene, held at Budapest in 1894, the
question of immunity was again discussed. Buchner[911] made a report in
which he specially insisted on the leucocytic origin of the alexins,
regarding this fact as particularly capable of reconciling the
bactericidal property of the body fluids with the theory of
phagocytosis. The alexins, however, secreted by the leucocytes, must, it
was assumed, carry out their principal function in the plasmas of the
blood and exudations. Phagocytosis would only intervene secondarily for
the purpose of ingesting the micro-organisms which had been already
killed or seriously injured by the alexins of the body fluids.
[Sidenote: [564]]
In his last summary of the question, presented to the International
Congress of Medicine at Paris in 1900, Buchner[912] maintains his theory
of leucocytic secretions. But he already takes one step more towards the
theory of phagocytosis, at least as regards natural immunity. He
consents to accept the fact “that phagocytic activity is in many cases
of decisive importance in overcoming the infective processes, especially
in those cases in which the secreted alexins were unable to bring about
more than a temporary attenuation of the vital functions of the
bacteria. Under these conditions the bacteria could only be modified in
so far as their chemical functions were transformed into a latent state,
from which they would be ready to regain their full vital activity
should it happen that the phagocytes were not there to prevent them from
doing so.” In any case this view is widely removed from the old theory,
according to which phagocytes were regarded as capable of ingesting dead
and inoffensive bacteria only.
A second adversary of the theory of phagocytosis, von Behring[913],
gives a place to this theory not only in certain examples of natural
immunity but even in some cases of acquired immunity, e.g. in the
immunity of sheep vaccinated against anthrax, an example I have already
cited in Chapter VIII (cf. _supra_, p. 242).
It would take too long to describe the change of opinion on the theories
of immunity that has taken place during recent years. I will content
myself with citing certain examples which shall be taken from the works
of declared adversaries of the theory of phagocytosis. Thus, Flügge, who
early declared against the cellular theory completely and categorically
and at the same time argued strongly in favour of the humoral theory,
has been gradually led to depart from his first position. We may follow
the steps of his conversion in the different editions of his _Outlines
of Hygiene_. In the first edition published in 1889 he expresses himself
in the following manner[914]: “Recent researches indicate the
probability, however, that the phagocytes in by far the greater majority
of cases seize the infective agents which, already dead, are not in a
condition suitable for the performance of a defensive function. On the
other hand, it is proved that the blood and blood plasma of warm-blooded
animals possess the property of destroying, very quickly, enormous
numbers of pathogenic bacteria,” ... etc. In the fourth edition of the
same work, published in 1897, we find at the corresponding place the
following passage[915]: “Recent researches indicate the probability,
however, that the theory of Metschnikoff ... is not in a position to
offer a complete explanation of the process of immunity.” This passage
is followed by a somewhat conciliatory and eclectic development of the
theory.
[Sidenote: [565]]
Let us take as a second example Günther’s _Introduction to the Study of
Bacteriology_, widely read both in the original and in translations. In
the first edition published in 1890[916] the theory of phagocytosis is
curtly dismissed as “being incapable of withstanding criticism.” In the
fifth edition of the same work, however[917], published in 1898, this
theory is no longer treated thus summarily. It is given a place amongst
the theories of immunity and an attempt, similar to that made by
Buchner, is made to reconcile it with the humoral theory.
A change in the same direction may also be observed in Charrin’s view.
In the first edition of his _Pathologie générale infectieuse_, this
observer[918] had already taken an eclectic view on this question of the
theories of immunity. But the function which he assigns to the
phagocytes is subsidiary and secondary, whilst to that of the humoral
properties is assigned a position of primary importance. In the second
edition of the same work, which appeared seven years later[919], the
importance of phagocytosis is recognised in a much larger measure, as
may be gathered from the following passages: “For my part, I have always
accepted phagocytosis: at the same time I have always accepted the
existence of special humoral properties. As early as 1888 I showed, _in
vivo_, that the germs are modified outside the cells; but I did not know
from what groups of anatomical elements these properties were derived, I
exaggerated their importance and it is the decision of this origin and
this importance that renders it possible to reconcile the two theories”
(p. 250). “After all, the defence rests upon these two great processes
or cellular activities, phagocytosis in the first line, and then humoral
influences, some of them bactericidal and injurious to the living germ,
others antitoxic and injurious to their secretions” (p. 253).
[Sidenote: [566]]
Whilst the theory of phagocytosis has been consolidated by the
demonstration: (1) that the phagocytes, in cases of immunity, ingest and
destroy the living and virulent micro-organisms without the latter
needing to be previously deprived of their toxins; (2) that the
phagocytes absorb toxic substances; (3) that the phagocytes contain
bactericidal cytases and produce fixatives; the humoral theories, in
spite of all the efforts made to defend them, could never be developed
as theories that were in the slightest degree of general application.
Certain observers who from the first were very sympathetic to the
humoral theories have attempted to give a complete summary of these
properties. Thus, Stern[920] and later Frank[921] have published reports
drawn up with great care and in a very impartial spirit on the works
treating of the properties of the body fluids and the part they play in
immunity. This is how they sum up the question. Stern came to the
conclusion that it is impossible “to demonstrate at all regularly the
existence of relations between the bactericidal action of the blood and
immunity in all the infective diseases. In some cases, however, these
relations are so marked that, for these examples, a causal bond between
the two factors is extremely probable.” Frank expresses himself in the
following manner: “It follows most clearly that the immunity of an
animal—immunity innate or acquired—corresponds with the bactericidal
property of the blood in certain exceptional cases only. The only
animal, absolutely susceptible to anthrax and whose blood is entirely
without any bactericidal power, that it is at present possible to cite,
is the mouse.” “The bactericidal action of the blood serum is
undoubtedly a fact of great biological importance; but equally certainly
it cannot be the general cause of immunity, whether innate or acquired.”
An attempt was made to give fresh life to the humoral theory, either by
assuming that the bactericidal substance is nothing but the eosinophile
or pseudo-eosinophile secretion of the leucocytes (Kanthack), or by
supposing that for the destruction of micro-organisms in the animal body
the intervention of the agglutinative substance dissolved and
distributed in the body fluids is essential (Max Gruber). These two
views were put forward in a tentative form and as preliminary
communications only; there is no possibility of raising them to the
dignity of theories, and of late years they have not been upheld.
It cannot be denied that not one of the humoral theories has been able
to retain its position or to stand against the numerous facts that have
been accumulated during recent years.
This extraordinary discrepancy between the bactericidal power of the
body fluids and immunity is explained by the circumstance that the
microbicidal substances exist in the living animal within phagocytes and
only escape from them when these cells have been injured. The fact, so
well demonstrated by Gengou, that the blood plasma is without any
bactericidal power has given the final blow to the microbicidal theory
of the body fluids and it can no longer be maintained.
[Sidenote: [567]]
The humoral theories, based on the antitoxic and protective power of the
body fluids, can claim only a very restricted application. These
properties are met with in acquired immunity only, and even there are
not constant. Many cases of acquired immunity against micro-organisms
are unaccompanied by any antitoxic power, and in several examples of
this immunity the body fluids do not exhibit any protective power.
There is only one constant element in immunity, whether innate or
acquired, and that is phagocytosis. The extension and importance of this
factor can no longer be denied.
It is clearly proved that phagocytes are susceptible cells which react
against morbific agents, whether organised or not. These cells ingest
micro-organisms and absorb soluble substances. They seize microbes
whilst these are still living and capable of exercising their noxious
effect and bring them under the action of their cellular contents, which
are capable of killing and digesting the micro-organisms or of
inhibiting their pathogenic action. Phagocytes act because they possess
vital properties and a faculty of exerting a fermentative action on
morbific agents. The mechanism of this action is not yet definitely
settled, and we can foresee that for future researches there will be a
vast and fertile field to be reached by pursuing this path.
The present phase of the question of immunity constitutes one stage only
in the development of biological science and one which is capable of
many improvements.
CHAPTER XVII
SUMMARY
Means of defence of the animal against infective agents.—Absorption of
micro-organisms.—Phagocytes, and their function in
inflammation.—The action of phagocytes in the absorption of
micro-organisms.—The cytases, phagocytic ferments.—The cytases are
closely bound up with the phagocytes.—The fixatives and their
function in acquired immunity.—The fixatives are excreted by the
phagocytes and pass readily into the fluids of the body.—Essential
mechanism of the action of the fixatives.—Adaptation of phagocytes
to destroy micro-organisms in acquired immunity.—Difference
between the fixatives and the agglutinins.—Antitoxins and their
analogy with the fixatives.—Hypotheses as to the origin of
antitoxins.—Cellular immunity is a fact of general
import.—Susceptibility and its rôle in immunity.—Applications of
the theory of immunity to medical practice.
[Sidenote: [568]]
When an animal remains unharmed in spite of the penetration of infective
agents it is said to be immune to the diseases usually set up by these
agents. This idea embraces a very great number of phenomena which cannot
always be sharply separated from allied phenomena. On the one hand,
immunity is closely connected with the process of cure, on the other, it
is related to the disease. An animal may be regarded as unharmed if the
penetration of a very dangerous virus sets up merely an insignificant
discomfort. Nevertheless, this discomfort is accompanied by morbid
symptoms, though they may be very slight. It is useless and impossible
to set up any precise limits between immunity and allied states.
Immunity presents great variability. Sometimes it is very stable and
durable; in other cases it is very feeble and transient. Immunity may be
individual or it may be generic. It may be the privilege of a race, of a
species.
Immunity is often innate, as is the case of the immunity which is called
natural. But it may also be acquired. This last category of immunity may
be developed either by natural means, after an attack of an infective
disease, or as a result of human intervention. The principal means of
obtaining artificial acquired immunity consists in the inoculation of
viruses and of vaccines.
[Sidenote: [569]]
Immunity is a phenomenon which has existed on this globe from time
immemorial. Immunity must be of as ancient date as is disease. The most
simple and the most primitive organisms have constantly to struggle for
their existence; they give chase to living organisms in order to obtain
food, and they defend themselves against other organisms in order that
they may not become their prey. When the aggressor in this struggle is
much smaller than its adversary the result is that the former introduces
itself into the body of the latter and destroys it by means of
infection. In this case it takes up its abode in its adversary in order
to absorb the contents of its host and to produce within it one or more
generations. The natural history of unicellular organisms, both
vegetable and animal, often presents to us these examples of primitive
infection.
But infection also has its counter. The attacked organism defends itself
against the little aggressor. It protects itself by interposing a
resistant membrane, or it uses all the means at its disposal to destroy
the invader. As a very large number of organisms, in order to obtain
nourishment, are obliged to submit their food to digestion by various
chemical substances, they utilise these substances in the struggle
against the infective agents. They digest them whenever they are able to
do so.
One of the most primitive of organisms, the plasmodium of the
Myxomycetes, which is composed of formless protoplasmic masses
intermediate between lower animals and plants, ingests foreign bodies of
various kinds. It often happens that it incorporates numerous bacteria
which are growing alongside it on rotten wood or elsewhere. The
plasmodium allows them to live for some time within its digestive
vacuoles. But in the end it digests them by means of its soluble
ferments, substances intermediate between pepsin and trypsin. Owing to
this digestive power the plasmodia are not attacked by bacterial
infections.
[Sidenote: [570]]
This example, taken from amongst the most simple organisms, may serve as
a prototype for the phenomena of immunity in general. At the
commencement of the study of this remarkable property of so many living
organisms it was thought that the pathogenic micro-organisms
encountered, within the refractory organism, a medium which did not
allow them to live, either because of the absence of certain nutritive
substances indispensable for their existence or because it contained
some substance injurious to micro-organisms. Very numerous and detailed
researches have demonstrated the incorrectness of these hypotheses.
There are, of course, certain pathogenic micro-organisms which are very
exacting as regards the medium in which they will grow. Some will
develop only in the presence of particular substances, whilst others are
extremely sensitive to the slightest traces of poisons. These, however,
are quite the exception. The great majority of pathogenic
micro-organisms belonging to the group of bacteria readily adapt
themselves to all kinds of culture media, and most of them live and
develop freely in the blood or other fluids of refractory organisms.
This, therefore, is not the cause of the immunity in such organisms. The
cause must be sought for amongst factors more closely connected with
life.
Wishing to penetrate more deeply into these phenomena the hypothesis was
put forward that the unharmed organism got rid of the infective
micro-organisms by expelling them to the outside along with the excreta.
It was maintained for a considerable time that the animal organism
possessed the means of causing pathogenic bacteria to pass into the
kidneys, whence they were eliminated by the urine. It had to be
acknowledged, however, that this elimination never takes place in cases
of immunity, and only comes into operation when the animal is ill and
the integrity of the renal filter is impaired.
The infective micro-organisms, after they have entered into the unharmed
organism, remain there for a longer or shorter period, and perish
without being expelled. This disappearance of the micro-organisms takes
place by the same mechanism that rids the plasmodium of those bacteria
which it has managed to ingest during its slow peregrinations over dead
leaves or rotten wood. The micro-organisms are absorbed into the
refractory organisms as the result of a true act of digestion. It is
very remarkable that the gastro-intestinal ingestion, so well provided
with means of rendering the most varied aliments soluble, is generally
incapable of digesting pathogenic or other micro-organisms. It is very
rare to meet with soluble ferments of the intestinal canal which are
capable of digesting microscopic organisms, especially bacteria.
Consequently this organ, so rich in digestive diastases, is generally
inhabited by a large number of bacteria and other micro-organisms.
[Sidenote: [571]]
Even in animals whose food contains large numbers of microorganisms,
_e.g._ the larvae of flies, the digestive juices are powerless to
destroy them. Nevertheless, there are organisms which feed exclusively,
or almost exclusively, on bacteria and which are quite capable of
digesting them. These are the Protozoa, such as the _Amoebae_ and
certain Infusoria, which, without any trace of a digestive tube, easily
bring about this result. _Amoebae_ can be grown on the surface of agar
by taking care to sow along with them bacteria for their nourishment. It
is only necessary to give them a single species of micro-organism, and
this may be selected from the pathogenic forms, such as the cholera
vibrio or the _Bacillus coli_. The _Amoebae_ ingest a number of these
bacteria in the living state. They then kill them and digest them in
their digestive vacuoles which contain, along with a little acid, a
ferment belonging to the trypsin group, the amoebodiastase.
The bodies of lower and higher animals, alike, are very rich in elements
which closely resemble the _Amoebae_. Sometimes these are to be found in
the epithelial cells of the digestive canal which put out protoplasmic
processes for the purpose of seizing food and transferring it to their
interior, where it is submitted to the action of digestive ferments.
Sometimes they are the cells disposed between the body wall and that of
the intestinal canal, which float freely in the fluids of the body or
are more or less fixed in the interstitial tissue. The animal kingdom
presents a great variety of these amoeboid elements, known under the
general name of phagocytes (cells capable of devouring solid bodies).
One of the most primitive arrangements of phagocytes is met with in
_Ascaris_ and its allies belonging to the group of the Nematoda. All the
organisation that these round worms possess consists merely of four, or
a few more, enormous cells attached to the body wall. These are
phagocytes which push out processes of enormous length, capable of
exploring the whole of the internal cavity of the body.
[Sidenote: [572]]
The majority of phagocytes circulate in the lymph and blood and pass
into the exudations. These white corpuscles have a comparatively uniform
structure in the Invertebrata and present themselves as small cells with
a nucleus and a protoplasm capable of amoeboid movements. In the
Vertebrata we meet with two great categories of white corpuscles, of
which one group resembles those of the Invertebrata in that they also
possess a single large nucleus and an amoeboid protoplasm. These are the
macrophages of the blood and of the lymph, and are intimately connected
with the macrophages of such organs as the spleen, lymphatic glands, and
bone marrow. Another group of white corpuscles in the Vertebrata is made
up of small amoeboid cells which are distinguished by having a nucleus
which, although single, is divided into several lobes. These are the
microphages whose chief peculiarity, the multi-lobed form of the
nucleus, must be regarded as an adaptation for the purpose of passing as
rapidly as possible through the walls of capillaries and small veins.
The diapedesis of the white corpuscles, their migration through the
vessel wall into the cavities and tissues, is one of the principal means
of defence possessed by an animal. As soon as the infective agents have
penetrated into the body, a whole army of white corpuscles proceed
towards the menaced spot, there entering into a struggle with the
micro-organisms. Aided by the special form of their nucleus the
microphages are the first to pass through the walls of the vessels. Each
of the several small lobes, into which the nucleus and its protoplasm is
divided, passes readily through the minute orifices between the
endothelial cells of the vessels. The macrophages follow the microphages
and become mixed in greater or less numbers with the exudations. But it
is not micro-organisms only which set up this inflammatory reaction
accompanied by the emigration and the accumulation of leucocytes. The
introduction of inert bodies and of aseptic fluids brings about the same
result. The phagocytes are, as a matter of fact, endowed with a special
susceptibility, which enables them to perceive exceedingly small changes
in the chemical or physical composition of the medium that surrounds
them.
The leucocytes, having arrived at the spot where the intruders are
found, seize them after the manner of the _Amoebae_ and within their
bodies subject them to intracellular digestion. This digestion takes
place in the vacuoles in which usually is a weakly acid fluid which
contains digestive ferments; of these a very considerable number are now
recognised.
Just as the _Amoebae_ and the Infusoria make a choice from amongst the
small organisms that surround them, so the leucocytes choose bodies
which are best suited to their use. The macrophages seize by preference
animal cells such as the blood corpuscles, the spermatozoa, and other
elements which are derived from animals. Among the infective
micro-organisms the macrophages have a predilection for those that set
up chronic diseases such as leprosy, tuberculosis, and actinomycosis and
also for those which are of animal nature. Into this last category come
the amoeboid parasites of malaria, Texas fever and the _Trypanosomata_.
The macrophages can also ingest the bacteria of acute diseases, but,
save in exceptional cases, their intervention is of little moment.
[Sidenote: [573]]
The microphages, on the other hand, appear to play their part specially
in acute infections. Their intervention against animal cells is _nil_,
or almost so. Thus they rarely seize the red corpuscles of the same or
of a foreign species of animal. They also appear to be repelled by
parasites of animal origin and by certain bacteria which set up chronic
diseases. Whilst the macrophages seize the bacilli of leprosy with great
avidity, the microphages ingest them only exceptionally.
The morphological and physiological differences between the two great
categories of mobile phagocytes (leucocytes), correspond to differences
in the composition of their soluble ferments. Just as the _Amoebae_
digest their prey by means of their amoebodiastase, a soluble ferment of
the group of trypsins, so the white corpuscles submit the foreign bodies
ingested by them to the action of what are now known as cytases. These
cytases (alexins or complements of other writers) are soluble ferments
which also belong to the trypsin group. They act in a medium which is
feebly acid, neutral, or feebly alkaline, and, like the amoebodiastase,
they are distinguished by a great sensitiveness to heat. When the
cytases are contained in fluids, a temperature of 55°–56° C. destroys
them rapidly and completely. When they are found in organs reduced to
the state of an emulsion, their sensitiveness diminishes and it is
necessary to raise the temperature to 58°–62° C. in order to destroy
their activity.
[Sidenote: [574]]
Bordet maintains that the cytases are very different in the various
species of animals, but that in the same species only one cytase exists.
Ehrlich and Morgenroth, on the other hand, hold that the same serum
contains several, sometimes many, different cytases. This question is
too difficult to be definitely solved at present. It appears to me very
probable that there exist, in the same species of animal, two different
cytases. One of these, the macrocytase which is found in the lymphoid
organs and in the serum of the blood, acts more particularly on animal
cells. Thanks to this substance an extract or maceration of the spleen,
omentum or lymphatic glands dissolves the red blood corpuscles more or
less readily; these extracts and macerations, however, are incapable of
destroying bacteria. When the macrophages seize the nucleated blood
corpuscles they digest them completely, not sparing even the nucleus, so
resistant to attack, but when the same phagocytes ingest such
micro-organisms as are most easily digested, such as the cholera vibrio,
their action is feeble. The vibrios, without any transformation into
granules, remain alive for some time and are destroyed and digested with
very great difficulty. The cytase of the microphages, or microcytase, is
distinguished by other properties. It destroys and digests easily many
micro-organisms, but has little or no action upon the red blood
corpuscles and other animal cells. The exudations which are rich in
macrophages, such as those of the lymphoid organs, are not at all or
only slightly bactericidal, but exhibit a solvent action on red blood
corpuscles. On the other hand, the exudations, which are composed in
great part of microphages, leave red blood corpuscles intact, but
readily destroy micro-organisms. Similar properties distinguish the bone
marrow, extracts and suspensions of which do not dissolve red
corpuscles, but attack micro-organisms. Now, we know that the bone
marrow is the principal seat of origin of the microphages.
Even after the addition of some of the specific fixative to the
microphagic exudations no solution of the red corpuscles is produced,
which demonstrates most clearly that the microcytase is really incapable
of attacking these animal cells.
We are, therefore, compelled to accept the existence of two different
cytases, of which one (the macrocytase) acts specially upon elements of
animal origin, and the other (the microcytase) acts principally on
micro-organisms. The indication of any more detailed differentiations is
impossible in the present state of our knowledge.
[Sidenote: [575]]
There are certain ferments which, during the life of the cells which
produce them, pass readily into the surrounding fluids. For instance,
sucrase can be recovered without difficulty from the culture fluid of
moulds and yeasts. The ferments of the intestinal digestion also pass
with great facility into the secreted fluids. Other soluble ferments, on
the other hand, remain very closely bound up with the cells which
manufacture them. Thus the zymase of the yeasts can only be freed from
the cells of these fungi with great difficulty, under the influence of
great pressure and under conditions which profoundly alter the cell. The
proteolytic ferment of the yeast is also very adherent to the cells of
these organisms. The fibrin-ferment, or plasmase of the white
corpuscles, is not secreted by these cells so long as they are quite
intact. But it is sufficient to subject them to unfavourable conditions
of existence to cause them to throw it out from their bodies. The
leucocytes, when removed from the animal, undergo a deterioration which
soon leads to the deposition around them of filaments of fibrin.
The cytases must also be grouped with the soluble ferments which are not
thrown off by the phagocytes so long as these remain intact. Immediately
these cells are injured, however, they allow a part of their cytases to
escape. In the blood, withdrawn from the animal, the white corpuscles
allow the plasmase to pass into the fluid, where it sets up the
coagulation of the fibrin and the formation of a clot. At the same time
these cells give up some of their cytases which communicate to the serum
its haemolytic and bactericidal properties. This fact is of the highest
importance in connection with the question of immunity. The best
demonstration of this has been furnished by a comparison of the
bactericidal power in the different parts of the body and in the body
fluids extracted from the animal.
When micro-organisms are introduced into those situations in the
refractory animal which contain pre-existent leucocytes, the leucocytes,
under the influence of the shock, undergo serious lesions, accompanied
by the throwing out of the cytases. Under these conditions the least
resistant micro-organisms (such as the cholera vibrio) exhibit
undeniable signs of deterioration: they become transformed into granules
and may even die in greater or less numbers. When, however, the
leucocytes are well protected and withstand the injection of the
micro-organisms without being profoundly altered, the extracellular
destruction of the micro-organisms does not take place. On the contrary,
a very rapid phagocytosis is produced which brings about the death and
intracellular digestion of these micro-organisms. Under these conditions
vibrios are also transformed into granules and perish, but only within
the leucocytes. The phenomena I have just mentioned are brought about in
the peritoneal cavity and in the blood vessels of refractory animals,
that is to say, in situations rich in leucocytes.
[Sidenote: [576]]
In the subcutaneous tissue, in the fluids of oedemas and in the anterior
chamber of the eye of these same refractory animals, the phenomena are
very different. As in these situations there are no pre-existing
leucocytes or their number is insignificant, the micro-organisms
introduced do not suffer serious injury; they continue to live up to the
moment when the leucocytes, having come up as the result of the
inflammatory reaction, seize them alive, kill them, and digest them
within their substance. Just as it is easy, in situations populated by
pre-existing leucocytes, to suppress the extracellular destruction of
the micro-organisms by preserving the phagocytes against injury or
phagolysis, so this same extracellular destruction is easily set up in
situations where leucocytes are absent. When, after exudations rich in
leucocytes have been injected into the subcutaneous tissue, we introduce
micro-organisms which are not very resistant, such as the cholera
vibrio, it is observed that these vibrios are destroyed outside the
cells, having first been transformed into granules.
There can be no doubt as to the conclusion to be drawn from these
various experiments. The microcytase is the substance which transforms
the vibrios into granules. It is within the microphages, when they
remain intact, that the vibrios undergo transformation. When, on the
other hand, the microphages are injured and allow the microcytase to
escape, the transformation of the vibrios into granules and their
partial destruction take place in the plasmas outside the phagocytes.
This conclusion is supported by comparative researches on the
bactericidal power of the serum and of the blood plasma outside the
animal. It is true that it is impossible to prepare a fluid which shall
in all respects be comparable to the plasma of the circulating blood.
There is, however, always a means of obtaining outside the animal a
fluid which approaches much more closely to blood plasma than does
serum. Gengou succeeded in preparing in tubes coated internally with
paraffin a fluid which coagulates very tardily, and which contains very
little fibrin-ferment. This fluid is found to be much less bactericidal
than is the blood serum of the same animal. It is, indeed, often found
to be entirely without bactericidal power, whilst the corresponding
serum is capable of destroying a large number of micro-organisms.
[Sidenote: [577]]
In the phenomena of the absorption of cells also a great number of facts
are met with which demonstrate that the macrocytase escapes from the
macrophages at the moment of their phagolysis only. For example, the
extracellular solution of the red corpuscles takes place easily in the
peritoneal fluid of animals prepared by a previous injection of the same
corpuscles. When the leucocytes of the peritoneal cavity are abandoned
to their fate, a marked phagolysis is produced and consequently a
solution of the red corpuscles in the fluid itself. When, on the other
hand, phagolysis is prevented, the macrophages remaining intact do not
allow their macrocytase to escape and the solution of the red corpuscles
takes place almost exclusively inside the phagocytes.
In certain animals the blood serum arrests the movements of their own
spermatozoa at once, whilst these remain quite motile in the animal
itself. This is due to the fact that the immobilising macrocytase is
contained within the macrophages and does not escape from them so long
as these cells remain intact. When, in such animals, their own
spermatozoa are introduced into the subcutaneous tissue, they remain
motile for a long time; when, on the contrary, the spermatozoa are
injected into the peritoneal cavity, where the leucocytes have not been
prepared, phagolysis is produced at once and the spermatozoa become
motionless immediately.
As all these data agree in demonstrating that the uninjured phagocytes
retain the cytases—which remain within them, and are not found in the
surrounding fluids,—we can readily understand the reason for the
differences between the phenomena of immunity and the bactericidal power
of the body fluids. The rat’s serum is capable of destroying a large
number of anthrax bacilli, although these rodents are certainly
susceptible to anthrax. The reason for this is that in the serum of the
rat the bacilli are destroyed by the microcytase which is set at
liberty, whilst in the body of the animal it remains enclosed within the
bodies of the living microphages. So long as these cells exhibit a
negative chemiotaxis against the anthrax bacillus, the micro-organism
remains in the plasma, where it is not interfered with. Thanks to this,
multiplication of the bacilli goes on in the body of the animal, the
micro-organism killing it after becoming generalised in the blood and in
the organs. The susceptibility of the leucocytes is, then, the cause of
the death of the rats from anthrax, the organism of these rodents being
unable to take advantage of its richness in bactericidal microcytase.
Another paradoxical fact is met with in guinea-pigs immunised against
Gamaleia’s vibrio (_Vibrio metchnikovi_). As demonstrated by von Behring
and Nissen, the blood serum of these guinea-pigs is very bactericidal
for the vibrio in question. A contact of less than an hour is quite
sufficient to destroy large numbers of the micro-organisms.
Nevertheless, when a small dose of a culture is injected subcutaneously
into these hypervaccinated guinea-pigs, the vibrios remain alive for
several days, up—indeed, to the moment when they are ingested and
destroyed by the leucocytes which come up in large numbers to the
menaced spot. This apparent contradiction is easily explained by the
fact that it is in the serum only that the vibrios encounter the
microcytase, which has escaped from the microphages at the time of the
formation of the clot and the separation of the serum.
[Sidenote: [578]]
Alongside those cases in which the serum of susceptible animals is found
to be very bactericidal, examples are not wanting where the blood and
the serum of refractory animals are entirely without this power. For
instance, the pigeon is refractory to Pfeiffer’s influenza bacillus, but
the blood of the pigeon forms the best culture medium for this
micro-organism. The dog is refractory to the anthrax bacillus, against
which the blood serum of the same animal is not at all bactericidal. The
cause of this absence of parallelism between immunity and the
bactericidal power of the serums must be sought in the difficulty with
which the cytases escape from the leucocytes, and also in the
modifications which they may undergo, once they are distributed in the
fluids.
[Sidenote: [579]]
In cases of natural immunity, the cytases rid the animal of the
micro-organisms without the slightest observable co-operation on the
part of other soluble ferments. It is impossible to settle definitely
even the question whether, in animals which enjoy this innate immunity,
there exists, alongside the microcytase, any ferments which come to its
aid. The conditions are quite otherwise in a very large number of cases
of acquired immunity. Here it is found, as a fairly general rule, that
in addition to the microcytases there exist other substances whose rôle
in the defensive action offered by the animal against micro-organisms is
very important. These substances are fixatives which co-operate in a
remarkable fashion with the bactericidal action of the cytases; but
whilst these latter injure the bacterial cell directly, the fixatives do
not interfere with its life. The bacteria, permeated by fixatives, may
even continue to reproduce themselves and, under certain conditions, to
invade the animal. The fixatives, then, are not bactericidal, but by
fixing themselves upon the micro-organisms they render them much more
susceptible to the bactericidal action of the microcytases. These latter
are further distinguished, in several other respects, from the cytases.
The fixatives must also be classed with the group of soluble ferments,
but they resist much higher temperatures than those which destroy the
cytases. Whilst the latter are quite destroyed at 55° C., the fixatives,
to be completely altered, must be heated to beyond 60° C. and even 65°
C. On the other hand, the fixatives are distinguished by a high
specificity which is never observed in the cytases. The majority of the
fixatives are incapable of fixing themselves upon more than a single
species of bacteria or upon a single class of animal cells, and only
certain of them can fix themselves upon allied species or cells, such as
the red corpuscles of several species of animals. In these cases, too,
there exists a sharp quantitative difference between the fixation on the
different formed elements. The same microcytases are, on the other hand,
able to attack all kinds of micro-organisms, and the same macrocytases
attack all kinds of animal cells.
We have seen that the cytases correspond to the zymase and to the
proteolytic diastase of the yeasts in the sense that all these soluble
ferments adhere with tenacity to the cells which produce them and
contain them. The fixatives, in this respect, approach sucrase
(invertin): these various soluble ferments pass readily into the fluids
which bathe the cells that produce them. The fixatives are found not
only in the blood serums, prepared outside the body, but also in the
blood plasma, whence they pass into the fluids of the exudations and
transudations. Whilst no cytases are found in the subcutaneous tissue,
or in the clear fluids of oedemas containing no, or almost no, cells,
fixatives are not absent from these various situations just indicated.
For this reason, when micro-organisms are introduced subcutaneously,
they are not found to be altered by the cytases, but it is easily seen
that they are permeated with fixatives. The same rule applies to the
fixatives of the animal cells. In the example we have cited, the
spermatozoa, in an animal whose serum renders these cells motionless,
remain quite motile in the epididymis and below the skin. From this fact
it may be concluded that these situations contain no free macrocytase.
It is sufficient, however, to add to these motile spermatozoa a drop of
normal serum containing macrocytase to stop their movements at once, the
fixative being well distributed in the plasma of the living animal. The
spermatozoa, then, were sensibilised by the fixative which was found in
both the epididymis and in the subcutaneous tissue.
[Sidenote: [580]]
The cytases are soluble ferments which are essentially intracellular:
the fixatives are, on the other hand, soluble ferments which are
humoral. These fixatives, however, although circulating in the plasmas,
are undoubtedly of cellular origin. This fact was first demonstrated by
Pfeiffer and Marx, who found the specific fixative of cholera vibrios in
the “haematopoietic organs,” that is to say, in the spleen, lymphatic
glands, and bone marrow, at a period when there was, as yet, none in the
blood. This fact has been extended to other examples of fixatives of
micro-organisms, and it cannot be questioned that the phagocytes produce
these soluble ferments. Under the influence of the introduction of
micro-organisms into the body, a phagocytic reaction is produced which
has, as a consequence, the digestion of these micro-organisms and the
production of corresponding fixatives. There is every reason to believe
that, in these cases, it is the microphages which, seizing and digesting
the micro-organisms, produce the fixatives.
