These experiments prove clearly that, in the action of the antitoxin on the toxin, there can no longer be any question of an actual destruction of the latter, a view which has been accepted by both von Behring and Ehrlich. But, as pointed out by Roux at the International Congress at Budapest in 1894, the manifestation of the toxic action of the venom after it has been heated along with antitoxin, may be reconciled with the view that the combination between the two substances, if such take place, must be very unstable. This same remark may be applied to Wassermann’s experiment. Therefore the great majority of observers, if not all, admit that the antitoxin combines with the toxin to form an innocuous and unstable substance which can be decomposed by heat and by other agents. The researches on the action of antitoxins in vitro have had a powerful influence in determining this view.
We have already in Denys and van de Velde’s[569] experiments an indication of the direct action of certain antitoxins. These observers showed that the serum of animals vaccinated against a Staphylococcus is capable of neutralising in vitro a particular toxin to which van de Velde gave the name of leucocidin. When it was added to a drop of the exudation from a rabbit, this leucocidin in a very short time destroyed the white corpuscles, by dissolving the cell content but leaving the nucleus untouched. When Denys and van de Velde prepared mixtures of leucocytes, leucocidin and antileucocidic serum in vitro, the white corpuscles retained their normal condition for a very long time. The leucocidin was, therefore, rendered inactive by the direct influence of the corresponding antitoxin. These facts have been confirmed by Bail[570] and other observers and even extended to certain other microbial toxins. Thus, the Bacillus pyocyaneus produces a leucocidin which kills the white corpuscles and dissolves their contents[571]. With the object of facilitating experiments with these leucocytic poisons and the corresponding antitoxic serums, Neisser and Wechsberg[572], of the Institute of Experimental Therapeutics at Frankfort, invented a method which allows us to observe the phenomena of the destruction of the leucocytes and of the antitoxic power in test tubes, without having recourse to a microscopical examination. They applied the fact, discovered by Ehrlich, that living formed elements reduce methylene blue and, depriving it of its oxygen, decolorise it. Leucocytes from aseptic exudations are introduced into tubes and a weak solution (2%) of methylene blue is poured on them. To prevent the re-oxidation of this colouringmatter by the oxygen of the air, the surface of the fluid is covered with a layer of liquid paraffin. If the leucocytes are living, the lower blue layer becomes decolorised in a short time (in about two hours); when the corpuscles are dead, decoloration does not take place. By adding to the mixture of leucocytes and colouring matter some leucocidin, alone or along with antileucocidic serum, it is possible not only to observe with the naked eye the phenomena which take place in these cases, but also to estimate to some extent the proportions of poison and counterpoison.
All these researches make it clear that the antitoxin acts directly on the leucocidin. Similar facts have been noted as regards certain other organic poisons and their antitoxins. Shortly after the discovery of antileucocidin by Denys and van de Velde, Kanthack made a communication to the Physiological Society in 1896[573], exhibiting tubes in which the coagulating action of Cobra venom on the blood had been prevented by the addition of antivenomous serum. Of all the experiments, however, made to prove the direct action of antitoxin on toxin, Ehrlich’s[574] have played the most important part in the study of this question. Ehrlich directed his attention to ricin which, as Kobert demonstrated, has the property of agglutinating the red corpuscles of defibrinated blood. This phenomenon can be easily observed in vitro. In tubes containing red blood corpuscles, the addition of ricin causes these corpuscles to agglutinate into clumps and to fall to the bottom of the tube, leaving a clear supernatant fluid. After adding progressively increasing quantities of antiricic serum to the tubes containing fluid blood and ricin, Ehrlich was able to demonstrate that small quantities of antiricin merely retarded the precipitation of the red corpuscles, whilst larger doses completely prevented it. Having studied the proportions of ricin and its antidote, necessary to retard and prevent the fatal poisoning of animals, Ehrlich was struck by the parallelism which is exhibited between the action of the antitoxin in the living animal and that in the test tubes.
The study of anticytotoxins, discussed in the fifth chapter, has furnished another opportunity of observing the action of antitoxins in vitro. Camus and Gley and H. Kossel were the first to observe the action in vitro of antitoxic serum against the ichthyotoxin of eel’s serum. Since this observation, this phenomenon has been repeatedly studied in the antihaemolysins and antispermotoxins. The antidiastatic serums also act in vitro and, as their effect can be demonstrated on soluble ferments placed in contact with unorganised bodies, such as gelatine and casein, the purely chemical character of the reaction is all the more strikingly shown. We are indebted to von Dungern, Briot and Morgenroth for accurate observations on this subject.
