CHAPTER IX

THE MECHANISM OF ACQUIRED IMMUNITY AGAINST MICRO-ORGANISMS

Cytases and fixatives.—Only the latter are augmented in the immunised organism.—Properties of the fixatives.—Difference between them and the agglutinative substances.—The part played by the latter in acquired immunity.—Protective property of the fluids of the immunised organism.—Stimulant action of the body fluids.—The protective power of serum cannot serve as a measure of acquired immunity.—Examples of acquired immunity in which the serums exhibit no protective power.—Phagocytosis in acquired immunity.—Negative chemiotaxis of leucocytes.—Theory of attenuation of micro-organisms by the fluids of immunised animals.—Refutation of this theory.—Phagocytosis acts without requiring any previous neutralisation of the toxins.—The origin of the fixative and protective properties of the body fluids.—The relation between these properties and phagocytosis.—The side-chain theory of Ehrlich and the theory of phagocytes.

[Sidenote: [263]]

Whilst, in natural immunity against micro-organisms, humoral phenomena play no prominent part, in acquired immunity these phenomena assume a much greater importance. The bactericidal power of the fluids of the body is, in natural immunity, reduced to a mere trace, for it has been demonstrated that the power of normal serums to destroy bacteria corresponds to no natural phenomenon of the living organism, but is dependent upon the presence of cytases which have escaped from the phagocytes at the time of the formation of the clot _in vitro_ and separation of the serum. The presence of the fixative, that other important element in immunity, has been demonstrated in the normal fluids only in rare cases and in small quantity. The agglutinative property of these fluids has likewise shown itself to be little developed and without any importance in natural immunity.

[Sidenote: [264]]

[Sidenote: [265]]

In acquired immunity against micro-organisms, on the other hand, we find that the bactericidal and agglutinative powers of the fluids of the body are very greatly increased. With the discovery that the bactericidal property was so highly developed in the serums of animals that had been vaccinated against vibrios arose the belief in the acquisition of a new and purely humoral property. R. Pfeiffer, especially, insisted on the fundamental difference between the power of the serum of immunised animals to transform the cholera vibrios into granules and the corresponding property of normal serums. In the first case Pfeiffer’s phenomenon exhibited marked specificity; in the second, it was much more general. A normal serum transforms into granules, indifferently, vibrios that are very distinct from one another; whilst the serum of an animal vaccinated against a particular species or race of vibrios gives Pfeiffer’s phenomenon with this species or race only. Bordet’s[364] researches have definitely settled this question. This investigator has shown that Pfeiffer’s phenomenon is produced, with all the usual serums, by means of the same substances, the cytases (alexine, or complement of Ehrlich). But in the serum of vaccinated animals there is added to these cytases the fixative (sensibilising substance of Bordet, immunising body or amboceptor of Ehrlich) which exhibits specific properties. Having thus carefully distinguished the two substances that set up the granular change in vibrios, Bordet shows that in vaccinated animals it is the fixative which increases in quantity, whilst the cytase remains pretty much in the same proportions as in the normal animal. He demonstrated, in fact, that when we take a very small dose of the serum of a vaccinated animal which by itself is incapable of transforming the vibrios into granules, about the same quantity of immunised serum or of normal serum must be added to it in order that Pfeiffer’s phenomenon may appear. The quantity of cytase, that soluble ferment which is necessary for the production of the phenomenon, is, therefore, about the same in the serum of a normal animal as in that of a well-vaccinated animal. Whilst the cytase does not increase as a result of vaccinal injections, the fixative, on the other hand, becomes more and more abundant. Consequently it is this second soluble ferment that impresses its characters on the blood serum and on some of the other fluids of the vaccinated animal. It has been pointed out in the preceding chapter that the fixative is found in the fluid of the oedema of vaccinated animals, although in less quantity than in their blood serum. It has also been mentioned that no fixative is found in the aqueous humour of well-vaccinated animals. It must be admitted that this ferment is not inseparably bound to the cells which produce it, as is the case with the cytases. I have already developed, at some length, the thesis that the cytases remain, in the normal animal, within the phagocytes, and only escape from them when these cells are destroyed, whether in the living animal (during phagolysis) or outside the animal (during the preparation of the serum). Gengou’s experiments with the plasma and the blood serum of normal animals have completely confirmed the fundamental observations that the cytases are not found free in the circulating blood. It is evident that the same law applies also to an animal that has acquired immunity. For this reason neither Pfeiffer’s phenomenon nor any analogous process that demands the action of cytases is ever produced in the anterior chamber of the eye, or in the subcutaneous tissue, or in oedema either active or passive. Further, it is in virtue of this same law that Pfeiffer’s phenomenon does not manifest itself even in the peritoneal cavity or in the blood vessels of vaccinated animals in which the phagocytes have been protected from phagolysis by previous injections of various fluids (physiological saline solution, broth, etc.). It would be very interesting to be able to demonstrate the absence of cytases in the fluids of immunised animals by experiments of the same order as those carried out by Gengou with the fluids of normal animals, but the obstacles to the realisation of this postulate are too great. We saw when discussing Gengou’s experiments that it is impossible to obtain _in vitro_ a fluid identical with the plasma of living blood. The greatest precautions in collecting the blood and in its after treatment are insufficient to prevent coagulation taking place sooner or later. It follows that, as there is always a considerable quantity of free fixative in the plasma of immunised animals, an infinitesimal quantity of microcytase, set free from the leucocytes, is sufficient for the production of Pfeiffer’s or any other analogous phenomenon. There must be a great improvement in the methods of preparation of plasmas outside the body before it will be possible to undertake successful researches on the above problem. For the present we must rest satisfied with other proofs, already numerous and very demonstrative, of the absence of free cytases in the normal plasmas of vaccinated animals.

[Sidenote: [266]]

The cytases being found in about the same quantity and presenting the same properties in all animals that enjoy immunity whether natural or acquired, it must be the fixative which specially distinguishes these two categories of immunity. Now, the fixative is found in the serum of perhaps all cases of acquired immunity. Bordet and Gengou have studied it by the method already mentioned (Chap. VII.). A certain quantity of micro-organisms of various species is introduced into the serum. If the cytases, present in the serum when the experiment was commenced, ultimately disappear from it, it indicates that this ferment has been absorbed by the bacteria, thanks to the fixative, which consequently should be found in the serum under observation. The presence or absence of the cytases can be demonstrated by the production or absence of Pfeiffer’s phenomenon with vibrios.

The application of this method enabled Bordet and Gengou[365] to satisfy themselves that the serum of animals immunised against several species of bacteria (plague bacillus, typhoid bacillus, bacillus of swine erysipelas, first anthrax vaccine, and _Proteus vulgaris_), really contains an appreciable quantity of fixative. It may, then, be accepted that the production of this substance is fairly constant in acquired immunity against bacteria, and that it constitutes one of the most important factors in such immunity.

The question has been raised: What is the nature of the substance to which the name of fixative is given? Pfeiffer and Proskauer[366] have attempted to solve this question by making use of a serum which acts against the cholera vibrio and which they obtained by vaccinating animals with this vibrio. They carried out a long series of experiments which led them to the conclusion that this substance, which they term “cholera antibody,” cannot be identified with any of the albuminoid substances of the serum. Further, the fixative is not represented by any of the salts or extractive substances of the serum, because these substances dialyse easily, whereas the cholera antibody does not pass through the dialysing membrane. The fixative is wholly precipitated by alcohol, and is regarded by Pfeiffer and Proskauer as belonging to the category of soluble ferments, an opinion which is certainly shared by many other investigators.

[Sidenote: [267]]

[Sidenote: [268]]

What is it that communicates to this ferment its remarkably specific character? Without being able to give a precise answer to this question, the authors just cited point out the analogy that exists between the cholera antibody and the soluble ferments of yeasts which have been studied by Emil Fischer. Some of these act only upon certain special sugars in a manner equally specific. From a logical point of view it might be permissible to attribute the specificity of fixatives to something borrowed from the species of micro-organism that has played a part in their production. It has long been recognised that in old cultures of the cholera vibrio these micro-organisms are transformed into spherical granules, the arthrospores of Hueppe, which closely resemble the granules produced in Pfeiffer’s phenomenon. There are, then, undoubtedly, vibrionic products which act much as do the microcytases, and it would be very interesting if we could find them in the bactericidal ferments of the animal body. An attempt of this kind was undertaken by Emmerich and Löw[367], who attribute the acquired immunity to a particular substance which they term “Nuclease-Immunproteïdin.” According to their hypothesis the microbial products which are produced in the animal during the period of vaccination—the nucleases—combine with proteid substances of the blood and organs to furnish the substance to which these authors have given such an elaborate name. In their most recent publication Emmerich and Löw even describe a method of producing this substance outside the animal body, by the action of ox blood, or better still pounded spleen, on the nuclease produced by the bacteria found in old cultures. To it they attribute the property of dissolving the various bacteria, of conferring immunity against and even of curing several infective diseases. But these authors do not say whether this remarkable substance is identical with, or analogous to, the antimicrobial ferments composed, as we have seen, of microcytase and fixative. It must be concluded that they look upon it as being similar to the alexine of Buchner, which is nothing more than a mixture of the two substances just named. Unfortunately the whole account given by Emmerich and Löw will do anything but gain over the reader, and in their publications no proof of their assertions can be found. Several of the facts advanced by them do not fall in with well-established data. Thus they speak of the complete lysis of the bacilli of swine erysipelas by their soluble “Erysipelase-Immunproteïdin” in vaccinated animals, a process that has never been demonstrated by them and which in no way accords with conscientious and carefully carried out observations. On the other hand, they cite facts which contradict one another. The “Pyocyanase-Immunproteïdin” is a substance which possesses an extraordinary bactericidal power, not only against the _Bacillus pyocyaneus_ but also against several other bacteria, _e.g._ the bacilli of anthrax, diphtheria, typhoid, and plague. This substance rapidly breaks up these bacteria, and cures diphtheria and experimental anthrax. But it is, at the same time, so affected by the invasion of the most common bacteria, such as _Bacillus subtilis_, that it is necessary to add antiseptics in order to preserve it. To these contradictions, inaccuracies, and uncertainties must be added further the advice, given by Emmerich and Löw to bacteriologists, not to attempt to reproduce their experiments, because they may easily fail, and I think that, in spite of the seductiveness of the attempt to attribute to bacterial products a share in the elaboration of antimicrobial substances, we must conclude not to follow these authors further. It is better to confess our ignorance of the chemical composition of these substances in general and of the fixatives in particular.

[Sidenote: [269]]

As the fixatives resist temperatures much higher than those which destroy the cytases, in this respect resembling the agglutinative substances so frequently found in the fluids of vaccinated animals, there has long been a tendency to identify them with these latter. It is indisputable that between the fixatives and the agglutinative substances the analogies are fairly numerous. Both are produced in quantity during the process of immunisation, and are found not only in the blood serum but also in the fluids of the living animal, especially in the fluids of exudations and transudations. Both dialyse through parchment more readily than do the cytases. Buchner[368] has demonstrated that his alexines (bactericidal substances of normal serum) will dialyse only where the lower fluid is pure water; dialysis is _nil_ when the distilled water is replaced by physiological saline solution. The fixatives and agglutinins, as demonstrated by Gengou[369] for the latter, pass almost completely through the dialyser in the case of pure water, and one-half still passes when the lower fluid approaches as nearly as possible to normal serum.

In spite of these analogies, however, the agglutinative property must be sharply distinguished from the fixative power of serums. In this fluid, derived from normal animals, the agglutinative property is often very marked when the power of fixing the cytases is totally, or in great part absent. Bordet and Gengou[370] have demonstrated also that feebly agglutinative serums of persons convalescent from typhoid fever may exhibit a great capacity for fixing the cytases. Other facts, to be mentioned later, confirm the real difference between the fixative and the agglutinative properties.

