Chapter XII: Artificial Immunity Against Toxins (2)

Just as, in the analysis of the influence of serums on the micro-organisms, it was found useful to study the action of certain fluids less complicated than the anti-infective specific serums, so we may utilise information furnished by the antitoxic action of fluids other than the true antitoxins. Cases are by no means rare in which normal serums exert a certain influence on toxins. Thus, Pfeiffer[581] noted that the normal blood serum of the goat has the power to prevent fatal poisoning by the cholera toxin. Freund, Grosz and Jelinek[582] observed an analogous action of solutions of nucleohiston on diphtheria intoxication and Kondratieff[583] demonstrated the same action of an extract of the spleen on the tetanus poison. Calmette[584], in collaboration with Deléarde, studied the influence of a whole series of fluids on abrin intoxication. Whilst physiological saline solution was absolutely incapable of preventing the death of animals, fresh broth exerted an undoubted antitoxic power. Amongst normal serums, ox serum exhibited a certain antirabic property. More, however, than the serums of normal animals, have those of animals immunised against various toxins other than abrin (antitetanus, antidiphtheria, antivenomous serums, &c.) been found to possess the power of preventing intoxication by abrin. These facts are connected with others of analogous nature, previously demonstrated by Calmette[585], of which I may cite the following: the serum of animals vaccinated against tetanus toxin is active, though to a less degree, against snake venom; the serum of rabbits vaccinated against rabies, a serum powerless to protect against this disease, is, however, very markedly effective against the same venom; the serum of animals immunised against snake venom is also antitoxic against scorpion venom (I have myself had the opportunity of confirming this fact on several occasions). In all these examples, the serums have proved to be less efficacious against poisons other than the toxin with which the animals that furnished the blood had been treated. Ehrlich[586], too, has demonstrated that animals vaccinated against robin (toxalbumin of _Robinia pseudacacia_) produce a serum, antitoxic not only against this poison but also against ricin. It need scarcely be added that in all these cases of non-specific action of serums derived from vaccinated animals, no question of any antitoxic effect of normal serums can enter. In all the experiments just summarised, the serums of normal animals, used as controls, were found to be inefficacious.

[Sidenote: [385]]

If, in the case of the non-specific action of serums, it were allowable to advance the hypothesis of a direct influence of these fluids on the toxins, it would still be impossible to sustain this view where broth fulfils the antitoxic _rôle_. This fluid, much simpler in composition than any serum, is an excellent culture medium for micro-organisms and one in which the toxins develop well and can be kept for a fairly long period. There is, therefore, not the slightest ground for assigning to it any direct antitoxic action, on the contrary, everything leads us to regard it as an indirect agent, which acts by stimulating the reaction of the animal organism. Here, then, the case would be quite analogous to that of the action of broth as a protective agent against certain bacterial injections, a subject already discussed in the tenth chapter. In this same category of indirect influences also, must be ranked the example of the antitoxic action of the blood of the crayfish against scorpion venom. I have demonstrated in a series of experiments that the fresh blood of the crayfish has the power to prevent fatal intoxication of mice by scorpion venom. Injected in a dose of from 1 to 1·25 c.c., several minutes or an hour before the injection of the rapidly fatal dose of scorpion venom, the crayfish’s blood exerts a very distinct preventive action. It might be supposed from this that the crayfish belongs to the group of animals insusceptible to scorpion venom. This, however, is not the case. The crayfish is very susceptible to this poison and succumbs to a quarter the dose necessary to kill a mouse. The blood of the crayfish is, therefore, completely ineffective as a protective to the crayfish itself, and only exerts its action when introduced into the body of the mouse. It might be concluded that it is only after it has been drawn from the crayfish that the blood acquires its antitoxic power. Experiment contradicts this supposition. Crayfish blood, when injected into another crayfish, in equal or greater amount than is necessary to protect a mouse, is incapable of preventing fatal intoxication by scorpion venom, although, here again, the crayfish received only one-quarter of the dose of venom used for the mice.