But the macrophages are also capable of producing these adjuvant
ferments. Even in normal animals the macrophagic organs, such as the
spleen, and especially the mesenteric glands, contain fixatives which
help in the solution of the red blood corpuscles. Into this group of
facts we must also place the production by the mesenteric glands, as
well as by certain other lymphoid organs, and the leucocytes of
exudations and the blood, of enterokynase,—the soluble ferment which
aids the digestive action of trypsin. This enterokynase is also a
species of fixative; it permeates the flakes of fibrin and renders them
much more accessible to the influence of the trypsins.
The fact that the enterokynase of the intestinal digestion corresponds
in so many respects to the fixatives which act in the absorption of
formed elements in general and of micro-organisms in particular,
furnishes a further proof that the destruction of micro-organisms in the
animal is an act similar to true digestion.
[Sidenote: [581]]
Phagocytes, those elements which accomplish the absorption of
micro-organisms and of animal cells, those holders of digestive cytases,
are also the manufacturers of fixatives. Having brought about this
absorption, the phagocytes set to work to elaborate large quantities of
fixatives, although they are unable to increase the amount of cytases in
any marked degree. The fixatives, produced in abundance, can be excreted
outside the phagocytes and pass into the blood plasma, and, with it,
into the fluids of exudations and transudations. But this excretion is
not an indispensable act for the functioning of the fixatives. As these
ferments prepare the way for the digestive action of the cytases, it is
necessary only that they should be able to fix themselves on the formed
elements before the latter. It is, therefore, easy to explain cases of
acquired immunity in which no fixatives are found in the body fluids.
Such examples are not rare, and are characterised by the absence of any
protective action on the part of the blood serum. In these cases, the
fixatives, whose existence is very probable, remain lodged within the
phagocytes, just as are the cytases. Within these digestive cells the
fixatives may quite well fulfil their preparatory rôle, this being
followed immediately by the action of the cytase. The same rule may
apply also to the cases of absorption in the unprepared animal, where
fixatives are not found in the blood serum, but where they are able to
act within phagocytes.
The excretion of fixatives into the plasmas, which constitutes the rule
in cases of acquired immunity, presents an analogy with the excretion of
pepsin into the blood. This soluble ferment can and does pass habitually
from the stomach into the blood and thence into the urine, where it is
often met with. As the pepsin, which only acts in an acid medium, cannot
be utilised in the alkaline blood plasma, it is evident that its
excretion is only the consequence of a too abundant over-production.
In recent years great attention has been paid to the essential mechanism
of the action of fixatives on the formed elements on the one hand, and
on the cytases on the other. According to Ehrlich, the fixatives are
bodies intermediate between the two. In possession of two haptophore
molecular groups, they are capable of entering into chemical combination
with the micro-organisms or the animal cells on the one hand, and with
the cytases on the other. It is for this reason that Ehrlich applies to
them the name of “amboceptors” or “intermediary substances.” Based on
analogous examples in organic chemistry, Ehrlich thinks that the
fixatives serve to introduce the cytases into the cells upon which they
have to act. Bordet does not share this view and maintains that the
action of the fixatives is not a chemical action in the proper sense of
the word, but is a kind of mordanting which sensibilises the formed
elements to the fermentative action of the cytases. According to him,
the fixatives have no affinity for the cytases and in no way serve them
as intermediaries, for which reason he gives to them the name of
sensibilising substances. The question is still under discussion, but we
may hope that it will soon enter into its final phase.
[Sidenote: [582]]
According to Ehrlich’s theory, the fixatives contain no product coming
from the micro-organisms or from the animal cells upon which they are
fixed. The fixatives are, according to him, side-chains or receptors,
produced in excess and expelled into the blood plasma by the cells which
produce them. Ehrlich does not tell us to what category these cells
belong; he maintains only that these cells must be in possession of
receptors endowed with a specific affinity for certain molecular groups
of micro-organisms and of animal cells. As soon as the receptors are
saturated by these molecular groups, the cells which make use of the
former for their nutrition produce them in superabundant quantity. The
cells of animals, treated with micro-organisms and their soluble
products, or with red blood corpuscles or any other kind of element of
animal origin, acquire the property of elaborating more and more of the
corresponding receptors, a large proportion of which are expelled into
the blood plasma.
The common point between Ehrlich’s theory and the view maintained in
this work consists in the admission of a cellular property which
develops more and more in proportion to the treatment of the animal by
formed elements of all kinds. As, in acquired immunity against
micro-organisms, the fixatives are most frequently found in the body
fluids, it must be concluded that, in all these cases, the cells which
produce them have become adapted by a kind of education to manufacture
increasing quantities of fixatives. But even in those examples of
acquired immunity where fixatives are not found in the plasmas, we must
accept a modification of the cells which resist the invasion of
micro-organisms. These changes in the cellular properties constitute,
therefore, the most general, and consequently the most important,
element in acquired immunity against micro-organisms.
As already mentioned Ehrlich does not assign any position to the cells
which exhibit these modifications. It must, however, be accepted that
they belong to the category of phagocytes. Indeed, the phagocytes put
themselves into most intimate contact with the micro-organisms and
foreign animal cells, and it is in the phagocytic organs that the
fixatives are found before they are met with in the blood plasma. It may
then be concluded that, in acquired immunity against micro-organisms,
the phagocytes become adapted to elaborate the fixatives in large
quantities, of which a portion is excreted into the body fluids, as has
been shown in many examples of such immunity.
[Sidenote: [583]]
The progressive adaptation of the phagocytes in intracellular digestion
can be demonstrated by the fact that in an immunised animal the
fixatives are found more especially in the phagocytic organs. The
leucocytes which digest gelatine exhibit in an even more distinct
fashion the modification of these cells in animals which have received
several injections of gelatine. The leucocytes of exudations, when the
fluid is removed, become much more fitted to digest the gelatine than
they were at first.
A similar adaptation is also observed in intestinal digestion, which may
serve as a fresh point of comparison between the intracellular digestion
of the phagocytes and the extracellular digestion in the intestines. The
pancreas, in order to secrete its soluble ferments, adapts itself to the
nature of the food which passes into the digestive canal.
The fixatives are not the only soluble ferments which appear in large
quantities in the fluids of the immunised animal. Very often there are
found along with them substances which agglutinate the micro-organisms
in animals which have received several injections of micro-organisms of
the same or an allied species. The same fact is observed in animals
treated with animal cells. Thus the fluids of animals injected with
blood corpuscles become agglutinative for these corpuscles.
[Sidenote: [584]]
The analogy between the agglutinins and the fixatives is so great that
for some time several observers assumed them to be one and the same
substance. This can no longer be upheld, for it is clearly demonstrated
that the property of the body fluids to agglutinate micro-organisms and
animal cells is different from that which brings about their permeation
by fixatives. The agglutinins resist the same temperatures as the
fixatives; both are specific to the same degree and pass equally from
the cells which produce them into the plasmas of the blood, lymph,
exudations, and transudations. The agglutinins capable of clumping the
formed elements into masses may, under certain conditions, render their
ingestion by the phagocytes more easy. In general, however, the part
played by the agglutinins in acquired immunity must be regarded as of
little importance, and for that reason we abstain from basing any theory
of this immunity on the agglutinative property of the body fluids.
Besides fixatives and agglutinins, the fluids of an animal which has
acquired immunity very probably possess other properties which must have
a greater or less function in acquired immunity. Thus, we are often
struck by the stimulating action of these fluids on the normal animal
into which they are introduced. This stimulation is especially
manifested against the phagocytic reaction.
As, in the majority of cases of acquired immunity, the blood serum
contains fixatives in considerable proportion, and as these fixatives
aid the action of the cytases in a remarkable fashion, we can readily
understand that the introduction of such a blood serum into a normal
animal, unprepared by any vaccination, may bring about a great
resistance against the corresponding pathogenic micro-organisms. The
fixatives, injected with the serum, fix themselves with avidity upon the
micro-organisms. These organisms may become a more ready prey to the
phagocytes and be destroyed very rapidly. In particular cases, where the
injection of microbial cultures sets up a phagolysis, enough cytases are
thrown out to affect the microbes already sensibilised by the fixative.
This is followed by a refractory condition of the animal proportionate,
in general, to the amount of fixative serum that is injected. This kind
of acquired immunity, conferred by serums or certain other body fluids
rich in fixative substances, has often received the name of passive
immunity. This term is only justified in those rare cases where the
introduced serum itself contains a sufficient amount of cytases to
destroy all the micro-organisms. Most often it is the normal animal
which has to furnish this bacteriolytic ferment. Now, as in phagolysis
the quantity given off is too small, it is to the co-operation of the
holders of cytases, that is to say, to the phagocytes, that the animal
must have recourse. The phagocytes, being susceptible cells, their
co-operation can only be counted upon in cases where they exhibit a
sufficient activity. When these elements are weakened by narcotics or by
any other cause, they become incapable of intervening with efficacy and
the animal falls a victim to the pathogenic micro-organisms, in spite of
the more than sufficient amount of fixatives that was introduced.
[Sidenote: [585]]
In natural or acquired immunity, it is the resistance of the animal
against the micro-organisms which plays the principal part. The
introduction of toxins ready prepared is only done under artificial
conditions, as in laboratory experiments. Hence we see that, under
natural conditions, it is against the penetration of the micro-organisms
that the animal must be protected. So soon as these producers of poisons
can no longer maintain themselves in the immunised animal their toxic
secretions do not come into play. It is for this reason that animals
vaccinated against pathogenic micro-organisms do not suffer from
intoxication, although they are by no means insusceptible to the
microbial poisons. It is a fact of the highest importance from the point
of view of immunity in general, that the resistance offered to
micro-organisms in no way implies insusceptibility to their poisons. The
view has frequently been expressed that, in acquired immunity at least,
the animal must first acquire immunity against the microbial toxins,
after which the micro-organisms, deprived of their principal weapon,
descend to the rank of inoffensive saprophytes. Such cases may be found,
but it is none the less true that immunity against micro-organisms may
be acquired independently of that against the toxins, and that this
constitutes the general rule.
Immunity is much more readily acquired against micro-organisms than
against their toxins. Hence, antimicrobial vaccination was accomplished
by science before that against their toxins. In the early researches on
this subject antitoxic immunity appeared to be very difficult of
attainment, and it was only after the discovery made by von Behring, who
inaugurated a new path in microbiology, that better results were
obtained. Von Behring not only succeeded in immunising animals against
some of the principal microbial toxins, he demonstrated the existence of
specific antitoxins in their body fluids.
This very unexpected conception of antitoxins at once took root in
science, for it has been possible, thanks especially to the remarkable
works of Ehrlich, to extend it to toxins of non-microbial origin. We are
already acquainted with a certain number of antitoxins which, however,
are not comparable in number to the other antibodies. Amongst these, the
fixatives have many points of analogy with the antitoxins. Like them,
they are resistant to heat: they exhibit also a fairly marked
specificity, and, like the fixatives, they are distributed in the
plasmas.
[Sidenote: [586]]
In the presence of so many points of similarity with the fixatives, one
is tempted to attribute to the two categories of antibodies the same
origin. The elaboration of antitoxins by the phagocytic elements,
accumulated in the blood and disseminated in the organs, appears, in
fact, to be very probable. Certain facts bearing on the absorption of
various toxins by the leucocytes, as well as the distribution of
antitoxins in the animal body, speak in favour of this view. On the
other hand, the impossibility of attributing the elaboration of
antitoxins to cells attacked by the corresponding toxins is quite in
harmony with the same hypothesis. This hypothesis is especially
supported by the numerous facts which prove the readiness with which the
leucocytes react against all kinds of poisons, microbial or other
toxins, as well as against organic and mineral poisons, such as the
alkaloids and the arsenical combinations. However, in spite of so many
data which speak in favour of the phagocytic origin of antitoxins, it
has been impossible to support this view by rigorous facts easy of
interpretation, such as those which science possesses in support of the
phagocytic origin of fixatives.
The antitoxins have acquired a very great importance in the artificial
cure of toxo-infective diseases, the aim in these cases being to
paralyse the action of the toxins already produced by the
micro-organisms and absorbed by the diseased animal. But their function
is less in the protection against diseases where the object to be
obtained is a reaction against the micro-organisms before these are able
to inundate the animal with their toxic secretions. It is for this
reason that the immunity against toxins must, in the study of immunity,
occupy a less preponderant place than does the immunity against
micro-organisms.
As the micro-organisms placed in the refractory animal ultimately
undergo a digestion by chemical substances elaborated by the phagocytes,
so also the toxins undergo a chemical modification due to the presence
of substances in the production of which the living elements of the
animal play a large part. The direct action of antitoxins on the toxins,
so well demonstrated, especially by Ehrlich’s investigations, does not,
however, exclude the intervention of living cells, which, though
sometimes not very manifest, is in other cases very marked.
[Sidenote: [587]]
The reaction of the living elements against the microbial toxins and
their allies leads to the production, and even the over-production of
antitoxins. According to Ehrlich, these elements are the receptors, or
side-chains, which, to a certain extent, pre-exist in the cells which
are capable of elaborating the antitoxins. On entering into combination
with the toxin molecules, the side-chains, which are indispensable for
the nutrition of the cells, are reproduced in very large numbers. After
having saturated, so to speak, the productive elements of the antitoxin,
the superfluous side-chains escape from the cell and pass into the
plasmas of the body fluids. This theory may be brought into harmony with
the other theory, which maintains that certain elements of the animal,
capable of acting on the complex molecules of microbial toxins and their
allies, produce special soluble ferments, which digest the toxins whose
introduction frequently excites the hypersecretion of the ferments. Here
we have something similar to the hypersecretion, by the glands of the
stomach, of pepsin, a part of which passes into the blood in order to
escape with the urine.
According to Ehrlich’s theory, the antitoxins are only capable of
neutralising the injurious action of toxins when the former are found
dissolved in the body fluids. The same receptors which fix the toxins in
the plasmas and thus prevent them from reaching the susceptible
elements, bring about an opposite result when they are found inside the
cells. In this latter case, the receptors, owing to their great affinity
for the toxins, attract them and allow them to pass into the cells, in
this way aiding the dangerous function of the toxophore group.
This is an ingenious idea, conceived to bring into harmony a certain
number of observed facts. In the present state of our knowledge it
cannot be subjected to rigorous experimental test. Many well-established
facts, however, are not in complete accord with this hypothesis.
According to it the antitoxic immunity resides exclusively in the body
fluids; the living cells, instead of acquiring immunity, become more and
more susceptible. Under these conditions it is difficult to conceive of
an immunity against poisons of the simplest organisms; nevertheless,
this certainly exists. A plasmodium, which becomes adapted to all kinds
of toxic substances, acquires an immunity against them, and this is due
to changes taking place in the living elements; it is not the result of
modifications in the toxic fluids which bathe them. This biological
adaptation is observed in the case of physical factors which may
interfere with the life of these primitive organisms.
On the other hand, it must be accepted that the living cells of a
complicated and higher organism may also acquire immunity against
toxins. The first example of this kind was shown in relation to the red
blood corpuscles of mammals vaccinated against the toxic serum of the
eel. Whilst the body fluids of immunised rabbits become antitoxic, their
red blood corpuscles, when completely freed from the serum, in certain
cases resist the action of the eel’s serum. It must be admitted that in
this example we have an acquired immunity of the cells similar to that
met with in lower organisms.
[Sidenote: [588]]
A second example of the immunity of the red corpuscles was observed by
Ehrlich and Morgenroth in goats prepared by injections of the blood of
other individuals of the same species. In this case, according to these
writers, no co-operation by antitoxin is met with. The body fluids of
the goats do not become capable of neutralising the toxin of the
haemolytic serum, whilst the red corpuscles themselves acquire an
immunity against this toxin, an immunity entirely cellular. Ehrlich
attempted to penetrate into the essential mechanism of the resistance of
the red blood corpuscles on the supposition that these corpuscles,
instead of reproducing their receptors, as when there is production of
antitoxin, get rid of them entirely. Deprived of receptors, they can no
longer be affected by the haemolytic cytase which, as Ehrlich maintains,
only penetrates into the red corpuscles owing to the affinity of the
intermediate substance (fixative) for the receptor. This hypothesis of
the mechanism of acquired cellular immunity scarcely accords with the
hypothesis of the special function attributed to the receptors in the
nutrition of the living elements.
Cellular immunity can be most easily demonstrated in relation to the red
corpuscles of the blood, as these elements are very numerous and are
capable of being isolated and freed from the fluid in which they are
bathed. For this reason, science does not as yet possess sufficiently
exact data on the immunity of other cells in higher animals. Many facts,
however, indicate that such immunity does exist. There are, indeed,
living elements which only acquire immunity with great difficulty and
very slowly. Such are the nerve cells, elements which are specially
susceptible. Von Behring has strongly insisted on the fact that in
animals subjected to repeated injections of bacterial toxins, the nerve
centres not only do not become accustomed to their injurious action, but
even acquire a hypersusceptibility which is often very great. The
observation is perfectly accurate, but it is none the less true that
this period of exaggerated susceptibility is followed by another, during
which the susceptibility becomes less marked and ends by giving place to
a true adaptation. We are, therefore, compelled to accept the fact that
even the nerve cells are no exception to the general rule, but are able
to acquire a diminished susceptibility to a poison.
[Sidenote: [589]]
Several facts of another series confirm this conclusion. In the study of
the action of the nervous system one frequently has occasion to observe
instances of adaptation. I will cite as an example the adaptation of
animals to spinal concussion studied by Lépine[922]. By percussing the
lumbar region of rabbits and guinea-pigs we may induce in them an
immediate paraplegia. This is transitory, and lasts at most for a few
hours. The phenomenon may be reproduced several times in the same
animal. “But,” remarks Lépine, “when these experiments are continued for
several days or several weeks, striking always at the same level, we
soon observe that the resistance of the animals to the blows increases
very rapidly, and that excitations which, in normal animals, produce
paraplegias of several hours’ duration, produce no effect upon those
which have been under experiment for several days.” We have in this
example a real adaptation of the spinal region when subjected to
concussion.
Similar facts are known to everyone as an experience of daily life. We
can become habituated more or less easily to all kinds of violent
sensations. Light and very intense noises which, at first, excite
exaggerated reflex actions are ultimately perceived without setting up
the least movement. Even in the psychical sphere habit dulls painful
feelings, and it is very probable that a whole gamut of adaptation,
starting from unicellular organisms which accustom themselves to live in
an unsuitable medium, up to cultured human beings who habituate
themselves to a disbelief in human justice, will be found to rest upon
one and the same fundamental property of living matter.
[Sidenote: [590]]
Regarded from this point of view, immunity becomes a very general
phenomenon, passing far beyond the resistance offered by the animal to
infective diseases. After all is said and done, it invariably reduces
itself to that cellular susceptibility [irritability] which governs so
many of the vital phenomena in plants and in animals. It is this
susceptibility which impels the branch towards the light and the root
towards the ground, and which guides the spermatozoon towards the ovum.
From the very commencement of embryonic life the cells derived from the
segmentation of the egg exhibit a marked susceptibility. Wilhelm
Roux[923] observed that the earliest cells of the frog embryo, if they
are separated by artificial intervention, guided by their positive
chemiotaxis again come together. In the formation of the tissues
cellular susceptibility plays an important undoubted rôle. The
prolongations of the nerve cells direct themselves towards the organs of
sense or towards the muscular fibres, according to their specific
susceptibility[924]. The mother-cells of the capillary vessels are also
guided by susceptibility, when they go towards a new-formed tissue, or
when they approach one another and come together in order to form a
vascular loop.
The phenomena of the organism which bear the sharpest impress of their
physical and chemical nature, also come under the influence of cellular
“sensations.” Thus, in gastro-intestinal digestion, the secretion of the
active juice is subordinated to the control of the nerve centres and
even of the psychic centres. The sight of various kinds of food
stimulates, unconsciously, by reflex action the activity of different
digestive glands. In the same way the contraction of the contents of the
cells of a plant subjected to plasmolysis, brings about the secretion of
acid in order to augment the osmotic pressure.
Susceptibility, whose part is so great in the phenomena of immunity,
taken as a whole, is a general property of living beings, regulated by a
common law. Thus, in the chemiotaxis of the lowest unicellular
organisms, as in the movements and the osmotic reaction of plants, there
is manifested the same psycho-physical law of Weber-Fechner which
regulates our own sensations.
[Sidenote: [591]]
All cells are able, by modifying their function under the direction of
susceptibility, to adapt themselves to changes in the surrounding
conditions. All living elements are able, therefore, to acquire a
certain degree of immunity. But, amongst all the cells of the animal
body, the elements which have retained most independence—the
phagocytes—most easily and first acquire immunity to infective diseases.
These are the cells which betake themselves to situations where
micro-organisms and their poisons make their appearance, and which
manifest a reaction against them. The phagocytes of the immune organism
ingest and destroy micro-organisms and absorb toxins and other poisons.
The final act of the reaction of the phagocytes is constituted by the
chemical or chemico-physical processes concerned in the digestion of the
micro-organisms, with the help of cytases, assisted by the fixatives; in
the defence offered against poisons the phagocytes must also exert a
chemical action. Before these phenomena come into play, however, the
phagocytes manifest phenomena which are purely biological, such as the
perception of chemiotactic and other sensations, the migration towards
menaced situations, the ingestion of micro-organisms and the absorption
of toxins, and finally the secretion of substances to be utilised in
intracellular digestion.
The immunity in infective diseases presents itself, therefore, as a
section of cellular physiology, and especially as a phenomenon concerned
in the absorption of micro-organisms. This absorption being carried out
by an act of intracellular digestion, the study of immunity comes into
the chapter on digestion regarded from the general point of view.
As in the struggle of the body of the animal against infective agents
the phagocytes play the principal part, it happens that in certain
diseases the micro-organisms in order to manifest their morbific effect
must be protected from the attacks of these defensive cells. It is for
this reason that the cholera vibrio, which is not very injurious when
introduced below the skin of the human subject, becomes very formidable
when it succeeds in gaining access to the digestive canal. Incapable of
maintaining a struggle against the phagocytes, the vibrio is able to
overcome in the stomach and in the intestines without difficulty the
obstacles which it here meets with. It is for this reason that the
channel of entrance of the micro-organisms at times plays such a
prominent rôle in immunity against infective diseases.
The question is often asked whether a theoretical study of immunity is
capable of rendering service in the search for means of conferring
immunity on the animal. It must not be forgotten that theory and
practice frequently march side by side, but that sometimes they advance
without very much regard for each other. Thus the first preventive
inoculations against snake-bite, small-pox, and pleuropneumonia,
attempted by laymen were evidently made independently of any theoretical
ideas of any kind, but were guided by the purest empiricism. On the
other hand, the theoretical researches on the nature and origin of
ferments led to the discovery of vaccinations by means of
micro-organisms and microbic products which have rendered immense
services to practical medicine.
[Sidenote: [592]]
[Sidenote: [593]]
The discovery of antitoxins, so rich in practical applications, was
influenced by theoretical researches on the mechanism of immunity. Von
Behring began his important series of investigations on this subject
with the study of the immunity of rats against the anthrax bacillus. It
did not suggest itself to anyone to suppose that this question could
have the slightest immediate practical interest; nevertheless, starting
from this investigation, von Behring, after giving up the theory of the
bactericidal property of the body fluids as a cause of immunity,
advanced, step by step, to the discovery of the antitoxic power of the
serums. When a study of the properties of the blood of animals treated
with the red corpuscles of another species was commenced, no one would
have suspected that these researches would end in the discovery of new
methods for the recognition of human blood in medico-legal researches,
or in the interests of hygiene for the determination of the source of a
milk. The cellular theory of immunity is, as yet, of too recent date for
us to claim the right to expect it to have amongst its assets methods
for purely practical application. Nevertheless, it has already been
found to be of service in the investigation of problems very closely
affecting medical practice. Lord Lister, the greatest surgeon of the
nineteenth century[925], asked himself how it was that wounds could heal
“by first intention under circumstances before incomprehensible.
Complete primary union was sometimes seen to take place in wounds
treated with water-dressing, that is to say, a piece of wet lint covered
with a layer of oiled silk to keep it moist. This, though cleanly when
applied, was invariably putrid within twenty-four hours. The layer of
blood between the cut surfaces was thus exposed at the outlet of the
wound to a most potent septic focus. How was it prevented from
putrefying as it would have done under such influence if, instead of
being between divided living tissues, it had been between plates of
glass or other indifferent material?” “How were the bacteria of
putrefaction kept from propagating in the decomposable film?
Metchnikoff’s phagocytosis supplied the answer. The blood between the
lips of the wound became rapidly peopled with phagocytes which kept
guard against the putrefactive microbes and seized them as they
endeavoured to enter. If phagocytosis was ever able to cope with septic
microbes in so concentrated and intense a form, it could hardly fail to
deal effectually with them in the very mitigated condition in which they
are present in the air. We are thus strongly confirmed in our conclusion
that the atmospheric dust may safely be disregarded in our operations;
and Metchnikoff’s researches, while they have illumined the whole
pathology of infective diseases, have beautifully completed the theory
of antiseptic treatment in surgery.” (_Rep. Brit. Ass._, p. 27.)
We may even attempt to increase phagocytosis in surgical operations,
especially in those on the peritoneal cavity, by there setting up an
artificial aseptic inflammation, by means of various substances,
innocuous in themselves, which attract a large number of leucocytes. In
laboratory practice this method is in daily use for the purpose of
increasing the resistance of an animal against intraperitoneal
injections of various micro-organisms, and Durham has suggested the
extension of the same method to human medicine. Certain surgeons have
already made attempts in this direction.
The application of the cellular theory of immunity to researches on new
micro-organisms of infective diseases has already been crowned with
success. Nocard and Roux have attempted to cultivate in the animal body
the virus of the pleuropneumonia of cattle. They selected the rabbit, an
animal naturally refractory against this infection. On the supposition
that, in this immunity, the phagocytes must play an important part as
destroyers of the presumed micro-organisms, the idea suggested itself to
them to withhold the virus from their voracity. With this object they
filled sacs of collodion or of reed pith with pleuropneumonia virus, and
introduced these sacs into the peritoneal cavity of rabbits. Some time
after this operation these investigators were able to demonstrate in the
contents of the sacs impregnated by the blood fluid of rabbits, immune
animals, the development of specific micro-organisms, the smallest
discovered up to the present. By means of cultivations of this
micro-organism, obtained in suitable media, they worked out a method of
vaccinating animals which, as mentioned in Chapter xv., has already
begun to give good results in veterinary practice. This method has thus
contributed to the prevention of diseases, a branch of knowledge which
has made such great advances since medicine became an exact science
under the inspiration of the discoveries and ideas of Pasteur.
[Sidenote: [594]]
Within a very short period immunity has been placed in possession not
only of a host of medical ideas of the highest importance, but also of
effective means of combating a whole series of maladies of the most
formidable nature in man and the domestic animals. Science is far from
having said its last word, but the advances already made are amply
sufficient to dispel pessimism in so far as this has been suggested by
the fear of diseases, and the feeling that we are powerless to struggle
against them.
LIST OF AUTHORITIES QUOTED.