[Sidenote: [270]]

The agglutination of bacteria was noted during the course of a series of researches on the acquired properties of the blood serum of vaccinated animals. Charrin and Roger[371], seeking to obtain a clear idea of the difference between the serum of normal animals and that of animals vaccinated against the _Bacillus pyocyaneus_, observed that this bacillus developed in the normal fashion in the former, but in the latter gave rise to special forms of growth. Instead of growing in the form of rods, it elongates into segmented filaments which become entangled and fall to the bottom of the tubes, leaving a supernatant limpid serum. I was able not only to confirm the accuracy of this observation for the _Bacillus pyocyaneus_, but to extend it to Gamaleia’s vibrio and to the pneumococcus[372]. In all these instances we have a modification of the bacteria developed in specific serums coming from vaccinated animals. Later, Bordet[373], during his researches on the bacteriolysis of vibrios _in vitro_, observed that these vibrios, when introduced into the blood serum of vaccinated animals, lose their movements and soon unite into more or less voluminous masses. This observation was confirmed by Gruber and Durham[374], who were the first to apply it in the specific diagnosis of bacteria. They showed that the agglutinating power of vaccinated animals, although not rigorously specific, might, nevertheless, be utilised for the differentiation of certain bacteria, especially the cholera vibrio and the typhoid bacillus. But, independently of this result, Gruber[375] essayed to formulate a theory of acquired immunity based on the agglutinative property of the serum. He accepted, in connection with the phenomenon of the destruction of the bacteria, Bordet’s hypothesis of the concurrent action of two substances, of which one, the bactericidal substance proper, is nothing but the alexine of Buchner, the second being that which agglutinates the bacteria. This agglutination, according to Gruber, results from the swelling of the bacterial membrane which becomes viscous and so leads to the cohesion of the bacteria and the formation of clumps. Thus transformed and rendered motionless, the bacteria succumb more readily to the destructive action of the alexine. It is supposed that the phagocytes do not intervene at all in these cases of acquired immunity, except in a purely secondary fashion when they ingest the bacteria already greatly weakened by the united action of the agglutinin and the alexine. The principal _rôle_ in this theory of immunity is thus given to the agglutinative substance, which is regarded as being a microbial product, modified by the macrophages and thrown into the blood.

[Sidenote: [271]]

The discovery of this agglutination of bacteria has acquired great importance, especially in connection with its application to the diagnosis of typhoid fever. Widal[376] succeeded in showing that typhoid bacilli agglutinate readily under the influence of blood serum and other fluids (milk, transudations, tears, etc.) derived from patients suffering from typhoid fever. As this phenomenon could be utilised for the early recognition of the disease, it began to be studied with great care and many interesting data concerning it have been collected. The general outcome of these researches accords with the conclusions drawn by Widal, and the serum-diagnosis of typhoid fever has taken an important place among the methods used for the recognition of this disease. This aspect of the question, however, does not interest us from the point of view of the problem of immunity which we now have under consideration, and we cannot here enter upon the study of the serum-diagnosis of typhoid fever and certain other diseases (cholera, tuberculosis, pneumonia). Moreover, we must refrain from any analysis of the hypotheses advanced to explain the mechanism of agglutination. A lively discussion has been carried on between the partisans of the chemical theory—according to whom the agglutinin acts directly on the agglutinable substance of the bacteria—and the advocates of the physical theory, led by Bordet[377], who attribute the agglutination to modifications in the molecular attractions which unite the agglutinable elements, be it between each other or with the surrounding fluid. At one time it was thought that Roger’s[378] observation that the cell membranes of _Oïdium albicans_, when cultivated in the specific serum of immunised animals, increased in volume and became greatly swollen, settled the question in favour of Gruber’s theory. But the objection formulated by Kraus and Seng[379], on the one hand, and by Bordet, on the other, dealt a severe blow to this view. As the serum employed by Roger was not deprived of its cytases (alexine), the viscosity of the membrane of the fungus could not be attributed to the agglutinin. When Bordet[380] demonstrated that the red blood corpuscles, under the influence of the serums, undergo an agglutination as marked as that seen in bacteria, it enabled us to study this phenomenon in the large red corpuscles of birds, in which no one has ever been able to demonstrate any viscosity of the corpuscular stroma. In a mixture of red corpuscles of bird and mammal, submitted to the action of a serum which agglutinates the former only, the red corpuscles of the mammal never unite with those of the bird, although this should undoubtedly take place if the membrane of the agglutinated corpuscles had really become viscous. All the facts collected up to the present are, therefore, in favour of Bordet’s physical theory in which an analogy between the phenomena of agglutination and of coagulation is traced.

[Sidenote: [272]]

The point that interests us more particularly in regard to agglutination is the relation of this phenomenon to immunity. We have already given (Chapter VII) the arguments which render it impossible for us to attribute to the agglutinative property of the fluids of the body any _rôle_, however unimportant, in natural immunity against micro-organisms. We must now study the importance of this property in the condition of acquired immunity, in which the agglutination of micro-organisms by the fluids of the body is much more frequent and active than in natural immunity.

[Sidenote: [273]]

The first question to be settled is the following: Is the agglutinative property really constantly present in the fluids of vaccinated animals? The blood serum of animals that have acquired immunity is unquestionably usually agglutinative as regards the corresponding micro-organism. This agglutination may be more or less pronounced, but it certainly exists in the great majority of cases. Nevertheless, examples can be cited in which, in spite of the refractory condition acquired as the result of immunisation, the serum exhibits not a trace of agglutinative power. Having demonstrated that several bacteria (_Bacillus pyocyaneus_, _Diplococcus pneumoniae_, _Vibrio metchnikovi_) develop in the serum of vaccinated animals in the form of elongated filaments more or less interlaced, I was quite prepared to allow that this fact might be of general import. But the study of a cocco-bacillus which produces the pneumo-enteritis of swine and which was isolated by Chantemesse during an epizootic at Gentilly, led me to believe that this was not the case. As this bacillus is characterised by great motility, I concluded[381] that it was identical with that of the hog cholera of American writers. Theobald Smith[382], to whom I sent a specimen and who is a competent authority on this question, refers it, however, to the species which produces swine plague. Knowing that the question of these two bacteria is not finally settled, it is impossible to come to an absolute decision in the matter. Fortunately, from the point of view of immunity, this is of no great importance. The point upon which I must lay stress is that the serum of rabbits vaccinated against the Gentilly bacillus, when sown with this cocco-bacillus, gave very abundant and uniformly turbid growths. In my researches, undertaken at a period when the rapid agglutination of micro-organisms added directly to the specific serum had not yet been recognised, I noted merely that the cocco-bacilli which grew in the blood serum of vaccinated rabbits presented their normal form and gave rise to a general turbidity of the fluid. Since then, however, it has often been observed that the mode of development of a micro-organism in a serum gives an even more delicate indication than does the agglutination properly so called, produced by the serum to which has been added an organism cultivated on its usual medium. Thus Pfaundler[383] saw that _Bacillus coli_ and _Proteus vulgaris_, which were not agglutinated by certain serums, developed in them in an unusual fashion and produced very long and interlacing filaments. When a serum is incapable of revealing its properties by agglutinative reaction properly so called, it is sown with the corresponding micro-organism and the development is then compared with that observed in a normal serum. Frequently a very marked difference is noted, the same organism growing into filaments in the specific serum and forming rods only in the normal serum. The first mode of development is sometimes designated “Pfaundler’s reaction.”

In the serum of rabbits vaccinated against the Gentilly cocco-bacillus, no filaments corresponding to those met with in the agglutinative reaction are formed, but bacilli are produced. In spite of this the animals that furnish the serum show a distinct resistance to infection. More recently, Karlinski[384] has studied the properties of the serums of animals treated with the cocco-bacilli of hog cholera and swine plague. He was able to demonstrate that blood serum from oxen that had received repeated injections of cultures or toxin of hog cholera, was not only incapable of killing the cocco-bacilli of the two swine diseases but it even “gave rise to no agglutination” of the two bacilli and did not arrest the motions of those of hog cholera. On the other hand, serums have been obtained from other species of animals (dog, pig) which brought about the typical agglutination of the cocco-bacillus of hog cholera[385].

[Sidenote: [274]]

In the preceding chapter, Gengou’s experiment on the serum of a dog that had been treated with a virulent culture of anthrax has already been cited. This serum did not agglutinate the bacillus, even of the first vaccine of Pasteur. Nevertheless, a second dog treated with an attenuated culture of this bacillus furnished an agglutinative serum. The immunisation of the first dog was carried very much further than that of the second, but the agglutinative properties were in inverse order. Sawtchenko, in his study of immunity against anthrax, demonstrated that the subcutaneous exudation from vaccinated rats does not agglutinate the bacillus which usually exhibits such a great tendency to collect into clumps.

Agglutination has been studied particularly carefully in typhoid fever. We know that after an attack of this disease, an acquired refractory condition is produced which lasts for a considerable period. In most cases the agglutinative power of the blood diminishes very rapidly, and disappears a few weeks after the commencement of convalescence. It is only in rare cases that it persists for years[386]. On the other hand, during the period of apyrexia which precedes the relapse in typhoid fever and during the period of relapse, the agglutinative power may manifest itself in a very marked degree. In an observation made on a case reported by Widal and Sicard[387], the agglutinative power was raised, two days before the relapse, to a ratio (1 : 150) it had never attained during the first attack. “The appearance of the relapse, two days after this observation”—these authors add—“renders it evident that the agglutinating reaction is independent of the state of immunisation.” Analogous cases have been pointed out repeatedly by several observers.

[Sidenote: [275]]

The examples cited show, on the one hand, that the serum of individuals endowed with acquired immunity may be without any agglutinative property, but, on the other, that this power may be highly developed in the serum of susceptible individuals. The argument based on these data may be corroborated by several other series of facts. Thus, Salimbeni[388] has pointed out that the cholera vibrio is not agglutinated in the fluids of immunised animals. The subcutaneous exudation of a horse treated with a large quantity of these vibrios does not agglutinate Koch’s vibrio except outside the body. When this exudation is drawn off shortly after the injection of the vibrios, the organisms render the fluid uniformly turbid. But a short exposure to the air is sufficient to bring about the agglutination of the vibrios in the same exudation. Guided by this observation, Salimbeni carried out comparative experiments on the action of the serum of vaccinated animals outside the body, in tubes deprived of oxygen and in others exposed to the air. In the former agglutination did not take place or was very incomplete, in the latter it soon came on. This fact accords perfectly with the observation of Pfeiffer’s phenomenon in the peritoneal cavity of guinea-pigs from which we withdraw a fluid containing granules that have resulted from perfectly isolated vibrios. In other micro-organisms a difference has been noted in this respect. Thus Gheorghiewsky has seen the agglutination of the _Bacillus pyocyaneus_ produced under the influence of the serum of vaccinated animals, even in tubes deprived of oxygen. Durham has made a similar observation in the case of the typhoid bacillus. When, however, Trumpp[389] wished to satisfy himself as to the agglutination of the same organism in the body of well-vaccinated guinea-pigs, he obtained only imperfect results. He concluded from his experiments “that the formation of typhoid clumps may precede the breaking down of the bacteria in the animal body itself, but only under certain conditions—when the degree of immunity of the animal is sufficiently high and when the bacilli introduced are not too numerous” (p. 130). In the case of the typhoid bacillus, a certain degree of agglutination is produced inside the animal body, but it is markedly increased in the fluids that have been withdrawn and exposed to the action of the air.