[Sidenote: [386]]

We are, therefore, compelled to believe that the crayfish’s blood is antitoxic for the mouse, not in virtue of its direct neutralising action on the venom, but owing to some indirect influence on the organism of the mouse. It is impossible to define, exactly, the mechanism of this action. We may suppose that the blood of the crayfish contains some substance which, by itself, is insufficient to prevent the intoxication, but which becomes active in the presence of some other substance, also inefficacious by itself, met with in the organism of the mouse. Here we should have something analogous to what is met with in immunity against micro-organisms where both fixatives and cytases intervene to bring about the destruction of micro-organisms. By making researches _in vitro_ on the action of the fluids on bacteria, we may easily observe certain phenomena which appear to indicate their direct influence. Take the case of the fluid of an oedema from an animal vaccinated against the cholera vibrio which renders this micro-organism motionless and agglutinates it _in vitro_; the oedema of an unvaccinated animal produces no such effect. If, however, we were to conclude from this fact that, in the oedema of the living animal or in its subcutaneous tissue, everything goes on as in the test-tube and that no other phenomenon of reaction against the vibrios is produced, we should fall into a grave error. It is extremely probable that, in the resistance of the living animal against the toxins, the phenomena are more complicated than are those observed _in vitro_. The example of the blood of the crayfish which prevents the poisoning of the mouse, without having any influence on that of the crayfish itself, may here serve as a guide to us. It is possible that, as in the struggle against the micro-organisms, we have here a co-operation of two substances, each one of which, by itself, is inactive. One of these substances would be found pre-existent in the blood of the crayfish, the other forming part of the organism of the mouse. Perhaps the action of this blood is even more complicated and only becomes active through the mediation of some constituent of the living cell.

Our study of immunity against toxins long ago revealed cases in which this resistance cannot be attributed simply to the antitoxic action of the body fluids. Animals vaccinated against living micro-organisms may succumb to infection in spite of the presence of a strong anti-infective power of the body fluids; similarly animals immunised against toxins may die from intoxication in spite of the antitoxins contained in their fluids. Facts of this order are not rare. Roux and Vaillard[587] on several occasions observed animals which died from tetanus although they had a large supply of antitoxin in their blood. Von Behring[588] and his collaborators, Knorr, Ransom, and Kitashima, also collected a large number of analogous facts. They showed that horses that have been treated for a long time with tetanus toxin and whose blood serum is very antitoxic, still experience marked illness after fresh injections of toxin and may even succumb, in spite of the presence of a large amount of antitoxin in their blood. In these cases the morbid phenomena are undoubtedly different from those typical of tetanus. Instead of the muscular contractions which characterise this disease, the above observers noted disturbance in the regulation of the body temperature, exudative inflammation around the point of inoculation, impairment of appetite and fall of body weight. Sometimes they observed muscular tremors and marked feebleness in the movements. These symptoms differing from those of typical tetanus, it may be asked whether this poisoning is not due to special substances other than tetanus toxin in the fluids injected. Von Behring does not think that this is the case, for he found that by adding antitetanus serum the formation of exudations at the seat of inoculation was suppressed. These exudations, then, must be attributed to the tetanus toxin.

[Sidenote: [387]]

In the cases where animals immunised against diphtheria toxin fall ill and even die as the result of fresh injections of toxin, in spite of the presence of a large quantity of antitoxin in their blood, we might also cast doubts on the diphtheritic character of the poisoning, because the clinical picture of this poisoning is not a very typical one. At the Pasteur Institute, where a large supply of antidiphtheria serum is prepared, we see, from time to time, horses, which have long been undergoing the process of immunisation and are furnishing a very good serum, suddenly fall ill and die from intoxication, without presenting any symptom of infective disease. On one occasion, there was actually quite a small epidemic of fatal poisonings as the result of the injection of a quantity of diphtheria toxin not exceeding the doses which had been well borne previously. Amongst the horses, inoculated with the same toxin, five of the best furnishers of serum died. The others, some of which were producing only a weak serum, remained unaffected.

Von Behring and Kitashima[589] have given a detailed history of a young horse which had become very susceptible as the result of vaccination with diphtheria toxin. It finally succumbed to the intoxication in spite of the presence of diphtheria antitoxin in its blood.

