Struck by the fact that antitoxins exert a specific action on the toxin which has been employed in the treatment of the animals that produce the serum, certain observers have sought an explanation on the hypothesis of a transformation of toxin into antitoxin. We have already seen that antitoxic action is not always absolutely specific; we have serums which prevent intoxication by various kinds of poisons, e.g. antitetanus serum, which is active against both tetanus toxin and snake venom. There is, however, a great quantitative difference between the influence of the antitoxin on the toxin with which the animals have been prepared and on a different poison. Thus, in the example just cited, in order to neutralise snake venom it is necessary to use a much larger quantity of antitetanus serum than against the toxin of tetanus. The classical example of the specific influence of antitoxins is the absolute inactivity of antidiphtheria serum against tetanus and the same non-effect of antitetanus serum against diphtheria intoxication. The most simple explanation of this specificity of action appeared to be the supposition that each antitoxin contains a part of the corresponding toxin, modified by the organism of the animal. H. Buchner^[603] advocates this hypothesis. I myself^[604] said “that it is probable that antitoxins, at least in great part, represent a modification of the toxins prepared by certain cells in the animal body; this product is then poured into the blood.” This view was stated as a “probability” and consequently contains no affirmation in the least definitive. I was, therefore, quite prepared to give it up under the weight of the crushing criticism formulated by several very distinguished observers. It was objected; first, that antitoxin is produced by animals in very great disproportion to the quantity of toxin they have received; secondly, that the animals which receive an injection of antitoxin eliminate it from their body much more rapidly than do those which prepare it in their own body; thirdly, that antitoxins are sometimes found in the blood of healthy animals, who have had no attack of the disease nor any injection of the specific toxin. Let us examine these objections more closely, objections all based on well-established facts.

It has been shown that the antitoxin produced by the animal is sufficient to neutralise a quantity of toxin much greater than that which was injected into the animals supplying the antitoxic serum. Knorr^[605], from his experiments, calculated that a horse reacts to one unit of toxin by the production of 100,000 units of antitoxin. This statement certainly does not allow us to affirm that all the antitoxin corresponds to toxin, but it does not eliminate the possibility that toxin, subjected to the influence of the cells of the animal body, may be found, in a modified form, in the product of these elements. This hypothesis would be quite sufficient to explain the very remarkable specificity of antitoxins.

If the toxin, in order to be modified by the living cells, must be subjected to some special action on the part of the latter, we can readily understand that this process must demand a greater or less length of time; this would lead to a much slower elimination of the antitoxin than in the case where it had been injected, ready prepared, into a normal animal. From the commencement of his researches on immunity against poisons, Ehrlich^[606] distinguishes two kinds of this immunity, an active immunity which is obtained as the result of the introduction of toxins into the animal, and a passive immunity, another form of the refractory condition which is set up by the injection of antitoxic serum formed in the actively immunised animal. Von Behring^[607] applies the term isopathic immunity to active immunity, and to passive immunity that of antitoxic immunity. It is generally admitted that the first kind of immunity is more slowly acquired, but that it persists for a much longer period than the second (passive or antitoxic immunity) which is acquired immediately after the introduction of the antitoxin, but which, on the other hand, lasts for a short time only. This view is supported by numerous observations on the very rapid disappearance of the refractory condition. According to von Behring the great difference in the duration of the isopathic and antitoxic immunities is only an apparent one. It is due to the fact that antitoxins are usually introduced along with the serum of different species which sets up a strong reaction and is rapidly eliminated from the animal. Thus the antitoxic serum of the horse is usually injected into small animals such as guinea-pigs, rabbits, and mice. When, however, von Behring injected horses with antitoxic serums from other horses, the antitoxic immunity lasted almost as long as in animals vaccinated with toxins. Ransom^[608] has developed this thesis in a work carried out in von Behring’s Institute at Marburg, and supports it by comparative researches which demonstrate the more rapid disappearance of the antitoxin when introduced with the serum of a different species than when introduced with that of the same species.

Even should we accept the current view on the greater duration of the antitoxic power of the blood in isopathic immunity, the hypothesis of the transformation of toxin by the cells of the animal is not necessarily invalidated. If a part of the toxin introduced into the animal remains stored for some time in an organ it is evident that only gradually can it be subjected to the transforming action of the cells. It is impossible, in the present state of our knowledge, to demonstrate this proposition, but we may invoke in its favour the prolonged persistence of red blood corpuscles when introduced into the body of a different species of animal (see Chapter IV). These corpuscles are in the end always completely digested but the process is of long duration.