[Sidenote: [276]]

It has been demonstrated, repeatedly, that the agglutination of micro-organisms by their specific serums does not prevent their growth and multiplication. These agglutinated organisms lose none of their virulence. Issaeff[390], working in my laboratory, carried out an investigation on this point in the case of the pneumococcus. He vaccinated rabbits against this organism and satisfied himself that the organism still grows well in the blood serum of such rabbits; but, instead of presenting the typical form of lanceolate diplococci, the pneumococcus, under these conditions, forms very long chains of true streptococci. Having filtered the cultures in order to get rid of the serum, he injected them into rabbits and mice and demonstrated that the pneumococci had retained to the full their initial virulence. Sanarelli[391] carried out corresponding experiments with Gamaleia’s vibrio, which, as we know, also forms chains in the serum of vaccinated animals. When filtered on a paper filter and washed with physiological saline solution, the vibrios were found to be just as virulent as were the control vibrios grown in the serum of susceptible animals. More recently, Mesnil[392] demonstrated the same point in connection with the bacillus of swine erysipelas. He experimented on cultures that were agglutinated after their formation and also on others agglutinated as they were growing. The fluid of the culture was decanted and replaced by fresh broth until the elimination of the serum was complete. Mice, inoculated with the washed clumps, died in the normal period, thus affording proof that “agglutination in no way alters the vitality and virulence of the bacillus of swine erysipelas” (p. 492).

We can readily understand, after the demonstration of these various facts, that it is impossible to maintain Max Gruber’s theory that the agglutinative power constitutes the fundamental basis of acquired immunity. Hence this writer, after publishing several preliminary notes in 1896, has not yet decided to give to his hypothesis a more extended development. Nor has any one else attempted to defend it.

[Sidenote: [277]]

It is probable that in certain special cases the immobilisation of very motile bacteria and their agglutination into clumps may facilitate the reaction of the animal organism, especially the rapidity of phagocytosis. Thus, Besredka[393] observed that guinea-pigs when inoculated with typhoid bacilli that had previously been mixed with the blood serum of normal animals survived. The most active amongst these serums was ox serum heated to 60° C. Guinea-pigs furnished a serum which was much less active. The resistance of guinea-pigs, inoculated into the peritoneal cavity, was in direct ratio to the agglutinated condition of the bacilli. Besredka lays stress on the facility with which the bacilli, when agglomerated into large clumps, were ingested by the phagocytes, and suggests that there is a certain stimulating action of the serums on the leucocytes. When he injected into guinea-pigs a mixture of typhoid bacilli and guinea-pig’s serum, made immediately before injection, his animals died from infection. But when he left the bacilli for some time in contact with the guinea-pig’s serum outside the body, and did not inject the mixture until after agglutination was complete, the inoculated animals usually survived. This experiment indicates the part played by agglutination in the resistance offered by the animal, and at the same time proves that in the body of the guinea-pig the agglomeration of the micro-organisms into clumps does not take place to the same degree as in the serum prepared in, and left in contact with, the air.

In any case, the data collected by Besredka cannot be put forward as an argument in favour of the essential part played by agglutination in acquired immunity, nor can they weaken the facts indicated as to the absence of agglutinative power in examples of acquired immunity and as to the virulence of the agglutinated micro-organisms. The part played by agglutination in this immunity is merely accidental and subordinate.

[Sidenote: [278]]

Special researches have been carried out with the object of defining, exactly, the origin of agglutinins in the body of an animal that has acquired immunity. Observers are unanimous in recognising that, of all parts of the organism, the blood is richest in agglutinin. This substance is found in the blood serum as well as in the plasma. From this (corroborated by the agglutinative property of other fluids, such as the pericardial fluid, oedemas very poor in formed elements, etc.) it follows that the agglutinin circulates in the blood and lymph of the living animal. Several observers, amongst whom I may cite Achard and Bensaude[394], Arloing[395], and Widal and Sicard[396], put to themselves the question whether, before passing into the blood, the agglutinin is not formed in the exudation developed at the seat of inoculation of the micro-organisms. Their conclusions were invariably negative; they were never able to find more agglutinins in these exudations than in the blood. Pfeiffer and Marx[397] had occasionally observed that their animals, inoculated with the cholera vibrio, early exhibited an agglutinative power in the spleen; but this result was not met with sufficiently constantly to enable them to draw a positive conclusion. A little later, van Emden[398] studied in detail the distribution of the agglutinative property in the body of an animal inoculated with _Bacillus aërogenes_. His researches led him to the conclusion that the spleen and the lymphoid organs must be regarded as the source of the agglutinins. Shortly after the inoculation of the bacilli, an extract of the spleen was more agglutinative than the blood or any of the other organs. In rabbits from which the spleen had been removed, the same _rôle_ was filled by the bone marrow and probably also by the lymphatic nodules. But this preponderance of the haematopoietic organs did not continue long, the blood soon becoming the most important seat of the agglutinative power.

The proof that this question of the origin of the agglutinins is a very delicate and difficult one is afforded by an investigation very carefully carried out by Gengou[399] on the agglutination of the attenuated anthrax bacillus (Pasteur’s first vaccine) by the fluids and organs of normal and prepared guinea-pigs. This observer was never able to obtain any confirmation of the results obtained by van Emden with another micro-organism. In Gengou’s guinea-pigs it was always the blood fluid which showed itself most agglutinative, the organs exhibiting merely a feeble and inconstant agglutinative power. As the extracts of leucocytes were always found to be markedly less active than the blood and the fluids of the exudations, Gengou was obliged to come to the conclusion that the agglutinins cannot be regarded as products of the cells of the animal body; this he sums up by saying that “in the increase of the agglutinative power of its blood the organism of the animal plays only a relatively passive part” (p. 337).

[Sidenote: [279]]

I think that, in spite of the facts established by Gengou, his conclusion can scarcely be regarded as final. The agglutinative property, developing in the animal body, must be attributed to some cellular influence, because we know that the prolonged sojourn of micro-organisms in the animal fluids is incapable of conferring on them this power. As Gengou’s experiments did not permit him to attribute the formation of agglutinin to any formed element, it must be concluded that, although perfectly exact, they were insufficient to solve the problem. Gengou killed his animals at a stage when their blood was already pretty strongly agglutinative. At this stage the organs only possessed it to a much more feeble degree. Perhaps, if he had examined his animals at an earlier stage, when the blood possessed a much less marked agglutinative power, he might have obtained a more powerful agglutination with an extract of the organs. In my researches on the resorption of cells, I observed, on several occasions, that the abdominal fluid of guinea-pigs which had received an injection of goose’s blood became agglutinative before the blood serum. Later, however, the blood exhibited a greater agglutinative power than did the peritoneal fluid. If to this fact we add the results of van Emden’s experiments, we shall be tempted to assign to the cells found in the peritoneal exudation and in the lymphoid organs a share in the production of the agglutinin. This question of the origin of the agglutinative power is, however, a very difficult one, and it is impossible, in the imperfect state of our knowledge, to express oneself in a more positive fashion. Fortunately, according to the whole of our data on this phenomenon, the part played by agglutination in immunity can only be very inconsiderable, and we may be allowed to consider our general problem without concerning ourselves over much about the origin of the agglutinative property.

Among the definite results obtained from the study of the agglutinins, it may be specially pointed out that these substances can in no way be identified with the fixatives. These latter were, for long, spoken of as _preventive substances_. They are so termed in the early papers of Jules Bordet treating upon this question. The explanation of this designation is that, for a series of years, the presence of the fixatives was revealed chiefly by the preventive or protective property of the media which contained them.

[Sidenote: [280]]

To gain a clear conception of this protective property, which occupies so important a place in the study of acquired immunity, we must go back to an epoch in our science when it was sought to prove that the fluids of the body played a part in the production of immunity. Shortly after the earliest researches on the bactericidal power of the blood had been made, the idea of applying the results obtained in this direction to the production of immunity in animals by means of injections of blood occurred. The first step in this direction was taken by Richet and Héricourt[400], who succeeded in vaccinating rabbits against a variety of staphylococcus by means of defibrinated dog’s blood. The dog is naturally refractory against this organism, and the blood of a normal dog exercised a certain vaccinal or protective influence on rabbits inoculated with the staphylococcus. But this action was much more marked when Richet and Héricourt employed the defibrinated blood of dogs which had previously received inoculations of the staphylococcus. Shortly after this observation, von Behring[401] made his discovery of antitoxins in the blood serum of animals immunised against tetanus and diphtheria toxins. In collaboration with Kitasato he demonstrated that the serum of these animals, when injected into normal animals, protected them against intoxication by the poisons of diphtheria and tetanus. This great discovery, which has been confirmed on all sides and extended to other poisons, gave rise to the view that a serum exerting any protective power depends solely on its property of impairing the action of the toxins. A more careful study of the phenomena which appear under the influence of the serums has, however, demonstrated the inaccuracy of this view. I was able to furnish the proof[402] that the blood serum of rabbits vaccinated against the micro-organism of the Gentilly pneumo-enteritis prevented normal rabbits from contracting a fatal infection. Nevertheless, the serum exerted no influence on the toxin of this micro-organism; the rabbits that received the minimal lethal dose of this toxin, mixed with serum from vaccinated rabbits, died, as did the control animals, from rapid poisoning. It was evident then that this serum, which prevented infection without in any way hindering intoxication, could not be classed in the category of antitoxic serums. We find ourselves, therefore, in the presence of a new property of the fluids of the body to which we have given the name of _protective_ or _anti-infective power_. We are driven to this conclusion the more as the serum in question was neither bactericidal nor agglutinative.

[Sidenote: [281]]

This discovery was soon confirmed by R. Pfeiffer[403] for the cholera vibrio. Animals vaccinated against this organism furnished Pfeiffer with a serum which, whilst not at all antitoxic, was distinctly protective when injected into normal guinea-pigs. It protected these animals from a fatal infection by the vibrio and, when injected into the peritoneal cavity, it set up the granular transformation of the cholera vibrios,—Pfeiffer’s phenomenon. Pfeiffer, for this reason, gave to the protective antivibrio serum the name of bactericidal serum. As the granular transformation was produced, under the influence of this serum, with cholera vibrios only and never with other species of vibrio, Pfeiffer gave to the active substance in the serum the name of _specific cholera antibody_. This substance, according to his theory, was formed in the animal body at the expense of an inactive antibody which became transformed into an active substance under the influence of the peritoneal endothelium.

The possibility of thus vaccinating susceptible animals by means of the serums of immunised animals, quite apart from any antitoxic power, was easily confirmed and extended to several other infective diseases. Pfeiffer and Kolle[404], Funck[405], Chantemesse and Widal[406] demonstrated it in connection with the experimental disease produced in animals by the typhoid bacillus; Loeffler and Abel[407] for the _Bacillus coli_, etc. The protective or anti-infective power of the serum and other fluids of immunised animals was soon recognised as a general property.

[Sidenote: [282]]

Pfeiffer and his collaborators, as well as many other investigators, laid special stress on the bactericidal character of these protective fluids. It was seen that the serums of immunised animals were often almost or completely incapable of killing the corresponding micro-organisms, but they were still regarded as bactericidal, because, when injected into the peritoneal cavity of normal animals, they set up the transformation of vibrios into granules, or, in the case of other bacteria, determined certain phenomena of extracellular destruction. Whilst carrying on researches in this direction, Fränkel and Sobernheim[408] discovered a fact of great importance. They found that the protective substance of the serum of animals vaccinated against the vibrios resisted heating to 70° C. When submitted to the influence of this temperature, the serum lost its bactericidal power completely, but remained quite as protective as the unheated serum, when injected into susceptible animals. This experiment, which has since been confirmed repeatedly, furnished us with a means of separating the bactericidal power from the protective power in cases where both were present in the same serum. Later, in the hands of Bordet, it proved to be of great service in connection with his researches on the concurrence of two substances in acquired immunity.