Abel, 443, 444, 445, 536
Abel. _See_ Loeffler
Achalme, 96
Achard and Bensaude, 264, 451
Adil Bey. _See_ Nicolle
Almquist, 178
Arloing, 264, 452
Arloing, Cornevin and Thomas, 471
Arnold, 411
Arthus, 95
Babes, 75, 348
Bach, 408, 410
Bail, 151, 185, 359
Balbiani, 13, 23, 133
Bardach, 150
Barthels, 507
Bary (de), 31, 32
Batzaroff, 411
Baumgarten, 138, 193, 521, 522, 524
Bayeux. _See_ Roger
Behring, 20, 152, 153, 205, 242, 290, 334, 335, 348, 350, 352, 367,
369, 374, 375, 378, 417, 526, 540, 561, 564, 567
Behring and Kitasato, 266, 344, 347, 354, 357, 493, 495
Behring and Kitashima, 42, 290, 368, 370, 373
Behring and Knorr, 355
Behring and Nissen, 211, 226, 526, 531
Bensaude, 439
Bensaude. _See_ Achard
Bernard, 59
Bernheim, 408
Bertrand. _See_ Phisalix
Besredka, 111, 191, 231, 263, 273, 318, 353, 390, 396
Besson, 170
Beumer and Peiper, 230
Biedl and Kraus, 44
Birch-Hirschfeld, 514
Bitter, 525
Bizzozero, 48, 177, 418, 428
Bjelooussoff, 55
Blagovestchensky, 323
Bolton, 205
Bordet, 22, 68, 79, 87, 90, 94, 95, 105, 107, 111, 112, 115, 123, 166,
179, 185, 193, 194, 196, 199, 215, 217, 223, 244, 251, 256, 257,
258, 282, 298, 302, 313, 320, 321, 535, 537
Bordet. _See_ Gengou
Bordet and Danysz, 467
Borrel, 478
Borrel. _See_ Roux, Yersin
Bouchard, 184, 232, 286, 323, 343, 427, 529
Bouchard and Charrin, 42, 528
Bourne, 327
Braun, 12
Brieger, 369
Brieger and Fränkel, 344
Briot, 109
Brücke, 66
Brunner, 45
Buchner, 87, 95, 184, 185, 188, 193, 255, 357, 362, 377, 412, 512, 527,
528, 530, 539, 540
Cahanescu, 430
Calmette, 334, 339, 345, 346, 347, 348, 358, 365, 386, 389, 395, 425,
489
Calmette. _See_ Yersin
Calmette and Deléarde, 365
Calmette and Salimbeni, 491
Camus and Gley, 110, 121, 360
Cantacuzène, 224, 225, 306
Castle. _See_ Davenport
Cattani, 446
Cayley, 484
Celakovsky, 30
Celli, 278
Centanni, 446
Chamberland, 470
Chamberland. _See_ Pasteur, Roux
Chantemesse, 259
Chantemesse and Widal, 230, 267, 319, 437
Chapeaux, 55, 56
Charrin, 232, 286, 287, 427, 428, 541
Charrin. _See_ Bouchard
Charrin and Gamaleia, 290, 343
Charrin and Gley, 446
Charrin and Lefèvre, 419
Charrin and Magnin, 427
Charrin and Roger, 232, 256
Chatenay, 393
Chauveau, 289, 446, 455, 511, 512
Chépowalnikoff, 59
Cherry. _See_ Martin
Cienkowski, 446
Cobbett, 205
Cohn, 23
Colombot. _See_ Sabrazès
Cornevin, 452
Cornevin. _See_ Arloing
Couch, 53
Courmont, 400
Courmont. _See_ Nicolas
Courmont and Doyon, 330, 386, 394
Curtis, 172
Czaplewski, 146, 147
Dallinger, 26
Danysz, 21, 25
Danysz. _See_ Bordet
Daremberg, 87
Darwin, 8
Davenport and Castle, 27
Davenport and Neal, 24
Decroly and Rousse, 396
Deléarde. _See_ Calmette
Delezenne, 61, 96, 98, 107, 116
Delezenne and Froin, 66
Delius and Kolle, 277
Dembinski, 147
Denys, 533
Denys and Havet, 151, 185
Denys and Kaisin, 151
Denys and Leclef, 243, 246, 283, 312
Denys and Marchand, 313
Denys and van de Velde, 359
Deutsch, 107, 293, 294, 537
Dienert, 26
Dieudonné, 139, 143, 147
Dinkelspiel. _See_ Nuttall
Doederlein, 429
Dönitz, 391
Dominici, 78
Dominici. _See_ Gilbert
Doyon. _See_ Courmont
Dreyer, 350
Duclaux, 26
Dujardin-Beaumetz, 478
Dungern (von), 91, 109, 123, 324
Durham, 256, 261, 569
Dzierzgowsky, 448, 449
Effront, 26
Ehrlich, 114, 115, 344, 346, 349, 356, 360, 361, 365, 378, 391, 392,
420, 449, 562, 563
Ehrlich and Hübener, 446, 452
Ehrlich and Lazarus, 76
Ehrlich and Morgenroth, 88, 89, 92, 95, 104, 114, 116, 124, 193, 194,
199, 268, 537, 538, 563
Ehrlich, Kossel and Wassermann, 496
Ehrlich and Wassermann, 356
Elmassian. _See_ Morax
Emden (van), 264
Emmerich, 237, 322, 527
Emmerich and di Mattei, 236, 527
Emmerich and Löw, 254
Emmerich and Mastbaum, 475
Ermengem, 420, 491
Errera, 39
Escherich. _See_ Klemensiewicz
Faber (Knud), 344
Fahrenholtz, 138
Fehleisen, 434
Fermi and Pernossi, 109
Ferran, 480
Fischer, 193, 213, 253
Fischl and Wunschheim, 445
Fleck, 413
Flügge, 43, 184, 525, 540
Fodor, 184, 525
Foerster, 380
Fontana, 333
Forssmann, 565
Frank, 35, 154, 542
Fränkel, 344, 347, 499, 534
Fränkel. _See_ Brieger
Fränkel and Sobernheim, 268
Frantzius, 425
Fraser, 345, 425
Frédéricq, 55, 57
Freudenreich, 323
Freund, Grosz and Jelinek, 365
Froin. _See_ Delezenne
Funck, 267, 319, 320, 456
Galeotti. _See_ Lustig
Gamaleia, 419
Gamaleia. _See_ Charrin
Garnier, 220, 304
Gaule, 515
Gautier, 400
Gengou, 19, 20, 146, 151, 157, 185, 190, 203, 242, 252, 255, 260, 264,
308, 543
Gengou and Bordet, 190
Geret. _See_ Hahn
Gheorghiewsky, 210, 234, 236, 261, 269, 301, 307, 359
Gibier, 137
Giessler, 37
Gilbert and Dominici, 424
Gilkinet, 172
Gley. _See_ Camus, Charrin
Glogner, 434
Goldschmidt, 411
Gottstein, 499
Gramatschikoff, 412
Grancher. _See_ Pasteur
Grawitz, 513, 515
Griffon. _See_ Landouzy
Grosz. _See_ Freund
Gruber, 224, 256, 262, 542
Gscheidlen. _See_ Traube
Guarnieri, 455
Guinon. _See_ Voisin
Günther, 541
Haeckel, 517
Haffkine, 480, 486–488
Hafkine, 17
Hahn, 188, 190
Hahn and Geret, 197
Hankin, 156, 187
Hardy. _See_ Kanthack
Harnack, 337
Häser, 507
Havet. _See_ Denys
Hayem, 47, 514
Hegeler, 196
Herbst, 565
Héricourt. _See_ Richet
Herzen, 62
Hess, 144, 149, 524
Hewlett. _See_ Thomson
Heymans. _See_ Lang
Heymans and Masoin, 396
Hildebrandt, 109, 119, 412
Himmel, 182
Hippocrates, 342
Hirsch and Mehring, 64
Hoffmann and Recklinghausen, 46
Horvath, 337
Hübener. _See_ Ehrlich
Hudalo, 436
Hueppe, 254
Hugenschmidt, 415
Issaeff, 219, 262, 287, 318, 320, 441
Issaeff. _See_ Pfeiffer
Jakowski, 42
Jeanselme, 411
Jelinek. _See_ Freund
Jenner, 507
Jetter, 193
Jona, 172
Joubert. _See_ Pasteur
Kaisin. _See_ Denys
Kanthack, 360, 542
Kanthack and Hardy, 185
Karlinsky, 134, 260
Kempner and Schepilewsky, 387
Kempner. _See_ Rabinowitsch
Kilborne. _See_ Smith
Kitasato. _See_ Behring
Kitashima. _See_ Behring
Klebs, 514
Klecki (von), 44
Klein, 324
Klemensiewicz and Escherich, 443
Klemperer, 271, 356, 411, 441, 449
Klipstein, 170
Knorr, 361, 362, 370, 375, 378, 383, 392, 443
Knorr. _See_ Behring
Koch, 137, 247, 278, 279, 283, 419, 425, 434, 436, 466, 514, 529
Kolle and Turner, 466, 467
Kolle. _See_ Delius, Pfeiffer
Kondratieff, 365
Kossel, 110, 121, 183
Kossel. _See_ Ehrlich
Kossiakoff, 25
Kovalevsky, 41, 133, 134, 209
Krafft-Ebing, 436
Krajouchkine, 465
Kraus and Seng, 258
Kraus. _See_ Biedl
Kretz, 371
Krikliwy, 46
Krompecher, 83
Krönig. _See_ Menge
Krukenberg, 30, 49, 55
Kübler, 458
Kupffer, 75
Kuprianow, 204, 340
Kurt, 499
Laehr, 413
Landouzy and Griffon, 451
Landsteiner, 100
Lang, Heymans and Masoin, 363
Langhans, 73, 84
Laschtschenko, 188
Laurent, 33, 35, 86
Laveran and Mesnil, 173, 248, 316
Lazarus, 272, 441
Lazarus. _See_ Ehrlich
Leber, 79, 96
Leclainche, 475, 476
Leclainche. _See_ Nocard
Leclainche and Vallée, 107, 171, 472, 523
Leclef. _See_ Denys
Le Dantec, 13
Lefèvre. _See_ Charrin
Leishman. _See_ Wright
Leo and Senator, 66
Lépine, 564
Lermoyez. _See_ Wurtz
Lesage, 47
Le Sourd. _See_ Widal
Leube, 67
Levaditi, 223
Levin, 159
Lewes, 53
Lewin, 337, 338
Lignières, 247, 279
Lindemann, 68
Lingelsheim, 193, 244, 312
Lister, 521, 530, 568
Loeffler, 7, 283, 513
Loeffler and Abel, 267
Löhr, 500
Lombard, 396
London, 94
Lorenz, 475
Löw. _See_ Emmerich
Lubarsch, 141, 151, 184, 529
Lustig and Galeotti, 490
Madsen, 349, 350
Madsen. _See_ Salomonsen
Magnin. _See_ Charrin
Maksutow. _See_ Pawlowsky
Malm, 149
Manfredi, 428
Mankowski. _See_ Podwyssozki
Marchand, 167
Marchand. _See_ Denys
Marchoux, 240, 276, 309, 311
Marie, 331, 382, 465
Marinesco, 75
Marmorek, 243, 312
Martel, 150, 159
Martin and Cherry, 361
Marx, 465, 476, 497
Marx. _See_ Pfeiffer
Masoin. _See_ Heymans, Lang
Massart, 34, 38, 39, 79, 281
Mastbaum. _See_ Emmerich
Mattei (di). _See_ Emmerich
Maupas, 16
Mehring. _See_ Hirsch
Melkich. _See_ Sawtchenko
Mendez, 470
Menge and Krönig, 429, 430
Mesnil, 55, 75, 78, 135, 139, 141, 143, 188, 209, 221, 238, 262, 270,
305, 307, 527
Mesnil. _See_ Laveran
Metchnikoff, 31, 55, 69, 70, 73, 100, 101, 116, 131, 137, 138, 146,
149, 151, 153, 154, 156, 160, 163, 180, 181, 185, 214, 221, 227,
237, 239, 241, 256, 259, 266, 271, 275, 286, 287, 290, 302, 304,
311, 377, 382, 385, 393, 396, 405, 426, 441, 520, 521, 522, 531,
532, 534
Metchnikoff (Mme), 20, 159, 193
Métin, 44
Miller, 414, 415, 418
Mitchell, 423
Morax and Elmassian, 409
Morgenroth, 109, 119, 331
Morgenroth. _See_ Ehrlich
Morishima, 390
Morse, 412
Mouton, 15
Moxter, 101, 185, 199
Müller, 17, 89, 114, 233
Myers, 68, 107
Myers. _See_ Stephens
Neal. _See_ Davenport
Néfédieff, 68
Neisser, 194, 196
Neisser and Wechsberg, 205, 298, 349, 359
Nencki, 419, 424, 427
Nencki and Sieber, 109, 355
Nencki, Sieber and Wyznikiewicz, 468
Netter, 503
Nicolas and Courmont, 353
Nicolas, Courmont and Prat, 353
Nicolle and Adil Bey, 279, 468
Nikanoroff, 348
Nissen. _See_ Behring
Nittis (de), 277, 288
Nocard, 148, 279, 494
Nocard and Leclainche, 461
Nocard and Roux, 130, 466, 478, 479, 569
Nolf, 94, 96
Nowakowski, 12
Nuttall, 107, 138, 150, 184, 192, 525, 527
Nuttall and Dinkelspiel, 107
Oken, 337
Opitz, 43, 44
Oppel, 231
Orlowski, 443, 444
Pagel, 507
Panum, 514
Pasteur, 2, 181, 208, 288, 322, 477, 508, 510, 511, 569
Pasteur, Chamberland and Roux, 469
Pasteur and Joubert, 144
Pasteur, Roux and Grancher, 208
Pasteur and Thuillier, 283, 473
Patella, 97
Pawloff, 59, 62, 65, 427
Pawlowsky, 44, 323
Pawlowsky and Maksutow, 348
Peiper. _See_ Beumer
Péré, 26
Pernossi. _See_ Fermi
Petruschky, 138
Pfaundler, 259
Pfeffer, 27, 38, 79
Pfeiffer, 130, 165, 185, 219, 221, 267, 269, 271, 277, 290, 301, 303,
320, 365, 438, 455, 532, 533, 534
Pfeiffer and Issaeff, 212, 533
Pfeiffer and Kolle, 230, 267, 274, 302, 319, 481
Pfeiffer and Marx, 185, 264, 291, 442
Pfeiffer and Proskauer, 253
Phisalix, 387, 425
Phisalix and Bertrand, 333, 337, 338, 345, 347
Pierallini, 218, 219
Plato, 181
Podwyssozki, 77
Podwyssozki and Mankowski, 456
Pollender, 11
Ponfick, 46
Portier, 96
Prat. _See_ Nicolas
Preobrajensky, 431
Prevôt, 374
Proskauer. _See_ Pfeiffer
Rabinowitsch and Kempner, 248, 316
Ransom, 351, 379, 382, 389
Ranvier, 409
Rauchfuss, 501
Recklinghausen (von), 514
Recklinghausen. _See_ Hoffmann
Remlinger, 447, 450
Répin, 420
Rhumbler, 15
Ribbert, 413, 428, 524
Richet and Héricourt, 266, 532
Rindfleisch, 514
Rochebrune (de), 506
Röden, 109
Roger, 243, 257, 287
Roger. _See_ Charrin
Roger and Bayeux, 414
Rogers, 468
Römer, 401
Roncali, 170
Roser, 515, 516
Ross, 129
Rossbach, 95
Rouget. _See_ Vaillard
Rousse. _See_ Decroly
Roux, 156, 347, 358, 497, 498, 530
Roux. _See_ Nocard, Pasteur
Roux (W.), 565
Roux and Borrel, 340, 383, 386, 391
Roux and Chamberland, 530
Roux and Vaillard, 347, 355, 356, 357, 367, 379, 432, 493
Roux and Yersin, 343
Ruffer, 427, 428, 523
Rysselberghe (van), 37, 39
Sabouraud, 406
Sabrazès and Colombot, 135
Sakharoff, 160, 177
Salimbeni, 222, 245, 261, 478
Salimbeni. _See_ Calmette
Salmon, 455
Salomon, 418
Salomonsen, 19
Salomonsen and Madsen, 346, 356, 370, 379, 380
Saltykoff, 272
Samoïloff, 63
Sanarelli, 262, 287, 415
Sanchez-Toledo, 170
Sarassewitch, 195
Sawtchenko, 21, 99, 156, 162, 227, 240, 260, 270
Sawtchenko and Melkich, 162, 227
Schattenfroh, 172, 188, 196
Schepilewsky. _See_ Kempner
Schiff, 62
Schimmelbusch, 42
Schoumow-Simanowski. _See_ Sieber
Schumacher, 451
Schütz, 283, 422
Schütz. _See_ Voges
Schütze, 107, 114
Schütze. _See_ Wassermann
Sclavo, 276, 310
Selander, 290
Senator. _See_ Leo
Seng. _See_ Kraus
Serpa Pinto, 506
Shaffer, 42
Sicard. _See_ Widal
Sieber. _See_ Nencki
Sieber and Schoumow-Simanowski, 419, 424
Skchiwan, 172
Slateano, 277
Slawyk, 501
Smith, 259
Smith and Kilborne, 247, 279
Sobernheim, 242, 276, 310, 441
Sobernheim. _See_ Fränkel
Soudakewitch, 75
Soulié, 460
Stadelmann, 97
Stahl, 30, 31
Stein, 12
Stephens and Myers, 360
Stern, 419, 542
Sticker, 411
Stöhr, 428
Stoudensky, 388, 394
Strassman, 499
Straus and Wurz, 417, 418
Stroganoff, 429
Takaki. _See_ Wassermann
Talma, 424
Tarassewitch, 86, 87, 98, 99
Tchistovitch, 68, 75, 106, 110, 120, 121, 122, 283, 413
Thiltges, 145, 147
Thomas, 452
Thomas. _See_ Arloing
Thomson and Hewlett, 410
Thuillier. _See_ Pasteur
Tizzoni, 357, 446
Tooth, 484
Torday, 498
Toussaint, 509
Trapeznikoff, 139, 145
Traube and Gscheidlen, 184
Trommsdorff, 23, 189
Trumpp, 261
Turner. _See_ Kolle
Uhlenhuth, 68, 107
Vaillard, 204, 335, 347, 356, 372, 447
Vaillard. _See_ Roux
Vaillard and Rouget, 169, 170
Vaillard and Vincent, 169, 394
Vallée, 289, 425
Vallée. _See_ Leclainche
Velde (van de). _See_ Denys
Viala, 465
Vincent. _See_ Vaillard
Vincenzi, 443
Virchow, 48, 519, 524
Voges, 238, 272
Voges and Schütz, 475
Voisin and Guinon, 502
Vries (de), 35
Wagner, 144
Waldeyer, 514
Wallgren, 168
Walter, 64
Walz, 193
Warlomont, 456
Washbourn, 485
Wassermann, 115, 191, 205, 231, 234, 273, 317, 318, 319, 322, 351, 358,
371, 441
Wassermann. _See_ Ehrlich
Wassermann and Schütze, 107
Wassermann and Takaki, 292, 382, 394
Wassilieff, 65
Watson-Cheyne, 323
Weber-Fechner, 27, 38, 566
Wechsberg. _See_ Neisser
Wecker, 502
Wehrmann, 417, 419, 424
Weichhardt, 118, 124
Weigert, 363, 379, 399, 424, 523
Werigo, 281
Wernicke, 276, 446, 447
Widal, 257
Widal. _See_ Chantemesse
Widal and Le Sourd, 439
Widal and Sicard, 260, 261, 264, 439, 440, 450
Wood. _See_ Woodhead
Woodhead and Wood, 323
Wright, 482
Wright and Leishman, 482
Wunschheim. _See_ Fischl
Wurtz and Lermoyez, 410
Wurz. _See_ Straus
Wyssokowitch, 43, 412, 485
Wyznikiewicz. _See_ Nencki
Yersin, 468
Yersin. _See_ Roux
Yersin, Borrel and Calmette, 487
Zabolotny, 95
Zeliony. _See_ Zilberberg
Ziegler, 519, 522
Zilberberg and Zeliony, 282
INDEX.
Abrin, 344, 345, 346, 401
Abrin intoxication, action of body fluids on, 365, 420;
leucocytic reaction against, 393, 401
Absorption. _See_ Resorption
Acari, mechanical action of, 3
Acclimatisation. _See_ Adaptation
Acid reaction inside phagocytes, 83, 182
Acid, secretion of, in osmosis, 37, 566
Acidophile microbian flora of stomach, 418
Actinians, digestion in, 53, 82, 85
Actinodiastase, 57, 197
_Actinophrys_, 14, 18
Adaptation. _See also_ Immunity
Adaptation to toxic substances, 21–27, 30, 342, 390;
to saline solutions, 23, 30, 515;
to physical conditions, 26, 30–31;
of plasmodia to arsenious acid, 31;
of pancreatic secretion to kind of food, 64, 65;
of phagocytes to destroy micro-organisms, 281, 558, 566;
of animals to spinal concussion, etc., 564;
of cells, 513
Addiment (syn. Complement), 95
Agglutination in natural immunity, 202, 206;
and phagocytosis, 202, 242, 245;
in the diagnosis of typhoid, 256, 257, 261, 439;
its mechanism, 257;
of red blood corpuscles by serums, 258;
of red blood corpuscles by ricin, 360;
does not prevent growth of micro-organisms, 262
Agglutinative power, transmission by heredity or suckling, 450;
not developed parallel with bactericidal power, 483
Agglutinins in immunity, 242, 245, 256–265, 295, 542, 559;
characters of, 255, 559;
origin of, in immunised animal, 263–265, 294;
difference between fixatives and, 255, 265, 559;
not the same as protective substances, 268, 269, 294
Albuminoid substances, resorption of, 106–127
Alexins. _See also_ Cytases
Alexins, 87–95, 96, 98, 184, 193, 255, 528, 533, 535, 539
Alimentary canal. _See_ Intestine
Alizarin sulpho-acid, 13, 83, 183
Alligator, 77, 143, 332, 401
Amboceptors (syn. fixatives), 91, 93, 297, 557
_Ammocoetes_, 77, 78
_Amoeba_, 14, 18, 23, 547, 549
Amoebo-diastase, 16, 197, 549
Amoeboid cells. _See_ Leucocytes and Phagocytes
Amphibia. _See_ Frog, Axolotl
Amylase, 95;
in the urine, 65
_Androctonus._ _See_ Scorpion
_Anopheles_ and malaria, 129
Antagonism between certain bacteria, 323
Anthrax, 11, 20, 21, 25, 41, 46, 180;
immunity of dog against, 149–151, 242;
acquired immunity of _Scolopendra_ against, 209;
natural immunity of white rat against, 526;
protective serums against, 20, 276, 309–311;
phagolysis in acquired immunity against, 280;
immunisation against, by means of other bacteria, 323;
infection by inhalation, 412;
by ingestion, 423;
immunity against, transmitted to offspring, 445, 447;
vaccinations against, 208, 241, 468–471;
method, 470;
statistics, 471;
vaccination against, by heated anthrax blood, 507;
vaccines against, 208, 470, 509;
phagocytosis in, 521, 523
Anthrax bacillus, action on rabies, 150;
bactericidal action of blood serums on, 20, 146, 150, 151, 156, 157,
240;
increasing the virulence of, 150;
attenuation of, 208, 288;
eosinophile transformation in, 198;
protective thickening of bacterial membrane in, 242;
agglutination of, 203, 242, 260, 264;
natural immunity against, 132–140, 143, 147, 149–159, 511, 512;
acquired immunity against, 239–242, 276, 277;
antagonism between, and certain bacteria, 323;
fate of, in Algerian sheep, 512;
destruction of, by defibrinated blood, 525
Anthrax, symptomatic: immunity against bacilli of, 171;
heredity of immunity against, 452;
vaccinations against, 471–473;
phagocytosis in, 523
Antiabrin, 401
Anti-arsenic serum, 390
Anticytases, 112
Anticytase serum, 115, 371
Anticytotoxins, 110, 118, 122, 127, 360
Antidiastase, 109
Antidiastatic serums, 361
Anti-enzymes, 109
Antifixative, 112
Antihaemolysins, 111
Antihaemotoxins, 111, 119, 122
Anti-infective. _See_ Protective
Antileucocidin, 359
Antineurotoxin, 116
Antirennet, 109
Antiricin, 360
Antisepsis, Nature replaces by asepsis, 432
Antiseptics. _See also_ Toxins and Adaptation
Antiseptics and foods, 26
Antiseptic action of the gastric juice, 417
Antispermofixative, 124
Antispermotoxins, 116, 122–126
Antistreptococcic serum, 243–245
Antitetanin, nervous origin of, 390
Antitoxic. _See also_ Protective
Antitoxic unit of Ehrlich, 373, 496;
action of non-specific and normal serums and of broth, 365;
function of the saliva, 417;
function of pepsin and other digestive ferments, 419, 424;
action of intestinal flora, 427;
property of the body fluids, 531 (_see_ Body fluids, Serums);
power of the blood of new-born children, 445
Antitoxins, natural, in normal blood, 111, 204, 444;
rarity in body fluids in natural immunity, 204, 532, 533;
development of, during immunisation, 354;
properties of, 354;
present in various fluids of immunised animal, 355, 531;
mode of action of, on toxins, 356–362, 371;
conditions acting in mixtures of, with toxins, 362;
immunity against toxins not in direct constant ratio to amount of,
367–376;
effect of using serum from same species, 379;
hypothesis as to nature and origin of, 377–402, 562;
probable part played by phagocytes in production of, 400–402;
rapid regeneration of, after bleeding, 379;
augmentation in production of, by pilocarpin, 380;
transmission of, by milk to offspring, 449;
analogy of, with fixatives, 561;
hypersecretion of, 563
Antivenomous property of blood of scorpion, 328;
action of serums, 334, 338;
serum, action of, 334, 338, 358, 360
Aqueous humour, bactericidal action of, 184, 192;
in immunised animals contains no fixative, 217, 222;
in immunised animals contains antitoxin, 355
Arsenic; adaptation to, 31, 343, 390;
protective serum against, 390;
leucocytic reaction against, 396–399;
as a remedy against microbial disease, 513
Arsenic acid, action of, on anthrax bacillus, 25
Arsenious acid, adaptation of plasmodia to, 31
Arthropoda. _See_ Clothes-moth, Crayfish, Crustacea, _Daphnia_,
_Scolopendra_, Scorpion, Spider, Tick
Arthrospores of Hueppe, 254
_Ascaris_, poor microbian flora in intestine of, 421;
phagocytic organs of, 547
Asepsis is Nature’s method, 432
Aspergillosis, 2, 4.
_See also_ Mycoses
Atrophic diseases, probably due to a parasite, 3
Atropin, reaction of rabbit and guinea-pig to, 395, 396
Attenuation. _See also_ Vaccination, Vaccines
Attenuation of micro-organisms and viruses, discovery and application
of, 208, 247, 288, 508;
of micro-organisms by the fluids of immunised animals, 286–289;
of toxins, 344
Autodigestion in yeast, 197
Autospermotoxins, 101
Autotoxins, 104
Axolotl, susceptible to tetanus toxin, 330
Bacilli, anaerobic, natural immunity against, 169, 170
_Bacillus aërogenes_, agglutination in, 264
_Bacillus chauraei._ _See_ Anthrax symptomatic
_Bacillus coli_ attacks potato, 35;
vaccination against, 267;
transformation of, into granules, 198;
modified growth on certain serums, 259
Bacillus of Doederlein, 429;
of Kiel water, 408
_Bacillus pyocyaneus_, 42, 180, 254, 528;
acquired immunity against, 210, 232–236, 301;
Pfeiffer’s phenomenon in, 234, 307;
special forms of growth in serums from vaccinated animals, 256;
agglutination of, 261, 307;
susceptibility to the toxins of, 290, 351;
action of specific serum on, 307, 358;
antagonistic to anthrax bacillus, 323;
immunisation against toxin of, 351;
a leucocidin from, 359;
action of liver on toxin of, 427;
heredity of immunity against toxin of, 446
_Bacillus ranicida_, 140
Bacteria. _See_ Micro-organisms
Bactericidal action of serum, influence of alkalinity or acidity on,
196;
function of the tears, 408
Bactericidal property. _See also_ Body fluids, Humoral theory, Serums
Bactericidal property: in blood and other fluids, 20, 146, 150, 151,
156, 157, 184–193, 211, 226, 233, 238, 240, 241, 243, 244, 512,
525–531, 542, 554;
of body fluids, theory of osmotic pressure, 193, 213;
of extracts of glands and exudations, 195;
of the saliva, 415;
absence of, from the intestinal ferments, 424, 567;
of serums, Wright’s method of testing, 483;
does not develop parallel with agglutinative, 483;
and immunity, absence of parallelism, 554
Bactericidal substance (alexin, complement, cytase): in blood and other
fluids, 184–193, 534;
source of, in body fluids, 185–193;
theory of leucocytic secretions, 187–191;
presence in body fluids due to phagolysis, 191;
is of phagocytic origin, 185, 192;
in body fluids, microphages source of, 187;
not resistant to heat, 268;
and so distinguished from protective substance, 268;
Pfeiffer’s theory of, 534
Bacteriolysis. _See_ Micro-organisms, destruction of
Bacteriolysis, analogy between haemolysis and, 537
Bat, immunity against tetanus of hibernating, 339
Baumès-Colles’ law in syphilis, 436
Behring’s “normal serum,” 496
Bile, function of, 60;
salts protective against snake venom, 388;
protective function of, 424
_Bipinnaria_, 70, 518
Blastomycetes. _See also_ Yeast-cells
Blastomycetes, resistance of _Daphnia_ to, 131, 404, 520;
fate of, in refractory organism, 172;
acidophile, 418
Blood, pepsin in the, 66, 563;
precipitins in the, 68, 106, 107, 568;
fate of effusions of, 73;
bactericidal power of, 184 (_see also_ Bactericidal, Serums);
natural antitoxins in normal, 111, 204, 444;
stimulant (protective) action of human, 271, 318;
immunity conferred by maternal, 447;
recognition of, in medico-legal research, 107, 568;
from convalescents, protective power of, 437, 441, 443;
agglutination of (_see_ Agglutination)
Blood corpuscles, resorption of red, 47, 50, 56, 57, 70, 72, 79–100,
537 (_see also_ Haemolysis);
fixation of cytase by red, 194;
agglutination of red, by serums, 258;
agglutination of red, by ricin, 360
Body fluids. _See also_ Bactericidal, Blood, Humoral theory, Serums
Body fluids, natural immunity and the composition of, 128–131, 146;
in natural immunity, absence of antitoxic property in, 204;
bactericidal power of, 184–193, 512, 525–531, 542 (_see also_ Body
fluids, Serums);
antitoxic power of the, 204, 531, 533, 543;
protective properties of, 266–280
_Boophilus bovis_, 247
Bordet’s sensibilising substance, 91, 199, 298, 535, 537, 557
Botulism, protective action of fats against toxin of, 387;
action of digestive diastases on toxin of, 420
Bouchard’s theory of acquired immunity, 232, 286;
of attenuating power of serums, 286–289
Bouillon de panse, 473
Bovidae, acquired immunity of, against Texas fever, 247, 279;
protection of, against tetanus, 494;
vaccination of, against rinderpest, 425, 466–468;
against rabies, 466;
against anthrax, 470;
against symptomatic anthrax, 471;
against pleuropneumonia, 477–479;
ancient methods against pleuropneumonia in, 506
Broth as a protective fluid, 320, 321, 365
Buccal cavity, microbial products in the protection of the, 416;
flora of, 414
Buchner’s theory of immunity, 512, 527
Calf lymph vaccine, method of preparation, 456
_Carassius._ _See_ Goldfish
Carmine, fixation of tetanus toxin by, 388, 394
Cattle. _See_ Bovidae
Cattle plague. _See_ Rinderpest
Cayman. _See_ Alligator
Cellular or histogenic immunity, 335, 336, 340, 563–565
Cellulosase, 86
Cerebral substance, action of emulsions of, on toxins, 386
Cerebral tetanus, 383, 391
Chemiotaxis. _See also_ Hyperleucocytosis, Susceptibility
Chemiotaxis in Infusoria, 19;
in plasmodia of the Myxomycetes, 30;
of duodenal mucous membrane, 64;
of phagocytes, 79, 108, 133, 167, 177, 280;
of leucocytes for rennet, &c., 119;
positive, in segmentation-cells of frog embryo, 565
Cholera antibody (fixative), 253, 267, 292
Cholera, Asiatic, protective power of blood of convalescents from, 441;
vaccinations against, 480–481
Cholera peritonitis, heredity of immunity against, 447, 448;
immunity of guinea-pig against, 533
Cholera toxin, alligator resistant to, 333;
immunisation against, 350;
action of normal serum of goat on, 365
Cholera vibrio. _See also_ Pfeiffer’s phenomenon, Vibrios
Cholera vibrio, adaptation of, to bactericidal substance, 23;
susceptibility of larva of Rhinoceros beetle to, 40, 133;
immunity of frog against, 142;
of guinea-pig against, 163, 533;
extracellular destruction of, 165, 212 (_see also_ Pfeiffer’s
phenomenon);
eosinophile transformation in, 198;
arthrospores of, 254;
agglutination of, 261, 264;
protective action of serums against, 268, 271, 318;
of human blood against, 271, 318;
immunity to, is not insusceptibility to its toxin, 290;
origin of protective property against, 291;
protective action of various fluids against, 320;
antagonism between certain bacteria and, 324;
in stomach, 419, 567;
susceptible to acids _in vitro_, 419;
in intestine, 423, 567;
serum from animals immunised against, 532
Cholesterin. _See also_ Fats
Cholesterin, fixation of toxins by, 387;
fixation of saponin by, 389
_Chytridium_, 12
Cicatrisation of plants, 34
Clasmatocytes, 78
Clavelée (la). _See_ Sheep-pox
Clavelisation against Sheep-pox, 460
Clothes-moths, micro-organisms absent from digestive canal of larvae of
certain, 420
_Coccobacillus prodigiosus._ _See under Micrococcus_
Cockchafer larva, 70, 326
Complement of Ehrlich, 88, 91, 193, 251, 297
Complementoids of Ehrlich, 115
Concussion, spinal, adaptation to, 564
Conjunctiva, elimination of micro-organisms by the, 408;
absorption of toxins by the, 409
Copula of P. Müller, 91
Cornea, protective resistance by the, 409
Crayfish, susceptible to certain toxins, 345;
blood of, antitoxic against scorpion venom, 366;
poor intestinal flora of, 421
Crickets and micro-organisms, 41, 133;
natural immunity against toxins in, 329
Crustacea. _See_ Crayfish, _Daphnia_
Crustacea, protective function of integument of, 404
_Cyprinus._ _See_ Goldfish
Cytase of Laurent, 86
Cytases (syn. alexins, complements), 93, 98, 123;
elaborated by phagocytes, 197, 252, 539, 549–556;
thrown out into plasmas during phagolysis, 95, 99, 102, 197, 252,
551–554;
bactericidal power of, 183, 184, 191, 193–198, 217 (_see also_
Bactericidal, Body fluids, Serums);
unity or plurality of, in same serum, 193, 197;
absorption of, 194, 200;
two kinds of, macrocytase and microcytase, 195, 296, 549;
characters of the, 197, 549;
enzymes other than, in phagocytes, 197;
in the immunised organism, 250–255, 296, 317, 554;
presence or absence of, how determined, 253;
Ehrlich’s and author’s views on, contrasted, 297;
compared with fixatives, 555
Cytotoxins, 105 (note), 110, 116
_Daphnia_, resistance of, to Blastomycetes, 131, 404, 520
Darwin on the extinction of the elephant, 8
Dermis, arrest of micro-organisms in the, 406
Desmon (of London), 91
Diastases. _See_ Digestive ferments, Ferments
Digestion in the higher animals, 49, 59–65;
psychical and nervous elements in, 62, 566;
extracellular, by secreted juices, 49, 58, 62;
the liver of the Mollusca as second organ of, 59;
in the tissues, 67;
and resorption closely related, 69, 85;
by macrophagic organs, 85, 150
Digestion, intracellular. _See also_ Phagocytes, Phagocytosis,
Resorption
Digestion, intracellular, 48, 85, 517, 518, 520;
in the Protozoa, 13, 30, 49;
in Planarians, 49, 71, 82;
in Actinians, 53, 82, 85;
in Sponges, 69, 517;
transition from, to digestion by secreted juices, 49, 58
Digestive ferments, antitoxic function of, 424;
action of, on toxin of botulism, 420
Diphtheria, 7, 41, 132, 204;
antitoxic power of blood of convalescents from, 443;
antitoxic power against, in blood of healthy persons, 444;
and in blood of new-born children, 445;
heredity of immunity against, 445, 447, 448;
influence of anticytase serum on, 371;
vaccinations against, 495–503;
serum against, 495;
standardisation and testing of this serum, 496–498;
its protective and antitoxic powers do not develop in equal ratio,
497;
its prophylactic use, 498–503;
accidents during treatment, 499, 502;
statistics, 500–503
Diphtheria toxin, increased susceptibility of immunised guinea-pig to,
290;
natural immunity of rat and mouse against, 204, 339;
natural immunity of frog against, 330;
immunisation against, 344, 347, 349, 353;
attenuation of, 344;
preventive action of nucleohiston on, 365;
action of, on brain of laboratory animals, 386;
sets up local lesions in the conjunctiva, 409;
pepsin destroys, 419
_Diplococcus pneumoniae._ _See_ Pneumococcus
Diseases, fear of, and pessimism, 1, 569;
atrophic, probably due to a parasite, 3;
mechanical element as etiological factor, 3;
toxic element as etiological factor, 4;
developed on the earth at a very early epoch, 8;
and extinction of species, 8;
infective, in multicellular plants, 29–39;
set up by Fungi. _See_ Fungi
Dog, immunity of, against anthrax, 149–151, 242;
action of anthrax bacillus on rabid, 150;
immunity of, against streptococci, 167;
naturally refractory against a staphylococcus, 266;
bactericidal action of blood of, on anthrax bacillus, 150, 151, 156;
digestion of gelatine by leucocytes of, 108;
enterokynase in lymphoid organs of, 61;
digestive fluids of, 62–65;
disinfecting power of small intestine of, 422;
phagocytosis in, 149, 151;
haematozoon in, 279
Domestic animals, immunisation of, against disease. _See_ Bovidae, Dog,
Goat, Horse, Pig, Sheep, Swine, Vaccines, Vaccinations
Dourine, 2, 247
_Drepanidium_, 515
Drugs, absorption of, by leucocytes, 400
Duodenum, chemiotaxis of mucous membrane of, 64
“Dust” cells, 75, 411–414
Eel’s serum. _See also_ Ichthyotoxin
Eel’s serum, toxic action of, 20, 111, 563;
and precipitins, 68, 106
Effusions of blood, fate of, 73
Ehrlich’s neutral red reaction, 13, 83, 181;
classification of leucocytes, 74, 76–78;
theory of side-chains or receptors, 120, 381–384, 538, 557, 562–563;
compared with theory of phagocytes, 296–299, 538, 558;
“immunising unit,” 373, 496
Elephant, extinction of, 8
Elimination of micro-organisms from the body, 43, 46;
by the epidermis, 406;
by the conjunctiva, 408;
by the nasal mucosa, 410
_Emys._ _See_ Turtle
Endo-enzymes, 197
Endotrypsin of yeast, 197
Enterokynase, 59, 98
Enzymes. _See_ Ferments
Eosinophile leucocytes, secretion by, in bacteriolysis, 187, 542
Eosinophile staining reaction, 198
Epidermis, exfoliation of the, 406
Ernst’s bacillus, immunity of frog against, 140
Erysipelas. _See_ Swine erysipelas
Erysipelas, immunity in, 434
Erysipelas streptococcus, protective action of, against anthrax, 323;
its use in malignant tumours, 434
Excretion. _See also_ Elimination
Excretion in relation to micro-organisms, 43, 432;
of pepsin in the urine, 65;
of pepsin in the blood, 66, 563
Exfoliation of the epidermis, 406
Exudations, bactericidal power of, 185, 193, 195
Farcy, slow evolution of, 406
Fats, protective action of, against toxins, 387
Ferments. _See also_ Intestinal, Digestive, Fibrin-ferment, Gastric
juice, Saliva, Trypsin
Ferments, Pasteur on the organised nature of, 2;
soluble (diastases or enzymes), in digestion, 49, 55, 57, 108, 109,
197;
antitoxic function of digestive, 424;
phagocytic, 197, 549–559;
hypersecretion of, 563
Fibrin ferment (plasmase), 95, 197, 550
Fishes. _See_ Goldfish
Fishes, phagocytosis in, 135
Fixatives (immunising body, or amboceptor, or sensibilising substance),
88, 92–95, 97, 98, 103–105, 199–202, 296;
synonyms of, 91;
analogy of, with enterokynase, 98;
presence of, in plasmas, 103, 112–114, 217;
in protective serums, 269, 438;
in mesenteric glands, 98;
in spermotoxins, 101;
origin of, 103, 294, 537, 556–559;
specificity of, 88, 105, 216, 251, 253, 296;
rarity of, in normal fluids, 199–201, 250;
method of determining whether present in a serum, 199;
absent from aqueous humour of immunised animals, 217, 222, 251;
in the immunised organism, 250–255;
properties of, 251, 253, 255, 554;
differ from agglutinative substances, 255, 265, 559;
relation of, to phagocytosis, 291, 295;
part played by, in Pfeiffer’s phenomenon, 251, 295;
and protective substances closely connected, 269, 294, 295, 561;
compared with cytases, 555;
mechanism of action of, 557
Food substances, absorption of, by other channel than alimentary canal,
67
Foods and antiseptics, 26
Foreign bodies, fate of, in organism, 46, 52, 55, 56, 517
Formed elements, resorption of the, 47, 67–105
Fowl, immunity of, against anthrax, 144, 159;
phagocytosis in, 144, 282;
bactericidal action of plasma of, on anthrax, 146;
blood serum of, and tetanus, 204;
immunity of, against tetanus, 204;
natural immunity of, against tetanus toxin, 335;
influence of removal of parts of brain and cord on tetanus in, 384
Fowl cholera, infection of laboratory animals with, 181;
vaccine against, 208;
phagocytosis in, 282;
action of exudations of fowls vaccinated against, 288;
acquired immunity against, 288, 508;
failure of bacillus of, to grow in certain media, 510
Friedländer’s bacillus prevents infection by anthrax, 323
Frog, phagocytosis in, 137, 142;
immunity of, against anthrax, 137;
against Ernst’s bacillus, 140;
against bacillus of mouse septicaemia, 141;
against cholera vibrio, 142;
acquired immunity of, against pyocyanic disease, 210, 301;
natural immunity of, against tetanus toxin, 330;
against diphtheria toxin, 330;
immunisation of, against abrin, 345;
absorption of tetanus toxin by brain of, 386
Frog embryo, positive chemiotaxis in segmentation-cells of, 565
Fungi, diseases set up by, 2, 4, 18, 32, 131, 135, 404
(_see also_ Aspergillosis, Mycoses)
Galactose. _See_ Milk-sugar
Gamaleia’s vibrio. _See Vibrio metchnikovi_
Gastric juice, antiseptic action of, 417;
psychic influence on, 63, 566.
