Opening
IMMUNITY
IN
INFECTIVE DISEASES
CAMBRIDGE UNIVERSITY PRESS WAREHOUSE, C. F. CLAY, MANAGER.
=London=: FETTER LANE, E.C. =Glasgow=: 50, WELLINGTON STREET.
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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.
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.
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.
[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).
[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.
[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.
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.
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.
[Sidenote: [55]]
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.
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.
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.
[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).
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.