The possibility of obtaining Pfeiffer’s phenomenon outside the body by “reactivating” the protective serum with peritoneal fluid or blood serum of normal unvaccinated animals has still further facilitated the study of the action of the two substances in acquired immunity. It was with the help of this method that Bordet was able to furnish so much valuable information on the subject of anticholera serums and, later, on that of haemolytic serums. The discovery by Ehrlich and Morgenroth[409] of the fixation by the sensitive elements of the heat-resisting (thermostabile) substance (that which resists a temperature of 65°–70° C.) constitutes a new and important acquisition to the study of acquired immunity. The discovery has been applied by Bordet to micro-organisms, and since then it has been found possible to study much more precisely the mode of action of specific protective serums.

[Sidenote: [283]]

Even before this last scientific advance had been made it was possible to determine the relations between the protective power and the agglutinative power of the fluids of animals that had acquired immunity. Both resist about the same temperatures; both are found in the blood plasma and pass into the fluids of exudations and transudations. But it may be affirmed with certainty, as already stated, that the two properties are quite distinct. Pfeiffer has laid great stress on the fact that highly protective serums often exhibit only a feeble agglutinative power and _vice versa_. During an investigation[410] into an epidemic of typhoid fever, he had occasion to study the serum of patients convalescent from this disease. The exact dosage of the two properties demonstrated that a slightly marked agglutinative property might be associated with a very powerful protective property. Gheorghiewsky[411] made similar observations on animals vaccinated against the _Bacillus pyocyaneus_. The serum of a goat, although more agglutinative, invariably proved to be less protective than that of a rabbit. A similar result was obtained with the serum of immunised guinea-pigs. “This shows distinctly”—concludes Gheorghiewsky—“that the property possessed by serums of agglutinating the _Bacillus pyocyaneus_ does not march parallel with the protective property” (p. 304). Analogous examples are sufficiently numerous to justify us in accepting the distinctiveness of the two properties of specific serums.

The protective or anti-infective substance is, therefore, not the same as the agglutinin. But are we justified in regarding it as identical with the fixative substance, or fixative (sensibilising substance, immunising or intermediary substance, or amboceptor)? From the fact that the fixative was at first rightly designated by Bordet as protective substance we should conclude in the affirmative. The question is an important one and merits close examination. The discovery of an exact method of determining the presence of fixatives has rendered it possible to ascertain whether these substances are always found in the protective fluids and also whether the presence of fixatives necessarily implies the protective power of the serums.

The first of these questions has been answered in the affirmative. All the protective serums studied from this point of view, by Bordet and Gengou, were found to be endowed with very distinct fixative properties. They also found the specific fixative in the serum of guinea-pigs immunised with the attenuated bacilli of the first vaccine of Pasteur. Now this serum is powerless to prevent the production of fatal infection in mice into which is simultaneously injected the bacillus of the first vaccine. Consequently a fixative fluid is not necessarily protective. This is in accordance with the fact that the micro-organisms that have absorbed the fixative may, nevertheless, retain their virulence. We have already cited the experiment of Mesnil that the bacilli of swine erysipelas, mixed with the specific serum and then deprived of this fluid, produce a fatal infection in mice. We have also drawn attention to the fact, demonstrated by Sawtchenko, that anthrax bacilli, obtained from the exudation of immunised rats, give rise to a fatal anthrax in normal guinea-pigs and rats. The experiments of Bordet and Gengou proved that there is absorption of the fixative substance by the bacilli of swine erysipelas and of anthrax when placed in contact with the specific serums of the immunised animals. In order that the protective power may manifest itself adequately, therefore, besides the fixative substance, some other factor capable of acting is also necessary.

[Sidenote: [284]]

In connection with my work on immunity against the micro-organism of swine pneumo-enteritis I was able to demonstrate that the serum of vaccinated rabbits, incapable of preventing the multiplication of the specific cocco-bacillus, is also powerless to deprive it of its virulence; it is without the power of causing its agglutination or of neutralising its toxin. In short, this serum appears to exercise no direct action on the micro-organism, yet, in spite of that, it prevents its pathogenic action. With these results before me, I was led to assume a certain stimulating action of the serum on the defensive elements of the animal organism and especially on the phagocytic system. The discovery of the fixative property of serums would lead us to believe that this stimulation was entirely useless, and that the permeation of micro-organisms by the fixative was amply sufficient to bring about their destruction and removal from the animal. A living micro-organism in its normal form, endowed with full virulence and provided with its fighting weapon, the toxin, but at the same time permeated by the fixative substance, might behave in the animal in some special way. It might excite a strong positive chemiotaxis of the leucocytes and be ingested and destroyed by these cells with greater facility. _A priori_, there would be nothing to object to in this view, but certain facts are opposed to it. Thus, in the case of micro-organisms just cited, we see bacteria, permeated not only with the fixative but also with cytases, capable of producing a fatal infection. We are thus compelled to accept the theory of an influence of protective serums not only on the micro-organisms but also on the organism of the animal into which they are introduced. As this influence manifests itself in the form of a strong phagocytosis, it is only natural that we should attribute it to the existence of a _stimulating action_ of the serums of vaccinated animals on the phagocytes of the normal animals. The detailed analysis of the mechanism of the immunity acquired as the result of the injection of these serums, as we shall attempt to prove in the following chapter, in many cases confirms this view.

[Sidenote: [285]]

The important part played by the stimulation of the phagocytic reaction in acquired immunity is supported by yet another series of facts and from a different side. It has been clearly established that not only the serum of immunised animals but also that of normal man and normal animals, themselves susceptible to the pathogenic action of the micro-organisms, protects the animal organism against infection. This fact was first demonstrated in connection with researches on the vaccination of guinea-pigs against the experimental peritonitis produced by the cholera vibrio.

G. Klemperer[412] was the first to observe that the blood of several individuals who had never had cholera was, nevertheless, in the case of guinea-pigs, protective against peritoneal infection by the cholera vibrio. He concluded therefrom that the individuals who had furnished this protective blood possessed immunity against cholera. Soon afterwards I[413] was able to extend analogous researches over a large number of persons and to show that the protective power of the blood is of very wide distribution in human beings. But, instead of assuming that all these individuals, whose fluids protect the guinea-pig from peritoneal infection, possess a natural immunity against cholera, I came to the conclusion that the protective power of the blood cannot be taken as a measure of the immunity of the individual from whom the blood was drawn. Here again I assumed a stimulant action of the human blood on the phagocytic reaction of the guinea-pig, looking upon it as quite natural that the blood, capable of exciting the reaction in an alien animal, might remain inactive in the body of the animal which furnished it.

[Sidenote: [286]]

R. Pfeiffer[414] has given much attention to the protective action of serums; he has laid special stress on the essential difference between the influence of normal serums and of those obtained from animals that have acquired immunity. Whilst, in order to obtain a protective effect with the normal blood or serum of man and animals, it is necessary to inject a considerable quantity (from 0·5 c.c. upwards), the specific serum, i.e. serum obtained from persons recovered from cholera or from animals vaccinated against the cholera vibrio, is active in a very minute dose. Sometimes the cholera peritonitis of the guinea-pig is prevented by a fraction of a milligramme of such serum[415]. Based on these facts, Pfeiffer has expressed the view that the normal serum acts by stimulating the natural powers of defence of the animal, whilst the specific serum exercises its influence in virtue of the property of causing the formation of a special secretion which acts only against the micro-organism which served for the production of the immunity. Pfeiffer and his collaborators have demonstrated that normal serums are protective, not only against the cholera vibrio, but also against several other micro-organisms, e.g. the typhoid bacillus. One of his pupils, Voges[416], believed that, in certain infections, the protective power of normal blood might be greatly exaggerated, and that, in these cases, the limit between the activity of normal and of specific serums might be almost completely effaced. He affirmed, especially, that very small doses (0·1 c.c.) of blood serum from a normal guinea-pig was quite sufficient to prevent, in other guinea-pigs, a fatal infection by the micro-organism of hog cholera and its allies. As this fact might be of general application I asked M. Saltykoff[417], who was working in my laboratory, to verify the statements of Voges. Several series of experiments demonstrated the incorrectness of the contention. The small doses of normal serum of guinea-pigs, indicated by Voges, were found to be absolutely incapable of protecting against the virus used by him in his experiments.

The fact that normal serums, injected in sufficiently large doses, exhibited an undoubted protective property, affords additional proof that this property cannot be identified with the fixative power. The latter was present in serums which were not protective; here, then, we have the inverse phenomenon and we see normal serums exercise their protective action although they contain no fixative. This follows from Bordet and Gengou’s experiments already described, according to which the cytases, placed in contact with micro-organisms in normal serums, remain free, simply because of the absence of fixatives.

We are led, then, from these demonstrations to recognise the presence of stimulins not only in specific serums, but also in normal serums. Between the two there is this difference that, when applied with the normal fluids, the stimulins alone act, whilst when injected with the serum of the animal enjoying acquired immunity the action of the stimulins is facilitated and reinforced by the fixatives or sometimes, perhaps, by the agglutinins.

[Sidenote: [287]]

[Sidenote: [288]]

The stimulating influence of certain normal serums may be so considerable that it may prevent infection by the micro-organism, injected at the same time in a dose many times more than lethal. Wassermann[418] protected guinea-pigs by injecting into the peritoneal cavity a quantity as great as 40 times the lethal dose of typhoid bacilli, by introducing at the same time and at the same place 3 c.c. of normal rabbit’s serum, heated to 60° C. Besredka[419], who confirmed this observation, has analysed its special mechanism. He showed that the serum exercises a very marked stimulating influence on the guinea-pig’s leucocytes, which then exhibit a truly extraordinary phagocytic activity. They are seen to act in the peritoneal fluid, but they are much more active in the region of the omentum, where the leucocytes gorge themselves with micro-organisms, devouring them by dozens. The stimulating action of the heated rabbit’s serum is exercised in a similar fashion if, instead of micro-organisms, grains of carmine be injected. Very shortly after the commencement of the experiment very little carmine is found outside the cells; it is all either ingested by individual leucocytes, if the grains are small, or surrounded by numerous leucocytes when the grains are massed together; this phagocytosis is most developed in the region of the omentum, exactly as in the case of typhoid bacilli.

These facts, which so clearly demonstrate the stimulating action of the normal rabbit’s serum, prove in another way that the stimulin resists heating to 60° C., and that, in this respect, it resembles the agglutinins and fixatives. This may afford us an indication as to the nature of the stimulating substance. The possibility of obtaining an antistimulin gives us another valuable indication. Wassermann, in the work we have just cited, showed that the serum of a rabbit, previously treated with guinea-pig’s serum and injected under the same conditions as in the experiment with normal rabbit’s serum, has completely lost its protective power. The typhoid bacilli multiply freely in the peritoneal cavity and the organism of the guinea-pig is incapable of opposing a sufficient resistance. Wassermann thinks that, in this case, the disease becomes grave because of the anticytase found in the serum of rabbits treated with guinea-pig’s blood. There is no doubt that this serum is really anticytasic. But as the free cytases found in the peritoneal cavity of a guinea-pig inoculated at the moment of phagolysis, become inactive under the influence of the anticytase and play merely a minor part, it is impossible to accept the German investigator’s interpretation. Indeed, Besredka has proved that, in this case, it is the antiphagocytic or antistimulant action of the rabbit’s serum which brings about the fatal issue in the case of the typhoid inoculation.

We have laid stress on the point that an animal, whose serum is protective when introduced into another animal, may itself not be refractory against the specific micro-organism. As regards the serum of normal unvaccinated animals this has been so fully demonstrated that nowadays no one doubts it. The question is more complicated in the case of animals that have acquired immunity. As in the great majority of cases the serum of these animals is found to be endowed with a very great protective power, it has been accepted as proved that the animal which furnishes it must itself possess great immunity. The degree of protective power has even been taken as the measure of the acquired immunity. Thus, the numerous attempts to vaccinate the human subject against typhoid fever, undertaken in consequence of the researches of Pfeiffer and Kolle[420], were based on the fact that in these cases the serum of vaccinated individuals acquires a great protective power. It was argued that if this power is present it can only be due to the acquired immunity of the individuals who furnish such a serum. Undoubtedly the protective property of the fluids and the resistance are often equal; but it is none the less true that there are cases where, in spite of this property being markedly developed, the animal that furnishes the protective serum is susceptible to the action of the micro-organism and may even succumb to infection therewith.