The same hypothesis will also explain a fact, first demonstrated by Roux and Vaillard^[609]. They have shown that after repeated bleedings of rabbits immunised against tetanus, the antitoxic property of the blood was soon raised to almost the same value as before. Salomonsen and Madsen^[610] have confirmed the fact of the regeneration of antitoxin after the bleeding of their animals (horses and goats) immunised against diphtheria. Those authors who do not accept the possibility of the transformation of toxins in the production of antitoxins, regard these facts as absolutely incompatible with the hypothesis which they attack. Thus, Weigert^[611] considers that the regeneration of antitoxin after bleeding can only be understood by accepting that antitoxin, like the blood, may be reproduced in the actively immunised animal without any fresh introduction of toxin. It is, however, just as simple, we think, to explain the fact in question by the hypothesis of a provision of toxin stored up in certain cells. This also is sufficient explanation of another observation made by Salomonsen and Madsen^[612], who showed that pilocarpin is capable of augmenting the production of antitoxin. Since it is the living cells which transform the toxin and excrete the antitoxin, it is quite natural to suppose that every factor which stimulates cell function may be capable of causing an increase of the product transformed by the cells.

The third argument invoked against the possibility of the transformation of toxins into antitoxins is based on the fact that the serum of normal horses has sometimes a certain degree of antitoxic power against diphtheria toxin. The horses have never suffered from diphtheria, therefore the antidiphtherin of their blood has nothing to do with diphtheria toxin. It is not known why the blood serum of certain untreated horses is from the first active against diphtheria toxin, whilst that of others exerts absolutely no action on the same poison. We know only that this property is far from being constant in the equine species. Perhaps it is acquired as the result of the penetration into the animal of some pseudo-diphtheria bacillus, whose frequency and number are very great. In order that the microbial products may give rise to the formation of antibodies, it is not at all necessary that the micro-organisms should produce an evident disease. Thus, to cite one example only, Foerster^[613] observed a considerable agglutinative power against the typhoid cocco-bacillus in the serum of a child which was found living among a family of typhoid patients but which, itself, presented no morbid symptom.

The criticism, directed against the hypothesis that modified toxin enters into the production of antitoxin, may not be sufficient to show the incorrectness of this view; it does not follow, however, that the view is right. In the present state of our knowledge it is impossible to solve the problem definitely, and as the hypothesis of transformation gives us the best idea of the specificity of the action of antitoxins, it has a right to be taken into consideration as much as any other.

Ehrlich^[614] has formulated another hypothesis to explain not only this specificity but the origin of antitoxins in general. This is the ingenious hypothesis of side-chains or of receptors, which has already been considered in other chapters of this work. It is now for the first time brought forward in relation to the antitoxins properly so-called, that is to say substances capable of preventing intoxication by microbial toxins. In order to make his hypothesis as clear as possible Ehrlich begins by explaining its bearing on the concrete example of tetanus antitoxin. “When we introduce into an animal a small quantity of tetanus toxin, it is easy to obtain exact proof that it is quickly fixed by the central nervous system, probably by the motor cells of the ganglia; that the central nervous system more than any other organ attracts the tetanus toxin and retains its toxic molecules very firmly.” There we have the side-chains of the protoplasm fulfilling this rôle and subjecting the living protoplasm to the prolonged action of the poison. Once it is combined, the side-chain becomes incapable of fulfilling its normal function, and there is induced on the part of the living elements the production of new chains of a similar character. Following the law that the reaction is stronger than the action, there is an over-production of these side-chains which finally so embarrass the cell which has developed them that they are excreted by it into the blood plasma. Once expelled into this plasma, they continue to manifest their affinity for the tetanus toxin, an affinity which must be even greater in the case where the chains are found in the blood than when they were connected with the cell. Owing to this affinity, these chains, now in the blood, fix the tetanus poison introduced into the animal and prevent it from reaching the susceptible nerve elements. Antitoxins, according to this hypothesis are, therefore, nothing but overplus side-chains poured into the body fluids. Ehrlich extends his theory to a whole series of bodies capable of causing the formation of antitoxins and antidiastases. “It is probable,” he says, “that all analogous bodies can only become toxic to the animal on condition that the animal is capable of fixing their toxophore groups in certain of the organs that are important for its life” (p. 17).