If, in these examples, we have any reason to doubt the specific nature of the intoxication, all doubt must give way before the case described by Brieger[590]. One of his goats, well immunised with tetanus toxin, which, for months, had furnished a good serum and even an antitetanus milk, after an injection, stronger than the preceding ones, was seized with tetanic contractions. These, becoming general, brought about the death of the animal with the symptoms of classic tetanus. The blood, drawn off after death, exhibited strong antitoxic power.

[Sidenote: [388]]

As the result of these observations von Behring formulated the theory of a hypersusceptibility acquired during immunisation. “Paradoxical as it may appear,” he writes[591], “there can no longer exist any doubt that horses which have acquired a high immunity as the result of treatment with tetanus toxin, present a histogenic hypersusceptibility of the organs which react against the tetanus toxin.” In support of this thesis von Behring compares the effect produced by this toxin on horses immunised with this same poison and on normal horses treated with antitoxic serum from other horses. The former, in spite of the fact that they contain in their blood 1,500 times more antitoxin than do the latter, are, nevertheless, less refractory to tetanus toxin. This feeble resistance is due, in von Behring’s opinion, to the much greater susceptibility of the living elements in the horses treated with repeated doses of the poison.

Von Behring’s theory of this form of acquired specific hypersusceptibility has been confirmed by several well-observed facts. These show that, in the animal subjected to treatment by toxins, phenomena of very diverse order are evolved simultaneously: on the one hand, cell reactions which bring about the production of antitoxins; on the other, an increase in the susceptibility of some of the living elements to the specific poison. We are, however, justified in asking if the great difference between the immunity of animals treated with toxin, and that of others treated with antitoxic serum, can be altogether attributed to this hypersusceptibility?

Let us examine in a little more detail some examples of this hypersusceptibility. We know that the guinea-pig is characterised by its great natural susceptibility to the toxins of tetanus and diphtheria. Small doses of these poisons are quite sufficient to produce in it a fatal intoxication. But it is possible to diminish greatly this feeble resistance of the guinea-pig by frequent injections of very small quantities of toxin. Knorr[592] increased their susceptibility to tetanus toxin by daily injections of one-tenth of a minimal lethal dose. The animals died before they had received the ten tenths of this dose. The hypersusceptibility produced under these conditions might be so great that one-fiftieth of the minimal lethal dose was capable of causing death. From these facts we can understand the great difficulty experienced in the earlier attempts to vaccinate guinea-pigs by means of unmodified toxin.

Von Behring and Kitashima[593] made analogous researches on the susceptibility of guinea-pigs to diphtheria toxin. By frequent injections of very small doses of this poison they succeeded in killing these animals with ¹⁄₄₀₀ of the minimal lethal dose _distributed over several injections_. They never succeeded in vaccinating guinea-pigs with increasing doses of pure diphtheria toxin. Their animals died even when they commenced with one-millionth of the minimal lethal dose.

[Sidenote: [389]]

Here, then, we have examples of the greatest hypersusceptibility that it is possible to observe. When we compare it with the changes in the antitoxic power of the blood, we find that these are even more marked. Thus, Salomonsen and Madsen’s horse, to which we have already referred, presented extraordinary oscillations in this power. After receiving, during the course of immunisation, a fresh dose of diphtheria toxin, the antitoxic value of its blood suddenly fell more than one-third (35%). In order to neutralise, completely, this dose of toxin, when injected into a normal animal mixed with antitoxic serum from this same horse, a very small quantity of the blood of the latter would have been sufficient. The injection into the immunised horse should have passed unperceived, as this animal contained in its body more than 50 litres of strongly antitoxic blood. Nevertheless the antitoxic power of this blood fell 12,000 times more than it ought to have fallen according to the calculation made upon the data just indicated. This fall is incomparably greater than the increase of susceptibility to toxin in the most significant examples reproduced above.