[Sidenote: [289]]

As the hypothesis just mentioned is of importance from a general point of view it must be supported by adequate proof. It was during the course of the vaccination of rabbits against the micro-organism of the pneumo-enteritis epidemic at Gentilly that I was first able[421] to assure myself of its accuracy. I noticed that some of these rabbits, although vaccinated, ultimately succumbed to pyaemia, set up solely by this micro-organism. They were consequently not refractory against the disease, and yet their blood serum, when injected into normal rabbits along with an absolutely fatal dose of micro-organisms, was found to be highly protective. This observation drove me to the conclusion that the protective power is not a function of immunity and cannot be received as a measure of this immunity. Analogous facts have since been demonstrated in certain other cases. Thus, Pfeiffer[422] on several occasions has found that guinea-pigs, highly immunised against the cholera vibrio, have succumbed after the injection of a moderate quantity of these organisms. “On post-mortem examination of these cases living vibrios were found in the peritoneal cavity, sometimes in considerable numbers; and yet minimal doses of the heart blood given to normal guinea-pigs caused in these animals a very marked breaking down of the vibrios.” Alongside these facts may be placed others, described in the preceding chapter, of well immunised animals dying from infection, after they had been weakened by opium, cold, or other lowering agent. It is clearly seen, then, that for the manifestation of acquired immunity it is necessary that the reaction of the living cell elements should take place without let or hindrance. When this reaction fails, the possession of even great protective power is insufficient to prevent the immunised animal from contracting a fatal infection.

[Sidenote: [290]]

[Sidenote: [291]]

If, in acquired immunity against micro-organisms, it is really the cell defence which plays the most important part, we can readily imagine cases where it by itself can confer immunity without calling in the co-operation of the protective power of the fluids. When in this connection we study the resistance of an animal against various pathogenic organisms, we note, first of all, the very great variability that exists in the production of the acquired humoral properties. In certain cases, as in vaccination against vibrios or typhoid bacilli, the serum very readily becomes not only protective, but agglutinative and fixative. In other cases these properties develop with difficulty and are only manifested after a long period of vaccination. Such is the case with anthrax. After the discovery of protective serums, numerous attempts were made to obtain a serum protective against the anthrax bacillus. Several observers failed in their attempts, others were more fortunate. Sclavo[423] and Marchoux[424] were the first to succeed in obtaining a protective serum from animals hyperimmunised against anthrax. They were able to show that the serum of sheep, treated first with vaccines and then repeatedly with anthrax virus, would protect rabbits against a fatal dose of the bacillus. Marchoux even obtained, with hyperimmunised rabbits, a serum which prevented normal rabbits from contracting fatal anthrax. Sobernheim[425] was less fortunate in his first experiments. He satisfied himself that the blood serum of cattle that had recovered spontaneously from anthrax or that had been vaccinated according to Pasteur’s method, was absolutely unable to protect small animals against the anthrax bacillus, and his hypervaccinated rabbits furnished serums of doubtful activity. It was only later that he succeeded[426] in obtaining better results; especially when he used sheep. Even then he found that in the production of the anti-infective property the individuality of the immunised animals had a dominant influence. Thus, in two sheep, treated in exactly the same way, the serum of one was found to be incapable of protecting a rabbit, whilst that of the other exhibited an undoubted, although feeble, protective power.

But what is of greater interest to us, from our point of view, is that guinea-pigs which have been vaccinated against anthrax and which enjoy a considerable immunity against this disease, exhibit no protective power. In a letter from Behring I learnt that this fact had for the first time been demonstrated by Wernicke in experiments carried out in the Hygienic Institute at Marburg. After repeated and painstaking attempts this observer succeeded in vaccinating guinea-pigs against enormous doses of virulent anthrax bacilli. The serum from the animals so immunised was, however, quite incapable of protecting normal guinea-pigs against a fatal infection. This result was the more extraordinary since Wernicke’s pigeons, likewise vaccinated against anthrax, gave a serum whose protective power was quite distinct. Realising the great importance of these facts I asked M. de Nittis[427] to repeat these experiments in my laboratory. The vaccination of pigeons is an easy matter, but that of guinea-pigs presents great difficulties. He succeeded, nevertheless, in vaccinating some of these rodents very highly, and this enabled him to compare the protective power of the blood serum in the two species. That of the vaccinated pigeon was found to be endowed with this power and protected guinea-pigs and mice against virulent anthrax. The serum of the immunised guinea-pigs, on the contrary, exhibited no protective property, just as in Wernicke’s experiments. The guinea-pigs and mice, into which this serum was injected at the same time as the anthrax bacilli, died even when attenuated anthrax was used. We have, then, in this case, an example of acquired immunity, independent of any protective power of the fluids of the body.

In the course of their researches on the bacillus isolated by R. Pfeiffer from persons attacked by influenza, Delius and Kolle[428] tried to vaccinate susceptible animals (guinea-pigs) against this minute organism and to immunise animals naturally refractory (dog, sheep, goat) against fairly large doses of cultures. They succeeded in vaccinating guinea-pigs against ten times the lethal dose, but never obtained any protective serum. Nor did the other animals that were treated furnish a protective serum. “From the whole of our experiments carried on for several years”—conclude Delius and Kolle—“it is quite evident that we were unable to produce any appreciable change in the blood by the use of those methods which have produced specific immunising serums against other bacteria such as the bacilli of diphtheria, cholera, typhoid fever, and ‘blue pus’” (p. 345). Slatineano undertook a detailed study of Pfeiffer’s bacillus in my laboratory, but he found it impossible to demonstrate any unquestionable protective effect exerted by the blood serum of vaccinated guinea-pigs upon normal guinea-pigs inoculated with a fatal dose of this organism. We are not justified, therefore, in classing this bacillus with the anthrax bacillus; we may, however, cite it as an argument illustrating the difficulty that is met with, in certain examples of acquired immunity, of discovering the protective power, when feeble and masked.

The inoculation with micro-organisms of animal nature causes the development of acquired immunity, but in this case the properties of the fluids of the body are but little in evidence or they may be even _nil_. Let us return to the example of the _Trypanosoma_ of the rat which excites in vaccinated animals a protective and weakly agglutinative power of the serum. This fluid, however, is usually found to be incapable even of rendering the flagellated parasites motionless.

[Sidenote: [292]]

The question of immunity against malaria has been much discussed. It is well known that a first attack of this disease, so far from conferring any immunity of the slightest durability, leaves a certain predisposition to another attack. In spite of this the study of malaria in various countries and in individuals belonging to different races has demonstrated that there does indeed exist a certain degree of acquired immunity against this disease. During recent years Koch[429] has paid special attention to this subject and has furnished us with very valuable data, based especially on a comparative study of the blood of children and adults. The frequency of Laveran’s parasite in the former and its rarity in the latter, have led him to the conclusion that infantile malaria sets up an immunity which persists in the adult. Moreover, it has been established that in malarial countries the indigenous inhabitants exhibit an attenuated form of the disease, unaccompanied by acute attacks, but with phenomena that are chronic and very slow in development.

In spite of the existence of a certain degree of acquired immunity against malaria, all attempts to demonstrate any protective action of the serum have been fruitless. Celli[430], indeed, injected, as a preventive, the blood serum of individuals who had recovered from malaria or of others who were bled during the period of defervescence following an acute crisis of this disease, but in every instance these injections were found to be useless in preventing an attack of malaria.

We can readily understand that in a disease which is exclusively human, such as malaria, it has not been possible to perform a sufficient number of experiments to decide the question of the protective property of the blood. In this respect we shall have greater chance of obtaining satisfactory data if we direct our attention to some analogous disease attacking one of the lower animals. Such a disease we have in Texas fever, occurring in the Bovidae, as the result of the action of an animal parasite, _Piroplasma bigeminum_, which invades the red blood corpuscles much as Laveran’s parasite invades those of the human subject.

[Sidenote: [293]]

[Sidenote: [294]]

As mentioned in the preceding chapter, Smith and Kilborne and Koch have demonstrated that the Bovidae may acquire a real immunity against Texas fever. Nicolle and Adil Bey[431] at Constantinople found indigenous races that exhibited a remarkable immunity against the _Piroplasma_. Having demonstrated this fact the idea occurred to them to inoculate these refractory cattle with very large quantities of virulent blood and to make use of the serum from animals so treated for the prevention of infection in susceptible races of Bovidae. This experiment gave negative results. Lignières[432] elaborated a special method of vaccinating susceptible Bovidae and was successful in obtaining very encouraging results. A commission of veterinary surgeons from Alfort[433] appointed to verify these observations came to the conclusion that “the vaccination as carried out by Lignières was absolutely effective.”

Lignières also carried out researches on the protective power of the blood serum of his immunised cattle. In a communication to the International Congress of Medicine, held in Paris in 1900, he stated that the injection of several hundred cubic centimetres of this fluid did not protect normal animals against infection. We must conclude, therefore, that, here also, we have another example of acquired immunity unaccompanied by the presence of any protective property of the blood fluid.

These results have received confirmation from a most authoritative source. Nocard has kindly communicated to me the fact that he has tried in vain to confer immunity on normal dogs into which he has injected blood serum coming from dogs that had recovered from the disease produced by a haematozoon closely allied to that of Texas fever or serum from sheep immunised with blood from the affected dogs.

Looking at the data we have just summarised as a whole, we are compelled to recognise that, on the one hand, the protective power of the body fluids may coincide with a susceptibility to the corresponding micro-organism, and that, on the other, real acquired immunity may exist without any manifestation of this humoral property, especially as, even in immunised animals, the acquired immunity often persists longer than does this property. It must be accepted then, that, in this immunity, there exists something other than the powers of the fluids of the body, that is to say, the factor which plays the predominant part is to be sought for in the cellular elements. We need only recall the many facts collected in the preceding chapter to be convinced that in acquired immunity phagocytosis is the most constant and most general phenomenon. We find it in cases where the humoral properties are the most marked, as well as in those in which they are only slightly developed or are entirely absent. We need not again discuss Pfeiffer’s phenomenon analysed in the preceding chapter. It is sufficient to mention that this example of the extracellular destruction of micro-organisms only occurs under limited and special conditions. It is observed only in cases where the injection is made into a situation rich in leucocytes which undergo phagolysis as a result of the sudden change brought about in their conditions of existence. Further, this phenomenon is observed only in connection with micro-organisms that are slightly resistant to the influence of the microcytases. In those cases in which we meet with Pfeiffer’s phenomenon, we also meet with a widely extended phagocytic reaction.

[Sidenote: [295]]

This reaction is most pronounced where the properties of the body fluids are only slightly developed or are absent. The study of acquired immunity against anthrax provides us with a very convincing proof of this. We have already cited the example of vaccinated rabbits and rats in which phagolysis is incomparably greater than in the susceptible control animals which contract a fatal anthrax. This rule is general. It is confirmed in the vaccinated sheep and guinea-pig. The absence, or feeble development, of the protective power of the blood or of the other humoral properties in no way, then, prevents the considerable change which is set up in the phagocytes of animals that have acquired immunity against anthrax. The negative chemiotaxis of the leucocytes, so marked in susceptible animals, is modified into positive chemiotaxis as the result of vaccination. This fact, one of fundamental importance, was first demonstrated for the immunity against anthrax, later being extended to other micro-organisms. Massart[434] studied the general subject and collected a series of data which led him to say that “vaccination effects an education of the leucocytes; these latter become so adapted that they can approach the virulent micro-organisms.” The best method of forming an estimate of the change which the leucocytes undergo is by injecting subcutaneously very virulent micro-organisms capable of setting up a generalised infection. The anthrax bacillus, Gamaleia’s vibrio, the streptococci and the cocco-bacilli of swine and fowl cholera are very suitable for such study. These micro-organisms, when inoculated subcutaneously into susceptible animals, set up a very slight local reaction or none at all, in the form of an exudation of transparent fluid almost entirely without leucocytes. The micro-organisms grow freely in these exudations and soon invade the animal. In vaccinated animals the local reaction is more marked and the exudation, very rich in leucocytes, is poor in fluid; the micro-organisms remain free for a very short time, being soon ingested by the leucocytes. Their destruction, inside these cells, takes a longer or shorter time according to circumstances; but in the end it is always complete.