Martin and Cherry[575] made use of a different method to demonstrate the direct action of antitoxins on toxins which exhibit their toxic power on the animal organism. They chose snake venom mixed with antivenomous serum. The mixtures were filtered under great pressure [50 atmospheres] through a film of gelatine, under the idea that, if the venom and antitoxin were not chemically combined, the former alone, owing to its much smaller molecules as compared with those of the antivenom, would pass into the filtered fluid. This fluid should, under these conditions, possess a toxic power for animals, when the mixture, used for filtration, was deprived of the larger molecules. Martin and Cherry left the venom and the antitoxic serum in contact for periods of varying length, before filtering the mixtures. As the result of a series of such experiments carried out according to this scheme, they found that the product of the filtration made after some minutes’ contact between the two substances, was distinctly toxic; whilst the filtrate obtained after a contact of half-an-hour was absolutely innocuous. From their observations these authors conclude that the antitoxin enters into chemical combination with the venom, but that the combination does not take place instantaneously, a certain amount of time being necessary for its accomplishment.
In addition to the time factor others have an influence on the combination between toxins and antitoxins, as is seen from Ehrlich’s[576] and Knorr’s[577] investigations. Both observers have shown that antitoxin neutralises the toxin more slowly in dilute solutions than in more concentrated form. For this reason, when animals are injected with very weak solutions, the toxin may manifest its action before it can be neutralised by the antitoxin; this may lead to erroneous conclusions. On the other hand, according to data furnished by these authors, temperature also exerts an influence on the combination. Lowering the temperature retards, whilst raising it accelerates the neutralisation of the toxins by the antitoxins. Insisting on the purely chemical character of the combination between these two substances, Ehrlich and Knorr adduce the fact that this combination, in cases where we have a complete neutralisation of the toxin, follows, most rigorously, the law of multiple doses, that is to say, in order to render innocuous a hundred doses of toxin we have only to take a hundred times the quantity of antitoxin.
The series of facts summarised above demonstrate distinctly that antitoxins act directly on toxins. But how can this result be reconciled with the observations given above according to which must be admitted the no less real influence of the organism of the living animal on intoxication by mixtures of antitoxin with toxin? Knorr[578] sought at first to minimise the importance of the facts brought forward by Buchner and Roux. He failed to corroborate Buchner’s results and found that the injection of mixtures, made with very large doses of tetanus toxin (20,000 times the minimal lethal dose) and corresponding quantities of antitetanus serum, brought about the same effect in guinea-pigs and mice. By modifying the quantity of antitoxin, he rendered the mixture equally innocuous or equally toxic for these two species. But the data given by Knorr are quite sufficient to prevent us from accepting his conclusion. In his experiments, as in those of Buchner, the guinea-pigs manifested a greater susceptibility and died from mixtures which, in mice, caused merely a tetanus of medium intensity.
Some have sought to explain Buchner’s experiment by assuming that the mixtures, lethal for the guinea-pig and innocuous for the mouse, owed their toxic action to the presence of the tetanus toxone and not of the true tetanus poison, the tetanospasmin. This hypothesis of toxones, as stated above, was put forward by Ehrlich as the outcome of his ingenious researches on the constitution of the diphtheria poison. As, however, the toxones must act differently from the toxins, we can only attribute to their action the results in those cases where the guinea-pigs die without presenting typical symptoms of true tetanus, that is to say without spasms. Now, in Buchner’s experiments, a much larger proportion of these animals, injected with the same mixtures as the mice, succumbed and exhibited the characteristic tetanic convulsions. Even in those cases, however, where the death of the guinea-pigs might be attributable to an intoxication by the toxone, the general result could not be altered. The toxones are, according to Ehrlich, manufactured by the micro-organisms in the culture media and form an integral part of the natural microbial poisons. Again, they are neutralised by antitoxic serums. If, therefore, in spite of there being the same quantity of toxones and of antitoxin in the mixtures, these mixtures become more toxic for the guinea-pig than for the mouse, we have an indication that some special change must take place in the animal to upset the conditions of toxicity.