[Sidenote: [390]]

As the fact above cited is not at all unique, it is probable that the phenomena which appear in the animal subjected to vaccination by toxins, must be much more complicated than is usually supposed. If the fresh injections of these poisons bring about a specific hypersensitiveness on the one hand, and on the other a great fall in antitoxic power, followed by its still more notable augmentation, it is evident that the introduction of toxins must give rise to a great perturbation in the cell functions. The general analogy between acquired immunity against micro-organisms and against toxins probably rests on similar bases. Kretz[594] has already advanced the hypothesis that, in antitoxic action, two factors, comparable to the cytases and fixatives in the antimicrobial action, co-operate. In the absence of one of these elements we can understand that the one which remains may be incapable of bringing about the neutralisation of the toxin. For this reason the antitoxic serum may act very differently in the organism of the animal which produces it and in that of a normal animal which receives it. An explanation which is adequate for the antitoxic action of the blood of the crayfish injected into mice serves equally well in the case of the antitoxic influence of the serums of animals which themselves succumb to intoxication.

Wassermann’s[595] experiments on the anticytase serums might appear to supply an argument against the hypothesis we are defending. Having shown that animals injected with antityphoid serum die of intoxication when serum which prevents the action of the cytases is introduced simultaneously, Wassermann put the question: May not the action of the antitoxins be prevented by this same anticytase serum? To solve this point he injected into guinea-pigs a mixture of antidiphtheria serum with toxin in excess and a fairly strong dose (3 c.c.) of anticytase serum, upon which we have already spoken (see Chapter VII). The animals, so treated, behaved exactly as did the animals used for control which received the same quantities of antitoxin and toxin but without the addition of anticytase serum. Wassermann concludes from these experiments that the exclusion of the cytase, contrary to what takes place with antimicrobial serums, in no way impedes the action of the antitoxins. This conclusion, which appears at first sight to be justified, cannot, however, be accepted, as the two examples chosen by Wassermann, typhoid infection and diphtheria intoxication, differ very profoundly from each other. In the former, we have an experimental typhoid peritonitis which kills the control animals in less than 24 hours, whilst the second is diphtheria in which the controls do not succumb until the sixth day after injection. The effect of the anticytase serum being only very transitory, it is quite natural that this should manifest itself in an infection of short duration and should not do so in a slow intoxication. Besides, Wassermann himself has shown that in several other cases of immunity against micro-organisms (the bacilli of influenza and of leprosy) the injection of his anticytase serums does not interfere with the perfect resistance of the animals. But even were it demonstrated that the cytases really play no part in immunity against toxins, the intervention of some other similar factor could always be evoked.

[Sidenote: [391]]

The analogy between immunity against micro-organisms and that against toxins may facilitate the study of the relations between the latter and the antitoxic power of the body fluids. In the preceding chapters we have described examples in which animals possess a protective power in their blood but are not refractory to the corresponding infection; on the other hand, we have cited cases in which acquired antimicrobial immunity exists without the blood presenting any appreciable protective power. The idea of measuring acquired immunity against micro-organisms by the measurement of the protective or agglutinative power of the blood must therefore be abandoned, and it is impossible to regard immunity against toxins as a function of the antitoxic property of the body fluids. As we have seen, animals completely refractory to tetanus, such as the cayman, whose immunity does not depend on the antitetanic power of the blood, develop antitoxin after the injection of toxin. A similar state of affairs, but less pronounced, has been demonstrated by Vaillard as occurring in the fowl. The fowl, in spite of its very marked natural immunity against tetanus, produces antitetanin as the result of the introduction into its body of tetanus toxin; the rabbit, on the other hand, a susceptible animal, may acquire a real immunity without the development of any antitoxic power in its fluids. An additional fact was noted by Vaillard[596]. He showed that the repeated inoculation of tetanus spores along with a small quantity of lactic acid, made below the skin of the tail of rabbits procured for them an immunity against tetanus toxin, although no antitoxic property appeared in their blood. In his experiments, one hundred volumes of blood serum were found to be incapable of neutralising a single minimal lethal dose of the toxin. The rabbit, however, still remains quite capable of developing antitetanic power in its fluids. All that is necessary is to inject into it some tetanus toxin heated to 60° C. or treated with Lugol’s iodo-ioduretted solution. As the outcome of his researches Vaillard concludes that the antitoxic property of the body fluids “is not sufficient ... for the general interpretation of acquired immunity, as it cannot be demonstrated in all animals which have become refractory.”