According to this theory tetanus antitoxin must pre-exist in the central nervous system of the normal animal. In the immunised animal, the side-chains must be reproduced in very great quantity in the nerve cells and pass thence into the circulation. Indeed, Wassermann, a supporter of this theory, made a search for tetanus antitoxin in the nerve centres of normal animals. In collaboration with Takaki^[615] he made the important discovery that the brain and spinal cord of small mammals (guinea-pigs and rabbits) when triturated with tetanus toxin prevent the manifestation of its toxic action in animals most susceptible to tetanus. The brain was always found to be more active than the spinal cord. The property of neutralising the toxin of tetanus belongs to the solid parts of the nerve centres; the fluid of the cerebral emulsion is incapable of exercising this action.

This discovery was soon confirmed. Ransom^[616] demonstrated it almost at the same time, and independently of Wassermann and Takaki; and the fact is indisputable. It remains to be seen whether the “antitoxin” of the nerve centres of normal animals is really the same as that which is found in the fluids of animals immunised against tetanus toxin, as is accepted by Wassermann and the other partisans of the side-chain theory. The former is characterised by a very local reaction; it is incapable of being dissolved and distributed through the body of the animal. This is shown by Marie’s^[617] experiments, and my own^[618], all carried out in my laboratory. All that is necessary is to introduce, beneath the dorsal surface of the thigh of a guinea-pig, a quantity of the cerebral substance sufficient to neutralise several times the lethal dose of toxin, and below the skin of the ventral aspect of the same thigh, a lethal dose of this toxin, when it will be found that the guinea-pig contracts a fatal tetanus. The antitoxic action of the nerve substance extends, therefore, for a short distance only; it is strictly local.

The view that the action of the substance of the pounded nerve centres is different from the neutralisation of the toxin by the antitoxin of the body fluids is further confirmed by the fact that the fixation of the tetanus poison by the cerebral substance is very transient. We have shown that a mixture of toxin and pounded cerebral substance, that does not produce any tetanic symptom when injected into the peritoneal cavity of guinea-pigs, sets up a grave tetanus when it is injected subcutaneously into the thigh. In the latter case the toxin becomes separated from the particles of the cerebral substance that had fixed it. Danysz^[619] convinced himself that the mixture of pounded brain with tetanus toxin when it is left in physiological saline solution, in distilled water, or in a 10% solution of sea salt, allows the tetanus toxin to pass into the macerating fluid. The fixation of the toxin to the cerebral substance is, therefore, more comparable to the mordanting of colouring matters by the tissues than to a real combination.

Observers who have repeated the experiments of Wassermann and Takaki have been greatly struck by the difference between the action of the pounded cerebral substance and that of the living brain upon the tetanus toxin. Whereas the former, taken from the guinea-pig, an animal very susceptible to tetanus, prevented intoxication when employed in minimal dose, the living brain of the same species was found to be incapable of neutralising the most minute quantities of toxin. On the other hand, Roux and Borrel^[620] have shown that the brain of rabbits, whether untreated or vaccinated against tetanus, was very susceptible to the action of the tetanus toxin. This toxin, injected directly into the brain, set up in both groups of rabbits a special and characteristic cerebral tetanus. On the other hand, when a little of the cerebral substance of the rabbits, mixed in vitro with tetanus toxin, was injected into other susceptible animals, these remained unaffected.

This great difference between the antitoxic action of the living brain and that of the pounded cerebral matter, on the one hand, and the rigorous localisation of the antitetanic influence of this cerebral substance, on the other, have suggested to several observers the idea that the brain cannot be regarded as the organ of formation of the true antitoxin, such as is found in the fluids of immunised animals. This view has been expressed by Roux and Borrel, Marie and ourselves. Knorr^[621] also shares this view, being struck by the fact that rabbits attacked by tetanus remain for weeks with contractions, but are incapable of producing in their nerve-cells sufficient antitoxin to disintoxicate them, although their blood is already loaded with dissolved antitoxin.

At this period it was generally supposed that, in accordance with Ehrlich’s theory, the hypothetical side-chains were capable, under certain conditions, not only of fixing the tetanus toxin, but also of neutralising it. It was said, therefore, that these chains, reproduced in large quantities in the cerebral cells, must exercise their neutralising action in the brain itself. Consequently, when it was seen that, in Roux and Borrel’s experiments on vaccinated rabbits, this organ was itself affected, it was concluded that the brain must not be regarded as the producer of the antitoxin.