Weigert[579] accepts the accuracy of Buchner’s experiment, which, indeed, can no longer be denied, but explains it on the hypothesis that there is some substance in the animal possessing a very great affinity for the toxin. This substance is supposed to be capable of decomposing the innocuous combination of the antitoxin with the toxin, just as heat does in Calmette’s and Wassermann’s experiments, described above. In both cases the toxin would be set free to exert its noxious action. Such a hypothesis is very probable, because it agrees with direct observation, but it compels us to accept some new phenomenon which is produced not in vitro, but in the living animal, and which carries on its work in a very different fashion in the guinea-pig and in the mouse.
In the present imperfect state of our knowledge it is very difficult to form any idea of the precise conditions which must intervene in the organism of the guinea-pig to cause the tetanus toxin to act in a mixture with antitoxin which is much more innocuous for the mouse. In order, however, to satisfy those who seek to understand these complex phenomena, it may be useful to cite another example of antitoxic action in which certain factors are distinguished by their simplicity.
Lang, Heymans and Masoin[580] have demonstrated that hyposulphite of soda prevents poisoning by prussic acid. This terrible poison becomes innocuous if we take care to introduce into the animal by any channel whatever (subcutaneously, intravenously, or by the stomach) a sufficient quantity of hyposulphite of soda. Under these conditions the sulphite is substituted for the hydrogen of the prussic acid, transforming the poison into sulphocyanic acid, which has no action on the organism. The hyposulphite of soda, then, acts as the antitoxin of the prussic acid, thanks to a chemical reaction of substitution between bodies of simple composition. We have never yet succeeded in reproducing this reaction in vitro, whilst in the animal body it is effected with very great ease. Consequently, we are quite justified in invoking special conditions in the body of the living animal; this, however, does not preclude the possibility of a transformation of the toxic substance into an innocuous substance through a chemical reaction. It is probable that analogous phenomena may also be met with in the action of true antitoxins on the microbial toxins or allied substances (venoms, vegetable toxalbumins).
The case of the destruction of micro-organisms, which is now more easily studied because it is possible to observe with the eye the fate of these organisms in the animal, is a further source of valuable information. The direct action of cytases on certain bacteria, such as the cholera vibrio, can be just as easily demonstrated in vitro as can the action of antiricin on ricin. If we proceeded to argue from this, a perfectly accurate observation, that the living animal plays no part in the destruction of the micro-organisms and that this destruction takes place always in a fashion analogous to Pfeiffer’s phenomenon in vitro, we should undoubtedly arrive at an erroneous conclusion. We know already, as has been indicated in previous chapters, that the granular transformation of vibrios is only part of a whole series of phenomena of destruction of micro-organisms, the great majority of which phenomena require more or less active intervention of the animal organism. In reality, matters usually go on in a very complicated fashion, in which direct and indirect actions are blended in varied proportions. In the examples described elsewhere, we see, alongside the granular transformation, an agglutination into clumps and immobilisation, and an ingestion and intracellular destruction of micro-organisms. The final phase, no doubt, is always a chemical or physico-chemical action, exerted against the micro-organism, but how varied are the means used to bring about this result! We may surely be allowed to suppose that analogous phenomena may take place in the action of antitoxins on the toxins.
Just as, in the analysis of the influence of serums on the micro-organisms, it was found useful to study the action of certain fluids less complicated than the anti-infective specific serums, so we may utilise information furnished by the antitoxic action of fluids other than the true antitoxins. Cases are by no means rare in which normal serums exert a certain influence on toxins. Thus, Pfeiffer[581] noted that the normal blood serum of the goat has the power to prevent fatal poisoning by the cholera toxin. Freund, Grosz and Jelinek[582] observed an analogous action of solutions of nucleohiston on diphtheria intoxication and Kondratieff[583] demonstrated the same action of an extract of the spleen on the tetanus poison. Calmette[584], in collaboration with Deléarde, studied the influence of a whole series of fluids on abrin intoxication. Whilst physiological saline solution was absolutely incapable of preventing the death of animals, fresh broth exerted an undoubted antitoxic power. Amongst normal serums, ox serum exhibited a certain antirabic property. More, however, than the serums of normal animals, have those of animals immunised against various toxins other than abrin (antitetanus, antidiphtheria, antivenomous serums, &c.) been found to possess the power of preventing intoxication by abrin. These facts are connected with others of analogous nature, previously demonstrated by Calmette[585], of which I may cite the following: the serum of animals vaccinated against tetanus toxin is active, though to a less degree, against snake venom; the serum of rabbits vaccinated against rabies, a serum powerless to protect against this disease, is, however, very markedly effective against the same venom; the serum of animals immunised against snake venom is also antitoxic against scorpion venom (I have myself had the opportunity of confirming this fact on several occasions). In all these examples, the serums have proved to be less efficacious against poisons other than the toxin with which the animals that furnished the blood had been treated. Ehrlich[586], too, has demonstrated that animals vaccinated against robin (toxalbumin of Robinia pseudacacia) produce a serum, antitoxic not only against this poison but also against ricin. It need scarcely be added that in all these cases of non-specific action of serums derived from vaccinated animals, no question of any antitoxic effect of normal serums can enter. In all the experiments just summarised, the serums of normal animals, used as controls, were found to be inefficacious.