_See_ Pepsin
Gelatine, resorption of, 107
Gentilly bacillus. _See_ Pneumo-enteritis
Gerbil, tubercle in, 22, 183
Goat, action of normal serum of, on cholera toxin, 365;
vaccination of, against rabies, 466;
acquired immunity in, 563
Goldfish, 72, 135
Goose septicaemia. _See Spirochaete anserina_
“Greek method” of variolisation against small-pox, 507
Gruber’s theory of immunity, 256, 262
Guinea-pig, immunity of, against spirilla, 160, 162;
against vibrios, 163, 211–227, 275, 287, 531, 533;
against streptococci, 165;
against tetanus bacillus, 169;
against symptomatic anthrax, 171;
against _Trypanosomata_, 173;
acquired immunity against spirilla of recurrent fever, 227–230;
against typhoid, 191, 230;
against _Bacillus pyocyaneus_, 234–236;
against anthrax, 276, 277;
phagocytosis in, 162, 163, 166, 170, 223;
hypersusceptibility of immunised, to diphtheria toxin, 290;
protective power of serum of immunised, 293;
effect of removal of spleen of, 293;
antivenomous action of serum of, 338;
immunisation of, against cholera toxin, 351;
increasing natural susceptibility of, to toxins, 369, 370;
reaction of, to atropin, 396
Haematopoietic organs. _See also_ Lymphoid organs
Haematopoietic organs as source of protective substance, 292–294
Haematozoa. _See_ _Piroplasma_, _Trypanosoma_
Haematozoon in dog closely allied to that of Texas fever, 279
Haemolysis. _See also_ Blood corpuscles, resorption of
Haemolysis, 79–100, 111, 112, 537;
the two substances which act in, 88, 98, 538;
analogy between bacteriolysis and, 537
Haemomacrophages, 76, 136
Haptophore atomic group in a toxin, 120, 350, 384
Hedgehog, natural immunity of, against poisons and venoms, 337
_Helix pomatia_, 70, 134
Heredity of immunity, 445–453, 513
_Herpestes._ _See_ Mongoose
Hibernation, effects on resistance to toxins, 339
_Hippocampus_, 135
Histogenic immunity, 336
(_see_ Immunity, cellular)
Hog cholera, resemblance of bacillus of, to that of pneumo-enteritis,
259;
serum of animals vaccinated against, 260;
agglutination in, 260;
protective action of serums against, 272;
susceptibility of vaccinated animals to the toxin, 290
Horse. _See also_ Diphtheria
Horse, acquired immunity against cholera vibrio, 222;
against streptococci, 244, 245, 313;
local reaction to tetanus toxin in, 352;
immunised, with poor yield in antitoxin, 373, 375;
reaction of, to one unit of toxin, 378;
antitoxic power of serum of normal, 380;
phagocytosis in, 245, 313;
antivenomous action of serum of, 338;
vaccination of, against rabies, 466;
vaccination of, against anthrax, 470;
protective serum against tetanus in, 493
Humoral phenomena in immunity, 184, 250, 290, 437–440, 525–531, 542,
543
Humoral theories of immunity, 184, 525–531, 542, 543;
attempts to reconcile with theory of phagocytes, 539
Humours. _See_ Body fluids, Serums
Hyperleucocytosis. _See also_ Chemiotaxis
Hyperleucocytosis during immunisation, 352, 393
Hypersecretion, 563 (_see_ Receptors)
Hypersusceptibility to toxins in immunised animals, 290, 368–374, 564
Hyphomycetes, diseases caused by, 2
Hypopyon, pus of, 96
Ichthyotoxin, 110, 120, 121, 122, 326, 360
(_see also_ Eel’s serum)
Immunisation. _See_ Immunity, acquired, artificial and temporary,
Vaccination
Immunisation against toxins, principal methods of, 345–350;
by unmodified toxins, 345–346;
by modified toxins, 347;
by mixtures of toxin and antitoxin, 348;
by toxones and toxoids, 349;
phenomena produced during, 352–354
Immunising body of Ehrlich, 91, 251;
unit of Ehrlich, 373, 496
Immunity, historical sketch, 505–543;
summary, 544–569;
by attenuated micro-organisms, 2;
predisposition or absence of, 7;
against infective diseases, 9;
definition of, 10;
against micro-organisms, 10, 41, 42, 128–206, 207–324;
against toxins, 10, 41, 42, 325–341, 342–402;
not same as against micro-organisms, 290, 351;
in unicellular organisms, 11–28;
in multicellular plants, 29–39;
in plants, action of manures on, 36;
in the animal kingdom, 40–66;
cellular or histogenic, 335, 336, 340, 563–565;
active (Ehrlich), 378 = isopathic immunity (von Behring);
passive (Ehrlich), 378, 453 = antitoxic immunity (von Behring);
passive against micro-organisms, 300–324, 560;
isopathic (von Behring), 378;
antitoxic (von Behring), 378;
of the skin, 403–407;
of the mucous membranes, 407–432;
susceptibility in, 565 (_see also_ Hypersusceptibility,
Susceptibility);
channel of entrance in, 567;
applications of theory of, to medical practice and to the research of
new organisms, 567–569
Immunity, natural: 10, 17, 18, 30;
amongst Invertebrates, 40, 131–135;
amongst Vertebrata, 41, 135–174;
against micro-organisms, 128–174, 175–206;
and composition of body fluids, 128–131;
against anaerobic bacteria, 169, 170;
part played by inflammation in, 176;
importance of microphages in, 177;
humoral theory of, 184;
agglutination in, 202, 206;
against toxins, 325–341
Immunity, acquired: 10, 19, 31;
against micro-organisms, 207–249, 250–299;
against vibrios, 211–227;
against pyocyanic disease, 210, 232–236, 301;
against spirilla of recurrent fever, 227–230;
against typhoid bacillus, 230;
against swine erysipelas, 236–239;
against anthrax, 239–242;
against streptococcus, 243–247;
against _Trypanosomata_, 247–249, 316;
against staphylococcus, 266
Immunity, rapid and temporary: against micro-organisms, 300–324;
conferred by specific serums, 301–317;
conferred by normal serums, 317–320;
conferred by fluids other than serums, 320–322;
conferred by non-specific micro-organisms, 322–324
Immunity, artificial, against toxins, 342–402;
against bacterial toxins, 343;
against vegetable toxins, 344, 365;
against snake venom, 345;
not in direct ratio to amount of antitoxin in body fluids, 367–376
Immunity acquired by natural means, 433–453;
acquired after recovery from infective diseases, 433–444;
acquired by heredity, 445–453;
conferred by maternal blood, 447;
by the yolk, 449;
by the milk of the mother, 449
Immunity, acquired: amongst Invertebrata, 209–210;
amongst Vertebrata, 210–249;
relation of Pfeiffer’s phenomenon to, 224;
Bouchard’s theory of, 232, 286;
double action of cytases and fixatives in, 250–255, 296, 554;
agglutinative substances in, 242, 245, 256–265, 295, 542, 559;
protective properties of body fluids in, 266–280;
phagocytosis in, 220, 223–226, 245, 280–286, 295;
origin of fixative properties in body fluids in, 294;
relation between fixatives and phagocytosis in, 291, 295;
humoral phenomena in, 184, 250, 290, 525–531, 542, 543;
bactericidal power of fluids in, 250;
Gruber’s theory of, 256, 262;
against micro-organisms, susceptibility to the specific toxin in,
289;
principal phenomena associated with, 295–296;
against micro-organisms in no ratio to protective power of blood,
372–374;
by suckling, mouse the only animal in which, 450, 452;
theory of exhaustion of nutrient medium as cause of, 510–512;
theory of presence of inhibitory substance, 511, 512;
theory of local inflammatory reaction, 512;
theory of adaptation of cells in, 513;
theory of phagocytes in, 514–525, 539–543;
theory of bactericidal power of body fluids, 525–531, 542, 543;
theory of antitoxic power of body fluids, 531;
theory of extracellular destruction of micro-organisms by leucocytic
secretions, 187–191, 533–537, 542;
theory of side-chains, 120, 381–384, 538, 557, 562–563;
present phase of the question of, 540–543
Immunproteidin of Emmerich and Löw, 254
Infection, agents, mechanical and other, that prevent or aid, 3–5,
170–173, 426
(_see also_ Diseases, Elimination, Micro-organisms)
Inflammation in immunity, 176, 512;
Cohnheim on, 518;
and phagocytosis, 516, 519–520, 547, 568
Influenza bacillus, cultivation of, in body fluids, 130, 554;
vaccination against, 277
Infusoria. _See also Trypanosoma_
Infusoria, 12–20, 23, 26, 326
Inoculation. _See_ Immunisation, Vaccination
Insects, natural immunity in, 132, 326, 329;
acquired immunity in, 209;
protective lining of digestive canal of, 421
Insusceptibility of cells of refractory animals, 341
Integument of Invertebrata, protective function of, 404
Intermediary body, 88, 91, 296, 557
Intestine, protective function of the, 422;
microbian flora of, 420;
antitoxic action of this flora, 427
Intestinal ferments, absence of microbicidal power from, 424, 567;
intestinal micro-organisms, favouring and retarding functions of,
426;
destruction of toxins by, 427
Invertebrata, natural immunity in the, 40, 131–135, 326–329;
acquired immunity in the, 209–210, 301;
immunisation of, by specific serums, 301;
protective function of integument of, 404
Iodine trichloride in immunisation, 347
Iron, absorption of, by leucocytes, 399
Irritability, part played by, 18, 27 (_see also_ Susceptibility);
in plants, 38
_Isaria_, resistance to infection by, 329
Koch’s phenomenon in tuberculosis, 437
Kupffer’s cells, 75
Leprosy, etiological factors in, 4
Leprosy bacillus, 75, 411
Leucocidin, and its neutralisation, 359
Leucocytes. _See also_ Phagocytes
Leucocytes (amoeboid cells) in resorption, 47, 73, 175, 514, 515;
adaptation of, to virulent bacteria, an education, 281;
various categories of, 74–79;
soluble ferments of, 95;
chemiotaxis of, 119, 177;
theory of bactericidal secretions by, 187–191, 533–537, 539, 540,
542;
action of leucocidin on, 359;
absorption of poisons by, 393–400;
situations where there are no pre-existing, 551
Lily of the valley, acquired immunity in, 513, 515
Liver, serum against cytotoxin acting on, 116;
protective function of the, 427;
of Mollusca an organ of second digestion, 59
Lizard, resistance of, to tetanus toxin, 332
Lugol’s solution in immunisation, 347
Lupus, slow growth of, 406
Lymphocytes. _See also_ Leucocytes, Phagocytes
Lymphocytes, 76, 78
Lymphoid organs. _See also_ Haematopoietic organs, Phagocytic organs
Lymphoid organs, protective function of the, 428;
as source of sensibilising substance (fixative), 537
Lymphomacrophages, 76
Macrocytase (alexin, complement), 86, 98, 105, 112, 196, 549;
analogy of, with actinodiastase, 86;
escape of, during phagolysis, 95, 99, 102, 552;
presence of, in spermotoxin, 101;
origin of, 103;
active for resorption of animal cells, 196, 197, 296;
in extracellular solution of red corpuscles, 552
Macrophages, 76, 77, 79, 547;
the part they play in resorption, 80–100, 176;
staining reactions of, 77;
in phagocytosis, 144, 148, 154, 157, 161, 162, 164, 173, 184, 228,
245, 321, 548;
act more especially in resorption of animal cells, 176, 196, 548;
but intervene specially against human tubercle bacillus in pigeon,
148;
against spirilla, 162, 177, 228;
and against streptococci, 245;
not source of bactericidal substance in body fluids, 187;
part played by, in arsenic poisoning, 397;
the principal source of antitoxin, 401;
of skin, reaction of, against micro-organisms, 407
Macrophagic organs, digestive property of, 85, 150
Malaria, immunity against, 129, 278;
protective action of serum in, 278;
immunity acquired after, 434
Manures, influence on plant diseases, 36
Marmot, immunity of hibernating, against tetanus, 339
Martin’s broth (bouillon de panse), 473
Massowah vibrio, acquired immunity against, 221;
action of specific serum on, 305
Mastzellen, 77
Membranes, protective secretion of, by bacteria, 21, 242
_Meriones shawii_, 22, 183
Mesenteric glands, 62, 85, 98, 195
Mesoderm, function of amoeboid cells of, 518
Microbicidal. _See_ Bactericidal
_Micrococcus prodigiosus_, 42, 45;
antagonistic to anthrax bacillus, 323;
action of vaginal mucus on, 430
Microcytase digests bacteria, 196, 197, 296, 550;
in immunity, 218;
escape of, during phagolysis, 218, 222, 230, 295, 554;
transforms vibrios into granules, 552;
action of, on _Vibrio metchnikovi_, 553
Micro-organisms, minuteness of certain pathogenic, 3;
variability in action of, 5;
staining reactions of, 13, 83, 181, 183, 198, 213;
immunity by attenuated, 2, 509;
pathogenic, in healthy persons, 7;
adaptation of, to toxic substances, 21, 25;
protective secretion of membranes by, 21, 242;
defence in plants against, 35;
defences of animals against, 545;
elimination of, from the body, 43, 46 (_see also_ Elimination);
resorption of, 46, 175, 546;
antidiastase against enzymes of, 109;
natural immunity against pathogenic, 128–174, 175–206;
acquired immunity against pathogenic, 207–249, 250–299, 300–324;
anaerobic, immunity against, 169, 170;
pathogenic animal, 2, 173, 247–249, 277–279, 316;
destruction of, an act of resorption, 175, 206 (_see_ Bacteriolysis);
presence of, in white corpuscles, 514;
adaptation of phagocytes to destroy, 558, 566;
mode of entry into phagocytes, 177;
digested by phagocytes, 181, 514–525, 536, 539–543 (_see_ Phagocytes,
Phagocytosis);
transformation into spherical granules, 198 (_see also_ Pfeiffer’s
phenomenon);
extracellular destruction of, 165, 212, 533–537, 542;
modified growth in serums from immunised animals, 256, 259 (_see
also_ Agglutination);
specific diagnosis of, by modified growth, 256, 259;
agglutination does not prevent growth of, 262;
changes which they undergo in immunised animal, 289;
attenuation of, 208, 286–289, 508;
adjuvant and retarding functions of, 170, 426;
antagonism between anthrax and certain, 323;
antagonism between cholera vibrio and certain, 324;
acidophile, 418;
exfoliation of epidermis to get rid of, 406;
localisation and arrest of, in the dermis, 406;
destruction of toxins by, 427
Microphages, 77, 78, 79, 148, 152, 154, 162, 164, 172, 185, 245, 548;
intervene specially against micro-organisms and in acute infections,
177, 196, 206, 549;
source of bactericidal substance in body fluids, 187, 195;
granular transformation of vibrios inside, 164, 165, 224
(_see also_ Pfeiffer’s phenomenon)
_Microsphaera_, 18
Milk, absorption of, 107;
precipitins in the differentiation of various kinds of, 107, 568;
of immunised animals, antitoxin in, 356;
immunity conferred by mother’s, 449, 450, 452;
transmission of agglutinative power by, 450
Milk-sugar, adaptation of yeasts to, 26
Mithridates, method of protecting himself against poisons, 343
Mollusca. _See also Helix_, _Phyllirhoë_, _Thetys_
Mollusca, natural immunity in, 134;
liver of, an organ of second digestion, 59
Mongoose, immunity of, against snake venom, 339
Monkeys, immunised, with poor yield in antitoxin, 373;
immunisation of, against diphtheria toxin, 373;
transient acquired immunity against recurrent fever, 434
_Monospora_, parasite of _Daphnia_ disease, 131, 404, 520
Morphia, adaptation to, 343
Mouse, infection of, by swine erysipelas, 270, 307, 476;
the only animal that acquires immunity by suckling, 450, 452;
acquired immunity of, against typhoid, 230;
natural immunity of, against diphtheria toxin, 204, 339
Mouse septicaemia, immunity of frog against, 141;
phagocytosis in, 283;
acquired immunity of rabbit against, 509
Mouth. _See_ Buccal cavity
Mucous membranes, immunity of the, 407–432;
elimination of micro-organisms by the nasal, 410;
protective function of the genital, 429
Mycoses, pulmonary, 413
(_see also_ Aspergillosis)
_Mygale._ _See_ Spiders
Myriapods. _See Scolopendra_
Myxomycetes, plasmodia of, 30, 545
Naegeli’s theory of immunity, 512
Nagana disease, 2, 4, 247, 316
(_see Trypanosoma_)
Narcosis. _See_ Opium
Nasal mucous membrane, elimination of organisms by, 410
_Nepenthes_, digestive juice of, 355
Nerve centres, susceptibility of, to toxins, 564
Neuroglia cells, their phagocytic function, 75
Neurotoxin, 116
Neutral red, reaction of, 13, 83, 181
Nuclein as a protective substance, 320;
vaccinal against plague, 490
Nucleohiston, preventive action of, on diphtheria toxin, 365
Nutrition, certain diseases of, probably due to a parasite, 3;
extra-buccal, 67, 69
_Oidium albicans_, growth of, in serum of immunised animals, 257
Omentum, glands of, 85;
bactericidal power of extracts of, 195;
phagocytosis of vibrios in, 224
Opium, its action on leucocytes, 225, 231, 236, 306, 307;
its influence on immunisation by specific serums, 306;
resistance of hedgehog to, 337
_Oryctes nasicornis._ _See_ Rhinoceros beetle
Osmotic pressure, adaptation of plants to, 37, 39, 566;
as cause of bactericidal action of body fluids, 193, 213
Ovum in the Graafian follicle, immunity acquired by the, 448
Oxalic acid, function of, in plants, 37, 566
Oxydases, 96
Pancreatic digestion, 60, 63, 65
Pancreatic juice, antitoxic power of, 424
Pancreatic secretion, its adaptation to kind of food, 64, 65
Paralysis, general progressive, and syphilis, 435
_Paramaecia_, 13, 16, 17, 19
Parasites in infective diseases, 2, 9
(_see also_ Micro-organisms)
Pasteur’s theory of exhaustion of nutrient medium, 510–512;
anthrax vaccines, 208, 470;
modification of Willems’ method against pleuropneumonia, 477;
vaccines against rabies, 462, 463–464;
and Thuillier’s vaccines against swine erysipelas, 208, 473, 509
Pepsin in the urine, 65, 97;
in the blood, 66, 563;
antitoxic function of, 419;
antiseptic action of, 417;
chemical composition of, 109
Pessimism and fear of disease, 1, 569
Peyer’s patches, 61;
protective function of, 428
_Peziza._ _See Sclerotinia_
Pfaundler’s reaction, 259
Pfeiffer’s phenomenon in cholera vibrio, 165, 192, 212–226, 251, 267,
268, 280, 301–307, 534–536;
in spirillum of recurrent fever, 229;
in typhoid bacillus, 230, 303, 304;
in _Bacillus pyocyaneus_, 234, 307;
different in immunised and in normal fluids, 251;
conditions for its manifestation, 252, 253, 295, 534
Pfeiffer’s theory of immunity, 534
Phagocytes (_See also_ Leucocytes), amoeboid cells with digestive
function, 7, 182, 547;
in Sponges, 69;
in Vertebrata, 73;
various categories of, 74–79;
of _Bipinnaria_ and _Phyllirhoë_, 70;
chemiotaxis of, 79, 108, 133, 167, 177, 280;
the source of the haemolytic ferment, 100;
of osseous fishes, 135;
of frog, 137;
ingest living and virulent bacteria, 142, 177, 179–181, 558, 566;
function of, 151, 157, 177, 181, 206, 547, 548, 566;
mode of entry of microorganisms into, 177;
acid reaction inside, 83, 182;
action of opium on, 225, 231;
theory of, and side-chain theory compared, 296–299, 538;
in defence of animal against poisons, 393–400;
in production of antitoxin, 400–402;
in the defence of the skin, 407;
attempts to reconcile theory of, with humoral theory, 539;
history of theory of, 514–525, 539–543;
stimulant action of, 532
Phagocytic crisis of Bordet, 314;
ferments, 549–558;
function of neuroglia cells, 75;
organs, 85, 150, 292, 293, 537;
of cricket, 133;
of _Ascaris_, 547
Phagocytosis in osseous fishes, 135;
in frogs, 137, 142;
in fowl, 144, 282;
in dog, 149, 151;
in rat, 154, 157;
in guinea-pig, 162, 163, 166, 170, 223;
in horse, 245, 313;
in rabbit, 159, 167, 169, 233, 239, 314;
effect of removal of spleen on, 150;
agents that prevent, 170–173 (_see also_ Opium);
neutralisation of toxins not necessary for, 205, 289;
and agglutination, 202, 242, 245;
ensures natural immunity, 206;
action of opium on, 225, 231, 236, 306, 307;
action of rabbit’s serum on, 231;
in acquired immunity, 220, 223–226, 245, 280–286, 295, 313;
relation to fixatives in acquired immunity, 291, 295;
in the immunity conferred by specific serums, 303–317;
history of, and of the theory of phagocytes, 514–525, 539–543;
its application in surgery, 568
Phagolysis, 80, 99, 165;
prevention of, 99, 218, 219, 220, 230, 252, 304;
its relation to extracellular destruction of bacteria and Pfeiffer’s
phenomenon, 218–220, 230, 280, 295, 534;
escape of cytases during, 95, 99, 102, 191, 197, 252, 551–554, 560
Philocytase, 91, 92
Phloridzin, its action on natural immunity, 150
_Phyllirhoë_, two modes of digestion in, 58;
resorption by phagocytes of, 70
Pig. _See also_ Swine
Pig, protection of, against tetanus, 493
Pigeon, immunity of, against anthrax, 146;
immunity of, against human tuberculosis, 147;
immunity of, against influenza bacillus, 130, 554;
its blood best culture medium for influenza bacillus, 130, 554;
susceptible to swine erysipelas, 476;
protective power of serum of, immunised against anthrax, 276, 277,
288;
vaccination of, against anthrax, 276, 277
Pilocarpin augments production of antitoxin, 380
_Piroplasma bigeminum_, 247, 279
Plague, bubonic, rapid immunisation by serum, 312;
protective influence of broth against, 321;
production of antitoxic serum by, 401;
infection by, through the nasal cavity, 409, 411;
vaccinations against, 486–492;
serum treatment in, 490–492;
immunity against, when acquired and duration, 488, 489;
statistics on vaccinations against, 488;
prophylactic treatment against, 491;
Reports of German and English Commissions on, 489
Planarians, digestion in, 49, 71, 82
Plants, immunity in multicellular, 29–39;
cicatrisation of, 34;
and osmotic pressure, 37, 39, 566;
ravages of _Sclerotinia_ amongst cultivated, 32;
action of manures on immunity of cultivated, 36;
function of oxalic acid in, 37, 566
Plasma, Gengou’s method of preparing, 157, 190
Plasmas. _See also_ Body fluids, Serums
Plasmas, presence of fixatives in, 103;
bactericidal power of, 190, 543
Plasmase (fibrin ferment), 95, 197, 550
Plasmodia, intracellular digestion in, 30, 545;
chemiotaxis of, 30;
adaptation of, to poisons, 30
Pleuropneumonia, bacterium of, 3, 130, 478, 569;
vaccinations against, 477–479;
action of serum from animals immunised against, 479;
vaccinal methods used by savage races against, 506
Pneumococcus, modified growth of, in serums from immunised animals,
256, 262;
vaccination against, 262;
attenuated by serums from vaccinated animals, 287;
agglutination of, 287
Pneumo-enteritis of swine, cocco-bacillus of, 259;
action of serum of vaccinated rabbits on bacillus of, 260, 266, 287,
532;
acquired immunity against, 260, 275, 311, 532
Pneumonia, fibrinous, relapses separated by periods of immunity, 434
Poisons. _See also_ Toxins
Poisons, absorption of, by leucocytes, 393–400
_Polyphagus euglenae_, 12
Potato attacked by _Bacillus coli_, 35
Precipitins in the blood serum, 68, 106, 107;
use of, in medico-legal investigations, 107, 568;
and in the differentiation of various kinds of milk, 107, 568
Predisposition or absence of immunity, 7
Preventive substances of Bordet (syn. fixatives), 266
Profetta, law of, 453
Protective or anti-infective property. _See also_ Antitoxic,
Antitoxins, Blood, Body fluids, Serums
Protective property, origin of, in serums and other fluids, 291–294;
differs from agglutinative, 268, 269, 294;
of blood and other fluids in convalescents, 437–444
Protective action of normal serums, 317–320;
of fats against toxins, 387;
of leucocytes against poisons, 393–400;
of flow of a fluid, 431
Protective function of the skin, 404–407;
in the respiratory channels, 411–414;
of the cornea, 409;
of the saliva, 415;
of the intestine, 422;
of the bile, 424;
of the liver, 427;
of the lymphoid organs, 428;
of the suprarenal capsules, 431;
in the urinary organs, 431
Protective substance resistant to heat, 268;
and so distinguished from bactericidal substance, 268;
closely connected with fixative substance, 269, 294, 295, 561
Protective vaccinations, 454–504
_Proteus vulgaris_, susceptibility of leucocytes to, 166, 179, 201,
282;
eosinophile transformation in, 198;
modified growth in certain serums, 259
Protozoa, intracellular digestion in the, 13, 30, 49;
adaptation of, to saline solutions, 23, 515;
and to physical conditions, 26
Prussic acid, antidote to, 363
Pseudo-diphtheria bacilli, 444
Pseudo-eosinophile leucocytes, secretion by, 187, 542
Pseudo-immunity or resistance, 320
Pus, ferment in, 96
Pyrogallic acid, its action on natural immunity, 150
Rabbit, immunity of, against anthrax bacillus, 159;
against streptococci, 167, 168;
against tetanus bacillus, 169;
against cholera vibrio, 424;
against pleuropneumonia, 569;
acquired immunity of, against pyocyanic disease, 232;
against swine erysipelas, 236–239, 527;
against anthrax, 239, 323;
against streptococcus, 243–247, 284–286, 312, 314;
against pneumo-enteritis, 260, 266, 275, 311, 532;
against pneumococcus, 262;
against a staphylococcus, 266;
against hog cholera, 290;
against mouse septicaemia, 509;
phagocytosis in, 159, 167, 169, 233, 239, 314, 569;
infection by streptococci in, 283;
action of serum of vaccinated, on bacillus of pneumo-enteritis, 287;
action of agglutinated pneumococci on, 287;
vaccinated against hog cholera susceptible to its toxin, 290;
immunised against anthrax by means of the erysipelas coccus, 323;
immunised against anthrax by products of _Bacillus pyocyaneus_, 323;
infection by anthrax prevented by Friedländer’s bacillus, 323;
brain of, very susceptible to action of tetanus toxin, 383;
reaction of, to atropin, 395
Rabies, action of anthrax bacillus on, 150;
action of normal ox serum on, 365;
action of bile on, 425;
heredity of immunity against, 446;
vaccinations against, 461–466;
statistics of vaccinations against, 464–466;
in domestic animals, vaccinations against, 466
Rat, immunity of, against anthrax bacillus, 152, 526;
against diphtheria bacillus, 204;
acquired immunity against _Trypanosomata_, 247–249, 316;
against anthrax, 240;
natural immunity of, against diphtheria toxin, 204, 339;
bactericidal ferment of phagocytes of, 20, 157;
phagocytosis in, 154, 157
Receptors, 93, 120, 296;
over-production of, 121, 296, 562;
antitoxic and philotoxic functions of, 120;
theory of, _see_ Side-chain theory
Recurrent fever. _See_ Spirilla, _Spirochaete obermeyeri_
Recurrent fever, transient acquired immunity against, 434
Rennet, 109, 119
Reptilia. _See_ Alligator, Turtle, Snake, Lizard
Reptilia, natural immunity of, against tetanus toxin, 331–334
Resistance to disease, 8–10.