Later, Ehrlich and his supporters, amongst whom I will name especially Weigert, have developed the theory of side-chains in a much more detailed fashion, leading to a different interpretation of several facts previously established. Ehrlich distinguishes in the toxin molecule a haptophore group which combines with the side-chain or the corresponding receptor of the living elements, and a toxophore group which produces the poisoning of the protoplasm. The side-chains, inactive for the toxophore group, neutralise only the haptophore group. Consequently, when these side-chains are numerous in the nerve elements which produce them, they may be a source of great danger to this living element, by attracting the toxic molecules. In this case, these chains, or receptors, serve to attract the poison, just as the badly adjusted lightning-conductor attracts lightning. For this reason rabbits vaccinated against tetanus become tetanic when the toxin is injected directly into the brain. It is only at a distance from the nerve centres that the receptors, excreted into the body fluids, fulfil their rôle of true antitoxins. There they combine with the haptophore group of the toxic molecule, leaving the toxophore group intact; this latter group, however, diverted from the nerve-cells, is incapable of exercising an injurious action.

From this point of view not only the cerebral tetanus of vaccinated rabbits, but also the hypersusceptibility of immunised animals, upon which von Behring has so strongly insisted, may be explained. The argument, drawn from these facts, against the nervous origin of tetanus antitoxin, loses, therefore, much of its weight. If we confront this hypothesis with the other data collected on the question, the solution of the problem becomes beset with great difficulties. Previous to the discovery made by Wassermann and Takaki, I attempted to solve the problem by removing from fowls portions of the brain and spinal cord, proposing to take advantage of the fact that birds, which are capable of producing antitoxins, withstand these operations fairly well. My hopes were not fulfilled; I could never keep my fowls alive long enough to complete the experiment. We must, therefore, for the present, be content with indirect arguments. If the nerve centres do really produce the tetanus antitoxin and excrete it into the blood, we ought at a given moment to find in these organs a greater quantity of this substance than in the blood and the other organs. The reader will recall the researches of Pfeiffer and Marx, and of Deutsch, who demonstrated the possession of a greater richness in protective substance by the phagocytic organs of animals, treated with micro-organisms, than by the blood serum. The same result might be obtained by a comparative investigation of the tetanus antitoxin in the nerve centres and the blood of animals immunised against tetanus. My experiments directed to this point have not been favourable to the hypothesis of the nervous origin of tetanus antitoxin.

In fowls, killed as soon as tetanus antitoxin began to appear in the blood, the brain and spinal cord did not exhibit the slightest antitoxic power^[622]. We might be tempted to explain this result as due to an accumulation of toxin in the nerve centres which would prevent the manifestation of the antitoxin. For this reason, in my later researches^[623], I made use of animals that had been long immunised, but whose blood was still antitoxic. I killed a fowl which had not received any toxin for about eight months, and a guinea-pig into which the last toxic injection had been made almost two years before the date of this experiment. After removing a portion of the brain the blood of these two animals was found to be more antitoxic than before the operation, which indicated that the source of the antitoxin was as yet uninjured. To ascertain whether this source was to be found in the nerve centres I made a comparative determination of the antitoxic power of the brain, of the spinal cord and also of several other organs, of the blood and of the exudations. The result was still negative. The nerve centres were found to be less antitoxic than the blood and other fluids of the body, and even less active than such organs as the liver and kidneys.