If, in the case of the non-specific action of serums, it were allowable to advance the hypothesis of a direct influence of these fluids on the toxins, it would still be impossible to sustain this view where broth fulfils the antitoxic rôle. This fluid, much simpler in composition than any serum, is an excellent culture medium for micro-organisms and one in which the toxins develop well and can be kept for a fairly long period. There is, therefore, not the slightest ground for assigning to it any direct antitoxic action, on the contrary, everything leads us to regard it as an indirect agent, which acts by stimulating the reaction of the animal organism. Here, then, the case would be quite analogous to that of the action of broth as a protective agent against certain bacterial injections, a subject already discussed in the tenth chapter. In this same category of indirect influences also, must be ranked the example of the antitoxic action of the blood of the crayfish against scorpion venom. I have demonstrated in a series of experiments that the fresh blood of the crayfish has the power to prevent fatal intoxication of mice by scorpion venom. Injected in a dose of from 1 to 1·25 c.c., several minutes or an hour before the injection of the rapidly fatal dose of scorpion venom, the crayfish’s blood exerts a very distinct preventive action. It might be supposed from this that the crayfish belongs to the group of animals insusceptible to scorpion venom. This, however, is not the case. The crayfish is very susceptible to this poison and succumbs to a quarter the dose necessary to kill a mouse. The blood of the crayfish is, therefore, completely ineffective as a protective to the crayfish itself, and only exerts its action when introduced into the body of the mouse. It might be concluded that it is only after it has been drawn from the crayfish that the blood acquires its antitoxic power. Experiment contradicts this supposition. Crayfish blood, when injected into another crayfish, in equal or greater amount than is necessary to protect a mouse, is incapable of preventing fatal intoxication by scorpion venom, although, here again, the crayfish received only one-quarter of the dose of venom used for the mice.
We are, therefore, compelled to believe that the crayfish’s blood is antitoxic for the mouse, not in virtue of its direct neutralising action on the venom, but owing to some indirect influence on the organism of the mouse. It is impossible to define, exactly, the mechanism of this action. We may suppose that the blood of the crayfish contains some substance which, by itself, is insufficient to prevent the intoxication, but which becomes active in the presence of some other substance, also inefficacious by itself, met with in the organism of the mouse. Here we should have something analogous to what is met with in immunity against micro-organisms where both fixatives and cytases intervene to bring about the destruction of micro-organisms. By making researches in vitro on the action of the fluids on bacteria, we may easily observe certain phenomena which appear to indicate their direct influence. Take the case of the fluid of an oedema from an animal vaccinated against the cholera vibrio which renders this micro-organism motionless and agglutinates it in vitro; the oedema of an unvaccinated animal produces no such effect. If, however, we were to conclude from this fact that, in the oedema of the living animal or in its subcutaneous tissue, everything goes on as in the test-tube and that no other phenomenon of reaction against the vibrios is produced, we should fall into a grave error. It is extremely probable that, in the resistance of the living animal against the toxins, the phenomena are more complicated than are those observed in vitro. The example of the blood of the crayfish which prevents the poisoning of the mouse, without having any influence on that of the crayfish itself, may here serve as a guide to us. It is possible that, as in the struggle against the micro-organisms, we have here a co-operation of two substances, each one of which, by itself, is inactive. One of these substances would be found pre-existent in the blood of the crayfish, the other forming part of the organism of the mouse. Perhaps the action of this blood is even more complicated and only becomes active through the mediation of some constituent of the living cell.