_See_ Immunity, Pseudo-immunity
Resorption of micro-organisms, 46, 175 (_see also_ Immunity, cellular,
Micro-organisms);
of the formed elements, 47, 67–105;
a true intracellular digestion, 85, 296;
of cells in the Invertebrata, 70;
of red corpuscles by phagocytes of the Vertebrata, 72, 80 (_see also_
Phagocytes, Phagocytosis);
part played by macrophages in (_see_ Macrophages);
and digestion closely related, 69, 85;
of spermatozoa, 84, 100;
of white corpuscles, 84 (_see also_ Leucocytes, Phagocytes);
of albuminoid substances, 106–127;
of cells and the phenomena in acquired immunity, 296
Respiratory channels, protection by the, 411–414;
absorption of poisons by the, 414
Rhinoceros beetle, natural immunity in larvae of, 132, 209, 326, 329;
susceptibility to cholera vibrio, 40, 133
Ricin, 344, 360, 446, 449
Rinderpest, action of bile on, 425, 466;
vaccinations against, 466–468;
Koch’s method of vaccination against, 466;
Kolle and Turner’s method of “simultaneous vaccinations” against, 467
Ring-worm, mechanical factor in, 4
Robin (toxalbumin of _Robinia pseudacacia_), 365;
serum of animals vaccinated against, antitoxic, 365;
heredity of immunity against, 446
_Saccharomyces._ _See_ Yeasts
Saline solution (physiological) as a protective fluid, 320, 365
Saliva, microbicidal property of the, 415;
antitoxic function of, on snake venom, 417;
psychic influence on flow of, 62, 566
Saponin, haemolytic action of, 389;
and cholesterin, 389;
and antisaponic power, 390
_Saprolegnia._ _See_ Fungi
Sarcinae as adjuvant organisms, 426
Sarcinae, acidophile, 418
_Sclerotinia_, pathogenic action of, 32
_Scolopendra_, acquired immunity in, against anthrax, 209
Scorpion, natural immunity of, against tetanus toxin, 326;
against its own poison, 327;
antivenomous property of blood of, 328;
supposed suicide of, 327
Scorpion serum, action of antivenomous serum on, 365
Scorpion venom, antitoxic action of crayfish blood against, 366
Scrofula in immunity against tuberculosis, 436
Secretion of bactericidal substance, theory of, 187–191, 533–537, 540,
542
Sensibilising substance of Bordet (fixative), 91, 199, 298, 535, 537,
557
Sensitiveness of plants to osmotic pressure, 37, 566
Septicaemia of goose. _See Spirochaete anserina_
Septicaemia of mouse. _See_ Mouse septicaemia
Septic vibrio, 170
Serums. _See also_ Blood, Body fluids, Humoral theory, Toxins
Serums, haemolysis by, 83, 87–95 (_see also_ Haemolysis);
effect of injections of, 68;
increasing haemolytic power of, 90;
isotoxic, 104;
absorption of, 106;
antihaemotoxic, 111, 112;
haemolytic or haemotoxic, 111, 112;
anticoagulating, 190;
anticytase, 115, 371;
antispermotoxic, 116, 122–126;
bactericidal properties of, 184, 190, 191, 192, 193, 206, 211, 226,
233, 238, 241, 243, 244, 260, 298, 554;
influence of alkalinity or acidity on bactericidal action of, 196;
agglutination of red blood corpuscles by, 258;
agglutination of bacteria by, 256–265, 380;
protective power of, in the immunised organism, 266–280, 287, 293,
295, 532;
differs from bactericidal power, 268;
and from agglutinative power, 268;
and is not a measure of acquired immunity, 271, 274, 275;
protective, may be only feebly antitoxic, 497;
modified growth of bacteria in immunised, 256, 259 (_see also_
Agglutination);
resistance to heat of protective substance of, 268;
fixatives in protective, 269, 438;
their origin, 294;
protective and fixative substances contrasted, 269;
relations of fixative and cytase in bactericidal action of, 298;
stimulating action of, 270–274, 301, 308–320, 365;
absence of protective power in specific, 270, 276–279;
origin of protective power in, 291–294;
theory of attenuation of micro-organisms by immune, 286–289;
inactive specific, rendered active by addition of normal serum, 215,
268, 298, 302, 317;
protective action of heated normal serum, 273, 318;
protective action of non-specific, against toxins, 365;
from convalescents, protective action of, 437–444;
temporary immunity against micro-organisms conferred by specific,
301–317;
conferred by normal, 317–320;
conferred by fluids other than, 320–322;
phagocytosis in the immunity conferred by specific, 303–306;
influence of opium on immunisation by specific, 306;
antivenomous action of, 334, 338, 358, 360, 361;
antitoxic action of non-specific and normal, 365, 380;
anti-arsenic, 390;
antileucocidic, 359;
antidiastatic, 361;
testing and standardisation of antitoxic, 376, 476, 496–498
Sheath, protective. _See_ Membrane
Sheep, natural immunity of, against anthrax, 159, 289;
acquired immunity of, against anthrax, 241–3, 289;
bactericidal action of blood serum of, 241, 286;
protective power of serum of, immunised against anthrax, 276;
immunised with blood from dog affected by a haematozoon, 279;
vaccination of, against sheep-pox, 460;
against rabies, 466;
against anthrax, 469;
protection against tetanus in, 493;
fate of anthrax bacilli in Algerian, 512
Sheep-pox (la clavelée), heredity of immunity against, 452;
vaccinations against, 460–461
Side-chains or receptors, theory of, 120, 381–384, 538, 557, 562–563;
compared with theory of phagocytes, 296–299, 538, 558
Silver, soluble salts of, absorbed by leucocytes, 400
Skin, immunity of the, 403–407;
protective function of the, 404–407;
phagocytes in the defence of the, 407
Small-pox, mortality from, in 18th century, 454;
vaccinations against, 454–460;
vaccination with calf lymph, 456;
with contents of pustule of cow-pox, 455;
vaccination statistics, 457–459
Snail. _See Helix pomatia_
Snake, natural immunity of, against snake venom, 333
Snake venom, natural immunity of snakes against, 333;
of hedgehog against, 337;
of mongoose against, 339;
artificial immunity against, 345, 347;
action of antivenomous serum on, 358, 360, 361;
of other specific serums on, 365;
of cerebral substance on, 386;
protective substances against, 387;
action of saliva on, 417;
action of bile on, 425;
vaccination methods of savage races against, 506
Spermatozoa, resorption of, 84, 100;
action of spermotoxin on, 101, 116, 125
Spermotoxin, 101, 116, 125
Spiders, natural immunity of, against tetanus toxin, 326
Spirilla, natural immunity against, 159;
acquired immunity against, 227–230, 434;
living in stomach of dog, 177;
acidophile, 418
_Spirochaete anserina_, 160
_Spirochaete obermeyeri_, 160;
acquired immunity against, 227–230;
Pfeiffer’s phenomenon in, 229
Spleen, function of, 62, 85;
action of extract of, on tetanus toxin, 365;
effect of removal of, 150, 293;
as source of fixative substance, 295, 537
Spleen and other haematopoietic organs as source of protective
substance, 292–294;
as source of agglutinins, 264;
are phagocytic organs, 85, 150, 292
Sponges, digestion of, 69, 517
Staining reactions of cells and micro-organisms, 13, 77, 83, 181, 183,
198, 213
Standardisation of antidiphtheria serums, 376, 496–498;
Ehrlich’s method, 496;
Pasteur Institute method, 496–497
Staphylococcus, acquired immunity against, 266, 532;
protective action of normal serum against, 319
_Staphylococcus pyogenes_ in vagina, 430
Stellate cells of Kupffer, 75
Stimulant action. _See also_ Body fluids, Protective
Stimulant action of serums, 270–274, 301, 308–320, 365;
of phagocytes, 532;
of normal fluids of the body, 559
Stimulins and their action in serums, 270–274
Stöhr’s phenomenon, 429
Stomach, acidophile microbian flora of, 418
Streptococci, protective sheath formed by, 22;
immunity against, 165, 179, 282, 284–286;
phagocytosis in immunity against, 245, 313;
acquired immunity against, 243–247, 313;
agglutination by serum of, 244, 245;
reaction of animal organism against, 245–247;
antitoxin against, 205;
and phagocytosis, 283;
action of specific serums on, 287, 288, 312;
protective action of various fluids against, 320, 321
Streptococcic serum, action of, on leucocidin, 359
Sturin, bactericidal action of, 183
Suprarenal capsules, protective function of, 431
Susceptibility. _See also_ Chemiotaxis, Hypersusceptibility,
Irritability, Sensitiveness
Susceptibility of immunised animals to the specific toxin, 289;
of frogs to tetanus toxin, 330;
diminution of, in immunised animals, 374–376;
in immunity, the part played by, 565;
cellular, a general property of living beings, 565–566
Swine. _See_ Pig, Pneumo-enteritis
Swine erysipelas, acquired immunity against, 236–239, 254, 283, 527;
agglutination of bacilli of, 262;
specific serum of, will not prevent infection, 270;
phagocytosis in, 283;
action of immune serums on bacillus of, 288, 289;
protective action of specific serum against, 307;
method of testing strength of serums against, 476;
vaccinations against, 473–477;
Pasteur’s method, 473;
Lorenz’s method, 475;
“serum-vaccinations” method, 475;
vaccines against, 208, 473, 509
Swine plague, 259, 260
_Synapta_, 518
Syphilis, immunity in, 435;
and general progressive paralysis, 435;
law of Profetta in immunity against, 453;
law of Baumès-Colles in, 436;
transmission of, 452
Tears, microbicidal function of the, 408
Testing of serums. _See_ Standardisation
Tetanolysin of Ehrlich, 349
Tetanospasmin, 362
Tetanus, immunisation against, 344, 347, 492–495;
cerebral, in rabbit, 383, 391;
difference between antitoxic action of living brain and that of
cerebral emulsion on, 383;
in fowl, 384;
no antitetanic power in serum of convalescents, 443;
vaccinations against, 492–495;
vaccines against, 493;
protective serum treatment against, 493–495
Tetanus antitoxin, hypothesis of nervous origin of, 381–385;
nature of, 355;
mode of action on toxin, 357, 381;
of nerve centres locally restricted in its action, 382
Tetanus bacillus, natural immunity against, 169, 204
Tetanus toxin, natural immunity of spiders and scorpions against, 326;
of larvae of _Oryctes_ and of cricket against, 329;
of frog against, 330;
of reptiles against, 331–334;
of fowl against, 335;
of hibernating animals against, 339;
attenuation of, 344;
localisation of, in vascular organs, 336;
brain of rabbit very susceptible to action of, 383;
fixation of, by substance of nerve centres, 382;
by certain parts of brain and cord, 386, 391;
by other cells, 391, 392;
action of emulsions of frog’s brain on, 386;
fixation of, by carmine, 388, 394;
absorption of, by leucocytes, 393–395;
action of extract of spleen on, 365;
toxone (tetanolysin) of, 349, 362;
local reaction to, in horse, 352;
heredity of immunity against, 446, 448, 450
Texas fever, acquired immunity of Bovidae against, 247, 279;
attenuation of parasite of, in the tick, 247;
haematozoon in dog closely allied to that of, 279
_Thetys_, 517
Thymus gland, immunising power of, 293
Tick, attenuation of parasite of Texas fever in, 247
Tonsils, protective function of, 428
Torulae as adjuvant organisms, 426
Toxins, immunity against, 10;
immunity of unicellular organisms against, 19;
adaptation of bacteria to, 21–27;
of yeasts to, 20, 26;
of plasmodia to, 30;
action of, on Infusoria, 19, 326;
composition of, 120;
neutralisation of, not necessary for phagocytosis, 205, 289;
immunity against micro-organism not same as against toxin, 251, 290;
protective fixation of, by nerve elements and other cells, 386–400;
methods of immunisation by modified and unmodified, 345–347 (_see_
Immunisation);
local reaction in immunisation against, 352;
action of normal serums on, 365;
of non-specific serums on, 365;
protective action of fats against, 387;
leucocytic reaction against, 393–400;
absorption of, by the conjunctiva, 409;
by the respiratory channels, 414;
destruction of, by the intestinal organisms, 427;
attenuation of, 344;
natural immunity against, 325–341;
artificial immunity against, 342–402;
against bacterial, 343;
against vegetable, 344, 365;
heredity of immunity in phanerogamic, 446, 449;
susceptibility of nerve centres to, 564
Toxoids, 349 (_see also_ Toxophore);
immunisation by, 350
Toxones, 349, 362;
method of immunisation by, 349
Toxophore atomic group in toxin (= toxoid), 120, 350, 384
_Trichinae_, mechanical action of, 3
Tristeza (syn. Texas fever), 247
_Tropidonotus._ _See_ Snake
_Trypanosoma_, 4, 129, 147;
_brucei_, 9;
_lewisi_, 173, 248
_Trypanosomata_, fate of, in refractory animal, 173;
acquired immunity against, 247–249, 316;
and agglutinative power, 278
Trypsin, antitoxic power of, 424
Tsetse fly, 4, 9, 129, 247
Tubercle bacillus, formation of sheath by, 22, 183
Tuberculin as a protective substance against cholera vibrio, 320
Tuberculosis, mechanical etiological factors in, 4
Tuberculosis, bacillus of, 22, 42, 143;
avian, 41, 148, 149, 182;
human, immunity of pigeon against, 147;
acquired immunity in, 436;
after scrofula, 436;
Koch’s phenomenon in, 437
Tumours, malignant, probability of discovery of parasite of, 3;
use of erysipelas streptococcus in, 434
Turtle, natural immunity of, against tetanus toxin, 332, 386
Typhoid, protective power of serum of convalescents from, 437–440;
its agglutinative power, 439;
serum-diagnosis of, 256, 257, 261, 439;
immunity against, not acquired by suckling, 450;
vaccinations against, 479, 481–486;
Wright’s vaccine against, 482;
bactericidal power of serum from persons immunised against, 483;
statistics of vaccinations against, 483–485
Typhoid bacillus, 23, 143, 191, 198, 203;
acquired immunity against, 230;
attenuated Pfeiffer’s phenomenon in, 230, 303, 304;
agglutination of, 260, 261, 380, 439;
resistance to agglutinated, 263;
protective action of serums against, 272–274, 293, 317, 319;
origin of protective substance against, 292;
of agglutinative property against, 294;
protective action of various fluids against, 320;
passes uninjured through stomach, 418;
transmission by suckling, of agglutinative power against, 450
Typhoid infection, experimental, in laboratory animals, 230, 267;
influence of anticytase serum on, 371;
uncertainty of, by ingestion, 423
Typhoid septicaemia, experimental, heredity of immunity against, 447
Tyrosin, protective action of, 387
Unicellular organisms, immunity in, 11–28;
infective diseases of, 12;
irritability of, 27;
adaptation of, to saline solutions, 23, 515
Unit, Ehrlich’s immunising, 373, 496
Urinary ferments, 66
Urinary organs, protective function in, 431
Urine as a protective fluid, 320, 431;
pepsin in the, 65;
amylase in the, 65
Vaccination. _See also_ Immunisation
Vaccinations, protective, 208, 241, 267, 454–504, 507;
with attenuated micro-organisms, 509
Vaccine against fowl cholera, 208
Vaccines against anthrax, 208, 470, 509;
against swine erysipelas, 208, 473, 509;
against rabies, 208, 462, 463–464;
against symptomatic anthrax, 471;
against small-pox, 455–457, 507;
against pleuropneumonia, 477;
against cholera, 481;
against plague, 487, 489, 490;
against tetanus, 493
Vaccinia, supposed micro-organism of, 455–456
Vagina, autopurification of, 429
Variolisation, early use of, 455, 507
Venom. _See_ Snake venom
_Ver blanc_, syn. cockchafer larva
Vibrio. _See also_ Cholera vibrio, Massowah vibrio, Septic vibrio
Vibrios, acquired immunity against, 211–227;
phagocytosis in immunity against, 220, 223–226;
granular transformation of, 164, 165, 192, 212–226 (_see_ Pfeiffer’s
phenomenon);
bacteriolysis (agglutination) of, 256;
susceptibility of animals vaccinated against, to the toxins, 290
_Vibrio metchnikovi_, acquired immunity against, 211, 226, 527, 531;
modified growth of, in serums from immunised animals, 156, 262;
action of, grown in serum of vaccinated animals, 287;
perishes in intestine of dog, 422;
action of microcytase on, in hypervaccinated guinea-pigs, 553
Viper. _See_ Snake, Snake venom
Viruses, attenuated, 208, 508;
vaccination with, whose nature is as yet unknown. _See_ Small-pox,
Sheep-pox, Rabies, Rinderpest
Vitellus of egg of immunised fowl, tetanus antitoxin present in, 356;
immunity conferred by, 449
Warlomont’s calf lymph vaccine, 456
Weber-Fechner, law of, 27, 38, 566
Willem’s method of vaccination against pleuropneumonia, 477;
Pasteur’s modification of, 477
Wright’s method of vaccination against typhoid, 482;
method of testing bactericidal power of body fluids, 483
Yeast-cells, adaptation of, to poisons, 20, 26;
to milk-sugar, 26;
destruction of injected, by phagocytes, 172;
Curtis’s pathogenic, 172;
endotrypsin of, 197;
autodigestion in, 197;
soluble ferments of, 253
Yeasts, diseases due to, 2
Yolk. _See_ Vitellus
Zymase, 197, 550
CAMBRIDGE: PRINTED BY JOHN CLAY, M.A. AT THE UNIVERSITY PRESS.
-----
Footnote 1:
_Deutsche med. Wchnschr._, Leipzig, 1884, SS. 499, 519.
Footnote 2:
_Mitth. aus d. K. Gesundheitsamte_, Berlin, 1884, Bd. II, S. 421.
Footnote 3:
“On the Origin of Species,” 6th ed., London, 1872, Chapter XI, p. 277.
Footnote 4:
_Vrtljschr. f. gerichtl. Med._, Berlin, 1855, S. 102.
Footnote 5:
“Ueber Chytridium,” in _Monatsber. d. Berliner Akad._, 1855, June, No.
14.
Footnote 6:
Cohn’s “Beiträge zur Biologie der Pflanzen,” Breslau, 1876, Bd. II, S.
210.
Footnote 7:
For the parasites of Infusoria, cf. Bütschli in Bronn’s “Klassen und
Ordnungen d. Thier-Reichs,” Leipzig, 1885—1889, Bd. I, SS. 872, 1823,
1944.
Footnote 8:
_Arch. d’anat. microsc._, Paris, 1898, t. II, p. 528.
Footnote 9:
Le Dantec, “Recherches sur la digestion intracellulaire,” Lille, 1891,
p. 53.
Footnote 10:
Ehrlich u. Lazarus, “Die Anämie,” in Nothnagel’s “Specielle Pathologie
u. Therapie,” Wien, 1898, Bd. VIII, I^{ter} Theil, S. 85; also
“Pathology of the Blood,” authorised English translation, Cambridge,
1900, p. 125.
Footnote 11:
_Arch. f. Entwickelungsmech._, Leipzig, 1898, Bd. VII.
Footnote 12:
_Compt. rend. Acad. d. Sci._, Paris, 1901, t. CXXXIII, p. 244.
Footnote 13:
_Arch. de zool. expér._, Paris, 1889, 2^{me} série, t. VII, p. 446.
Footnote 14:
_Ann. de l’Inst. Pasteur_, Paris, 1890, t. IV, p. 148.
Footnote 15:
“Leçons sur la pathologie comparée de l’inflammation,” Paris, 1892, p.
24; “Lectures on the comparative pathology of inflammation,”
authorised translation into English, London, 1893, p. 20.
Footnote 16:
“Leçons sur la pathologie comparée de l’inflammation,” p. 21; English
edition, p. 17.
Footnote 17:
_Monatsber. d. Berl. Akad. d. Wissensch._, 1881, p. 388.
Footnote 18:
_Compt. rend. du Congrès internat. de Méd. tenu à Paris en 1900._
Section de bactériologie et de parasitologie.
Footnote 19:
“Sur l’immunité naturelle des organismes monocellulaires contre les
toxines” _Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 465.
Footnote 20:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 801.
Footnote 21:
“Ueber die Ursache der Immunität von Ratten gegen Milzbrand,” in the
_Centralbl. f. klin. Med._, Bonn, 1888, no. 38.
Footnote 22:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 872.
Footnote 23:
_Ann. de l’Inst. Pasteur_, Paris, 1900, t. XIV, p. 641.
Footnote 24:
Metchnikoff, _Virchow’s Archiv_, 1884, Bd. XCVII, S. 510.
Footnote 25:
“Contribution à l’étude du sérum antistreptococcique,” _Ann. de
l’Inst. Pasteur_, Paris, 1897, t. XI, p. 177, Planche V.
Footnote 26:
_Arch. f. Hyg._, München u. Leipzig, 1900, Bd. XXXIX, S. 31.
Footnote 27:
“Entwickelungsgeschichte der mikroskopischen Algen und Pilze,” _Nov.
Acta Acad. Caes. Leop. Carol._, 1854, t. XXIV, p. 1.
Footnote 28:
“Action des sels sur les infusoires,” _Arch. d’anat. microsc._, Paris,
1898, t. II, p. 595.
Footnote 29:
“On the acclimatisation of organisms to poisonous chemical
substances,” _Arch. f. Entwickelungsmech._, Leipzig, 1895, Bd. II, S.
564.
Footnote 30:
_Ann. de l’Inst. Pasteur_, Paris, 1887, t. I, p. 465.
Footnote 31:
_Monit. scient. du D^r Quesnerille_, 1890, 1891, 1892, 1894.
Footnote 32:
“Traité de Microbiologie,” Paris, 1898, t. I, p. 238.
Footnote 33:
_Ann. de l’Inst. Pasteur_, Paris, 1896, t. X, p. 417.
Footnote 34:
_Ann. de l’Inst. Pasteur_, Paris, 1900, t. XIV, p. 139.
Footnote 35:
_Journ. R. Micr. Soc._, London, 1880, III, p. 1.
Footnote 36:
Davenport and Castle, _Arch. f. Entwickelungsmech._, Leipzig, 1895,
Bd. II, S. 227.
Footnote 37:
_Untersuch. a. d. physiolog. Inst. d. Univ. Heidelberg_, 1878, Bd. II,
S. 273.
Footnote 38:
_Flora_, Marburg, 1892, Bd. LXXVI, S. 182.
Footnote 39:
_Botan. Ztg._, Leipzig, 1884, S. 161.
Footnote 40:
[Stahl used plasmodia which had spread themselves on a substratum of
wet filter paper applied to the inside of glass vessels, its lower
edge touching the surface of the experimental fluid at the bottom of
the vessel (Translator).]
Footnote 41:
The italics are M. Metchnikoff’s.
Footnote 42:
“Vergleichende Morphologie u. Biologie der Pilze, Mycetozoen u.
Bacterien,” Leipzig, I^{te} Aufl., 1884; also authorised English
translation, Oxford, 1887.
Footnote 43:
_Botan. Ztg._, Leipzig, 1886, SS. 377, 393, 409, 433, 449, 465.
Footnote 44:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 44.
Footnote 45:
“La cicatrisation chez les végétaux,” _Mém. couron. de l’Acad. roy. de
Belgique_, Bruxelles, 1898, t. LVII.
Footnote 46:
Cf. Frank, “Die Krankheiten der Pflanzen,” Breslau, 2^{te} Aufl.,
1895, Bd. I, S. 43.
Footnote 47:
“Recherches expérimentales sur les maladies des plantes,” _Ann. de
l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 1.
Footnote 48:
“Réaction osmotique des cellules végétales,” _Mém. couron. de l’Acad.
roy. de Belgique_, Bruxelles, 1899.
Footnote 49:
“La cicatrisation,” _l.c._, p. 61.
Footnote 50:
_Untersuch. a. d. botan. Inst. zu Tübingen_, Leipzig, 1884, Bd. I, S.
363.
Footnote 51:
“Recherches sur les organismes inférieurs,” _Bull. de l’Acad. de
Belgique_, 1888, 2^e série, t. XVI, V, 12.
Footnote 52:
_L.c._, p. 40.
Footnote 53:
[Probably a surface growth on a sloped agar tube (Transl.).]
Footnote 54:
“Etude expérimentale sur les glandes lymphatiques des invertébrés,”
_Mélanges biol. de l’Acad. d. sc. de St-Pétersb._, 1894, t. XIII, p.
458.
Footnote 55:
“Ueber grünen Eiter,” Volkmann’s _Samml, klin. Vortr._, No. 62,
Leipzig, 1893.
Footnote 56:
“Processus chimiques dans les intestins de l’homme,” _Arch. d. sc.
biol. de St-Pétersb._, 1892, t. I, p. 539; _Ztschr. f. Hyg._, Leipzig,
1893, Bd. XV, S. 474.
Footnote 57:
Cited by Schimmelbusch, _l.c._
Footnote 58:
_Compt. rend. Acad. d. Sc._, Paris, 1892, t. II, p. 1226.
Footnote 59:
_Berl. klin. Wchnschr._, 1901, S. 163.
Footnote 60:
_Ztschr. f. Hyg._, Leipzig, 1898, Bd. XXIX, S. 548.
Footnote 61:
“Fermente und Mikroparasiten” in Ziemssen u. Pettenkofer’s “Handbuch
der Hygiene,” Leipzig, 1883.
Footnote 62:
“Ueber die Schicksale der in’s Blut injicirten Mikroorganismen,”
_Ztschr. f. Hyg._, Leipzig, 1886, Bd. I, S. 1.
Footnote 63:
_Ztschr. f. Hyg._, Leipzig, 1897, Bd. XXVI, S. 353.
Footnote 64:
_Arch. f. exper. Path._, Leipzig, 1897, Bd. XXXIX, S. 39.
Footnote 65:
_Ztschr. f. Hyg._, Leipzig, 1898, Bd. XXIX, S. 528.
Footnote 66:
_Ztschr. f. Hyg._, Leipzig, 1900, Bd. XXXIII, S. 261.
Footnote 67:
_Ann. de l’Inst. Pasteur_, Paris, 1900, t. XIV, p. 415.
Footnote 68:
_Berl. klin. Wchnschr._, 1891, S. 505.
Footnote 69:
_Vratch_ (in Russian), St Petersburg, 1896, Nos. 8, 12.
Footnote 70:
_Centralbl. f. d. med. Wissensch._, Berlin, 1867, No. 31.
Footnote 71:
_Virchow’s Archiv_, 1869, Bd. XLVIII, S. 1.
Footnote 72:
_Compt. rend. Acad. d. Sc._, Paris, 1884, t. XCVIII, p. 749.
Footnote 73:
_Compt. rend. Soc. de biol._, Paris, 1900, p. 553.
Footnote 74:
_Virchow’s Archiv_, 1852, Bd. IV, S. 536.
Footnote 75:
“Handb. d. klin. Mikroskopie,” 1887, S. 108; _Gaz. med. lombarda_,
1871 and 1872; _Wien. medic. Jahrbücher_, 1872, S. 160.
Footnote 76:
“Grundzüge einer vergl. Physiologie der Verdauung,” Heidelberg, 1882.
Footnote 77:
G. H. Lewes, “Sea-side Studies,” Edin. and London, 1858, p. 216.
Footnote 78:
_Zool. Anz._, Leipzig, 1880, Jahrg. III, S. 261, and 1882, Jahrg. V,
S. 310.
Footnote 79:
Metchnikoff, _Ann. de l’Inst. Pasteur_, Paris, 1893, t. VII, p. 348.
Footnote 80:
_Bull. Acad. roy. de Belg._, Brux., 1893, t. XXV, p. 262, and _Arch.
de Zool. expér._, Paris, 1893, 3^{me} série, t. I, p. 139.
Footnote 81:
“Etudes de physiologie sur les Actinies,” Charkoff, 1895 (in Russian).
Footnote 82:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 352.
Footnote 83:
Address delivered before the _Société des médecins russes_ at St
Petersburg. _Gaz. clin. de Botkine_, 1900.
Footnote 84:
“Physiologie du suc intestinal,” Saint-Pétersbourg, 1899 (Thesis, in
Russian).
Footnote 85:
_Arch. d. sc. biol._, St.-Pétersb., 1893, t. II, p. 698.
Footnote 86:
Cf. _Bull. Acad. de méd._, Paris, 1901, p. 17.
Footnote 87:
_Arch. d. sc. biol._, St.-Pétersb., 1899, t. VII, p. 1.
Footnote 88:
_Arch. d. sc. biol._, St.-Pétersb., 1893, t. II, p. 219.
Footnote 89:
_Virchow’s Archiv_, 1893, Suppl. to Bd. CXXXI, S. 142. The question of
urinary ferments is summarised in Neubauer u. Vogel’s “Analyse des
Harns,” Wiesbaden, 10^{te} Aufl., 1898, S. 599.
Footnote 90:
_Compt. rend, du XIII^e Congrès internat. de Méd._, Paris, 1901.
Leube, “Ueber extrabuccale Ernährung,” in “Deutsche Klinik am Eingange
d. XX. Jahrhunderts,” Wien u. Leipzig, 1901, I, S. 64.
Footnote 91:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 406.
Footnote 92:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 225.
Footnote 93:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1900, I^{te} Abt.,
Bd. XXVIII, S. 237.
Footnote 94:
_Deutsche med. Wchnschr._, Leipzig, 1900, S. 734.
Footnote 95:
_Ann. de l’Inst. Pasteur_, Paris, 1900, t. XIV, p. 49.
Footnote 96:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 17.
Footnote 97:
The resorption of the red blood corpuscles by the phagocytes of larvae
of starfish (_Bipinnaria_) and of _Phyllirhoë_ has been described in
my paper on intracellular digestion in the Invertebrates in _Arb. a.
d. Zool. Inst. d. Univ. Wien_, 1883, Bd. V, Hft. 2, S. 141.
Footnote 98:
I have only been able to discover the haemolytic property of the
serums of _Cyprinus_ after the third injection of guinea-pig’s blood.
Footnote 99:
_Virchow’s Archiv_, 1870, Bd. XLIX, S. 66.
Footnote 100:
Soudakewitch, _Ziegler’s Beitr. z. path. Anat._, Jena, 1888, Bd. II,
S. 129, and Babes, “Untersuchungen über den Leprabacillus,” Berlin,
1898, S. 58.
Footnote 101:
Marinesco, _Compt. rend. Soc. de Biol._, Paris, 1896, p. 726.
Footnote 102:
_Arch. f. mikr. Anat._, Bonn, 1899, Bd. LIV, S. 254.
Footnote 103:
Ehrlich u. Lazarus, “Die Anaemie,” in Nothnagel’s “Specielle
Pathologie u. Therapie,” Wien, 1898, Bd. VIII, I^{ter} Theil, S. 49.
Cf. the authorised English translation, “Histology of the Blood,”
Cambridge, 1900, p. 74.
Footnote 104:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 301.
Footnote 105:
_Arch. de méd. expér._, Paris, 1901, t. XIII, p. 1.
Footnote 106:
_Fortschr. d. Med._, Berlin, 1888, Bd. VI, S. 460; “Die Entstehung der
Entzündung,” Leipzig, 1891.
Footnote 107:
_Journ. publ. par la Soc. roy. d. Sc. méd. et nat. de Bruxelles_,
1890, 3 Feb.
Footnote 108:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 742.
Footnote 109:
Krompecher (_Centralbl. f. Bakteriol. u. Parasitenk._, I^{te} Abt.,
Jena, 1900, Bd. XXVIII, S. 588) has obtained a serum which was even
capable of altering the nuclei of the red corpuscles of the frog.
These nuclei must be much less resistant than those of the red blood
corpuscles of birds, such as the goose, fowl and pigeon.