In support of the hypothesis of the nervous origin of tetanus antitoxin there remains, then, only the fact of the retarding action of the cerebral substance upon tetanus. In the absence of other arguments this assumes a preponderating importance. We have seen that this action is based on a fleeting and not very firm fixation of the toxin by certain parts of the brain and the cord. Are we justified in regarding this as comparable to the more stable fixation observed in living animals susceptible to tetanus intoxication? Soon after Wassermann and Takaki’s discovery I pointed out that the pounded brain of frogs mixed with tetanus toxin does not prevent animals, into which this mixture is injected, from contracting fatal tetanus. This observation was confirmed by Courmont and Doyon^[624], in several series of experiments carried out under various conditions. They found that “the brain of the frog, heated or unheated, when mixed with tetanus toxin even for several hours, at the temperature of the laboratory or at 38° C., even in considerable doses, does not possess any neutralising property.” This fact would not be in any way wonderful if we had to do with an animal insusceptible to tetanus; but in the frog, as we have said in the preceding chapter, this is far from being the case. In the cold it does not readily become tetanic, but above 25°–30° C. it becomes very susceptible. The tortoise, which is very refractory to this intoxication, has a brain which, when pounded and mixed with tetanus toxin, exerts a certain preventive power over susceptible animals. Nevertheless, the brain of the living frog, as demonstrated by Morgenroth, absorbs this toxin. There is, therefore, a difference between the absorption of the tetanus poison by the living elements and by the pounded cerebral substance. A similar result is obtained with several other toxins. Diphtheria poison is very toxic when injected directly into the brain of the guinea-pig or rabbit. Even the rat, as demonstrated by Roux and Borrel^[625], is readily affected by this toxin under these conditions. Doses which when inoculated subcutaneously are well borne by the rat, when introduced into the brain set up a fatal intoxication in this animal. And yet the brain, when pounded and mixed with diphtheria toxin, can never protect susceptible animals from intoxication. Numerous attempts to reproduce Wassermann and Takaki’s experiment with the diphtheria poison have always been unsuccessful. Attempts to obtain the same result with snake venom have also given negative results. Calmette^[626] made several experiments with emulsions of rabbit’s brain and snake venom with the object of ascertaining whether the elements of the nervous system possess against venom the same properties as against tetanus toxin. “None of these emulsions”—concludes Calmette—“exhibited either the slightest protective or antitoxic power in vitro. There is, therefore, no analogy of action between what takes place in the nerve elements against tetanus toxin and against venom.” Nevertheless venom, like diphtheria toxin and tetanus toxin in the frog, exerts an undoubted action on the nerve centres.

Again, the protective fixation of poisons to the cerebral substance is not the exclusive privilege of tetanus toxin. Kempner and Schepilewsky^[627] obtained the same result with the toxin of botulism (produced by van Ermenghem’s anaerobic micro-organism which sets up intoxication of intestinal origin in certain cases of poisoning by food). The brain and spinal cord of the guinea-pig, when triturated with physiological salt solution and mixed with botulinic toxin, prevents intoxication in susceptible animals, exactly as in Wassermann and Takaki’s experiments with tetanus.

When Kempner and Schepilewsky wished to obtain some idea as to the substance or substances in the nerve centres which fix the toxin of botulism and thus prevent poisoning, they found that lecithin and cholesterin, mixed with this toxin or injected separately and simultaneously, protected mice just as completely as did the cerebral substance. On the other hand, they found a difference as regards the two substances when injected before the toxin was introduced; they were then unable to prevent poisoning, though the cerebral substance exerted an undoubted protective influence. Kempner and Schepilewsky also showed that heating altered the preventive action of lecithin and cholesterin less than it did that of cerebral emulsion.

These observers extended their researches to the protective action of fats and demonstrated that olive oil when emulsified and neutralised with soda and mixed with twice and even four times the lethal dose of botulinic toxin, prevented the contraction of a fatal poisoning by mice. Tyrosin also protected mice against this intoxication, not only when injected simultaneously with the poison, but even when introduced into the animal 24 hours before the poison was administered. Kempner and Schepilewsky conclude “that not only with the substance of the nerve centres, but also with various other substances, they were able to obtain a certain protective effect against the toxin of botulism” (p. 221). Their experiments with cholesterin and tyrosin were suggested to them by the previous researches of Phisalix^[628] who demonstrated that the bile salts, as well as the two substances I have just mentioned, would protect animals against the venom of the viper.

Bearing all these facts in mind, it appears to be probable that in the above cases it is principally the fatty matters of the nerve centres that temporarily fix these toxins, and allow the animal organism to divert the poisons from their morbific action. From this point of view, it is interesting to note that the toxic action of the tetanus poison can also be prevented by other substances than the emulsion of the nerve centres. Thus Stoudensky^[629] demonstrated, in an investigation carried out in Roux’s laboratory, that carmine fixes the tetanus toxin and prevents its action on the guinea-pig. As in the case of the cerebral substance, this fixation by carmine is very unstable. When the carmine that has fixed the tetanotoxin is macerated in distilled water it gives up the poison to the water which is then capable of producing tetanus. Such fixation does not end, any more than in the case of the cerebral substance, in the destruction or disappearance of the toxin. Carmine if first dissolved or macerated in water (especially if heated) loses its fixative power and can no longer prevent tetanus poisoning. Sterilisation, at 120°, 100° and even at 60° C., of the carmine, suspended in physiological salt solution, caused it to lose its protective action, although dry heat applied to it in closed tubes did not destroy this power.