Our study of immunity against toxins long ago revealed cases in which this resistance cannot be attributed simply to the antitoxic action of the body fluids. Animals vaccinated against living micro-organisms may succumb to infection in spite of the presence of a strong anti-infective power of the body fluids; similarly animals immunised against toxins may die from intoxication in spite of the antitoxins contained in their fluids. Facts of this order are not rare. Roux and Vaillard[587] on several occasions observed animals which died from tetanus although they had a large supply of antitoxin in their blood. Von Behring[588] and his collaborators, Knorr, Ransom, and Kitashima, also collected a large number of analogous facts. They showed that horses that have been treated for a long time with tetanus toxin and whose blood serum is very antitoxic, still experience marked illness after fresh injections of toxin and may even succumb, in spite of the presence of a large amount of antitoxin in their blood. In these cases the morbid phenomena are undoubtedly different from those typical of tetanus. Instead of the muscular contractions which characterise this disease, the above observers noted disturbance in the regulation of the body temperature, exudative inflammation around the point of inoculation, impairment of appetite and fall of body weight. Sometimes they observed muscular tremors and marked feebleness in the movements. These symptoms differing from those of typical tetanus, it may be asked whether this poisoning is not due to special substances other than tetanus toxin in the fluids injected. Von Behring does not think that this is the case, for he found that by adding antitetanus serum the formation of exudations at the seat of inoculation was suppressed. These exudations, then, must be attributed to the tetanus toxin.
In the cases where animals immunised against diphtheria toxin fall ill and even die as the result of fresh injections of toxin, in spite of the presence of a large quantity of antitoxin in their blood, we might also cast doubts on the diphtheritic character of the poisoning, because the clinical picture of this poisoning is not a very typical one. At the Pasteur Institute, where a large supply of antidiphtheria serum is prepared, we see, from time to time, horses, which have long been undergoing the process of immunisation and are furnishing a very good serum, suddenly fall ill and die from intoxication, without presenting any symptom of infective disease. On one occasion, there was actually quite a small epidemic of fatal poisonings as the result of the injection of a quantity of diphtheria toxin not exceeding the doses which had been well borne previously. Amongst the horses, inoculated with the same toxin, five of the best furnishers of serum died. The others, some of which were producing only a weak serum, remained unaffected.
Von Behring and Kitashima[589] have given a detailed history of a young horse which had become very susceptible as the result of vaccination with diphtheria toxin. It finally succumbed to the intoxication in spite of the presence of diphtheria antitoxin in its blood.
If, in these examples, we have any reason to doubt the specific nature of the intoxication, all doubt must give way before the case described by Brieger[590]. One of his goats, well immunised with tetanus toxin, which, for months, had furnished a good serum and even an antitetanus milk, after an injection, stronger than the preceding ones, was seized with tetanic contractions. These, becoming general, brought about the death of the animal with the symptoms of classic tetanus. The blood, drawn off after death, exhibited strong antitoxic power.
As the result of these observations von Behring formulated the theory of a hypersusceptibility acquired during immunisation. “Paradoxical as it may appear,” he writes[591], “there can no longer exist any doubt that horses which have acquired a high immunity as the result of treatment with tetanus toxin, present a histogenic hypersusceptibility of the organs which react against the tetanus toxin.” In support of this thesis von Behring compares the effect produced by this toxin on horses immunised with this same poison and on normal horses treated with antitoxic serum from other horses. The former, in spite of the fact that they contain in their blood 1,500 times more antitoxin than do the latter, are, nevertheless, less refractory to tetanus toxin. This feeble resistance is due, in von Behring’s opinion, to the much greater susceptibility of the living elements in the horses treated with repeated doses of the poison.
Von Behring’s theory of this form of acquired specific hypersusceptibility has been confirmed by several well-observed facts. These show that, in the animal subjected to treatment by toxins, phenomena of very diverse order are evolved simultaneously: on the one hand, cell reactions which bring about the production of antitoxins; on the other, an increase in the susceptibility of some of the living elements to the specific poison. We are, however, justified in asking if the great difference between the immunity of animals treated with toxin, and that of others treated with antitoxic serum, can be altogether attributed to this hypersusceptibility?
Let us examine in a little more detail some examples of this hypersusceptibility. We know that the guinea-pig is characterised by its great natural susceptibility to the toxins of tetanus and diphtheria. Small doses of these poisons are quite sufficient to produce in it a fatal intoxication. But it is possible to diminish greatly this feeble resistance of the guinea-pig by frequent injections of very small quantities of toxin. Knorr[592] increased their susceptibility to tetanus toxin by daily injections of one-tenth of a minimal lethal dose. The animals died before they had received the ten tenths of this dose. The hypersusceptibility produced under these conditions might be so great that one-fiftieth of the minimal lethal dose was capable of causing death. From these facts we can understand the great difficulty experienced in the earlier attempts to vaccinate guinea-pigs by means of unmodified toxin.