Footnote 110:
Some years ago it was proposed to give the name of cytase to the
ferments which digest cellulose. Thus Laurent, in the work analysed in
the second chapter, applies it to the ferment secreted by the bacilli
which attack the vegetable membrane. We think that the cellulose
ferment should be designated by the name of _cellulosase_ and that the
name of cytase would be more suitable for a soluble ferment which
digests the cells.
Footnote 111:
_Arch. de méd. expér._, Paris, 1891, t. III, p. 720.
Footnote 112:
_Verhandl. d. X. Congr. f. inn. Med._, Wiesbaden, 1892.
Footnote 113:
_München. med. Wchnschr._, 1900, S. 1193.
Footnote 114:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 273; 1901, t. XV,
p. 312.
Footnote 115:
_Berl. klin. Wchnschr._, 1899, SS. 6 and 481.
Footnote 116:
Ehrlich and Morgenroth, “Ueber Haemolysine,” II, _Berl. klin.
Wchnschr._, 1899, S. 481. The following are the combinations found by
these observers: heated calf’s serum with normal serum dissolves the
red blood corpuscles of the guinea-pig; heated rabbit’s serum plus
sheep’s serum dissolves the red blood corpuscles of the sheep; heated
serum of rabbit with the addition of goat’s serum dissolves the red
corpuscles of the goat; heated sheep’s serum with guinea-pig’s serum
produces haemolysis of the red corpuscles of the guinea-pig.
Footnote 117:
_Centralbl. f. Bakteriol. u. Parasitenk._, I^{te} Abt., Jena, 1901,
Bd. XXIX, S. 175.
Footnote 118:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 688.
Footnote 119:
Among the synonyms of this substance, resistant to the action of heat,
we may mention the following: haemolytic antibody, preventive
substance, immunising body (Immunkörper of Ehrlich), amboceptor
(Ehrlich), philocytase (Metchnikoff), desmon (London), copula (P.
Müller).
Footnote 120:
_München. med. Wchnschr._, 1900, S. 677.
Footnote 121:
_Ann. de l’Inst. Pasteur_, Paris, 1900, t. XIV, p. 656.
Footnote 122:
_Arch. d. sc. biol._ (russes), 1901, t. VIII, pp. 281 and 323.
Footnote 123:
_München. med. Wchnschr._, 1900, S. 1193.
Footnote 124:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 688; 1899, t. XIII,
p. 273.
Footnote 125:
_Berl. klin. Wchnschr._, 1899, SS. 6 and 481.
Footnote 126:
_Berl. klin. Wchnschr._, 1900, S. 682.
Footnote 127:
_Deutsche med. Wchnschr._, Leipzig, 1890, S. 389.
Footnote 128:
_Arch. russes d. path._, etc., St.-Pétersb., 1900, t. IV, p. 402.
Footnote 129:
“Die Entstehung der Entzündung,” Leipzig, 1891, S. 508.
Footnote 130:
_Compt. rend. Soc. de Biol._, Paris, 1899, p. 568.
Footnote 131:
“Les Oxydases dans la série animale,” Paris, 1897.
Footnote 132:
Stadelmann, _Ztschr. f. Biol._, München, 1887, Bd. XXIV, S. 226; 1888,
Bd. XXV, S. 208; Patella, _Ann. univ. di med. e chir._, Milano, 1887.
(Cited by Huppert in Neubauer u. Vogel’s _Analyse des Harns_, x^{te}
Aufl., Wiesbaden, 1898, S. 599.)
Footnote 133:
_Centralbl. f. Bakteriol. u. Parasitenk._, I^{te} Abt., Jena, 1901,
Bd. XXIX, S. 531.
Footnote 134:
Sawtchenko (_Arch. russes de Path._, etc., St. Pétersb., 1901, t. XI,
p. 455) has observed that leucocytes, after they have absorbed the
specific fixative, acquire the property of ingesting red blood
corpuscles with extraordinary rapidity. Tarassewitch was able to
confirm this fact.
Footnote 135:
_Centralbl. f. Bakteriol. u. Parasitenk._, I^{te} Abt., Jena, 1899,
Bd. XXV, S. 546.
Footnote 136:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 738.
Footnote 137:
_Deutsche med. Wchnschr._, Leipzig, 1900, S. 61.
Footnote 138:
_Ann. de l’Inst. Pasteur_, Paris, 1900, t. XIV, p. 369.
Footnote 139:
_Ibid._, p. 577.
Footnote 140:
_Berl. klin. Wchnschr._, 1900, S. 453.
Footnote 141:
We have given a sketch of the actual state of this question of cell
poisons or cytotoxins in the _Revue générale des sciences pures et
appliquées_, 1901, p. 1.
Footnote 142:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 413.
Footnote 143:
Deutsch, _Compt. rend. XIII congrès internat. de Méd. de Paris_, and
_Centralbl. f. Bacteriol. u. Parasitenk._, I^{te} Abt., Jena, 1901, t.
XXIX, S. 661; Uhlenhuth, _Deutsche med. Wchnschr._, Leipzig, 1901, S.
82; Wassermann u. Schütze, _Berl. klin. Wchnschr._, 1901, S. 187;
[Nuttall and Dinkelspiel, _Journ. of Hyg._, Cambridge, 1901, Vol. I,
p. 367; Nuttall, _Brit. Med. Journ._, London, 1902, I, p. 825].
Footnote 144:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 240.
Footnote 145:
_Ztschr. f. Hyg._, Leipzig, 1901, Bd. XXXVI, S. 5.
Footnote 146:
[Myers, _Lancet_, London, 1900, II, p. 98, and _Centralbl. f.
Bakteriol. u. Parasitenk._, I^{te} Abt., Jena, 1900, Bd. XXVIII, S.
237.]
Footnote 147:
_Compt. rend. Soc. de biol._, Paris, 1901, p. 51.
Footnote 148:
_Zeitschr. f. physiol. Chem._, Strassburg, 1901, Bd. XXXII, S. 291.
Footnote 149:
_Virchow’s Archiv_, Berlin, 1893, Bd. CXXXI, S. 32.
Footnote 150:
_Zeitschr. f. Hyg._, Leipzig, 1894, Bd. XVIII, S. 83.
Footnote 151:
_München. med. Wchnschr._, 1898, 15 August.
Footnote 152:
_Centralbl. f. Bakteriol. u. Parasitenk._, I^{te} Abt., Jena, 1899,
Bd. XXVI, S. 349 and 1900, Bd. XXVII, S. 721.
Footnote 153:
“Étude sur la présure et l’antiprésure.” Sceaux, 1900. (_Thèse d. l.
Faculté d. Sc. de Paris_, no. 4.)
Footnote 154:
_Arch. internat. de Pharmacodyn._, Bruxelles et Paris, 1898, t. III
and IV.
Footnote 155:
_Berl. klin. Wchnschr._, 1898, S. 152.
Footnote 156:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 406.
Footnote 157:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 785.
Footnote 158:
_Ibid._ 1899, t. XIII, p. 285.
Footnote 159:
_Ibid._, Paris, 1900, t. XIV, p. 270.
Footnote 160:
_Berl. klin. Wchnschr._, 1900, S. 684. Ehrlich, “Croonian Lecture,”
_Proc. Roy. Soc. London_, 1900, Vol. LXVI, p. 424.
Footnote 161:
_Berl. klin. Wchnschr._, 1901, S. 570.
Footnote 162:
_Deutsche med. Wchnschr._, Leipzig, 1900, S. 431.
Footnote 163:
_Centralbl. f. Bakteriol. u. Parasitenk._, I^{te} Abt., Jena, 1901,
Bd. XXIX, S. 175.
Footnote 164:
_Berl. klin. Wchnschr._, 1901, S. 251.
Footnote 165:
_Ztschr. f. Hyg._, Leipzig, 1901, Bd. XXXVI S. 190.
Footnote 166:
_Ann. de l’Inst. Pasteur_, Paris, 1900, t. XIV, p. 5.
Footnote 167:
_Ibid._, p. 583.
Footnote 168:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 833.
Footnote 169:
_Centralbl. f. Bakteriol. u. Parasitenk._, I^{te} Abt., Jena, 1899,
Bd. XXVI, S. 352.
Footnote 170:
_Virchow’s Archiv_, Berlin, 1893, Bd. CXXXI, S. 5.
Footnote 171:
_Klin. Jahrb._, Jena, 1897, Bd. VI, S. 299; “Croonian Lecture,” _Proc.
Roy. Soc. London_, 1900, Vol. LXVI, p. 424. Ehrlich, Lazarus u.
Pincus, “Leukaemie, etc.” in Nothnagel’s _Specielle Pathologie u.
Therapie_, Wien, 1901, Bd. VIII, Schlussbetrachtungen, S. 163.
Footnote 172:
Bordet, _Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 499; von
Dungern, _München. med. Wchnschr._, 1900, S. 678.
Footnote 173:
_Berl. klin. Wchnschr._, 1901, S. 255.
Footnote 174:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 833.
Footnote 175:
_Brit. Med. Journ._, London, 1897, II, p. 1786; 1898, I, p. 550. _Ann.
de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 136.
Footnote 176:
_Ztschr. f. Hyg._, Leipzig, 1893, Bd. XIII, S. 357.
Footnote 177:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 240.
Footnote 178:
_Virchow’s Archiv_, Berlin, 1884, Bd. XCVI, S. 177.
Footnote 179:
[English translation, pp. 83–86.]
Footnote 180:
_Bull. Acad. d. sc. de St Pétersb._, 1894, t. XIII, p. 437.
Footnote 181:
_Compt. rend. Acad. d. sc._, Paris, 1886, t. CIII, p. 952.
Footnote 182:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1889, Bd. V, S. 5.
Footnote 183:
_Ann. de l’Inst. Pasteur_, Paris, 1894, t. VIII, p. 696.
Footnote 184:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 301.
Footnote 185:
Cohn’s “Beiträge zur Biologie der Pflanzen,” Breslau, 1876, Bd. II, S.
300.
Footnote 186:
_Compt. rend. Acad. d. sc._, Paris, 1882, t. XCIV, p. 1605.
Footnote 187:
_Virchow’s Archiv_, Berlin, 1884, Bd. XCVII, S. 502.
Footnote 188:
_Centralbl. f. klin. Med._, Bonn, 1888, S. 516.
Footnote 189:
“Untersuch. über d. Immunität d. Frosches gegen Milzbrand,” Ziegler’s
_Beitr. z. path. Anat._, Jena, 1888, Bd. III, S. 357.
Footnote 190:
“Beiträge z. Kritik der Metschnikoff’schen Phagocytenlehre,” Inaug.
Diss., Königsberg, 1889.
Footnote 191:
_Ztschr. f. Hyg._, Leipzig, 1888, Bd. IV, S. 353.
Footnote 192:
_Virchow’s Archiv_, Berlin, 1888, Bd. CXIV, S. 466.
Footnote 193:
_Ann. de l’Inst. Pasteur_, Paris, 1891, t. V, p. 362.
Footnote 194:
_Arb. a. d. k. Gsndhtsamte_, Berlin, 1894, Bd. IX, S. 497.
Footnote 195:
_Ziegler’s Beitr. z. path. Anat._, Jena, 1890, Bd. VIII, S. 203.
Footnote 196:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1889, Bd. VI, SS. 481
and 529; _Fortschr. d. Med._, Berlin, 1896, Bd. VIII, S. 665; _Ztschr.
f. klin. Med._, Berlin, 1891; “Ueber Immunität u. Schutzimpfung,”
Schneidemühl’s _Thiermed. Vorträge_, 1892, Bd. II.
Footnote 197:
_Bull. Acad. de méd._, Paris, 1878, p. 440.
Footnote 198:
_Virchow’s Archiv_, Berlin, 1887, Bd. CIX, S. 365.
Footnote 199:
_Ann. de l’Inst. Pasteur_, Paris, 1890, t. IV, p. 570.
Footnote 200:
_Ann. de l’Inst. Pasteur_, Paris, 1891, t. V, p. 362.
Footnote 201:
_Ztschr. f. Hyg._, Leipzig, 1898, Bd. XXVIII, S. 189.
Footnote 202:
“Untersuchungen ü. die Immunität d. Tauben,” Königsberg, 1889;
_Ziegler’s Beitr. z. path. Anat._, Jena, 1890, Bd. VII, S. 49.
Footnote 203:
_Ann. de l’Inst. Pasteur_, Paris, 1890, t. IV, p. 38; p. 65.
Footnote 204:
_Ztschr. f. Hyg._, Leipzig, 1892, Bd. XII, S. 348.
Footnote 205:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 426.
Footnote 206:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 561.
Footnote 207:
_Arch. de méd. expér. et d’anat. path._, Paris, 1889, t. I, p. 325.
Footnote 208:
_Virchow’s Archiv_, Berlin, 1887, Bd. CIX, S. 365.
Footnote 209:
_Ann. de l’Inst. Pasteur_, Paris, 1890, t. IV, p. 520.
Footnote 210:
_Ann. de l’Inst. Pasteur_, Paris, 1900, t. XIV, p. 13.
Footnote 211:
_Ann. de l’Inst. Pasteur_, Paris, 1889, t. III, p. 577.
Footnote 212:
_Ztschr. f. Hyg._, Leipzig, 1888, Bd. IV, S. 353.
Footnote 213:
_Ann. de l’Inst. Pasteur_, Paris, 1887, t. I, p. 43.
Footnote 214:
“Untersuchungen ü. die Ursachen der angeborenen u. erworbenen
Immunität,” Berlin, 1891, S. 111.
Footnote 215:
_La Cellule_, Lierre et Louvain, 1893, t. IX, p. 337.
Footnote 216:
“Zur Lehre von den Geschswülsten und Infectionskrankheiten,”
Wiesbaden, 1899.
Footnote 217:
_Centralbl. f. Bacteriol, u. Parasitenk._, I^{te} Abt., Jena, 1900,
Bd. XXVII, SS. 10 und 517.
Footnote 218:
_La Cellule_, Lierre et Louvain, 1894, t. X, p. 7.
Footnote 219:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 68.
Footnote 220:
_Virchow’s Archiv_, Berlin, 1884, Bd. XCVII, S. 516.
Footnote 221:
_Centralbl. f. klin. Med._, Bonn, 1888, No. 38.
Footnote 222:
“Infectionsschutz und Immunität” in Eulenberg’s “Real-Encyclopädie d.
ges. Heilkunde,” III^{te} Aufl. (_Encyclop. Jahrbücher_), Wien, 1900,
Bd. IX, S. 196.
Footnote 223:
_Centralbl. f. Bacteriol. u. Parasitenk._, Jena, 1888, Bd. IV, SS.
710, 737.
Footnote 224:
_Ann. de l’Inst. Pasteur_, Paris, 1890, t. IV, p. 193.
Footnote 225:
_Centralbl. f. Bacterial. u. Parasitenk._, Jena, 1891, Bd. IX, SS.
336, 372.
Footnote 226:
_Ann. de l’Inst. Pasteur_, Paris, 1891, t. V, p. 479.
Footnote 227:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 865.
Footnote 228:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 232.
Footnote 229:
_Ann. de l’Inst. Pasteur_, Paris, 1891, t. V, p. 145.
Footnote 230:
“Om Mjältbrand hos Höns,” Stockholm, 1897.
Footnote 231:
_Ann. de l’Inst. Pasteur_, Paris, 1891, t. V, p. 564.
Footnote 232:
_Arch. russes de pathol._ etc., St Pétersb., 1900, t. IX, p. 578; and
Sawtchenko et Melkich, _Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV,
p. 502.
Footnote 233:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 448.
Footnote 234:
_Ztschr. f. Hyg._, Leipzig, 1894, Bd. XVIII, S. 1.
Footnote 235:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 177.
Footnote 236:
_Ann. de l’Inst. Pasteur_, Paris, 1896, t. X, p. 104.
Footnote 237:
_Arch. de méd. expér. et d’anat. path._, Paris, 1898, t. X, p. 253.
Footnote 238:
_Ziegler’s Beitr. z. path. Anat._, Jena, 1899, Bd. XXV, S. 206.
Footnote 239:
_Ann. de l’Inst. Pasteur_, Paris, 1891, t. V, p. 1; 1892, t. VI, p.
385; 1893, t. VII, p. 755.
Footnote 240:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 179.
Footnote 241:
_Ann. de l’Inst. Pasteur_, Paris, 1900, t. XIV, p. 202.
Footnote 242:
_Centralbl. f. Bacteriol. u. Parasitenk._, Jena. 1897, Bd. XXI, S.
147.
Footnote 243:
_Arch. de méd. expér. et d’anat. path._, Paris. 1897, t. IX, p. 881.
Footnote 244:
_Arch. f. Hyg._, München u. Leipzig, 1896, Bd. XXVII, S. 234.
Footnote 245:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 770.
Footnote 246:
_Ibid._, 1896, t. X, p. 448.
Footnote 247:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 673.
Footnote 248:
_Arch. f. mikr. Anat._, Bonn, 1893, Bd. XLII, S. 146.
Footnote 249:
_Ztschr. f. Hyg._, Leipzig, 1899. Bd. XXXI, S. 507. See review by
Podwyssotsky in the _Arch. russes de Path._, St Pétersb., 1899, t.
VIII, p. 257.
Footnote 250:
_Arb. a. d. zool. Inst. d. Univ. Wien_, 1883, tom. V, S. 160.
Footnote 251:
_Biol. Centralbl._, Erlangen, 1883–4, Bd. III, S. 562.
Footnote 252:
_Compt. rend. Acad. d. sc._, Paris, 1880, t. XC, p. 952.
Footnote 253:
_Ann. de l’Inst. Pasteur_, Paris, 1887, t. I, p. 325.
Footnote 254:
_Arch. f. mikr. Anat._, Bonn, 1900, Bd. LVI, S. 868.
Footnote 255:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 928.
Footnote 256:
“Leçons sur la pathologie comparée de l’Inflammation,” Paris, 1892, p.
193; authorised English translation, London, 1893, p. 162.
Footnote 257:
_Arch. f. Physiol._, Leipzig, 1894, S. 200.
Footnote 258:
_Jahresb. d. schles. Gesellsch. f. vaterl. Cultur_, Breslau, 1874.
Footnote 259:
_Deutsche med. Wchnschr._, Leipzig, 1886, S. 617; 1887, S. 745.
Footnote 260:
_Ztschr. f. Hyg._, Leipzig, 1888, Bd. IV, S. 208.
Footnote 261:
_Ztschr. f. Hyg._, Leipzig, 1888, Bd. IV, S. 353.
Footnote 262:
“Les microbes pathogènes,” Paris, 1892.
Footnote 263:
_Arch. f. Hyg._, München u. Leipzig, 1890, Bd. 10, S. 84; _Centralbl.
f. Bakteriol. u. Parasitenk._, Jena, 1889, Bd. V, S. 817, and Bd. VI,
SS. 1, 561; 1890, Bd. VIII, S. 65.
Footnote 264:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1889, Bd. VI, S. 481;
_Ztschr. f. klin. Med._, Berlin, 1891, Bde XVIII, XIX.
Footnote 265:
_Ann. de l’Inst. Pasteur_, Paris, 1889, t. III, p. 670.
Footnote 266:
_La Cellule_, Lierre et Louvain, 1894, t. X, p. 7.
Footnote 267:
_München, med. Wchnschr._, 1894, S. 717.
Footnote 268:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 462.
Footnote 269:
_Ztschr. f. Hyg._, Leipzig, 1898, Bd. XXVII, S. 272.
Footnote 270:
_Deutsche med. Wchnschr._, Leipzig, 1899, S. 687.
Footnote 271:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 68.
Footnote 272:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1892, Bd. XII, SS.
777, 809; 1893, Bd. XIV, S. 852.
Footnote 273:
_Proc. Roy. Soc. London_, 1892, Vol. LII, p. 267; _Phil. Trans._,
London, 1894, (B) Vol. 185, pt. I, p. 279.
Footnote 274:
_München. med. Wchnschr._, 1894, S. 717 and 1897, S. 1320.
Footnote 275:
_Arch. f. Hyg._, München u. Leipzig, 1895, Bd. XXV, S. 105; 1897, Bd.
XXVIII, S. 312. _Berl. klin. Wchnschr._, 1896, S. 864.
Footnote 276:
_Arch. f. Hyg._, München u. Leipzig., 1897, Bd. XXXI, p. 1; 1899, Bd.
XXXV, S. 135. _München. med. Wchnschr._, 1898, SS. 353, 1109.
Footnote 277:
_Arch. f. Hyg._, München u. Leipzig, 1900, Bd. XXXVII, S. 290.
Footnote 278:
_Arch. f. Hyg._, München u. Leipzig, 1901, Bd. XL, S. 382.
Footnote 279:
_Arch. f. Hyg._, München u. Leipzig, 1895, Bd. XXV, S. 105; _Berl.
klin. Wchnschr._, 1896, S. 864.
Footnote 280:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 232.
Footnote 281:
_Ibid._, p. 129.
Footnote 282:
_Deutsche med. Wchnschr._, Leipzig, 1901, S. 4.
Footnote 283:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 209.
Footnote 284:
Since Nuttall’s first paper appeared a certain bactericidal action of
the aqueous humour has been observed. This fact should be taken into
consideration in the study of the question of the phagocytic origin of
the bactericidal substance of the body fluids. If this substance
really comes from the phagocytes, it should not be found in the
transparent aqueous humour that contains no, or almost no, leucocytes.
Now this fluid sometimes destroys a certain number of micro-organisms.
This apparent contradiction is explained by the fact that the
bactericidal action may be exercised by all kinds of fluids, such as
physiological salt solution, nutritive broths, etc. The bactericidal
property of the aqueous humour comes into this category. Its action
is, as a rule, much more feeble than the action of serums and
exudations and is not modified by heating to 55°–56° C. In certain
aqueous humours, a little cytase, or true bactericidal substance, may
come into play, for we find aqueous humours which coagulate and which,
when centrifugalised, show a small deposit of leucocytes. These
results have been obtained by Mme. Metchnikoff.
It must not be forgotten also that, even in the bactericidal action of
blood serums, a certain factor is the change of medium which the
micro-organisms experience with the plasmolytic phenomena which
follow. But it is not possible to ascribe to this factor the whole of
the bactericidal property of serums and exudations, as is done by
Baumgarten (_Arb. a. d. pathol.-anat. Inst. zu Tübingen_, 1899, Bd.
III, S. 1, and _Berl. klin. Wchnschr._, 1900, SS. 136, 162, 192), and
his pupils Jetter and Walz supported by A. Fischer (_Ztschr. f. Hyg._,
Leipzig, 1900, Bd. XXXV, S. 1). The idea of reducing the destruction
of bacteria in serums and exudations to the effect of osmotic pressure
has been recently elaborately analysed by v. Lingelsheim (_Ztschr. f.
Hyg._, Leipzig, 1901, Bd. XXXVII, S. 131). With great justness he
comes to the conclusion that “the existence in extravascular blood or
in serum, of bactericidal substances acting as soluble ferments can
now no longer be denied” (p. 167). In studying this question we must
not lose sight of the fact that these bactericidal substances
(alexines, complements, or cytases) give rise to the production in the
animal organism of antagonistic substances as described by us in the
5th Chapter.
Footnote 285:
_Verhandl. d. Congresses f. inn. Med._, Wiesbaden, 1892, S. 273.
Footnote 286:
_Ann. de l’Inst. Pasteur_, Paris, 1900, t. XIV, p. 257; 1901, t. XV,
p. 312.
Footnote 287:
_Berl. klin. Wchnschr._, 1900, SS. 453, 677.
Footnote 288:
_Deutsche med. Wchnschr._, Leipzig, 1900, S. 790.
Footnote 289:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 303.
Footnote 290:
_Arch. f. Hyg._, München u. Leipzig, 1899, Bd. XXXV, S. 199.
Footnote 291:
_Arch. f. Hyg._, München u. Leipzig, 1901, Bd. XL, S. 375.
Footnote 292:
_Ztschr. f. Biol._, München u. Berlin, 1900, Bd. XL, S. 117.
Footnote 293:
_Sitzungsb. d. naturforsch. Gesellsch. zu Marburg_, 1900.
Footnote 294:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 295.
Footnote 295:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1899, I^{te} Abt.,
Bd. XXVI, S. 344.
Footnote 296:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 289.
Footnote 297:
_Ann. de l’Inst. Pasteur_, Paris, 1896, t. X, p. 107.
Footnote 298:
_Arch. internat. de Pharmacodyn._, Gand et Paris, 1899, t. VI, p. 299;
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 642.
Footnote 299:
_Compt. rend. Soc. de biol._, Paris, 1891, p. 464.
Footnote 300:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1894, Bd. XVI, S.
415.
Footnote 301:
Article “Immunität” in the 3rd edition of Eulenburg’s
_Real-Encyclopädie_, Wien, 1896.
Footnote 302:
_Deutsche med. Wchnschr._, Leipzig, 1894, S. 120 (of Vereins-Beilage).
Footnote 303:
[_Journ. Exper. Med._, New York, 1896, Vol. I, p. 543.]
Footnote 304:
[_Journ. Path. and Bacteriol._, Edin. and London, 1896, Vol. III, p.
328; _Lancet_, London, 1899, Vol. II, p. 332; _Centralbl. f.
Bakteriol. u. Parasitenk._, Jena, 1899, I^{te} Abt., Bd. XXVI, S.
548.]
Footnote 305:
_Ztschr. f. Hyg._, Leipzig, 1901, Bd. XXXVI, S. 299.
Footnote 306:
_Arch. de zool. expér._, Paris, 1895, 3^e série, t. III, p. 591.
Footnote 307:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 314.
Footnote 308:
_Ztschr. f. Hyg._, Leipzig, 1890, Bd. VIII, S. 412.
Footnote 309:
_Ztschr. f. Hyg._, Leipzig, 1894, Bd. XVII, S. 355, and _Deutsche med.
Wchnschr._, Leipzig, 1896, SS. 97, 119.
Footnote 310:
_Ztschr. f. Hyg._, Leipzig, 1900, Bd. XXXV, S. 1.
Footnote 311:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 433.
Footnote 312:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 462.
Footnote 313:
“Contribution à l’étude du sérum chez les animaux vaccinés,” _Ann.
Soc. d. sc. nat. et méd. de Bruxelles_, 1895, t. IV.
Footnote 314:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 308.
Footnote 315:
_Ztschr. f. Hyg._, Leipzig, 1894, Bd. XVI, S. 287.
Footnote 316:
_Deutsche med. Wchnschr._, Leipzig, 1896, S. 120.
Footnote 317:
_Centralbl. f. Bakteriol. u. Parasitenk._, I^{te} Abt., Jena, 1896,
Bd. XX, S. 761.
Footnote 318:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 767.
Footnote 319:
_Ann. de l’Inst. Pasteur_, Paris, 1896, t. X, p. 375.
Footnote 320:
_Ann. de. l’Inst. Pasteur_, Paris, 1898, t. XII, p. 199.
Footnote 321:
_Ann. Soc. d. sc. méd. et nat. de Bruxelles_, 1895, t. IV.
Footnote 322:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 894.
Footnote 323:
_München. med. Wchnschr._, 1896, SS. 277 and 310.
Footnote 324:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 273.
Footnote 325:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 288.
Footnote 326:
[_Trans. Seventh Internat. Congr. of Hyg. and Demogr._ London, 1892,
Vol. II. p. 179;] _Ann. de l’Inst. Pasteur_, Paris, 1891, t. V, p.
465.
Footnote 327:
_Arch. russes de Pathol._, etc., St Pétersb., 1900, t. IX, p. 584;
Sawtchenko et Melkich, _Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV,
p. 503.
Footnote 328:
_Ztschr. f. Hyg._, Leipzig, 1896, Bd. XXI, S. 203.
Footnote 329:
_Ibid._ 1887, Bd. II, S. 110.
Footnote 330:
_Ann. de l’Inst. Pasteur_, Paris, 1892, t. VI, p. 755.
Footnote 331:
_Ztschr. f. Hyg._, Leipzig, 1901, Bd. XXXVII, S. 173.
Footnote 332:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 209.
Footnote 333:
_Compt. rend. Soc. de biol._, Paris, 1889, pp. 250, 330, 627; 1890,
pp. 203, 332, 195.
Footnote 334:
“Les microbes pathogènes,” Paris, 1892.
Footnote 335:
_Centralbl. f. Bakteriol. u. Parasitenk._, I^{te} Abt., Jena, 1900,
Bd. XXVIII, S. 577.
Footnote 336:
_Ztschr. f. Hyg._, Leipzig, 1896, Bd. XXII, S. 263.
Footnote 337:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 298.
Footnote 338:
_Fortschr. d. Med._, Berlin, 1888, Bd. VI, S. 729.
Footnote 339:
_Ann. de l’Inst. Pasteur_, Paris, 1889, t. III, p. 289.
Footnote 340:
_Arch. f. Hyg._, München u. Leipzig, 1891, Bd. XII, S. 275.
Footnote 341:
_Ztschr. f. Hyg._, Leipzig, 1896, Bd. XXII, S. 515; _Deutsche med.
Wchnschr._, Leipzig, 1898, S. 49; _Ztschr. f. Hyg._, Leipzig, 1898,
Bd. XXVIII, S. 38.
Footnote 342:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 481.
Footnote 343:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 295.
Footnote 344:
_Virchow’s Archiv_, Berlin, 1884, Bd. XCVII, S. 502.
Footnote 345:
_Virchow’s Archiv_, Berlin, 1888, Bd. CXIV, S. 465.
Footnote 346:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 805.
Footnote 347:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 881.
Footnote 348:
_Ann. de l’Inst. Pasteur_, Paris, 1887, t. I, p. 42.
Footnote 349:
_Ztschr. f. Hyg._, Leipzig, 1888, Bd. IV, S. 353.
Footnote 350:
_Ztschr. f. Hyg._, Leipzig, 1899, Bd. XXXI, S. 89.
Footnote 351:
_Arch. internat. de Pharmacodyn._, Gand et Paris, 1899, Vol. VI, pp.
303, 338.
Footnote 352:
“Infectionsschutz und Immunität” in Eulenburg’s “Real-Encyclopädie d.
ges. Heilkunde,” III^{te} Aufl. (_Encyclop. Jahrbücher_), Wien, 1900,
Bd. IX, S. 202.
Footnote 353:
_Compt. rend. Soc. de biol._, Paris, 1891, p. 538; 1895, pp. 124, 224;
_Rev. de méd._, Paris, 1892.
Footnote 354:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 593.
Footnote 355:
_La Cellule_, Lierre et Louvain, 1895, t. XI, p. 175; _Bull. Acad.
roy. de méd. de Belg._, Bruxelles, 1895, No. 11.
Footnote 356:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 194.
Footnote 357:
_Arch. internat. de Pharmacodyn._, Gand et Paris, 1899, Vol. VI, p.
73; Behring’s “Beitr. z. experim. Therapie,” 1899, Bd. I.
Footnote 358:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 192.
Footnote 359:
_Bulletin No. 1, Bureau of Animal Industry, U.S. Dep. of Agric._,
Washington, 1893.
Footnote 360:
“Reisebericht über Rinderpest etc.,” Berlin, 1898.
Footnote 361:
_Rec. de méd. vét._, Paris, juillet, 1900, and _Ann. de l’Inst.
Pasteur_, Paris, 1901, t. XV, p. 121.
Footnote 362:
_Ztschr. f. Hyg._, Leipzig, 1899, Bd. XXX, S. 251.
Footnote 363:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 673.
Footnote 364:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 462.
Footnote 365:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 289.
Footnote 366:
_Centralbl. f. Bacteriol. u. Parasitenk._, Jena, 1896, I^{te} Abt.,
Bd. XIX, S. 191.
Footnote 367:
_Ztschr. f. Hyg._, Leipzig, 1901, Bd. XXXVI, S. 9.
Footnote 368:
_München. med. Wchnschr._, 1892, SS. 119, 982.
Footnote 369:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 647.
Footnote 370:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 289.
Footnote 371:
_Compt. Rend. Soc. de Biol._, Paris, 1889, p. 667.
Footnote 372:
_Ann. de l’Inst. Pasteur_, Paris, 1891, t. V, p. 473.
Footnote 373:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 462.
Footnote 374:
_München. med. Wchnschr._, 1896, S. 285 [cf. also Durham, _Journ.
Path. and Bacteriol._, Edin. and London, 1897, Vol. IV, p. 13, and
1901, Vol. VII, p. 240; _Brit. Med. Journ._, London, 1898, Vol. II, p.
588].