In many respects carmine, which is derived especially from the adipose body of the cochineal insect, exerts an antitoxic influence analogous to that of maceration with the nerve centres. If fats play a special part in this action, we can readily understand how a brain, such as that of the frog, poor in fatty matters, cannot fix the tetanus toxin and prevent its morbific action. In any case the fact that certain substances of diverse nature, acting on toxins, exert an influence similar to that of the pounded mass of the nerve centres, does not allow us to accept Wassermann and Takaki’s experiment as proving the nervous origin of tetanus antitoxin. The analogy with the facts bearing on the anticytotoxins, collected and described in the fifth chapter, also tells against this hypothesis. We would here remind the reader that the two constituent parts of the antispermotoxin, the anticytase and the antispermofixative, develop in castrated animals and are consequently produced outside the spermatozoa, elements susceptible to the spermotoxin. The facts collected concerning the antihaemotoxins indicate also that these substances have some other origin than the red blood corpuscles.

This latter supposition appears to be in contradiction to Ransom’s^[630] very interesting researches on the haemolytic action of saponin, carried out in Meyer’s laboratory at Marburg. This glucoside, owing to its property of fixing itself on the stroma of these corpuscles dissolves the red corpuscles of many vertebrates. The cholesterin of this stroma combines with the saponin, as the result of which the red corpuscles become altered and allow the haemoglobin to diffuse. But this same substance, cholesterin, which causes the poison to penetrate into the red blood corpuscles, prevents the solution of these elements when they are bathed in blood-serum. This fluid, in fact, acts as the antitoxin to saponin and does so just because it contains cholesterin. The cholesterin of the serum, fixing the saponin, prevents it from affecting the red corpuscles, thus fulfilling the function of a well fitted lightning conductor. On the other hand, when the cholesterin of the stroma of these corpuscles is linked on to the saponin, it renders them the disservice of a defective lightning conductor. The accord between these facts and the postulates of Ehrlich’s theory led Ransom to suppose that in the haemolysins and antihaemolysins, cholesterin perhaps played a similar part. His experiments convinced him that this was not the case. As it is generally accepted, after Calmette’s^[631] experiments and according to Ehrlich’s view, that the alkaloids and the glucosides in general are incapable of setting up the formation of antitoxins, we might regard the attempts to find an antisaponin and to settle whether it is identical with cholesterin as useless. But in regard to these delicate questions we must be careful not to give too great weight to a priori arguments. It was believed until quite recently that substances with very complex molecules, such as the albuminoids, toxins and soluble ferments, must always give rise to the production of antibodies in the animal; whilst the simpler substances whose chemical nature was better defined could never lead to this. Facts acquired in recent years have led to a modification of this view. In our fifth chapter we have already spoken of the fruitless attempts of Ehrlich and Morgenroth to obtain certain antifixatives. And yet the fixatives, as is shown by the results of the researches of Bordet and myself, belong to the category of substances which are quite capable of setting up the formation of antibodies. Again, certain mineral poisons, quite unexpectedly, gave rise to the development of the counterpoison in the animal body. This fact forced itself upon Besredka^[632] in his researches on the adaptation to arsenic made in my laboratory. His experiments were undertaken for the purpose of studying the mechanism of the refractory condition against a poison, apart from any antitoxic action whatever, which, according to previous investigations, seemed excluded. This action, however, was exhibited in such a degree that it could not be ignored. The serum of animals immunised against arsenious acid was found to possess both protective and antitoxic properties against a dose of this poison killing a rabbit in 48 hours. It is true that Morishima^[633], in a research carried out in Heyman’s laboratory at Ghent, has thrown doubt upon these results. His objections, however, cannot refute the statements of Besredka which rest on very precise and numerous experiments which I witnessed. Morishima left out of account several important circumstances and carried out his experiments without any continuous check by means of control animals. It must be said also that the resistance of the rabbit against arsenic depends on many different factors and that, at certain seasons, it is much more difficult to adapt them to the poison than at others. It is only by numerous researches extending over a very long period that we can arrive at precise and conclusive results.