[Sidenote: [296]]

The difference as regards phagocytic reaction between susceptible and vaccinated animals, such as I have just described, has been generally recognised by many observers. A few opponents are still found, however, who consider that they are justified in affirming that the negative chemiotaxis of the susceptible animal does not exist and that, consequently, vaccination can in no way change it into positive chemiotaxis. Werigo made himself the spokesman of this view, which he has maintained in several papers[435]. Instead, however, of introducing the virulent micro-organisms into the subcutaneous tissue of susceptible animals he injected them directly into the veins. Using cultures of the anthrax bacillus and of the cocco-bacillus of fowl cholera he injects these into the venous system of normal rabbits. The animals soon die from general infection. If, however, these animals are killed shortly after inoculation, it is found on examination of sections that many of the micro-organisms have been ingested by the leucocytes. Werigo concludes from these facts that in the higher animals the chemiotaxis is always positive; but that it ends in the destruction of the micro-organisms in the vaccinated animals, never bringing about this result in susceptible animals. Taking all the data on this question into consideration, it is easy to convince oneself that this view cannot be accepted as correct, for not only the definite phenomena observed below the skin but also the no less demonstrative process appearing in the peritoneal cavity prove most clearly the existence of this negative chemiotaxis of the leucocytes. I need only recall Bordet’s experiment on the fate of streptococci and _Proteus vulgaris_ when injected together into the peritoneal cavity of guinea-pigs. Whilst the _Proteus_ bacilli at the end of a very short time are all ingested by the leucocytes, the streptococci remain free in the peritoneal fluid up to the death of the animal. The leucocytes which exhibit a positive chemiotaxis as regards the former, manifest a negative chemiotaxis as regards the streptococci.

[Sidenote: [297]]

In spite of the great force of these arguments, the discovery of a means of reconciling the results obtained from the inoculation of micro-organisms subcutaneously or into the peritoneal cavity, with those observed after they had been injected into the blood vessels would be of great interest, and Zilberberg and Zeliony[436] have undertaken a series of experiments with this object. Following Werigo they made use of the cocco-bacilli of fowl cholera, and found, in accordance with his observations, that the intravenous injection of these organisms, obtained from cultures in nutrient media, causes a very marked phagocytosis of the cocco-bacilli. When, however, they injected into the veins of rabbits cocco-bacilli that had been grown in the peritoneal fluid of other rabbits, they found the micro-organisms free in the blood plasma and observed only a very restricted phagocytosis in the microphages of the liver. It follows from these experiments that the ingestion of the cocco-bacilli, in Werigo’s experiments, was dependent on the presence of a large number of attenuated micro-organisms which were present in the cultures that he employed for his injections. Alongside these organisms, slightly or not virulent, were others, endowed with their normal pathogenic activity and quite numerous enough to set up a fatal infection. When Zilberberg and Zeliony replaced cultures on agar by the peritoneal exudation which contained virulent cocco-bacilli almost exclusively, the phagocytosis in rabbits, injected into the veins, was found to be almost suppressed. With the object of establishing whether the absence of the phagocytic reaction, in this case, really depended on negative chemiotaxis on the part of the leucocytes, the above cited observers performed the following experiment. They injected into the vein of a rabbit, already affected with a generalised infection by the cocco-bacillus of fowl cholera, an innocuous culture of a saprophytic staphylococcus. Post-mortem examination showed that these cocci were almost entirely ingested by the same phagocytes which refused so energetically to seize the cocco-bacilli. This experiment, analogous to that of Bordet on streptococcus and _Proteus_, compels us to reject Werigo’s conclusions as to the absence of negative chemiotaxis in the phagocytes of the higher animals. I ought to add that the work of Zilberberg and Zeliony was in part executed in my laboratory so that I was able to convince myself by ocular demonstration of the complete accuracy of their statements.

Independently of these observers and even before their work appeared, Th. Tchistovitch[437] published an interesting study on the same question. He injected very virulent streptococci into the ear vein of rabbits. These micro-organisms set up a generalised and fatal infection in which phagocytosis was completely absent or nearly so. Here again was manifested a negative chemiotaxis of the phagocytes, which, henceforth, could no longer be questioned.

[Sidenote: [298]]

In certain infective diseases terminating fatally a very marked phagocytosis is observed even in susceptible animals. The most typical example of this is furnished by swine erysipelas and mouse septicaemia. We know from the researches of Koch[438], followed by those of Loeffler[439], Schütz[440] and others, that in animals which have died from these two diseases the leucocytes are gorged with small specific bacilli. A method of vaccinating animals against the micro-organism of swine erysipelas was worked out by Pasteur and Thuillier[441] and was afterwards studied by many observers. Thanks to this method it has been possible to demonstrate the phenomena which may be observed in vaccinated animals (especially rabbits). Here also a phagocytosis takes place, even more rapid and more complete than in susceptible animals. What is more important, the intracellular digestion of the ingested bacilli is followed by the total destruction of the micro-organisms in the vaccinated animals, though in the normal animals this digestion is very imperfect.

[Sidenote: [299]]

The acquisition of immunity against micro-organisms is, therefore, due not only to the change from negative to positive chemiotaxis, but also to the perfecting of the phagocytic and digestive powers of the leucocytes—a general superactivity and adaptation of the phagocytic reaction of the immunised animal is produced. This conclusion, based upon a large number of well-established facts and in complete harmony with the whole of the data at our disposal concerning acquired immunity, has been attacked by Denys and Leclef[442] in their work on the streptococcus. They base their opposition upon experiments made _in vitro_ on the action of serums and leucocytes on this micro-organism. They have compared the bactericidal power of mixtures of the serums of normal and of vaccinated rabbits with leucocytes isolated from exudations from these two groups of animals. The leucocytes, whether derived from normal or from vaccinated rabbits, when mixed with normal serum were equally incapable of ingesting and destroying the streptococci. When mixed with blood serum from vaccinated rabbits, however, the two kinds of leucocytes exhibited a very marked phagocytic reaction. Denys and Leclef conclude from this that phagocytosis, although an important factor in immunity, plays merely a secondary part and is dependent on the humoral properties. The experiments and views of these two observers have been generally received by the partisans of the bactericidal theory of the body fluids as an actual proof of this theory. We cannot agree. Researches extending over a long period have shown us that the study of phagocytosis _in vitro_ can give only a very inexact and imperfect idea of the course of the phenomena in the living animal. Usually the leucocytes taken from the exudations, although amoeboid, no longer fulfil their phagocytic functions at a time when in the animal they would ingest micro-organisms with the greatest rapidity. As a general rule, existence outside the living body weakens them very considerably. But in some cases, rare it is true, the leucocytes although inactive in the animal exhibit intense phagocytosis when introduced into a hanging drop of fluid from an exudation or even of urine. In any case it is very hazardous to infer from phenomena which appear under these artificial conditions what takes place in the living animal. The value of the experiments of Denys and Leclef is still further marred by the fact that they mixed the leucocytes with blood serum. They appear to have lost sight of the fact that this fluid is far from corresponding to that which bathes the leucocytes in the living animal. The serums contain leucotoxin in greater or less quantity and it is not to be wondered at that the leucocytes when mixed with normal rabbit’s serum should perish very rapidly. Further, the serum of vaccinated rabbits is agglutinative (this fact, however, was not sufficiently elucidated in 1894 when the researches of Denys and Leclef were made) and the clumping of streptococci might simulate their destruction. In a word, the experiments of these observers have been carried out under such conditions that it is impossible to base upon them a refutation of data obtained in the living animal. Moreover, in the description of the phenomena which appear in the subcutaneous tissue of rabbits inoculated with the streptococcus, Denys and Leclef provide us with arguments against their own view.

[Sidenote: [300]]

These observers introduce the same quantity of streptococci below the skin of the ear of normal and of vaccinated rabbits. In the first there is soon produced a very marked oedema of the ear, in which may be seen a number of streptococci and of leucocytes that have not ingested any micro-organisms. In the second the oedema does not develop, but at the seat of invasion a number of leucocytes come up and these soon ingest the streptococci. As we see, the phenomena manifest themselves here just as they do with the anthrax bacillus and many other micro-organisms when under analogous conditions. Denys and Leclef, indeed, recognise that, below the skin of the ear of vaccinated rabbits, the small quantity of exudation fluid is not sufficient to enable us to accept it as capable of exerting any considerable influence as regards humoral properties. Nevertheless, they think that the “serum” of this fluid may exercise a certain action, but they furnish no proof of this, and seem to ignore the fact that the plasma of the subcutaneous exudation is far from being identical with blood serum obtained outside the animal. At present it is well known that this latter fluid contains cytases which are absent from the plasmas. Now, the feeble bactericidal action, if this really exists as regards the streptococcus, must be attributed to the microcytase which has escaped from the leucocytes at the time of the preparation of the serum.

To sum up, the example studied by Denys and Leclef clearly comes under the general law of phagocytic reaction in acquired immunity against micro-organisms. It is impossible to deny that the superactivity of the phagocytes which is always found in this immunity, although readily observed, cannot be demonstrated in a rigorous fashion outside the fluids which bathe the cells. There are, however, very important analogies which may be invoked in favour of this thesis. We have already cited in our fifth chapter Delezenne’s experiments on the digestion of gelatine by the leucocytes of the dog, which show in the most demonstrative fashion that these cells accustom themselves to bring about this digestion more and more quickly and this quite independently of any humoral influence.

For some time past there has been no doubt as to the fundamental fact that the phagocytes in immunised animals seize and destroy living micro-organisms. Several attempts have been made to show that such destruction of these bacteria takes place solely by the body fluids, and that the phagocytes intervene only as “scavengers” to carry off the dead bodies of the micro-organisms. The numerous observations, described in the preceding chapter, absolve us from again entering into a discussion of this question. Moreover, the majority of these opponents now recognise that micro-organisms are ingested in a living state by the phagocytes of immunised animals. Some, however, have expressed the opinion that these living micro-organisms, before becoming the prey of the phagocytes, must undergo some preliminary attenuation of virulence through the action of the body fluids. Hence the theory of the attenuating power of the fluids of the body, maintained especially by Bouchard and his pupils. During the course of our exposition of the facts concerning acquired immunity, we have several times had occasion to speak of the virulence of micro-organisms in the immunised animal. Here, therefore, we may confine ourselves to a brief summary of the observations collected on this point.

[Sidenote: [301]]

Having observed that the anthrax bacillus, when developed in the blood of immunised sheep, was incapable of giving fatal anthrax to rabbits, I expressed[443] the opinion that under these conditions its virulence had become attenuated. Later, analogous changes were shown by Charrin[444] in the _Bacillus pyocyaneus_ when cultivated in the serum of immunised animals. Bouchard[445], generalising on these data, arrived at the following theory of vaccination. “The inoculation of a strong virus into a vaccinated animal is equivalent to the inoculation of an attenuated virus. The attenuation, however, instead of being done beforehand in the laboratory, is brought about in the tissues of the vaccinated animal” (p. 18). Charrin and Roger[446] upheld this view, and the latter offered several new arguments in support of it. He observed that animals inoculated with pneumococci and streptococci grown in the blood serum of vaccinated animals, contracted a transient and benign disease merely, whilst the control animals, inoculated with the same micro-organisms, cultivated in normal serum, always died from generalised infection.