[Sidenote: [392]]

[Sidenote: [393]]

The facts I have just mentioned were demonstrated early in our study of the antitoxic power of the animal organism. Since then a large number of analogous data have been collected. Recently, von Behring and Kitashima[597] have had to abandon the immunisation of monkeys against diphtheria toxin because of the poor yield in antitoxin which they obtained. The blood of one of their monkeys that had acquired a resisting power against very large doses of diphtheria toxin showed only a very moderate antitoxic power. In establishments where antitoxic serums are prepared on a large scale the workers have become convinced that the yield of antitoxin has no direct constant ratio to the immunity of the animal. This has been demonstrated repeatedly at the stables of the Pasteur Institute. Thus, of two horses, treated at the same time and in exactly the same way with diphtheria toxin, one furnished a very good antitoxic serum which was maintained at 200 units Ehrlich, rising up to 400 units, whilst the other never reached 150 units[598]. And yet both these animals possessed the same immunity against diphtheria toxin. They tolerate considerable doses of toxin and react merely by a slight or insignificant rise in temperature. In another series of horses, which have been immunised for nearly seven years, one remained capable of yielding a large quantity of antitoxin, seeing that the value of its serum oscillated between 200 and 300 units. After five years of this state of things the antitoxic power began to fall considerably, without, however, any corresponding loss of immunity. Indeed, an injection of 250 c.c. of toxin (of which 0·002 c.c. was sufficient to kill a guinea-pig) began, at the commencement of the present year, to be borne without the least febrile reaction. An attempt was made to raise the antitoxic power of the blood by making intravenous injections of toxin and of diphtheria culture, but in vain. The yield of antitoxin continued to fall and it became necessary to employ this horse for another purpose than the preparation of antidiphtheria serum. This is by no means an isolated example. Of a large number of treated horses it frequently happens that certain individuals, without being particularly susceptible to a given toxin, are found to be incapable of producing any corresponding antitoxin[599].

In presence of the fact that animals very resistant to toxins may possess no, or only an insignificant antitoxic power in their fluids, and that, on the other hand, animals in which this property is highly developed may succumb to intoxication, it may be readily understood that immunity against toxins and the antitoxic power of the body fluids may be two distinct conditions. Von Behring has clearly demonstrated the fact of the cellular hypersensitiveness of the animal immunised against the corresponding toxin and has laid great stress upon this fact. He came[600] to the conclusion that “the immunity of the tissues and the production of antitoxin follow a parallel course in their development so slightly that, in spite of an abundant accumulation of antitoxin, the susceptibility of the elements of the tissues may increase in an extraordinary fashion.” If, during the course of immunisation, this susceptibility can increase so greatly, it is probable _à priori_ that under certain circumstances it might also diminish notably. After demonstrating “that in time the antitoxin disappears from the blood of animals immunised with toxins without any consequent disappearance of immunity,” von Behring formulated the conclusion that in these animals “the living elements of the animal, which were previously susceptible to the poisons, have acquired an insusceptibility towards the same substances.” This result fully accords with the facts of the change of the negative chemiotaxis of phagocytes into positive chemiotaxis for micro-organisms during the acquisition of anti-infective immunity.

[Sidenote: [394]]