Footnote 375:
_Wien. klin. Wchnschr._, 1896, SS. 183, 204.
Footnote 376:
_Bull. Soc. méd. d. hôp._, Paris, 1896, 26 juin [_Semaine méd._,
Paris, 1896, p. 259].
Footnote 377:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 225.
Footnote 378:
_Rev. gén. d. sc. pures et appliq._, Paris, 1896, t. VII, p. 770.
Footnote 379:
_Wien. klin. Wchnschr._, 1899, S. 1.
Footnote 380:
_Ann. de. l’Inst. Pasteur_, Paris, 1898, t. XII, p. 688.
Footnote 381:
_Ann. de l’Inst. Pasteur_, Paris, 1892, t. VI, p. 289.
Footnote 382:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1894, Bd. XVI, S.
235.
Footnote 383:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena. I^{te} Abt., 1898,
Bd. XXIII, SS. 9, 71, 131.
Footnote 384:
_Ztschr. f. Hyg._, Leipzig, 1898, Bd. XXVIII, S. 406.
Footnote 385:
_Fifteenth Ann. Rep. of the Bureau of Animal Industry for 1898_,
Washington, 1899, p. 348, Pl. XI.
Footnote 386:
Widal et Sicard, _Bull. et Mém. Soc. méd. d. hôp._, Paris, 1896, p.
684 [_Semaine méd._, Paris, 1896, p. 514].
Footnote 387:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 411.
Footnote 388:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 277.
Footnote 389:
_Arch. f. Hyg._, München u. Leipzig, 1898, Bd. XXXIII, S. 124.
Footnote 390:
_Ann. de l’Inst. Pasteur_, Paris, 1893, t. VII, p. 260.
Footnote 391:
_Ann. de l’Inst. Pasteur_, Paris, 1893, t. VII, p. 225.
Footnote 392:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 481.
Footnote 393:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 209.
Footnote 394:
_Arch. de méd. expér. et d’anat. path._, Paris, 1896, 1^{re} série, t.
VIII, p. 759.—Bensaude, “Le phénomène de l’agglutination des
microbes,” Paris, 1897, p. 252.
Footnote 395:
_Compt. rend. Soc. de biol._, Paris, 1897, p. 104.
Footnote 396:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 376.
Footnote 397:
_Ztschr. f. Hyg._, Leipzig, 1898, Bd. XXVII, S. 272.
Footnote 398:
_Ztschr. f. Hyg._, Leipzig, 1899, Bd. XXX, S. 19.
Footnote 399:
_Arch. internat. de Pharmacodyn._, Gand et Paris, 1899, vol. VI, p.
299.
Footnote 400:
_Compt. rend. Acad. d. Sc._, Paris, 1888, t. CVII, p. 750.
Footnote 401:
Behring u. Kitasato, _Deutsche med. Wchnschr._, Leipzig, 1890, S.
1113.
Footnote 402:
_Ann. de l’Inst. Pasteur_, Paris, 1892, t. VI, p. 299.
Footnote 403:
_Ztschr. f. Hyg._, Leipzig, 1894, Bd. XVI, S. 268; 1894, Bd. XVIII, S.
1.
Footnote 404:
_Ztschr. f. Hyg._, Leipzig, 1896, Bd. XXI, S. 203; _Deutsche med.
Wchnschr._, Leipzig, 1896, SS. 185, 735.
Footnote 405:
“La sérothérapie de la fièvre typhoïde,” Bruxelles, 1896.
Footnote 406:
_Bull. Soc. méd. d. hôp._, Paris, 1893, 27 janvier.
Footnote 407:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1896, I^{te} Abt.,
Bd. XIX, S. 51; _Festschr. z. 100 jähr. Stiftungsfeier d. med. chir.
Friedr. Wilh. Instituts_, 1895.
Footnote 408:
_Hygien. Rundschau_, Berlin, 1894, IV Jahrg., SS. 97, 145.
Footnote 409:
_Berl. klin. Wchnschr._, 1899, S. 6.
Footnote 410:
“Typhusepidemien und Trinkwasser,” Jena, 1898, S. 26.
Footnote 411:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 298.
Footnote 412:
_Berl. klin. Wchnschr._, 1892, S. 970.
Footnote 413:
_Ann. de l’Inst. Pasteur_, Paris, 1893, t. VII p. 411.
Footnote 414:
_Ztschr. f. Hyg._, Leipzig, 1894, Bd. XVI, S. 268.
Footnote 415:
See Lazarus, _Berl. klin. Wchnschr._, 1892, S. 1072.
Footnote 416:
_Ztschr. f. Hyg._, Leipzig, 1896, Bd. XXIII, S. 149.
Footnote 417:
_Ann. de l’Inst. Pasteur_, Paris, 1902, t. XVI, p. 94.
Footnote 418:
_Deutsche med. Wchnschr._, Leipzig, 1901, S. 4.
Footnote 419:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 209.
Footnote 420:
_Ztschr. f. Hyg._, Leipzig, 1896, Bd. XXI, S. 203.
Footnote 421:
_Ann. de l’Inst. Pasteur_, Paris, 1892, t. VI, p. 300.
Footnote 422:
_Ztschr. f. Hyg._, Leipzig, 1895, Bd. XIX, S. 82.
Footnote 423:
[_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1895, Bd. XVIII, S.
744]; _Rir. d’Ig. e San. Pubbl._, Torino, 1896, t. VII, nos. 18–19;
_ibid._ 1901, t. XII, p. 212.
Footnote 424:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 785.
Footnote 425:
_Ztschr. f. Hyg._, Leipzig, 1897, Bd. XXV, S. 301.
Footnote 426:
_Ztschr. f. Hyg._, Leipzig, 1899, Bd. XXXI, S. 89.
Footnote 427:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 769.
Footnote 428:
_Ztschr. f. Hyg._, Leipzig, 1897, Bd. XXIV, S. 327.
Footnote 429:
_Deutsche med. Wchnschr._, Leipzig, 1900, S. 781.
Footnote 430:
“La Malaria secondo le nuove recherche,” Roma, 1899, p. 86 [translated
into English by Eyre from the 2nd Italian edition under the title
“Malaria according to the new researches,” London, 1900]. “Die
Malaria” [German translation of same] in Behring’s “Beiträge z. exper.
Therapie,” 1900, Bd. I, Hft. 3.
Footnote 431:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 343.
Footnote 432:
“La ‘Tristeza’ ou Malaria bovine dans la République Argentine,” Buenos
Ayres, 1900, p. 142.
Footnote 433:
_Bull. Soc. centr. de méd. vétérin._, Paris, 1900, séances des 12 et
26 juillet.
Footnote 434:
_Ann. de l’Inst. Pasteur_, Paris, 1892, t. VI, p. 321.
Footnote 435:
_Ann. de l’Inst. Pasteur_, Paris, 1894, t. VIII, p. 1; _Arch. de méd.
expér._, Paris, 1898, t. X, p. 725; _Arch. russes de Path._ &c., St
Pétersb., 1898.
Footnote 436:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 615.
Footnote 437:
_Ann. de l’Inst. Pasteur_, Paris, 1900, t. XIV, p. 802.
Footnote 438:
“Untersuchungen über die Aetiologie der Wundinfectionskrankheiten,”
Leipzig, 1878. [Translated into English in the New Sydenham Society’s
Series, London, 1880, Vol. LXXXVIII, under the title “On Traumatic
Infective Diseases.”]
Footnote 439:
_Arb. a. d. K. Gsndhtsamt._, Berlin, 1885, Bd. I, S. 46.
Footnote 440:
_Arb. a. d. K. Gsndhtsamt._, Berlin, 1885, Bd. I, S. 57.
Footnote 441:
_Compt. rend. Acad. d. sc._, Paris, 1883, t. XCVII, p. 1163.
Footnote 442:
_La Cellule_, Lierre et Louvain, 1895, t. XI, p. 177.
Footnote 443:
_Ann. de l’Inst. Pasteur_, Paris, 1887, t. I, p. 42.
Footnote 444:
_Compt. rend. Soc. de biol._, Paris, 1889–1891.
Footnote 445:
“Essai d’une théorie de l’infection.” Berlin, 1890.
Footnote 446:
Charrin, _Compt. rend. Soc. de biol._, Paris, 1890, pp. 203, 332;
Roger, _ibid._, 1890, p. 573, and _Rev. gén. d. sc. pures et appliq._,
Paris, 1891, p. 410.
Footnote 447:
_Ann. de l’Inst. Pasteur_, Paris, 1893, t. VII, p. 273.
Footnote 448:
_Ann. de l’Inst. Pasteur_, Paris, 1893, t. VII, p. 231.
Footnote 449:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 177.
Footnote 450:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 481.
Footnote 451:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 769.
Footnote 452:
_Compt. rend. Acad. d. sc._, Paris, 1880, t. XC, p. 1033.
Footnote 453:
_Compt. rend. Soc. de biol._, Paris, 1899, p. 432.
Footnote 454:
_Compt. rend. Acad. d. sc._, Paris, 1880, t. XC, p. 1526.
Footnote 455:
_Compt. rend. Soc. de biol._, Paris, 1890, p. 294.
Footnote 456:
_Ann. de l’Inst. Pasteur_, Paris, 1890, t. IV, p. 563.
Footnote 457:
_Ann. de l’Inst. Pasteur_, Paris, 1892, t. VI, p. 295.
Footnote 458:
_Berl. klin. Wchnschr._, 1891, p. 157.
Footnote 459:
_Ztschr. f. Hyg._, Leipzig, 1898, Bd. XXVII, S. 272.
Footnote 460:
_Berl. klin. Wchnschr._, 1898, S. 209.
Footnote 461:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 689.
Footnote 462:
Ehrlich, Lazarus u. Pinkus, “Leukaemie, etc.” in Nothnagel’s
“Specielle Pathologie u. Therapie,” Wien, 1901, Bd. VIII, I Theil, III
Heft. Schlussbetrachtungen. S. 163.
Footnote 463:
_München. med. Wchnschr._, 1901, p. 697.
Footnote 464:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 315.
Footnote 465:
_Ann. de l’Inst. Pasteur_, Paris, 1896, t. X, p. 193.
Footnote 466:
_Ztschr. f. Hyg._, Leipzig, 1896, Bd. XXI, S. 203.
Footnote 467:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 773.
Footnote 468:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 453.
Footnote 469:
_Ann. de l’Inst. Pasteur_, Paris, 1896, t. X, p. 371.
Footnote 470:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 290.
Footnote 471:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 312.
Footnote 472:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 492.
Footnote 473:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 289.
Footnote 474:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 800.
Footnote 475:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1899, I Abt., Bd.
XXVI, S. 428.
Footnote 476:
_Ztschr. f. Hyg._, Leipzig, 1899, Bd. XXXI, S. 110.
Footnote 477:
_Ann. de l’Inst. Pasteur_, Paris, 1892, t. VI, p. 308.
Footnote 478:
Marmorek, _Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 593.
Footnote 479:
_La Cellule_, Lierre et Louvain, 1895, t. XI, p. 175, and _Bull. Acad.
roy. de méd. de Belg._, Bruxelles, 1895.
Footnote 480:
_Bull. Acad. roy. de méd. de Belg._, Bruxelles, 1896.
Footnote 481:
_Habilitations-Schrift_, Marburg, 1899, and in von Behring’s “Beiträge
zur experimentellen Therapie,” 1899, Bd. I, S. 40.
Footnote 482:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 177.
Footnote 483:
_Ztschr. f. Hyg._, Leipzig, 1899, Bd. XXX, S. 251.
Footnote 484:
Laveran, “Titres et travaux scientifiques,” Paris, 1901, p. 39. _Ann.
de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 673.
Footnote 485:
_Deutsche med. Wchnschr._, Leipzig, 1900, S. 285.
Footnote 486:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 225.
Footnote 487:
_Deutsche med. Wchnschr._, 1901, S. 4.
Footnote 488:
_Ztschr. f. Hyg._, Leipzig, 1894, Bd. XVI, S. 287.
Footnote 489:
“La Sérothérapie de la fièvre typhoïde,” Bruxelles, 1896, p. 69.
Footnote 490:
_Ztschr. f. Hyg._, Leipzig, 1901, Bd. XXXVII, S. 199.
Footnote 491:
_Compt. rend. Acad. d. sc._, Paris, 1877, t. LXXXV, p. 107.
Footnote 492:
_Arch. f. Hyg._, München u. Leipzig, 1887, Bd. VI, S. 442.
Footnote 493:
_Virchow’s Archiv_, Berlin, 1887, Bd. CVIII, S. 494.
Footnote 494:
_London Medical Record_, 1887.
Footnote 495:
_Compt. rend. Acad. d. sc._, Paris, 1889, t. CVIII, p. 713.
Footnote 496:
_Ann. d. Microgr._, Paris, 1889, p. 465.
Footnote 497:
_Compt. rend. Acad. d. sc._, Paris, 1889, t. CIX, p. 985.
Footnote 498:
_Ann. de l’Inst. Pasteur_, Paris, 1890, t. IV, p. 689.
Footnote 499:
_Ztschr. f. Hyg._, Leipzig, 1894, Bd. XVIII, S. 177.
Footnote 500:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1893, Bd. XIII, S.
426.
Footnote 501:
_Proc. Roy. Soc. London_, 1887, Vol. XLII, p. 17.
Footnote 502:
_Compt. rend. Soc. de biol._, Paris, 1893, pp. 294, 618; 1898, p. 344.
“Le tétanos,” Paris, 1899, p. 25.
Footnote 503:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 597.
Footnote 504:
_Arch. internat. de Pharmacodyn._, Gand et Paris, 1900, Vol. VII, p.
265.
Footnote 505:
“Traité sur le venin de la vipère,” Florence, 1781.
Footnote 506:
_Arch. de physiol. norm. et path._, Paris, Année XXVI, 1894, p. 423.
Footnote 507:
“Le venin des serpents,” Paris, 1896, p. 40.
Footnote 508:
“Allgemeine Therapie der Infectionskrankheiten,” Berlin u. Wien, 1899,
S. 992.
Footnote 509:
_Compt. rend. Soc. de biol._, Paris, 1891, p. 462; _Ann. de l’Inst.
Pasteur_, Paris, 1892, t. VI, p. 229.
Footnote 510:
Article: _Infectionsschutz und Immunität_ in Eulenburg’s
“Real-encyclopädie d. ges. Heilkunde” (Encyclop. Jahrbücher), Wien,
1900, Bd. IX, S. 203.
Footnote 511:
_Deutsche med. Wchnschr._, Leipzig, 1898, S. 373.
Footnote 512:
_Vrach_, St Petersburg, 1897, p. 964.
Footnote 513:
_Compt. rend. Soc. de biol._, Paris, 1899, p. 77; _Bull. Muséum d.
hist. nat._, Paris, 1895, t. I, p. 294.
Footnote 514:
_Deutsche med. Wchnschr._, Leipzig, 1898, S. 629.
Footnote 515:
_Compt. rend. Soc. de biol._, Paris, 1895, p. 639.
Footnote 516:
_Bull. Muséum d’hist. nat._, Paris, 1896, t. II, p. 100.
Footnote 517:
“Le venin des serpents,” Paris, 1896, p. 43.
Footnote 518:
The temporary immunity of the marmot (amongst mammals) against tetanus
toxin must be considered separately. According to Billinger and Dönitz
the marmot is insusceptible to this poison during its winter sleep.
But once it is awakened it readily contracts tetanus. H. Meyer, Halsey
and Ransom have observed the same fact in hibernating bats that have
been waked up. In these cases the immunity is dependent on the low
temperature which approximates these examples to that of the natural
immunity of the frog against the same toxin.
Footnote 519:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1894, Bd. XVI, S.
415.
Footnote 520:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 225.
Footnote 521:
“Allgemeine Therapie der Infectionskrankheiten,” Berlin u. Wien, 1899,
S. 982.
Footnote 522:
“Essai d’une théorie de l’infection,” Berlin, 1890; “Les microbes
pathogènes,” Paris, 1892, p. 33.
Footnote 523:
_Ann. de l’Inst. Pasteur_, Paris, 1888, t. II, p. 629; 1899, t. III,
p. 273.
Footnote 524:
_Berl. klin. Wchnschr._, 1890, S. 717.
Footnote 525:
_Berl. klin. Wchnschr._, 1890, No. 11.
Footnote 526:
_Berl. klin. Wchnschr._, 1890, No. 49.
Footnote 527:
_Deutsche med. Wchnschr._, Leipzig, 1890, SS. 1145, 1245.
Footnote 528:
_Deutsche med. Wchnschr._, Leipzig, 1891, SS. 976, 1218.
Footnote 529:
_Compt. rend. Soc. de biol._, Paris, 1894, p. 111.
Footnote 530:
_Compt. rend. Soc. de biol._, Paris, 1894, pp. 120, 204. [Cf. also
Fraser, _Brit. Med. Journ._, London, 1895, Vol. I, p. 1309 and II, p.
416; _Nature_, London, 1896, Vol. LIII, p. 571.]
Footnote 531:
_Ann. de l’Inst. Pasteur_, Paris, 1896, t. X, p. 683.
Footnote 532:
“Le venin des serpents,” Paris, 1896, p. 54.
Footnote 533:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 316.
Footnote 534:
_Berl. klin. Wchnschr._, 1890, No. 49.
Footnote 535:
_Ann. de l’Inst. Pasteur_, Paris, 1892, t. VI, p. 225.
Footnote 536:
_Compt. rend. Acad. d. sc._, Paris, 1894, t. CVIII, p. 288; _Compt.
rend. Soc. de biol._, Paris, 1894, p. 111.
Footnote 537:
_Deutsche med. Wchnschr._, Leipzig, 1890, SS. 1145, 1245.
Footnote 538:
_Bull. Acad. de méd._, Paris, 1895, t. XXXIV, p. 216.
Footnote 539:
_Ztschr. f. Hyg._, Leipzig, 1896, Bd. XXI, S. 485.
Footnote 540:
“On the preparation of a potent antidiphtheria serum,” St Pétersbourg,
1897 (in Russian) [cf. _Berl. klin. Wchnschr._, 1897, S. 720].
Footnote 541:
“Allgemeine Therapie der Infectionskrankheiten,” Berlin u. Wien, 1899,
S. 1093.
Footnote 542:
_Deutsche med. Wchnschr._, Leipzig, 1898, S. 597.
Footnote 543:
_Ztschr. f. Hyg._, Leipzig, 1897, Bd. XXIV, S. 425.
Footnote 544:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, pp. 568, 801.
Footnote 545:
_Ztschr. f. Hyg._, Leipzig, 1901, Bd. XXXVI, S. 325.
Footnote 546:
_Compt. rend. du Congrès internat. de méd. de Paris_ (Section de
bactériologie et parasitologie), 1901, p. 40.
Footnote 547:
_Compt. rend. du Congrès internat. de méd. de Paris_ (Section de
bactériologie et parasitologie), 1901, p. 45; _Ztschr. f. Hyg._,
Leipzig, 1901, Bd. XXXVII, S. 250.
Footnote 548:
_Deutsche med. Wchnschr._, Leipzig, 1895, S. 457.
Footnote 549:
_Ann. de l’Inst. Pasteur_, Paris, 1896, t. X, p. 257.
Footnote 550:
_Ztschr. f. Hyg._, Leipzig, 1896, Bd. XXII, S. 312.
Footnote 551:
“Allgemeine Therapie der Infectionskrankheiten,” Berlin u. Wien, 1899,
S. 1052.
Footnote 552:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 318.
Footnote 553:
_Arch. de méd. expér. et d’anat. path._, Paris, 1897, t. IX, p. 770.
Footnote 554:
_Journ. de physiol. et de path. gén._, Paris, 1900, t. II, p. 973.
Footnote 555:
_Deutsche med. Wchnschr._, Leipzig, 1890, S. 1113.
Footnote 556:
“Die praktischen Ziele der Blutserumtherapie,” Leipzig, 1892, S. 52.
Footnote 557:
_Ztschr. f. physiol. Chem._, Strassburg, 1901, Bd. XXXII, S. 318.
Footnote 558:
_Ann. de l’Inst. Pasteur_, Paris, 1893, t. VII, p. 81.
Footnote 559:
_Ztschr. f. Hyg._, Leipzig, 1892, Bd. XII, S. 183.
Footnote 560:
_Ztschr. f. Hyg._, Leipzig, 1894, Bd. XVIII, S. 248.
Footnote 561:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 324.
Footnote 562:
_Compt. rend. Soc. de biol._, Paris, 1891, p. 462; _Ann. de l’Inst.
Pasteur_, Paris, 1892, t. VI, p. 229.
Footnote 563:
_Arch. f. exper. Path. u. Pharmakol._, Leipzig, 1893, Bd. XXXI, S.
371.
Footnote 564:
_Berl. klin. Wchnschr._, 1893, S. 1266.
Footnote 565:
_München, med. Wchnschr._, 1893, S. 480.
Footnote 566:
_Ann. de l’Inst. Pasteur_, Paris, 1894, t. VIII, p. 725.
Footnote 567:
“Le venin des serpents,” Paris, 1896, p. 58.
Footnote 568:
_Ztschr. f. Hyg._, Leipzig, 1896, Bd. XXII, S. 263.
Footnote 569:
_La Cellule_, Lierre et Louvain, 1896, t. XI, p. 359; _Ann. de l’Inst.
Pasteur_, Paris, 1896, t. X, p. 580.
Footnote 570:
_Arch. f. Hyg._, München u. Leipzig, 1897, Bd. XXX, S. 348.
Footnote 571:
Gheorghiewsky, _Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p.
298.
Footnote 572:
_Ztschr. f. Hyg._, Leipzig, 1901, Bd. XXXVI, S. 330.
Footnote 573:
[At a meeting held at St Bartholomew’s Hospital, London, cited by
Stephens and Myers in _Journ. Path. and Bacteriol._, Edin. and London,
1898, vol. V, p. 280.]
Footnote 574:
_Fortschr. d. Med._, Berlin, 1897, Jahrg. XV, S. 41.
Footnote 575:
_Proc. Roy. Soc. London_, 1898, Vol. LXIII, p. 423.
Footnote 576:
_Klin. Jahrbuch._, Berlin, 1897, Bd. VI, S. 13 [of reprint].
Footnote 577:
_Fortschr. d. Med._, Berlin, 1897, Jahrg. XV, S. 657; _München. med.
Wchnschr._, 1898, S. 321.
Footnote 578:
“Experimentelle Untersuchungen über die Grenzen der
Heilungsmöglichkeit des Tetanus,” Marburg, 1895, SS. 14, 21.
Footnote 579:
Lubarsch u. Ostertag’s “Ergebnisse d. allgem. Pathologie u. patholog.
Anatomie,” Wiesbaden, IV Jahrg. (for 1897), S. 121.
Footnote 580:
_Arch. internat. de Pharmacodyn._, Gand et Paris, 1896, Vol. III, p.
77.
Footnote 581:
_Ztschr. f. Hyg._, Leipzig, 1895, Bd. XX, S. 210.
Footnote 582:
_Centralbl. f. inn. Med._, Leipzig, 1895, Jahrg. XVI, SS. 913, 937.
Footnote 583:
_Arch. f. exper. Path. u. Pharmakol._, Leipzig, 1896, Bd. XXXVII, S.
191.
Footnote 584:
_Ann. de l’Inst. Pasteur_, Paris, 1896, t. X, p. 703.
Footnote 585:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 225.
Footnote 586:
“Die Werthbemessung d. Diphtherieheilserums” (_Klin. Jahrb._, Berlin,
1897, p. 20 of reprint).
Footnote 587:
_Ann. de l’Inst. Pasteur_, Paris, 1893, t. VII, p. 98.
Footnote 588:
_Deutsche med. Wchnschr._, Leipzig, 1893, S. 1253; “Allgemeine
Therapie der Infectionskrankheiten” in Eulenburg u. Samuel’s “Lehrb.
d. allg. Therapie,” Berlin u. Wien, 1899, Bd. III, S. 1051.
Footnote 589:
_Berl. klin. Wchnschr._, 1901, S. 157.
Footnote 590:
_Ztschr. f. Hyg._, Leipzig, 1895, Bd. XIX, S. 109.
Footnote 591:
“Allgemeine Therapie der Infectionskrankheiten,” in Eulenburg u.
Samuel’s “Lehrb. d. allg. Therapie,” Berlin u. Wien, 1899, Bd. III.
Footnote 592:
“Experimentelle Untersuchungen über die Grenzen der
Heilungsmöglichkeit des Tetanus,” Marburg, 1895, SS. 18, 19.
Footnote 593:
_Berl. klin. Wchnschr._, 1901, S. 157.
Footnote 594:
_Ztschr. f. Heilk._, Berlin, 1901, Bd. XXII, S. 1.
Footnote 595:
_Ztschr. f. Hyg._, Leipzig, 1901, Bd. XXXVII, S. 194.
Footnote 596:
_Compt. rend. Soc. de biol._, Paris, 1891, p. 464.
Footnote 597:
_Berlin klin. Wchnschr._, 1901, S. 157. The idea of immunising monkeys
against diphtheria was suggested to von Behring by the fact that the
immunity conferred by serums was the more durable the nearer the
relation between the serum used and the blood of the species which
receives the protective injection. Von Behring supposed that the
diphtheria antitoxin, introduced into the human body, would be
maintained there longer, if the antitoxic serum injected came from
monkeys, species much nearer man than is the horse, the usual source
of antidiphtheria serum. The immunity conferred by this horse serum is
generally of very short duration.
Footnote 598:
Ehrlich’s antitoxic unit is adopted by most investigators not only in
Germany, but also in other countries. This unit corresponds to 1 c.c.
of serum capable of neutralising 100 lethal doses of a standard toxin,
i.e. that used to establish the first standard of antitoxin. The serum
must be injected after being mixed _in vitro_ with the toxin. The
neutralisation must be complete and give rise to no symptom of
intoxication.
Footnote 599:
These observations were communicated to me by M. Prevôt, the director
of the serotherapeutic station of the Pasteur Institute at Garches.
Footnote 600:
_Deutsche med. Wchnschr._, Leipzig, 1893, SS. 1253, 1254.
Footnote 601:
Article “Immunität” in Eulenburg’s _Realencyclopädie_, III^{te}
Aufl., Wien, 1896; see also his “Allgemeine Therapie d.
Infectionskrankheiten,” in Eulenburg u. Samuel’s “Lehrb. d. allg.
Therapie,” Berlin u. Wien, 1899, Bd. III, SS. 996, 997.
Footnote 602:
_Op. cit. supra_ p. 370, S. 19.
Footnote 603:
_München. med. Wchnschr._, 1893, S. 380.
Footnote 604:
“Immunität” in Weyl’s “Handbuch der Hygiene,” Jena, 1897, Bd. IX, S.
48.
Footnote 605:
_München. med. Wchnschr._, 1898, p. 321.
Footnote 606:
_Deutsche med. Wchnschr._, Leipzig, 1891, SS. 976, 1218; [_Ztschr. f.
Hyg._, Leipzig, 1892, Bd. XII, S. 183].
Footnote 607:
“Allgemeine Therapie der Infectionskrankheiten” in Eulenburg u.
Samuel’s “Lehrbuch der allgemeine Therapie,” Berlin u. Wien, 1899, Bd.
III, S. 997.
Footnote 608:
_Journ. Path. and Bacteriol._, Edin. and London, 1900, Vol. VI, p.
180.
Footnote 609:
_Ann. de l’Inst. Pasteur_, Paris, 1893, t. VII, p. 82.
Footnote 610:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 763.
Footnote 611:
_Op. cit. supra_, p. 363, IV Jahrg., S. 122.
Footnote 612:
_Compt. rend. Acad. d. sc._, Paris, 1898, t. CXXVI, p. 1229.
Footnote 613:
_Ztschr. f. Hyg._, Leipzig, 1897, Bd. XXIV, S. 514.
Footnote 614:
“Die Werthbemessung des Diphtherieheilserums” (_Klin. Jahrb._, Berlin,
1897, Bd. VI), SS. 13–17 of reprint.
Footnote 615:
_Berl. klin. Wchnschr._, 1898, S. 5.
Footnote 616:
_Deutsche med. Wchnschr._, Leipzig, 1898, S. 68.
Footnote 617:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 91.
Footnote 618:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, pp. 81, 263.
Footnote 619:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 156.
Footnote 620:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 225.
Footnote 621:
_München. med. Wchnschr._, 1898.
Footnote 622:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 801.
Footnote 623:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 81.
Footnote 624:
_Compt. rend. Soc. de biol._, Paris, 1898, p. 602.
Footnote 625:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 238.
Footnote 626:
_l.c._ p. 343.
Footnote 627:
_Ztschr. f. Hyg._, Leipzig, 1898, Bd. XXVII, S. 213.
Footnote 628:
_Compt. rend. Acad. d. sc._, Paris, 1897, p. 1053; and 1898, p. 431;
_Compt. rend. Soc. de biol._, Paris, 1897, p. 1057; and 1898, p. 153.
Footnote 629:
_Ann. de l’Insl. Pasteur_, Paris, 1899, t. XIII, p. 126.
Footnote 630:
_Deutsche med. Wchnschr._, Leipzig, 1901, S. 194.
Footnote 631:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 244.
Footnote 632:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 465.
Footnote 633:
_Arch. internat. de Pharmacodyn._, Gand et Paris, 1900, vol. VII, p.
65.
Footnote 634:
_Semaine méd._, Paris, 1899, p. 411.
Footnote 635:
_Deutsche med. Wchnschr._, Leipzig, 1897, S. 428.
Footnote 636:
_l.c._ p. 229.
Footnote 637:
_Compt. rend. Congrès internat. de Médicine de Paris_, Section de
bactériologie et de parasitologie, Paris, 1891, p. 30.
Footnote 638:
_München. med. Wchnschr._, 1898, S. 321.
Footnote 639:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 808.
Footnote 640:
“Les réactions leucocytaires, vis-à-vis de certaines toxines,” Paris,
1894.
Footnote 641:
_Ann. de l’Inst. Pasteur_, Paris, 1891, t. V, p. 1.
Footnote 642:
“Cinquantenaire de la Société de Biologie,” Volume jubilaire, Paris,
1899, p. 202.
Footnote 643:
The rapid disappearance of poisons from the blood is proved also by
the experiments of von Behring, Dönitz, Decroly and Rousse (_Arch.
internat. de Pharmacodyn._, Gand et Paris, 1899, t. VI, p. 211) on
snake venom and diphtheria and tetanus toxins, as likewise by those of
Heymans and Masoin (_Ibid._, 1901, t. VIII, p. 1) on the malonic and
pyrotartaric nitrites. These poisons, within a few minutes of their
injection into the veins, are absorbed by the cell elements.
Footnote 644:
“Contribution à l’étude physiologique du leucocyte,” Paris, 1901, p.
39.
Footnote 645:
_Ann. de l’Inst. Pasteur_, Paris, 1894, t. VIII, p. 719.
Footnote 646:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, pp. 49, 209.
Footnote 647:
See Besredka, _op. cit._, p. 50, for its approximate composition and
distinction from ordinary yellow trisulphide.
Footnote 648:
_Arb. d. pharmak. Instit. z. Dorpat_, 1893, 1894, Bde VII-X.
Footnote 649:
_Ann. de l’Inst. Pasteur_, Paris, 1894, t. VIII, p. 719.
Footnote 650:
Lubarsch u. Ostertag’s _Ergebnisse d. allg. Path._, Jahrg. IV for
1897, Wiesbaden, 1899, S. 107.
Footnote 651:
_Arb. d. pharmak. Instit. z. Dorpat_, 1893, Bd. IX, S. 27.
Footnote 652:
Communication to the XIIIth Intern. Congress of Medicine in Paris,
1900.
Footnote 653:
“Les toxines microbiennes et animales,” Paris, 1896.
Footnote 654:
In Bouchard’s _Traité de Pathologie générale_, Paris, 1900, t. III,
2^{me} partie, article “Inflammation.”
Footnote 655:
Römer’s recent researches (_Arch. f. Ophth._, Leipzig, 1901, Bel. LII,
S. 72) on anti-abrin accord very well with our hypothesis. He was able
to demonstrate that the spleen, the bone-marrow, and the conjunctiva
of the eye, when submitted to the influence of abrin, contain a
notable quantity of anti-abrin. Now these three organs are very rich
in phagocytes.
Footnote 656:
_Virchow’s Archiv_, Berlin, 1884, Bd. XCVI, S. 192.
Footnote 657:
_Arch. de Biol._, Gand et Leipzig, 1893, t. XIII, p. 245.