[Sidenote: [302]]

The discovery of the protective property of serums has thrown a new light upon these experiments. We must now ask ourselves: Does the innocuousness of micro-organisms depend not on the attenuation of the virus, but rather on the protective action of the serum itself? When, in the course of my researches on the Gentilly cocco-bacillus, I found that this organism, cultivated in the serum of vaccinated rabbits, became much less pathogenic than when it was grown in the serum of normal rabbits, I set myself to answer this question. Simple filtration through paper was sufficient to rid the organism of the serum in which it had grown. The inoculation of cocco-bacilli thus treated proved at once that their virulence was in no degree modified, and that it was the intervention of the serum that prevented the micro-organism from setting up the rapidly fatal disease. Issaeff[447], who, in my laboratory, carried out the investigation, was able to extend this to the pneumococcus. He obtained agglutinated cultures in the serum of vaccinated rabbits, and he compared their activity by injecting them (1) with, and (2) without their culture medium. The difference was very marked. In the first case the infection produced was much slower in its course than in the second. The virulence of the washed pneumococci was found to be the same whether they came from a culture in normal serum or from one in immunised serum. Sanarelli[448] obtained the same result with Gamaleia’s vibrio. The vibrios when grown in the serum of vaccinated guinea-pigs proved to be very virulent so soon as they were freed from the fluid in which they were grown. Later, similar demonstrations were given by Bordet[449] and Mesnil[450] with respect to streptococci and to the bacilli of swine erysipelas. We must, then, conclude that we have here to do with a general law. Some experiments made by de Nittis[451] might seem to indicate an exception to such a law. He observed that anthrax bacilli when grown in the serum of vaccinated pigeons lost a part of their virulence. It must not be forgotten, however, that he grew his cultures under special conditions; the bacillus was grown for several days at 42° C., this in itself being quite sufficient to bring about a certain attenuation of virulence.

The theory of the attenuating action of the body fluids, based on the attenuation of the virus in the serum of vaccinated animals, can no longer be maintained, as it is a well-established fact that the serum, obtained outside the body, is a fluid differing in character and properties from the plasma of the living animal. We have seen up to what point this demonstration has shaken the theory of the bactericidal action of the body fluids.

[Sidenote: [303]]

It cannot be doubted that a micro-organism may undergo a certain weakening in virulence, as well as in certain other functions, in the body of the animal that has acquired immunity. But the question must be put: Is this effect obtained as the result of humoral or of cellular action? As a general rule, exudations obtained from vaccinated animals, and containing living micro-organisms, are found to be virulent when inoculated directly into susceptible animals. This fact was established by Pasteur[452] when he first carried out his researches on acquired immunity against fowl cholera. He showed that the exudations of vaccinated fowls set up a fatal disease in normal fowls, without there being the least evidence of any attenuation of the micro-organism. The same applies to the Gentilly cocco-bacillus and to the anthrax bacillus in a very great majority of examples. De Nittis observed that the exudations of immunised pigeons produced a fatal infection in the guinea-pig and in the mouse. In the immunised guinea-pig, on the other hand, he found that the exudations soon became innocuous for these animals. This alteration, however, must be attributed not to the body fluids (which exhibit no protective or attenuating power) but to the action of the cells.

With the object of gaining some idea of the changes that the micro-organisms undergo in the immunised animal, Vallée[453] carried out a series of experiments on rabbits vaccinated against the bacillus of swine erysipelas. He enclosed these bacilli in sacs of collodion which he introduced into the peritoneal cavity of susceptible rabbits and of others that were hyperimmunised. The bacillus developed well in both cases. It gave homogeneous non-agglutinated cultures in the sacs placed in normal animals, whilst in the sacs introduced into the peritoneal cavity of hyperimmunised rabbits the bacilli grew into agglutinated filaments. This proves that the wall of the sacs permitted of the passage of the active substances elaborated in the immunised animal. Different from the point of view of agglutination, the cultures likewise exhibited a considerable difference in their pathogenic activity. The cultures developed in the sacs in hyperimmunised rabbits were found to be much more virulent than those grown in the sacs in control animals. This augmentation of virulence depends, probably, on the influence of the active substances which pass through the walls of the sacs. In any case, this experiment affords further confirmation of the impossibility of maintaining the theory of the attenuation of micro-organisms by the fluids of an animal enjoying acquired immunity.

[Sidenote: [304]]

Since the discovery of the antitoxic property of the fluids of the body, it has been accepted that its manifestation was indispensable for the acquisition of immunity. It was thought that in order to get rid of pathogenic micro-organisms the animal had first to develop the means of neutralising their toxins. These substances once prevented from exerting their toxic action, the micro-organisms were left without their weapon of attack and found themselves reduced to the condition of simple saprophytes. It was accepted, therefore, that an effective antitoxic power was always to be found in the fluids of animals that had acquired immunity. Against this explanation, however, are certain established facts. Chauveau[454] had observed that Algerian sheep, whose natural immunity was further strengthened by considerable doses of anthrax bacilli, exhibited a susceptibility to injections of anthrax blood quite as marked as that of normal sheep. The immunity against the virus, then, did not progress _pari passu_ with that against the poison. Later, Charrin and Gamaleia[455] furnished important data on this subject. They showed that animals vaccinated against the _Bacillus pyocyaneus_ and the vibrios of Koch and Gamaleia were even more susceptible to intoxication by the soluble products of these micro-organisms than were normal animals which had acquired no immunity against the corresponding bacteria. Shortly afterwards this observation was confirmed by Selander[456], in his work on hog cholera, carried out under Roux’s direction. Rabbits vaccinated against the cocco-bacillus of this disease resisted infection by the virus, but died as a result of the exhibition of the same doses of toxin that killed normal rabbits. I[457] was able not only to verify this, but to add to it the further fact that the blood serum of vaccinated rabbits, although markedly protective against infection, exercised not the slightest antitoxic action.

When, later, R. Pfeiffer set himself to study the immunity of animals against the cholera vibrio, he, along with his collaborators, was able to furnish numerous data confirming the hypothesis that animals thoroughly vaccinated against this vibrio had not thereby become more resistant to its toxin and that their anti-infective serum exhibited no antitoxic power. These results have been confirmed repeatedly and must be regarded as fully established.

[Sidenote: [305]]

Von Behring here recognised a general law which, with the aid of his collaborators, he attempted to develop. We owe to him the knowledge that the susceptibility, augmented as regards the toxins, of animals vaccinated against micro-organisms, might even serve in doubtful cases to reveal the presence of their bacterial poisons. Culture products when deprived of micro-organisms often set up no poisoning in normal animals susceptible to infection. From this fact it is generally concluded that the toxin is not present in the products in question. But animals of the same species when immunised against infection by the micro-organism, owing to their “hypersusceptibility,” react much more delicately and allow of the demonstration of the presence of bacterial poisons in fluids inactive for unvaccinated animals.

In collaboration with Kitashima[458], von Behring immunised guinea-pigs against the diphtheria bacillus, and demonstrated that two or three injections of diphtheria toxin were quite sufficient to render these animals refractory to infection by the diphtheria bacillus though they became more susceptible to intoxication. Von Behring considers that this augmentation of susceptibility to the diphtheria poison may be a means of rendering the local reaction of the living elements at the point of introduction of the bacilli more active.

In any case, it is beyond question that acquired immunity against microbial infection is quite independent of the resistance against the toxins of the corresponding micro-organism. An antitoxic manifestation of any kind, therefore, cannot be regarded as necessary for the development of immunity against the micro-organism.

Of all the humoral properties developed in acquired immunity against micro-organisms, the fixative power and the protective power are the most constant. It might naturally be suggested, as a result of this observation, that these two powers are indispensable for the manifestation of phagocytosis for the purpose of destroying and of ridding the animal of the pathogenic organisms. It is quite possible to understand how, under these conditions, the idea has been put forward that anti-infective acquired immunity is the result of two different factors: in the first place, a humoral property independent of the phagocytes and, in the second place, the phagocytes themselves. But the part played by these cells cannot be accepted as purely secondary—a view which has been advanced and defended again and again. This question is of such importance that it is reasonable to ask whence come the humoral properties, such as the fixative power and the protective power, factors of such far-reaching influence in anti-infective immunity?

[Sidenote: [306]]

Thanks to the work of several investigators this question may now be answered. Pfeiffer and Marx[459] first supplied important information concerning the origin of the protective property. Into rabbits they made subcutaneous inoculations of cholera vibrios, killed by heat (70° C.), and then examined, most minutely, the protective power of the blood and of extracts from various organs. Examining, separately, the protective power of the serum and that of the layer of leucocytes deposited in tubes, Pfeiffer and Marx were unable to find any marked difference. Nor did they ever obtain any definite effect with leucocytes collected from pleuritic exudations. From these observations they concluded that the leucocytes of the blood could not be regarded as the source of the protective substance (or “cholera antibody”). At a period when the serum as yet exhibited an insignificant protective power or none at all, the extract from the spleen often exerted an action of the most marked character. In an experiment in which the rabbit was killed 48 hours after the injection of the vibrios, 0·3 c.c. of the serum was incapable of preventing fatal infection of a guinea-pig, whereas 0·03 c.c. of an extract of the spleen exerted a marked protective effect. From this and similar experiments, Pfeiffer and Marx conclude that the spleen is the principal source of the protective substance. In order to verify this observation they injected killed cholera cultures into rabbits which had previously been deprived of their spleens, but the asplenic rabbits still produced the same amount of protective substance, and these two observers were led to conclude that the lymphatic glands and the bone-marrow might also serve as the sites of origin of this substance.

It is only during the first few days, however, that these organs exhibit a protective power greater than that of the blood. Three or four days after the injection of the vibrios the blood serum becomes richer in protective substance; the organs contain much less of it. This condition is maintained for some time, after which the blood in turn begins to get impoverished.

[Sidenote: [307]]

Pfeiffer and Marx put to themselves the question: Is the marked protective power of the spleen due to the production of preventive substance by this organ, or is it to be explained by an accumulation in the spleen of this substance manufactured elsewhere? With the object of obtaining an answer to this question they injected protective serum from other individuals into rabbits, when they found that the protective substance showed not the slightest tendency to accumulate in the spleen. These authors were compelled to conclude, therefore, that the spleen and other haematopoietic organs (lymphatic glands and bone-marrow) are the real seats of the production of the protective substance. We may add that these organs are also the phagocytic organs _par excellence_, that is to say, the centres which serve not only for the development of phagocytes but which contain a large number of the adult elements capable of exercising the phagocytic function.

Almost simultaneously with Pfeiffer and Marx, A. Wassermann[460], in collaboration with Takaki, undertook similar researches on the origin of the substance protective against the typhoid cocco-bacillus. The outcome of this work was that “it was the bone-marrow, the spleen, and the lymphatic system, including the thymus gland, which exhibited immunising power against the bacillus of typhoid fever, whilst the other organs, the blood, brain, spinal cord, muscles, liver, kidney, etc., did not at this stage show any marked specific property.”

[Sidenote: [308]]

As these observations on the production of protective substance in the phagocytic organs was one of essential importance in connection with the problem of acquired immunity, I asked M. Deutsch[461], working in my laboratory, to carry out a series of experiments on this subject. Using guinea-pigs, he injected into the peritoneal cavity cultures of the typhoid bacillus killed by heat (66° C.). A few days later the serum had become distinctly protective. At this stage, and even before the appearance of this property in the blood, Deutsch killed some of his animals and carefully measured the protective power of the extract of the various organs. He began by confirming the result obtained by Pfeiffer and Marx as to the non-production of the protective substance in the peritoneal exudation. Usually this fluid was insufficient to protect normal guinea-pigs against typhoid infection. In a few experiments only was the exudation found to be as protective as the blood serum; in most of the others, the blood serum was much more active than the fluid of the exudation. The spleen was the organ which exhibited the greatest protective power, and in nearly one-half of the cases it was more active than was the blood. The bone-marrow sometimes gave analogous though much less marked results. The spleen consequently must be looked upon as the principal seat of the production of the protective substance.