Later, von Behring[601] changed his opinion. Whilst still accepting the change of cellular susceptibility in the direction of hypersensitiveness in animals immunised against toxins, he refused to admit the change in the opposite direction. The cells, according to him, never lose any of their susceptibility, so that acquired immunity against toxins cannot be obtained otherwise than by means of antitoxins capable of neutralising the poison in a susceptible or hypersusceptible animal. This new theory von Behring upheld in several papers and it is met with in his most recent publications. Nevertheless, certain well-established facts compel us to accept an immunity against toxins as coming about as the result of a diminution of the susceptibility of the vaccinated animal. Parallel with his researches on the increase of the susceptibility of guinea-pigs to tetanus toxin, researches discussed above, Knorr[602] describes analogous experiments on rabbits. When these animals are injected with fractions of the minimal lethal dose, frequently repeated, the rabbit not only does not become hypersusceptible to tetanus but exhibits a greater and greater insusceptibility. Whilst guinea-pigs, treated according to this method, die from tetanus before they have reached the minimal lethal dose, rabbits, as the result of frequent injections of small quantities of tetanus toxin, become capable of resisting five times the lethal dose (for normal rabbits) without exhibiting the slightest symptom of illness. Against the attribution of this result to the acquired insusceptibility of the living animals it might be objected that the immunity, in this case, may depend on the antitoxic power of the fluids of the body, developed with great rapidity. Such an objection cannot be raised in the case of horses which become insusceptible to toxins after a long period of vaccination. The horse whose history was given above, when discussing the diminution of antitoxic power, may serve as an example. At the commencement of its vaccinal period, in 1894, it reacted to the injection of 10 c.c. of diphtheria toxin by a rise of temperature of 1° C. Four years later, when its blood had become very antitoxic (350 units per c.c.), it was necessary to inject 350 c.c. of toxin to obtain the same rise of temperature. Quite recently, having now lost the greater part of its humoral antitoxic power, this horse exhibited no rise of temperature after an injection of 250 c.c. of strong diphtheria toxin. The diminution of the specific susceptibility is produced in this case in a most marked fashion; it is not therefore to the antitoxic property of the body fluids that this case of immunity must be attributed.

[Sidenote: [395]]

The insusceptibility acquired against poisons of different kinds is observed also in cases where the adaptation is not accompanied by the production of humoral antitoxic properties, as in the immunity of frogs against abrin. This form of immunity may be traced through the organic series down to such lowly developed organisms as the plasmodium of the Myxomycetes, which as we have seen readily becomes adapted to different poisons (see Chapter II).

It can be clearly seen, then, that immunity against toxic substances is a very complex phenomenon which it is impossible to reduce simply to an antitoxic function of the fluids of the body. For this reason we cannot accept a theory which would confine this kind of immunity within the narrow limits of a simple reaction between two substances, a reaction quite comparable to that observed in a test-tube. Attempts have been made to determine with almost mathematical precision the conditions under which it is possible to communicate to the animal a resistance against microbial toxins and formulae have been constructed to define these conditions. But the application of these formulae has been found to be a much more difficult matter. In Prussia, with the sanction of the Government, regulations have been enacted as to the procedure to be followed in the testing of antitoxic serums, and a paragraph has been added which requires a post-mortem examination of the guinea-pigs employed for this purpose in the case of diphtheria antitoxin. “The dead animals,” says this instruction, “must be submitted to a post-mortem examination, and special attention must be directed to the presence of any pre-existing diseases (tuberculosis, pseudotuberculosis, pneumonia) which may have induced hypersusceptibility in the animals under experiment.” Do we not see in this a proof of the important intervention of the organism of the living animal which may modify the results of calculations based upon too rigorous formulae? It must not be forgotten, too, that in addition to the three diseases named in the instructions, we have a number of other factors which may influence the receptivity and the resistance of animals. We have already cited Roux and Vaillard’s experiments which demonstrated that even animals which have been previously subjected to vaccinal inoculations against certain micro-organisms, exhibit a hypersusceptibility to mixtures of toxins with antitoxins.

In view, then, of this complexity of the phenomena of acquired immunity against toxins, it would be very important could we learn something of the nature and origin of antitoxins. Unfortunately, as we shall see, these questions are, as yet, far from having received a satisfactory solution.

[Sidenote: [396]]

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

[Sidenote: [397]]

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

[Sidenote: [398]]

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

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

[Sidenote: [399]]

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

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

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

[Sidenote: [400]]

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

[Sidenote: [401]]

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

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

[Sidenote: [402]]

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

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

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

[Sidenote: [403]]

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

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

[Sidenote: [404]]

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

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

[Sidenote: [405]]

[Sidenote: [406]]

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

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

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

[Sidenote: [407]]