From these observations there is every inducement for us to attempt to ascertain whether, by subjecting animals to repeated injections of saponin, it is possible to augment the antisaponic power of their blood-serum and whether, if this takes place, the antitoxic action is due to a rise in the amount of cholesterin in this serum. I therefore requested Besredka to carry out some experiments bearing on this point. Guinea-pigs, injected with progressive doses of saponin for more than two months, at the end of this period showed no increase in the antisaponic power of their serum. They followed the rule established by Ehrlich; they developed no antitoxin against a glucoside. Moreover, they gave us no new information as to the origin of these antibodies.

In his first memoir in which the theory of side-chains is treated, Ehrlich insists on the nervous origin of antitetanin as an example of the production of antitoxins by animals susceptible to poisons. Now, however, that he has come to distinguish haptophore and toxophore groups in the toxic molecule, it is to the side-chain, which fixes the first group, that Ehrlich attributes prime importance. “The formation of antitoxins”—he says^[634] in the opening address at his Institute at Frankfort—“would, therefore, be absolutely independent of the action of the toxophore elements.” In other words, for a cell to be capable of producing antitoxin, it is not at all necessary that it should be susceptible to the toxic influence of the poison; it is only necessary that it should possess receptors, or side-chains, capable of combining with the haptophore group of the toxin. Thus it is possible, as we have described above, to produce antitoxins, with modified toxins whose toxic action is nil or almost so, but which have retained their power of combining with antitoxic substances. According to Ehrlich, these modified toxins are toxoids, in which the toxophore group is completely destroyed; “whilst the haptophore group, the producer of immunising substances, is retained in its integrity.” It is evident then that, under such conditions, the tetanus antitoxin might be developed elsewhere than in the nerve centres. For that it would be sufficient that outside the nerve cells there should be other living elements capable of fixing the tetanus toxin, or, to use Ehrlich’s phraseology, elements, possessing side-chains, having an affinity for the haptophore group of the tetanus poison.

Dönitz^[635] has already expressed the view that in the rabbit the tetanus toxin may be fixed not only by the nerve elements but also by the various other cells.

The existence of such cells, outside the nervous system, is not merely hypothetical. It is shown very clearly in Roux and Borrel’s experiments on cerebral tetanus. In order to produce this disease in the rabbit, it is sufficient to introduce a very small dose of toxin directly into the brain. When inoculated subcutaneously with much larger quantities of the same tetanus poison, the rabbit remains in good health or exhibits merely a slight and transient tetanus. “The resistance of the rabbit against the tetanus toxin, injected under the usual conditions”—conclude Roux and Borrel^[636]—“is not due, then, to a relative insusceptibility of the nerve centres, but to the fact that much of the poison introduced does not reach the nerve cells and is destroyed in some part of the animal.” In the guinea-pig, as shown by the same investigators, the difference of the dose of tetanus poison, necessary to produce fatal tetanus by intracerebral or by subcutaneous injection, is minimal or nil, from which it may be argued that in this very susceptible animal there is no destruction of toxin outside the nerve centres and that the whole of the poison introduced makes its way without hindrance as far as these organs. Ehrlich, in his report to the International Congress of Medicine in Paris (August, 1900), accepted these results, as seen from his tenth and eleventh propositions: “The receptors exist, sometimes in certain tissues only, sometimes in the majority of the organs (action of tetanus poison in the guinea-pig and in the rabbit),” “... the presence of numerous receptors in the organs of less vital importance may bring about—thanks to a kind of diversion of the toxin molecules—a diminution in the susceptibility of the animal to this toxin^[637].” We must here recall the differences between the susceptibility of the guinea-pig and that of the rabbit to small doses of tetanus toxin frequently repeated as in Knorr’s experiments already referred to. The guinea-pig, subjected to these injections, dies in a tetanic condition long before it has received the minimal lethal dose for this species when injected in a single dose. The rabbit, on the other hand, is very tolerant of repeated doses and even rapidly acquires an immunity against five minimal lethal doses for the rabbit (injected at once). Knorr explained this difference as due to the hypersusceptibility of the nerve centres in the guinea-pig and to their acquired insusceptibility in the rabbit. The experiments of Roux and Borrel on the cerebral tetanus of rabbits vaccinated against tetanus, have demonstrated that this insusceptibility is not produced in these animals. We must, therefore, seek some other explanation. In rabbits subjected to small repeated doses, the poison is more and more prevented by certain living elements from reaching the nerve centres. Further, it is neutralised by the antitoxin which is rapidly produced. We find from Knorr’s^[638] researches that in rabbits antitoxin appears in the blood in cases where, affected with a transitory tetanus, their limbs remain contracted for weeks. In guinea-pigs, affected with the same form of tetanus, antitoxin in appreciable quantity is never found, even after complete recovery. All these facts accord with the hypothesis that there exist, outside the nervous system, certain living cells which absorb the tetanus toxin and produce antitoxin. Rabbits and fowls possess this property in a much greater degree than do guinea-pigs. The fowl, according to Knorr, develops “a large quantity of antitoxin, whilst the tetanic symptoms are still augmenting.” In this animal, as we have been able to show^[639], a portion of the tetanus toxin is absorbed by the leucocytes. By exciting aseptic exudations in fowls into which I had previously injected this toxin, I was able to convince myself that these exudations, much richer in leucocytes than was the blood, were also much more tetanigenic than was the blood. I observed also a more or less pronounced leucocytosis after the injection of non-lethal doses of tetanus toxin into fowls. It is possible that the leucocytes were actual agents in protecting the animal against the penetration of this poison to the susceptible nerve centres.