Having confirmed this observation of Pfeiffer and Marx and of Wassermann and Takaki, Deutsch tried to obtain the protective property in guinea-pigs deprived of their spleens. The experiment was quite successful, and here again his result agreed with that obtained by Pfeiffer and Marx. Guinea-pigs from which the spleen had been removed developed the protective property just as well as did the control animals; in the former the bone-marrow was found to be specially active.

When Deutsch, instead of removing the spleen from his guinea-pigs before the injection of the micro-organisms, did so some (3–5) days afterwards, there often occurred a marked diminution in the amount of the protective substance produced. We must conclude, therefore, that soon after inoculation there appears in the spleen a phenomenon which is associated with the development of the protective power. The most simple explanation of these facts is that the micro-organisms injected into the peritoneal cavity and soon afterwards seized by the phagocytes (for the most part by the microphages), are carried to the phagocytic organs, particularly the spleen, lymphatic glands, and bone-marrow. In those animals whose spleens are left intact a large number of these microphages loaded with micro-organisms make their way into this organ, a fact confirmed by direct observation. When the spleen is removed the microphages must necessarily betake themselves to other phagocytic organs. As the micro-organisms undergo intracellular digestion in the phagocytes, it is very difficult, if not impossible, to follow them for any length of time after they have been ingested, but the analogy with the phenomena of the resorption of red blood corpuscles, described in Chapter IV, justifies us in concluding that in the case of micro-organisms matters go on in much the same way. These organisms, seized at the seat of inoculation by the phagocytes, are transported by these cells, in their peregrination through the organs, into the general circulation. The interpretation I have just given has been accepted by Deutsch.

[Sidenote: [309]]

This observer wished also to come to some conclusion as to the origin of the agglutinative property so well developed in the fluids of animals inoculated with the typhoid cocco-bacillus. He did not succeed in solving this question, but he was able to demonstrate the undoubted difference between this property and the protective power. The facts brought forward by Deutsch must, therefore, be ranged alongside the many others, reported on above, which demonstrate in the most conclusive fashion that these two powers of the body fluids are essentially distinct.

Such concordant results obtained by all investigators who have studied the origin of the protective power warrant the conclusion that it is the elements of the phagocytic organs, that is to say, the phagocytes themselves, which produce the protective substance. But it will be asked: Can we therefore accept the fixative substance or fixative as being derived from the same source? When the experiments I have just summarised were carried out the fixatives were not as yet sufficiently known and were confounded with the protective substances. Nevertheless, there can be no doubt as to what the answer to the question just put must be. In the account of the experiments of Pfeiffer and Marx we find very precise statements as to the granular transformation of the vibrios. Thus, they observed on several occasions that an extract of the spleen set up this transformation in a particularly distinct and rapid fashion at a period when the blood and serum, used in a much stronger dose, were incapable of producing the same effect. Now, as Pfeiffer’s phenomenon is a visible manifestation of the action of the specific fixative, it cannot be doubted that the spleen is really the principal seat of development of the fixative substance before it makes its appearance in the blood.

Before concluding this chapter we must review very briefly the principal phenomena associated with acquired immunity against micro-organisms. The extracellular destruction of these parasites takes place in the living animal under special conditions only, when the phagocytes suffer a temporary injury (phagolysis) and allow their microcytases to escape. These latter by no means represent attributes of the body fluids, as is even yet maintained by some writers. These soluble ferments are connected with the phagocytes and represent the ferments of intracellular digestion. The cytases undergo no modification during the process of immunisation and correspond to those which act in natural immunity.

[Sidenote: [310]]

The agglutinative substance often present in the normal fluids of the body becomes much more developed in those of immunised animals. It is truly humoral, as it circulates in the plasmas and passes into the fluid exudations and transudations. But the part played by it in immunity is very restricted.

The protective and fixative properties, most often closely connected with each other, are very markedly developed in an animal enjoying acquired immunity. They may act upon the micro-organisms which become permeated by the fixative substance, or upon the infected animal by stimulating its defensive reaction, but they are incapable of affecting the vitality or virulence of the micro-organism. The two properties (protective and fixative) reside in the fluids of the body, but they are functions of the cell products. The elements of the phagocytic organs (spleen, bone-marrow, lymphatic glands), or phagocytes, produce the specific protective and fixative substances which pass thence into the plasmas.

The phagocytic reaction is very general in acquired immunity. The phagocytes which have a very imperfect antimicrobial function or none at all, become, as the result of vaccination, much more active. They exhibit a very marked positive chemiotaxis and acquire the faculty of digesting micro-organisms in a greatly intensified degree. It is with the increase of this digestive power that we have connected the over-production by the phagocytes of the fixative and protective substances which are excreted in large quantities by these cells and pass into the fluids of the animal. As these substances are phagocytic products it may be readily conceived that in certain examples of acquired immunity the animal overcomes the micro-organisms without the protective substances being found in the fluids. It is sufficient that it is in the possession of the phagocytes, which may retain it within themselves and not throw it off into the circulation.

[Sidenote: [311]]

From this account it will be seen that the phenomena, in acquired immunity against micro-organisms, are merely a more or less stereotyped copy of those that are presented in the animal after the resorption of cells. There, also, we have intracellular digestion with over-production of specific fixatives, part of which are excreted and thus pass into the plasmas. In the resorption of cells there is also a double action of cytases and fixatives; but in this case the macrocytases intervene, whilst in the resorption of micro-organisms this function is performed by the microcytases. The fixatives in the two cases are very different from the point of view of their action, for they are specific; but the cells which act in their production belong, in both cases (resorption of animal cells and of micro-organisms), to the category of phagocytes.

It is often maintained that the theory I have just summarised is fundamentally opposed to the theory of side-chains or receptors formulated by Ehrlich[462]. This view I cannot accept. Applied to acquired immunity against micro-organisms this theory may be summed up as follows. The micro-organisms, when inoculated in a non-lethal but immunising dose, combine with certain cells of the animal. The receptors of the micro-organisms find corresponding receptors in these cells, but, when once combined, the receptors of the cells become incapable of fulfilling their normal nutritive function. The cells, thus deprived of their receptors, reproduce such an enormous quantity of them that a portion is excreted into the surrounding medium and passes into the plasmas. These receptors, originating from cells, but which have become constituent parts of the body fluids, are nothing but the fixatives or intermediary bodies, or the amboceptors of Ehrlich. On a fresh arrival of the same micro-organisms, they meet with, in the fluid of the exudations, numerous amboceptors which combine with the corresponding receptors of the micro-organisms, without, however, destroying them or interfering with their vitality. As these amboceptors possess still a second affinity, that for the molecules of the cytases, or the “complements” of Ehrlich, the micro-organisms can be placed in contact with these soluble ferments. Without the intervention of the fixatives, the combination of the body of micro-organisms with the cytase can never take place, because the receptors of the micro-organisms are not adapted to those of the cytases. When the molecules of these ferments are found in the plasmas in a free state, they can be attacked by the corresponding group of the amboceptors.

[Sidenote: [312]]

Let us compare the theory we have just sketched with that described further back. The micro-organisms, inoculated with a non-lethal but immunising dose, are, as we have seen, ingested by the phagocytes and afterwards digested within them. This intracellular digestion is followed by the over-production of the specific fixative, of which a part is excreted and passes into the plasmas. These are the results of the well-established experimental data described in this chapter. Ehrlich’s theory is in no way in opposition to this; it simply attempts to penetrate more deeply into the mechanism of the phenomena observed as taking place between the micro-organism and the cell. The act which we simply term intracellular digestion is divided by Ehrlich into its constituent parts. According to him there is a combination of the fixative, on the one hand, with the molecule of the micro-organism, on the other, with that of the soluble ferment or cytase. According to Ehrlich it is the amboceptors of the cells which become detached in order to furnish the fixative that circulates in the plasmas. For us there is simply an over-production of one of the two ferments of intracellular digestion, without defining more exactly what constituent part of this ferment passes into the circulation. The two theories may supplement each other but are in no way contradictory in principle. There is only a single important point wherein they do not accord. Ehrlich thinks that the cytases are always free in the body fluids and that the cells, in order to exert a digestive action on the micro-organisms, must previously seize their molecules by means of one of the groups of their amboceptors. We, on the contrary, have developed the idea that the cytases are only free in the animal during phagolysis and that under normal conditions the cytases remain closely bound up with the phagocytes. This statement is based upon a large number of well-established experimental facts and must therefore be accepted as proved. It does not, however, affect any fundamental principle of Ehrlich’s theory. On the other hand the bases of Ehrlich’s theory affect none of the main features of the theory I have developed. The doctrine which regards acquired immunity as a particular case of resorption may be reconciled with the conception of amboceptors. But it accords equally well with Bordet’s conception, according to which the fixatives act not as intermediary substances between the micro-organism and the cytase, but as substances which sensitise the micro-organisms for the penetration of the digestive ferment. This delicate question has not yet been definitely settled, but Bordet’s experiments described in Chapter IV are greatly in favour of this view.

[Sidenote: [313]]

Neisser and Wechsberg[463] have tried to obtain some idea of the manner in which the fixatives act on the micro-organisms and have recorded a series of very interesting facts. They have shown that these substances only bring about the destruction of bacteria when they are in certain relations with the cytase. Mixtures of fixatives and cytases in which the former are found in excess not only do not kill the micro-organisms but even allow them to develop abundantly. To attain this result Neisser and Wechsberg mixed constant quantities of bacteria and normal serum containing cytase with variable quantities of the serum of immunised animals heated to 56° C. As we know, this specific serum, as the result of being thus heated, is deprived of its cytases, but may be readily made active again by the addition of normal, unheated serum. This paradoxical fact, demonstrated by Neisser and Wechsberg can, in their opinion, be explained only by Ehrlich’s theory of amboceptors. When these bodies with double affinities are found in too large quantity as regards the cytase, it may happen that one part only of those which combine with the receptors of the micro-organisms succeed in linking to themselves the molecules of the active ferment. The amboceptor being by itself incapable of destroying the micro-organism, can be injurious to it only on condition that it brings cytase. Consequently as the amount of this cytase is too small for the much larger number of amboceptors we can readily conceive that the micro-organisms may profit thereby and remain alive. This interpretation is certainly very ingenious, but nothing proves that it corresponds with the real state of things. Neisser and Wechsberg have themselves observed that the serum of the normal goat can also prevent the bactericidal action of the cytase. In this case, however, they suggest the intervention of an anticytase of this normal serum. The same explanation might perhaps serve also to explain the preventive action of the serum of immunised animals. We know that anticytases are found frequently enough in the various serums and that they undergo great variations, according to the conditions present in the animals furnishing the blood.

[Sidenote: [314]]

In any case, it is evident that the theory of receptors must in no way be regarded as the antithesis of the theory of phagocytosis. This latter quite retains its right to affirm that, in acquired immunity against micro-organisms, phagocytes play the most general and important part. They hold back the cytases which are capable of ridding the animal of micro-organisms from destroying them. It is further these same cells that produce and excrete the fixative and protective substances. The free fixatives may attack the micro-organisms in the body fluids but they are incapable of depriving them of life or even of virulence. The cytases, after escaping from the phagocytes, may certainly, in collaboration with the fixatives, destroy a certain number of the micro-organisms, but only in special cases met with, no doubt, but only rarely, under natural conditions. On the other hand, the phagocytes in the animal which enjoys acquired immunity constantly fulfil the function of seizing the micro-organisms and of submitting them in their interior to the combined action of fixatives and cytases.

Acquired immunity, like natural immunity against micro-organisms, presents merely special phases of intracellular digestion.