Von Behring and Kitashima[593] made analogous researches on the susceptibility of guinea-pigs to diphtheria toxin. By frequent injections of very small doses of this poison they succeeded in killing these animals with ¹⁄₄₀₀ of the minimal lethal dose distributed over several injections. They never succeeded in vaccinating guinea-pigs with increasing doses of pure diphtheria toxin. Their animals died even when they commenced with one-millionth of the minimal lethal dose.
Here, then, we have examples of the greatest hypersusceptibility that it is possible to observe. When we compare it with the changes in the antitoxic power of the blood, we find that these are even more marked. Thus, Salomonsen and Madsen’s horse, to which we have already referred, presented extraordinary oscillations in this power. After receiving, during the course of immunisation, a fresh dose of diphtheria toxin, the antitoxic value of its blood suddenly fell more than one-third (35%). In order to neutralise, completely, this dose of toxin, when injected into a normal animal mixed with antitoxic serum from this same horse, a very small quantity of the blood of the latter would have been sufficient. The injection into the immunised horse should have passed unperceived, as this animal contained in its body more than 50 litres of strongly antitoxic blood. Nevertheless the antitoxic power of this blood fell 12,000 times more than it ought to have fallen according to the calculation made upon the data just indicated. This fall is incomparably greater than the increase of susceptibility to toxin in the most significant examples reproduced above.
As the fact above cited is not at all unique, it is probable that the phenomena which appear in the animal subjected to vaccination by toxins, must be much more complicated than is usually supposed. If the fresh injections of these poisons bring about a specific hypersensitiveness on the one hand, and on the other a great fall in antitoxic power, followed by its still more notable augmentation, it is evident that the introduction of toxins must give rise to a great perturbation in the cell functions. The general analogy between acquired immunity against micro-organisms and against toxins probably rests on similar bases. Kretz[594] has already advanced the hypothesis that, in antitoxic action, two factors, comparable to the cytases and fixatives in the antimicrobial action, co-operate. In the absence of one of these elements we can understand that the one which remains may be incapable of bringing about the neutralisation of the toxin. For this reason the antitoxic serum may act very differently in the organism of the animal which produces it and in that of a normal animal which receives it. An explanation which is adequate for the antitoxic action of the blood of the crayfish injected into mice serves equally well in the case of the antitoxic influence of the serums of animals which themselves succumb to intoxication.
Wassermann’s[595] experiments on the anticytase serums might appear to supply an argument against the hypothesis we are defending. Having shown that animals injected with antityphoid serum die of intoxication when serum which prevents the action of the cytases is introduced simultaneously, Wassermann put the question: May not the action of the antitoxins be prevented by this same anticytase serum? To solve this point he injected into guinea-pigs a mixture of antidiphtheria serum with toxin in excess and a fairly strong dose (3 c.c.) of anticytase serum, upon which we have already spoken (see Chapter VII). The animals, so treated, behaved exactly as did the animals used for control which received the same quantities of antitoxin and toxin but without the addition of anticytase serum. Wassermann concludes from these experiments that the exclusion of the cytase, contrary to what takes place with antimicrobial serums, in no way impedes the action of the antitoxins. This conclusion, which appears at first sight to be justified, cannot, however, be accepted, as the two examples chosen by Wassermann, typhoid infection and diphtheria intoxication, differ very profoundly from each other. In the former, we have an experimental typhoid peritonitis which kills the control animals in less than 24 hours, whilst the second is diphtheria in which the controls do not succumb until the sixth day after injection. The effect of the anticytase serum being only very transitory, it is quite natural that this should manifest itself in an infection of short duration and should not do so in a slow intoxication. Besides, Wassermann himself has shown that in several other cases of immunity against micro-organisms (the bacilli of influenza and of leprosy) the injection of his anticytase serums does not interfere with the perfect resistance of the animals. But even were it demonstrated that the cytases really play no part in immunity against toxins, the intervention of some other similar factor could always be evoked.