The great susceptibility of leucocytes to microbial toxins serves to indicate that these cells are of some importance in the struggle of the animal against these poisons. Their injection usually sets up a marked hyperleucocytosis of the blood. On this point Chatenay^[640], working in my laboratory, has carried out a series of experiments on animals poisoned by bacterial (tetanus and diphtheria), phanerogamic (ricin and abrin) and animal (snake venom) toxins. He was able to demonstrate a striking analogy between them and the phenomena which occur in bacterial infections. When death takes place at the end of a very short period, the number of leucocytes markedly diminishes; if the animal lives beyond 24 hours or resists completely, a hyperleucocytosis, often of very marked character, is produced. In the guinea-pig, which is so susceptible to tetanus, the leucocytosis observed occurs even after injections of quantities of tetanus toxin equal to several lethal doses, and it is only after the introduction of an amount equal to one hundred times the lethal dose that the number of leucocytes remains stationary or shows a diminution. Here we have something analogous to what takes place against the anthrax bacillus in the same animal. The penetration of this deadly organism sets up a marked leucocytosis, but the accumulated leucocytes are incapable of seizing the bacilli or of preventing their noxious action. In other species of animals, such as the rabbit and the fowl, the intervention of the leucocytes against the anthrax bacillus, as well as against the tetanus toxin, is more effective.

If this toxin, instead of being injected in solution, be introduced along with the bodies of the micro-organisms which contain it, the struggle on the part of the animal takes place under more favourable conditions and even very susceptible animals may afford evidence that they offer a high resistance. Vaillard and Vincent^[641] have shown that if we inject living tetanus bacilli, or the spores of these bacilli, deprived of free toxin, into guinea-pigs a great accumulation of leucocytes, which prevent the production of infection and intoxication by devouring the bacilli and their spores, takes place. The toxin contained in the ingested bacilli remains innocuous; this affording evidence of the protective part played by the leucocytes against the toxin. The same interpretation may be offered to explain the survival of animals very susceptible to tetanus, when the tetanus poison, mixed with pounded cerebral substance or with carmine powder, is injected. In these mixtures the toxin, as mentioned above, becomes attached to certain substances of the triturated brain or to the grains of carmine. This fixation is very unstable, the toxin is readily set free; but, when introduced into the body of the animal, the mixture induces a great accumulation of leucocytes which seize the cerebral particles and the grains of carmine and along with them take possession of the toxin. Wassermann and Takaki’s experiments and those of Stoudensky are easily explained if we assume two protective acts: the first of these consists in fixing the toxin, thus preventing it from diffusing and rapidly reaching the living nerve cells; the second is the absorption of the toxin fixed by the leucocytes,—cells endowed with receptors for the haptophore group of the toxin, but insusceptible to its toxophore group. When one of the two factors is absent, tetanus cannot be prevented. It is for this reason that in Courmont and Doyon’s experiments with emulsion of the frog’s brain, mixed with tetanus toxin, the inoculated animals died from tetanus in spite of an accumulation of leucocytes. This fact affords additional proof that, under these conditions, the toxin does not become anchored to the particles of the pounded cerebral substance, this anchoring being a condition necessary for the effective reaction of the leucocytes.