Footnote 658:
_Ann. de dermat. et de syph._, Paris, 1900, t. X, p. 729.
Footnote 659:
von Graefe’s _Arch. f. Ophth._, Leipzig, 1894, Bd. XL, S. 130.
Footnote 660:
Deutschmann’s _Beitr. z. Augenheilk_, Hamburg u. Leipzig, 1893, Hft.
VIII.
Footnote 661:
_op. cit. supra._
Footnote 662:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 210.
Footnote 663:
_Arch. d’anat. microsc._, Paris, 1898, t. II, pp. 44, 177.
Footnote 664:
[_Med.-Chir. Trans._, London, 1895, Vol. LXXVIII, p. 239]; _The
Lancet_, London, 1896, Vol. I, p. 86; _Brit. Med. Journ._, London,
1896, Vol. I, p. 137.
Footnote 665:
_Compt. rend. Soc. de biol._, Paris, 1893, p. 756.
Footnote 666:
_München. med. Wchnschr._, 1896, S. 730.
Footnote 667:
Batzaroff, “La pneumonie pesteuse expérimentale,” _Ann. de l’Inst.
Pasteur_, Paris, 1899, t. XIII, p. 385.
Footnote 668:
“La Lèpre,” Paris, 1894.
Footnote 669:
_München. med. Wchnschr._, 1897, S. 1063.
Footnote 670:
_Presse méd._, Paris, 1899, 8 avril.
Footnote 671:
“Untersuchungen über Staubinhalation,” Leipzig, 1885.
Footnote 672:
“Eingangspforten der Infectionsorganismen,” Berlin, 1881.
Footnote 673:
_Mitth. aus der Brehmer’schen Heilanstalt_, 1899, S. 297.
Footnote 674:
“Experim. Unters. ii. d. Eindringen path. Microorganismen,”
Königsberg, 1888, [and in Ziegler’s _Beitr. z. path. Anat._, Jena,
1888, Bd. II, S. 411].
Footnote 675:
_Arch. f. Hyg._, München u. Leipzig, 1887, Bd. VIII, S. 145.
Footnote 676:
Baumgarten’s _Arb. auf d. Geb. d. path. Anat._ etc., Braunschweig,
1892, Bd. I, S. 450.
Footnote 677:
“Der Untergang pathog. Schimmelpilze im Körper,” Bonn, 1887.
Footnote 678:
“Die acute Entzündung der Lunge,” Bonn, 1886.
Footnote 679:
“Ueb. d. Untergang des Staphylococcus,” etc., Bonn, 1887.
Footnote 680:
_Ann. de l’Inst. Pasteur_, Paris, 1889, t. III, p. 337.
Footnote 681:
_Compt. rend. Soc. de biol._, Paris, 1897, p. 265.
Footnote 682:
“Die Mikroorganismen der Mundhöhle,” Leipzig, 2^{te} Aufl., 1892.
Footnote 683:
“La saliva umana,” Siena, 1891, and _Centralbl. f. Bakteriol. u.
Parasitenk._, Jena, 1891, Bd. X, S. 817.
Footnote 684:
_op. cit._
Footnote 685:
_Ann. de l’Inst. Pasteur_, Paris, 1896, t. X, p. 545.
Footnote 686:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 510.
Footnote 687:
“Allgemeine Therapie der Infectionskrankheiten,” in Eulenburg u.
Samuel’s “Lehrb. d. allg. Therapie,” Berlin u. Wien, 1899, Bd. III, S.
980.
Footnote 688:
_Arch. de méd. expér. et d’anat. path._, Paris, 1889, t. I, p. 370.
Footnote 689:
_Deutsche med. Wchnschr._, Leipzig, 1885, no. 49.
Footnote 690:
Amongst this acidophile flora one species merits particular attention.
This is a spirillum, discovered by Bizzozero in the mucous membrane of
the stomach of the dog. Salomon (_Centralbl. f. Bakteriol. u.
Parasitenk._, Jena, 1896, Bd. XIX, S. 433) has studied this organism,
not only in the dog, but in the cat and Norway rat. Multiplying on the
mucous membrane, the very mobile spirillum penetrates into the
epithelial cells or is met with inside vacuoles. These latter being in
communication with the external medium, the spirilla can readily
penetrate by the openings. This fact has, then, nothing in common with
phagocytosis, where it is the cell which ingests the micro-organisms
by means of its amoeboid movements.
Footnote 691:
Von Volkmann’s _Samml. klin. Vortr._, Leipzig, 1898, no. 38, S. 290.
Footnote 692:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 510.
Footnote 693:
_Compt. rend. Soc. de biol._, Paris, 1892, p. 153.
Footnote 694:
_Compt. rend. Soc. de biol._, Paris, 1897, p. 830, and Charrin, “Les
défenses naturelles de l’organisme,” Paris, 1898, p. 128.
Footnote 695:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1898, Bd. XXIII, SS.
840, 880.
Footnote 696:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 517.
Footnote 697:
_Deutsche med. Wchnschr._, Leipzig, 1891, SS. 976, 1218.
Footnote 698:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1896, Bd. XIX, S.
442.
Footnote 699:
_Berl. klin. Wchnschr._, 1900, S. 553.
Footnote 700:
_Compt. rend. Acad. d. sc._, Paris, 1880, t. XCI, p. 86.
Footnote 701:
_Fortschr. d. Med._, Berlin, 1888, Bd. VI, S. 809.
Footnote 702:
_Compt. rend. Soc. de biol._, Paris, 1894, p. 38.
Footnote 703:
_Brit. Med. Journ._, London, 1897, Vol. II, p. 595.
Footnote 704:
_Compt. rend. Soc. de biol._, Paris, 1898, p. 1057.
Footnote 705:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 345.
Footnote 706:
_Deutsche med. Wchnschr._, Leipzig, 1897, SS. 225, 241.
Footnote 707:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1898, Abt. I, Bd.
XXIII, S. 782.
Footnote 708:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 506.
Footnote 709:
Perhaps the intestinal micro-organisms also play a part in the
immunity of the animal against Entozoa. Many of the examples of this
immunity are very striking. Certain intestinal worms can live only in
the digestive canal of a single or of a very small number of species
of animals. When we feed rabbits with a quantity of the cysticerci of
the pig these pass living into the small intestine and are there
transformed into true scolices. But, instead of reproducing
themselves, they are expelled and never give rise to the development
of taeniae. The immunity against intestinal parasites has never been
made the object of special study, and it is only as a pure hypothesis
that I offer this suggestion as to the part played by the
micro-organisms of the intestinal flora.
Footnote 710:
_Ann. de l’Inst. Pasteur_, Paris, 1894, t. VIII, p. 549.
Footnote 711:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 802.
Footnote 712:
_Compt. rend. Soc. de biol._, Paris, 1897, p. 545.
Footnote 713:
_Arch. de Sci. biol._, St Pétersbourg, 1892, t. I.
Footnote 714:
“Les défenses naturelles de l’organisme,” Paris, 1898.
Footnote 715:
_Deutsche med. Wchnschr._, Leipzig, 1885, S. 197.
Footnote 716:
_Centralb. f. d. med. Wissensch._, Berlin, Jahrg, 1885, S. 801.
Footnote 717:
_Gior. internaz. d. sc. med._, Napoli, 1886, p. 318.
Footnote 718:
_Quart. Journ. Micr. Sc._, Lond., 1890, Vol. XXX, n.s., p. 481.
Footnote 719:
_Virchow’s Archiv_, 1884, Bd. XCVII, S. 211.
Footnote 720:
“Bakteriologie des weiblichen Genitalkanals,” Leipzig, 1897.
Footnote 721:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 842.
Footnote 722:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 699.
Footnote 723:
_Ann. de l’Inst. Pasteur_, Paris, 1893, t. VII, p. 65.
Footnote 724:
“Die Etiologie des Erysipels,” Berlin, 1883.
Footnote 725:
_Deutsche med. Wchnschr._, Leipzig, 1900, SS. 781, 801.
Footnote 726:
_Virchow’s Archiv_, 1900, Bd. CLXII, S. 222.
Footnote 727:
Address given at the XIIth International Congress of Medicine at
Moscow, 1897.
Footnote 728:
See Hudalo, _Ann. de dermat. et de syph._, Paris, 1891, t. II, pp.
353, 470.
Footnote 729:
_Deutsche med. Wchnschr._, Leipzig, 1891, S. 101.
Footnote 730:
_Ann. de l’Inst. Pasteur_, Paris, 1892, t. VI, p. 773.
Footnote 731:
_Ztschr. f. Hyg._, Leipzig, 1896, Bd. XXI, S. 213.
Footnote 732:
_Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV, p. 289.
Footnote 733:
_Bull. et mém. Soc. méd. d. hôp. de Paris_, 1901, 20 juin, p. 624.
Footnote 734:
“Le phénomène de l’agglutination des microbes,” Paris, 1897, p. 76.
Footnote 735:
_Presse méd._, Paris, 1896, No. 83.
Footnote 736:
_Deutsche med. Wchnschr._, Leipzig, 1892, S. 827.
Footnote 737:
_Berl. klin. Wchnschr._, 1892, S. 1072; 1893, S. 1241.
Footnote 738:
_Berl. klin. Wchnschr._, 1892, S. 1267.
Footnote 739:
_Ztschr. f. Hyg._, Leipzig, 1894, Bd. XVI, S. 308.
Footnote 740:
_Ztschr. f. Hyg._, Leipzig, 1893, Bd. XIV, S. 42.
Footnote 741:
_Hyg. Rundsch._, Berlin, 1895, S. 145.
Footnote 742:
_Ann. de l’Inst. Pasteur_, Paris, 1893, t. VII, p. 417.
Footnote 743:
_München. med. Wchnschr._, 1898, S. 363.
Footnote 744:
_Deutsche med. Wchnschr._, Leipzig, 1898, S. 247.
Footnote 745:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1893, Bd. XIII, S.
153.
Footnote 746:
_Deutsche med. Wchnschr._, Leipzig, 1894, SS. 899, 936.
Footnote 747:
_Deutsche med. Wchnschr._, Leipzig, 1895, S. 400.
Footnote 748:
_Ztschr. f. Hyg._, Leipzig, 1895, Bd. XIX, S. 408.
Footnote 749:
_Prag. med. Wchnschr._, 1896.
Footnote 750:
_Ann. de l’Inst. Pasteur_, Paris, 1888, t. II, p. 69.
Footnote 751:
_Ztschr. f. Hyg._, Leipzig, 1892, Bd. XII, S. 183; Brieger u. Ehrlich,
_Deutsche med. Wchnschr._, 1892, S. 393.
Footnote 752:
_Ztschr. f. Hyg._, Leipzig, 1894, Bd. XVIII, S. 57.
Footnote 753:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1893, Bd. XIII, S.
81; _Deutsche med. Wchnschr._, Leipzig, 1892, S. 394.
Footnote 754:
_Compt. rend. Acad. d. sc._, Paris, 1893, t. CXVII, p. 365; _Rev. gén.
d. sc. pures et appliq._, Paris, 1896, p. 1.
Footnote 755:
_Festschr. z. 100-jahr. Stiftungsf. d. med. chir. Friedr.
Wilhelms-Instituts_, Berlin, 1895.
Footnote 756:
_Ann. de l’Inst. Pasteur_, Paris, 1896, t. X, p. 65.
Footnote 757:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 129.
Footnote 758:
_Arch. d. Sci. biol._, St Pétersbourg, 1901, t. VIII, p. 211.
Footnote 759:
_Arch. d. Sci. biol._, St Pétersbourg, 1901, t. VIII, p. 421.
Footnote 760:
_Compt. rend. Soc. de biol._, Paris, 1897, p. 804.
Footnote 761:
_Compt. rend. Soc. de biol._, Paris, 1897, p. 950.
Footnote 762:
_Semaine méd._, Paris, 1896, p. 303.
Footnote 763:
_Ztschr. f. Hyg._, Leipzig, 1901, Bd. XXXVII, S. 323.
Footnote 764:
“Le charbon bactérien,” Paris, 1883, p. 184.
Footnote 765:
_Compt. rend. Acad. d. sc._, Paris, 1868, t. LXVI, pp. 289, 317, 359.
Footnote 766:
_Virchow’s Archiv_, 1872, Bd. LV, S. 229.
Footnote 767:
_Monatssch. f. prakt. Dermat._, Hamburg, 1887; “Die Protozoen als
Krankheitserreger,” Jena, 1891, S. 184.
Footnote 768:
_Arch. per le sc. med._, Torino, 1892, t. XVI, p. 403.
Footnote 769:
_Ann. de l’Inst. Pasteur_, Paris, 1897, t. XI, p. 289.
Footnote 770:
_Deutsche med. Wchnschr._, Leipzig, 1901, S. 130; _Brit. Med. Journ._,
London, 1901, Vol. I, p. 448.
Footnote 771:
_Deutsche med. Wchnschr._, Leipzig, 1901, S. 261.
Footnote 772:
“Die Geschichte der Pocken und der Impfung,” von Coler’s _Bibliothek_,
Berlin, 1901.
Footnote 773:
_Lancet_, London, 1901, Vol. II, p. 796.
Footnote 774:
_Médecine moderne_, Paris, 1896, p. 441.
Footnote 775:
Nocard et Leclainche, “Les maladies microbiennes des animaux,” 2^e
édition, Paris, 1898, pp. 464, 469.
Footnote 776:
Report by Viala in the _Ann. de l’Inst. Pasteur_, Paris, 1901, t. XV,
p. 445. There will be found in Marie’s work, “La rage” (_Collection
des aides-mém._, Paris, 1900), many details on antirabic vaccination.
Footnote 777:
According to Krajouchkine, in the _Arch. d. Sci. biol._, St
Pétersbourg, 1901, t. VIII, p. 349.
Footnote 778:
According to Marx in _Klin. Jahrb._, Berlin, 1900, Bd. VII, S. 1.
Footnote 779:
_Ann. de l’Inst. Pasteur_, Paris, 1888, t. II, p. 341.
Footnote 780:
_Compt. rend. Acad. d. sc._, Paris, 1881, t. XCIII, p. 284.
Footnote 781:
_Deutsche med. Wchnschr._, Leipzig, 1897, SS. 225, 241.
Footnote 782:
_Ztschr. f. Hyg._, Leipzig, 1898, Bd. XXIX, S. 309.
Footnote 783:
_Ann. de l’Inst. Pasteur_, Paris, 1899, t. XIII, p. 319; 1901, t. XV,
p. 715.
Footnote 784:
_Rec. de méd. vét._, Paris, 1901, pp. 48, 115.
Footnote 785:
_Report on an experim. Investig. of the method of Inoculation against
Rinderpest_, Calcutta, 1900; _Ztschr. f. Hyg._, Leipzig, 1900, Bd.
XXXV, S. 59.
Footnote 786:
Nencki, Sieber and Wyznikiewicz, _Arch. internat. de Pharmacodyn._,
Gand et Paris, 1899, vol. V, p. 475.
Footnote 787:
J. Mendez, _Anal. d. Circ. Med. Argent._, Buenos Aires, 1901, t. XXIV,
Nos. 5, 6.
Footnote 788:
On the methods of vaccination against anthrax see Chamberland, “Le
charbon et la vaccination charbonneuse,” Paris, 1883.
Footnote 789:
“Le charbon bactérien,” Paris, 1883; 2^e édition, 1887.
Footnote 790:
_Ann. de l’Inst. Pasteur_, Paris, 1900, t. XIV, pp. 202, 513.
Footnote 791:
_Compt. rend. Acad. d. sc._, Paris, 1883, t. XCVII, p. 1163.
Footnote 792:
_Arch. f. Hyg._, München u. Leipzig, 1891, Bd. XII, S. 275.
Footnote 793:
_Deutsche thierärztl. Wchnschr._, Karlsruhe, 1893, Bd. I, SS. 41, 85;
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1893, Bd. XIII, S.
357; _Deutsche Ztschr. f. Thiermed._, Leipzig, 1894, Bd. XX, S. 1.
Footnote 794:
_Rev. vét._, Toulouse, 1900, t. LVII, p. 346.
Footnote 795:
_Rev. vét._, Toulouse, 1901, t. LVIII, p. 149.
Footnote 796:
_Deutsche thierärztl. Wchnschr._, Karlsruhe, 1901, No. 6.
Footnote 797:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 240; _Cinquanten.
d. l. Soc. d. biol._, Paris, 1899, p. 440; Dujardin-Beaumetz, “Le
microbe de la péripneumonie,” Thèse de Paris, 1900.
Footnote 798:
In 1884, in the Department of the Basses-Pyrenées, the Willems’ method
of inoculation was carried out on 1354 Bovidae; of this number 10 died
and 45 lost their tails completely. In 1901, in the same department,
2800 Bovidae were inoculated with pure cultures, only 1 died and 9
lost their tails.
Footnote 799:
“L’inoculation préventive contre le choléra morbus asiatique”
(translated from the Spanish), Paris, 1893.
Footnote 800:
“Anti-cholera Inoculations in India,” _Indian Med. Gaz._, Calcutta,
1895, No. 1. [Also Report to the Gov. of India, Calcutta, 1895.]
Footnote 801:
_Ann. de l’Inst. Pasteur_, Paris, 1893, t. VII, p. 579.
Footnote 802:
_Deutsche med. Wchnschr._, Leipzig, 1896, S. 735.
Footnote 803:
Wright and Leishman, _Brit. Med. Journ._, London, 1900, Vol. I, p.
122; [Wright, “A short treatise on anti-typhoid inoculation,” London,
1904].
Footnote 804:
_Brit. Med. Journ._, London, 1901, Vol. I, p. 84.
Footnote 805:
_Lancet_, London, 1901, Vol. I, p. 399.
Footnote 806:
_Lancet_, London, 1901, Vol. I, p. 1272.
Footnote 807:
_Brit. Med. Journ._, London, 1900, Vol. I, p. 1456.
Footnote 808:
_Gaz. clin. de Botkine_, St Pétersb., 1899, p. 1911 (in Russian).
Footnote 809:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 589.
Footnote 810:
_Brit. Med. Journ._, London, 1897, Vol. I, p. 1461; _Indian Med.
Gaz._, Calcutta, 1897, Vol. XXXII, p. 201.
Footnote 811:
“Joint Report on the Epidemic of Plague in Lower Damaun,” Bombay,
1897.
Footnote 812:
_Arb. a. d. K. Gsndhtsamte_, Berlin, 1899, Bd. XVI, S. 331.
Footnote 813:
“Report of the Indian Plague Commission,” London, 1901, Vol. V,
Chapter IV.
Footnote 814:
_Ibid._ Chapter IV, p. 81.
Footnote 815:
See Calmette, “Rapport sur les vaccinations contre la peste,” _Compt.
rend. d. X Congr. internat. d’hyg. de Paris_, 1900.
Footnote 816:
_Deutsche med. Wchnschr._, Leipzig, 1897, SS. 227, 289.
Footnote 817:
_Ann. de l’Inst. Pasteur_, Paris, 1899. t. XIII, p. 902.
Footnote 818:
_Bull. Acad. roy. de méd. de Belg._, Bruxelles, 1900. 27 Octobre.
Footnote 819:
_Bull. Acad. de méd._, Paris, 1895, t. XXXIV, p. 407; _ibid._, 1897,
t. XXXVIII, p. 109; _Compt. rend. XII Congr. Internat. de Méd. à
Moscou_, 1897, t. VII, p. 244.
Footnote 820:
_Deutsche med. Wchnschr._, Leipzig, 1893, S. 390.
Footnote 821:
Ehrlich, Kossel u. Wassermann, _Deutsche med. Wchnschr._, Leipzig,
1894, S. 353; _Klin. Jahrb._, Berlin, 1897, Bd. VI.
Footnote 822:
_Compt. rend. X Congr. internat. d’hyg. et de démogr._, Paris, 1900.
Footnote 823:
_Ztschr. d. Hyg._, Leipzig, 1901, Bd. XXXVIII, S. 372.
Footnote 824:
_Deutsche med. Wchnschr._, Leipzig, 1895, S. 408.
Footnote 825:
_Deutsche med. Wchnschr._, Leipzig, 1895, SS. 426, 443, 464.
Footnote 826:
_Deutsche med. Wchnschr._, Leipzig, 1895, S. 758.
Footnote 827:
_Berl. klin. Wchnschr._, 1896, S. 602.
Footnote 828:
_Berl. klin. Wchnschr._, 1896, S. 516.
Footnote 829:
_Therap. Monatsh._, Berlin, 1896, S. 269.
Footnote 830:
_Deutsche Vrtljschr. f. öff. Gsndhtspflg._, Brnschwg., 1897, Bd. XXIX,
Heft 1.
Footnote 831:
See the report by Löhr in _Jahrb. f. Kinderh._, Leipzig, 1896, Bd.
XLIII, S. 67.
Footnote 832:
See Slawyk, _Deutsche med. Wchnschr._, Leipzig, 1898, S. 35.
Footnote 833:
“Les progrès dans l’application du sérum antidiphthérique,” St
Pétersbourg, 1898, p. 105 (in Russian).
Footnote 834:
_Vrach_, St Pétersbourg, 1900, p. 1178 (in Russian).
Footnote 835:
_Chron. méd. d. gouvern. de Kherson_, 1896, No. 5, p. 160 (in
Russian).
Footnote 836:
_Chron. méd. d. gouvern. de Kherson_, 1896, No. 19, p. 743.
Footnote 837:
_Bull. et mém. Soc. méd. des Hôp. de Paris_, 1901, p. 585.
Footnote 838:
_Bull. Soc. d. Pédiatr. de Paris_, 1901, mai et juin.
Footnote 839:
_Compt. rend. Acad. d. sc._, Paris, 1896, t. CXXII, p. 441.
Footnote 840:
_Compt. rend. Acad. d. sc._, Paris, 1885, t. C, p. 659.
Footnote 841:
This pamphlet has been reprinted in the _Rec. de méd. vét._, Paris,
1886, p. 624.
Footnote 842:
Barthels, “Die Medicin der Naturvölker,” Leipzig, 1893, S. 128; Pagel,
“Einführung in die Geschichte der Medicin,” Berlin, 1898, S. 313.
Footnote 843:
Häser, “Lehrbuch der Geschichte der Medicin,” 3^{te} Aufl., Jena,
1881, Bd. II S. 1075.
Footnote 844:
See Vallery-Radot, “La Vie de Pasteur,” Paris, 1900, p. 427.
Footnote 845:
_Compt. rend. Acad. d. sc._, Paris, 1880, t. XC, pp. 939, 952, 1030;
t. XCI, pp. 571, 673.
Footnote 846:
_Compt. rend. Acad. d. sc._, Paris, 1880, t. XC, p. 247.
Footnote 847:
_Compt. rend. Acad. d. sc._, Paris, 1880, t. XC, p. 1526.
Footnote 848:
_Compt. rend. Acad. d. sc._, Paris, 1880, t. XCI, p. 536.
Footnote 849:
_Compt. rend. Acad. d. sc._, Paris, 1880, t. XCI, p. 680.
Footnote 850:
“Die Naegeli’sche Theorie d. Infectionskrankheiten,” Leipzig, 1877;
“Eine neue Theorie über Erziel. v. Immunität,” München, 1883.
Footnote 851:
_Virchow’s Archiv_, 1881, Bd. LXXXIV, S. 87.
Footnote 852:
_Mitth. a. d. k. Gsndhtsamte_, Berlin, 1881, Bd. I, S. 134.
Footnote 853:
_Compt. rend. Soc. de biol._, Paris, 1870, p. 115; _Gaz. hebd. de
méd._, Paris, 1871, p. 291.
Footnote 854:
Abstract in _Schmidt’s Jahrb._, Leipzig, 1872, Bd. CLX, S. 97.
Footnote 855:
“Beiträge zur pathologische Anatomie der Schusswunden,” Leipzig, 1872.
Footnote 856:
_Archiv f. Gynaek._, Berlin, 1872, Bd. III, S. 293.
Footnote 857:
Cohn’s _Beitr. z. Biol. d. Pflanzen_, Breslau, 1876, Bd. II, S. 300.
Footnote 858:
_Virchow’s Archiv_, 1874, Bd. LX, S. 347.
Footnote 859:
_Virchow’s Archiv_, 1877, Bd. lxx, S. 546; 1881, Bd. LXXXIV, S. 87.
Footnote 860:
_Archiv f. Physiol._, Leipzig, 1881, S. 308, Taf. V.
Footnote 861:
“Beiträge zur Biologie niederster Organismen,” Marburg, 1881.
Footnote 862:
Roser, “Ueber Entzündung und Heilung,” Leipzig, 1886.
Footnote 863:
“Methoden der Bacterienforschung,” 4^{te} Aufl., Wiesbaden, 1889, S.
10.
Footnote 864:
“Die Radiolarien,” Berlin, 1862.
Footnote 865:
“Die Kalkschwämme,” Berlin, 1872.
Footnote 866:
_Arb. a. d. zool. Inst. d. Univ. Wien_, 1883, Bd. V, S. 141.
Footnote 867:
_Biol. Centralbl._, Erlangen, 1883, Bd. III, S. 560.
Footnote 868:
_Virchow’s Archiv_, 1884, Bd. XCVI, S. 177.
Footnote 869:
_Virchow’s Archiv_, 1884, Bd. XCVII, S. 502.
Footnote 870:
_Rep. Brit. Ass. Adv. Sci._, London, 1896, p. 26; _Rev. Scient._,
Paris, 17 Octobre, 1896, p. 493.
Footnote 871:
_Berl. klin. Wchnschr._, 1884.
Footnote 872:
_Ztschr. f. klin. Med._, Berlin, 1888, Bd. XV, S. 1.
Footnote 873:
_Virchow’s Archiv_, 1888, Bd. CXIV, S. 465; _Ann. de l’Inst. Pasteur_,
Paris, 1890, t. IV, p. 35.
Footnote 874:
“Lehrb. d. pathol. Anat.,” Jena, 3^{te} Aufl.; _Beitr. z. path.
Anat._, Jena, 1889, Bd. V, S. 419.
Footnote 875:
_Fortschr. d. Med._, Berlin, 1887, Bd. V, S. 732; _Ibid._, 1888, Bd.
VI, SS. 83, 809.
Footnote 876:
_Virchow’s Archiv_, 1885, Bd. CI, S. 12.
Footnote 877:
_Deutsche med. Wchnschr._, Leipzig, 1890, S. 690.
Footnote 878:
_Virchow’s Archiv_, 1887, Bd. CIX, S. 365.
Footnote 879:
_Deutsche med. Wchnschr._, Leipzig, 1886, S. 617; _Arch. f. Hyg._,
München u. Leipzig, 1886, Bd. IV, S. 129.
Footnote 880:
_Ztschr. f. Hyg._, Leipzig, 1888, Bd. IV, S. 353.
Footnote 881:
_Ztschr. f. Hyg._, Leipzig, 1888, Bd. IV, S. 223.
Footnote 882:
_Ztschr. f. Hyg._, Leipzig, 1888, Bd. IV, S. 318.
Footnote 883:
_Centralbl. f. klin. Med._, Bonn, 1888, No. 38.
Footnote 884:
_Ztschr. f. Hyg._, Leipzig, 1890, Bd. VIII, S. 412.
Footnote 885:
_Fortschr. d. Med._, Berlin, 1887, Bd. V, S. 653.
Footnote 886:
_München. med. Wchnschr._, 1887.
Footnote 887:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1891, Bd. X, S. 727.
Footnote 888:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1890, Bd. VIII, S.
65.
Footnote 889:
“Les microbes pathogènes,” Paris, 1892.
Footnote 890:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1889, Bd. VI, SS.
481, 529.
Footnote 891:
“Ueber bacteriologische Forschung,” Berlin, 1890.
Footnote 892:
“The present position of antiseptic surgery,” Berlin, 1890.
Footnote 893:
_München. med. Wchnschr._, 1891, SS. 551, 574.
Footnote 894:
_Ann. de l’Inst. Pasteur_, Paris, 1891, t. V, p. 517.
Footnote 895:
_Ann. de l’Inst. Pasteur_, Paris, 1887, t. I, p. 561.
Footnote 896:
_Ann. de l’Inst. Pasteur_, Paris, 1891, t. V, pp. 465, 534.
Footnote 897:
_Ann. de l’Inst. Pasteur_, Paris, 1892, t. VI, p. 289.
Footnote 898:
_Compt. rend. Acad. d. sc._, Paris, 1888, t. CVII, pp. 690, 748.
Footnote 899:
_Ztschr. f. Hyg._, Leipzig, 1894, Bd. XVI, S. 268.
Footnote 900:
_Ztschr. f. Hyg._, Leipzig, 1894, Bd. XVIII, S. 1; cf. also Pfeiffer
u. Issaeff, _ibid._, 1894, Bd. XVII, S. 355.
Footnote 901:
_Deutsche med. Wchnschr._, Berlin, 1896, SS. 97, 119.
Footnote 902:
“Schutzimpfung und Impfschutz,” Marburg, 1895.
Footnote 903:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 433.
Footnote 904:
_Ann. de l’Inst. Pasteur_, Paris, 1895, t. IX, p. 462; 1896, t. X, pp.
104, 193.
Footnote 905:
_Centralbl. f. Bakteriol. u. Parasitenk._, Jena, 1896, I^{te} Abt.,
Bd. XX, S. 766.
Footnote 906:
It would clearly be wrong to take one’s stand, in a purely scientific
question, on a national point of view. But it is a still greater
mistake to look at matters, in the investigation of problems which
concern science only, from a personal point of view. This, however, is
what has happened several times in the discussion of phagocytosis.
Certain discontented students have attempted to avenge themselves by
publishing works and criticisms directed against the theory of
phagocytosis. Having no doubt as to the motive for these publications
I consider myself fully justified in not referring to them in this
book, in which I have taken an exclusively scientific point of view,
and in which I have endeavoured to weigh as carefully as possible all
criticisms and objections that have been directed against me.
Footnote 907:
_Ztschr. f. Hyg._, Leipzig, 1898, Bd. XXVII, S. 272.
Footnote 908:
_Ann. de l’Inst. Pasteur_, Paris, 1898, t. XII, p. 688; 1899, t. XIII,
p. 273.
Footnote 909:
_Berl. klin. Wchnschr._, 1899, S. 6.
Footnote 910:
_Brit. Med. Journ._, London, 1892, Vol. I, pp. 373, 492, 591, 604. A
very short summary of this discussion was given in the _Deutsche med.
Wchnschr._, Leipzig, 1892, S. 296.
Footnote 911:
_München. med. Wchnschr._, 1894, S. 717.
Footnote 912:
_München. med. Wchnschr._, 1900, S. 1193.
Footnote 913:
_Encyclop. Jahrbücher_, Wien, 1900, Bd. IX, S. 203.
Footnote 914:
“Grundriss der Hygiene,” Leipzig, 1889, S. 487.
Footnote 915:
“Grundriss der Hygiene,” Leipzig, 4^{te} Anfl., 1897, S. 507.
Footnote 916:
“Einführung in das Studium der Bakteriologie,” Leipzig, 1890, S. 146.
Footnote 917:
“Einführung in das Studium der Bakteriologie,” 5^{te} Aufl., 1898, S.
275.
Footnote 918:
“Traité de médecine” de Charcot, Bouchard, et Brissaud, 1891, t. I,
pp. 219–230.
Footnote 919:
“Traité de médecine...,” 2^e éd., 1898, t. I, pp. 250–254.
Footnote 920:
_Centralbl. f. allg. Path. u. path. Anat._, Jena, 1894, Bd. V, S. 212.
Footnote 921:
Lubarsch u. Ostertag’s “Ergebnisse d. allg. Path. u. path. Anat.,”
Wiesbaden, 1895, I. Abt., S. 384.
Footnote 922:
_Compt. rend. Soc. de biol._, Paris, 1900, p. 385.
Footnote 923:
“Ueber die Selbstordnung der Furchungszellen,” in _Berichte d.
naturwiss. Vereins zu Innsbruck_, 1893, Bd. XXI.
Footnote 924:
Herbst, _Biol. Centralbl._, Erlangen, 1894, 1895, Bde. XIV, XV;
Forssmann, Ziegler’s _Beitr. z. path. Anat._, Jena, 1898, Bd. XXIV, S.
56.
Footnote 925:
“The Relations of Clinical Medicine to Modern Scientific Development,”
a discourse delivered at Liverpool in September, 1896. _Rev. scient._,
Paris, 1896, 4^e sér. t. VI, p. 481; [_Rep. Brit. Ass. Adv. Sci._,
London, 1896, p. 3; _Brit. Med. Journ._, London, 1896, Vol. II, p.
733].
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