The analogy between immunity against micro-organisms and that against toxins may facilitate the study of the relations between the latter and the antitoxic power of the body fluids. In the preceding chapters we have described examples in which animals possess a protective power in their blood but are not refractory to the corresponding infection; on the other hand, we have cited cases in which acquired antimicrobial immunity exists without the blood presenting any appreciable protective power. The idea of measuring acquired immunity against micro-organisms by the measurement of the protective or agglutinative power of the blood must therefore be abandoned, and it is impossible to regard immunity against toxins as a function of the antitoxic property of the body fluids. As we have seen, animals completely refractory to tetanus, such as the cayman, whose immunity does not depend on the antitetanic power of the blood, develop antitoxin after the injection of toxin. A similar state of affairs, but less pronounced, has been demonstrated by Vaillard as occurring in the fowl. The fowl, in spite of its very marked natural immunity against tetanus, produces antitetanin as the result of the introduction into its body of tetanus toxin; the rabbit, on the other hand, a susceptible animal, may acquire a real immunity without the development of any antitoxic power in its fluids. An additional fact was noted by Vaillard[596]. He showed that the repeated inoculation of tetanus spores along with a small quantity of lactic acid, made below the skin of the tail of rabbits procured for them an immunity against tetanus toxin, although no antitoxic property appeared in their blood. In his experiments, one hundred volumes of blood serum were found to be incapable of neutralising a single minimal lethal dose of the toxin. The rabbit, however, still remains quite capable of developing antitetanic power in its fluids. All that is necessary is to inject into it some tetanus toxin heated to 60° C. or treated with Lugol’s iodo-ioduretted solution. As the outcome of his researches Vaillard concludes that the antitoxic property of the body fluids “is not sufficient ... for the general interpretation of acquired immunity, as it cannot be demonstrated in all animals which have become refractory.”
The facts I have just mentioned were demonstrated early in our study of the antitoxic power of the animal organism. Since then a large number of analogous data have been collected. Recently, von Behring and Kitashima[597] have had to abandon the immunisation of monkeys against diphtheria toxin because of the poor yield in antitoxin which they obtained. The blood of one of their monkeys that had acquired a resisting power against very large doses of diphtheria toxin showed only a very moderate antitoxic power. In establishments where antitoxic serums are prepared on a large scale the workers have become convinced that the yield of antitoxin has no direct constant ratio to the immunity of the animal. This has been demonstrated repeatedly at the stables of the Pasteur Institute. Thus, of two horses, treated at the same time and in exactly the same way with diphtheria toxin, one furnished a very good antitoxic serum which was maintained at 200 units Ehrlich, rising up to 400 units, whilst the other never reached 150 units[598]. And yet both these animals possessed the same immunity against diphtheria toxin. They tolerate considerable doses of toxin and react merely by a slight or insignificant rise in temperature. In another series of horses, which have been immunised for nearly seven years, one remained capable of yielding a large quantity of antitoxin, seeing that the value of its serum oscillated between 200 and 300 units. After five years of this state of things the antitoxic power began to fall considerably, without, however, any corresponding loss of immunity. Indeed, an injection of 250 c.c. of toxin (of which 0·002 c.c. was sufficient to kill a guinea-pig) began, at the commencement of the present year, to be borne without the least febrile reaction. An attempt was made to raise the antitoxic power of the blood by making intravenous injections of toxin and of diphtheria culture, but in vain. The yield of antitoxin continued to fall and it became necessary to employ this horse for another purpose than the preparation of antidiphtheria serum. This is by no means an isolated example. Of a large number of treated horses it frequently happens that certain individuals, without being particularly susceptible to a given toxin, are found to be incapable of producing any corresponding antitoxin[599].
In presence of the fact that animals very resistant to toxins may possess no, or only an insignificant antitoxic power in their fluids, and that, on the other hand, animals in which this property is highly developed may succumb to intoxication, it may be readily understood that immunity against toxins and the antitoxic power of the body fluids may be two distinct conditions. Von Behring has clearly demonstrated the fact of the cellular hypersensitiveness of the animal immunised against the corresponding toxin and has laid great stress upon this fact. He came[600] to the conclusion that “the immunity of the tissues and the production of antitoxin follow a parallel course in their development so slightly that, in spite of an abundant accumulation of antitoxin, the susceptibility of the elements of the tissues may increase in an extraordinary fashion.” If, during the course of immunisation, this susceptibility can increase so greatly, it is probable à priori that under certain circumstances it might also diminish notably. After demonstrating “that in time the antitoxin disappears from the blood of animals immunised with toxins without any consequent disappearance of immunity,” von Behring formulated the conclusion that in these animals “the living elements of the animal, which were previously susceptible to the poisons, have acquired an insusceptibility towards the same substances.” This result fully accords with the facts of the change of the negative chemiotaxis of phagocytes into positive chemiotaxis for micro-organisms during the acquisition of anti-infective immunity.
Later, von Behring[601] changed his opinion. Whilst still accepting the change of cellular susceptibility in the direction of hypersensitiveness in animals immunised against toxins, he refused to admit the change in the opposite direction. The cells, according to him, never lose any of their susceptibility, so that acquired immunity against toxins cannot be obtained otherwise than by means of antitoxins capable of neutralising the poison in a susceptible or hypersusceptible animal. This new theory von Behring upheld in several papers and it is met with in his most recent publications. Nevertheless, certain well-established facts compel us to accept an immunity against toxins as coming about as the result of a diminution of the susceptibility of the vaccinated animal. Parallel with his researches on the increase of the susceptibility of guinea-pigs to tetanus toxin, researches discussed above, Knorr[602] describes analogous experiments on rabbits. When these animals are injected with fractions of the minimal lethal dose, frequently repeated, the rabbit not only does not become hypersusceptible to tetanus but exhibits a greater and greater insusceptibility. Whilst guinea-pigs, treated according to this method, die from tetanus before they have reached the minimal lethal dose, rabbits, as the result of frequent injections of small quantities of tetanus toxin, become capable of resisting five times the lethal dose (for normal rabbits) without exhibiting the slightest symptom of illness. Against the attribution of this result to the acquired insusceptibility of the living animals it might be objected that the immunity, in this case, may depend on the antitoxic power of the fluids of the body, developed with great rapidity. Such an objection cannot be raised in the case of horses which become insusceptible to toxins after a long period of vaccination. The horse whose history was given above, when discussing the diminution of antitoxic power, may serve as an example. At the commencement of its vaccinal period, in 1894, it reacted to the injection of 10 c.c. of diphtheria toxin by a rise of temperature of 1° C. Four years later, when its blood had become very antitoxic (350 units per c.c.), it was necessary to inject 350 c.c. of toxin to obtain the same rise of temperature. Quite recently, having now lost the greater part of its humoral antitoxic power, this horse exhibited no rise of temperature after an injection of 250 c.c. of strong diphtheria toxin. The diminution of the specific susceptibility is produced in this case in a most marked fashion; it is not therefore to the antitoxic property of the body fluids that this case of immunity must be attributed.
The insusceptibility acquired against poisons of different kinds is observed also in cases where the adaptation is not accompanied by the production of humoral antitoxic properties, as in the immunity of frogs against abrin. This form of immunity may be traced through the organic series down to such lowly developed organisms as the plasmodium of the Myxomycetes, which as we have seen readily becomes adapted to different poisons (see Chapter II).
It can be clearly seen, then, that immunity against toxic substances is a very complex phenomenon which it is impossible to reduce simply to an antitoxic function of the fluids of the body. For this reason we cannot accept a theory which would confine this kind of immunity within the narrow limits of a simple reaction between two substances, a reaction quite comparable to that observed in a test-tube. Attempts have been made to determine with almost mathematical precision the conditions under which it is possible to communicate to the animal a resistance against microbial toxins and formulae have been constructed to define these conditions. But the application of these formulae has been found to be a much more difficult matter. In Prussia, with the sanction of the Government, regulations have been enacted as to the procedure to be followed in the testing of antitoxic serums, and a paragraph has been added which requires a post-mortem examination of the guinea-pigs employed for this purpose in the case of diphtheria antitoxin. “The dead animals,” says this instruction, “must be submitted to a post-mortem examination, and special attention must be directed to the presence of any pre-existing diseases (tuberculosis, pseudotuberculosis, pneumonia) which may have induced hypersusceptibility in the animals under experiment.” Do we not see in this a proof of the important intervention of the organism of the living animal which may modify the results of calculations based upon too rigorous formulae? It must not be forgotten, too, that in addition to the three diseases named in the instructions, we have a number of other factors which may influence the receptivity and the resistance of animals. We have already cited Roux and Vaillard’s experiments which demonstrated that even animals which have been previously subjected to vaccinal inoculations against certain micro-organisms, exhibit a hypersusceptibility to mixtures of toxins with antitoxins.
In view, then, of this complexity of the phenomena of acquired immunity against toxins, it would be very important could we learn something of the nature and origin of antitoxins. Unfortunately, as we shall see, these questions are, as yet, far from having received a satisfactory solution.