spoken of as the method of vaccination by _toxones_. This name,
“toxone,” was first applied by Ehrlich[542] to a product developed by
the diphtheria bacillus in culture media, a product less and differently
toxic than is the true diphtheria toxin, yet capable of neutralising
antitoxin. The idea of toxones presented itself to Ehrlich in connection
with a fundamental fact noted by him, namely, that when to a non-toxic
mixture of diphtheria toxin and antitoxin there is added one and even
several lethal doses of the former, the animal is not affected. To make
it succumb to intoxication it is sometimes necessary to add more than 20
lethal doses of toxin. To explain this paradoxical result, Ehrlich
formulated the hypothesis that, in the soluble products of the
diphtheria bacillus there exist two poisons: (1) the true toxin which
exhibits a very strong affinity for antitoxin, and (2) the toxone which
possesses less avidity for this antibody. When to an inactive mixture of
the products of diphtheria bacilli and of antitoxin, there is added a
fresh quantity of these same products, the added toxin, owing to its
greater affinity, replaces the toxone of the previous combination. In
the mixture to which is added one or several lethal doses of diphtheria
poison, the toxone only is found free, all the toxin being combined with
the antitoxin, and, as the toxone is only feebly toxic, the animal
resists without suffering any serious illness.
Madsen[543] adopted the theory of the diphtheria toxone, and affirmed
that this substance poisons but slowly, produces neither early nervous
symptoms nor loss of hair, but excites slight oedema at the point of
inoculation and late paralyses. Susceptible animals may die from
toxones, but very much later than as the result of poisoning by the
toxins.
Ehrlich’s pupils have extended the theory of toxones to other bacterial
poisons. Thus Madsen[544] has described a similar toxone in tetanus
poison—the tetanolysin of Ehrlich—which dissolves the red blood
corpuscles, and Neisser and Wechsberg[545] refer to a toxone in the
poison produced by the staphylococcus.
Ehrlich also describes _toxoids_ as occurring in diphtheria poison. The
toxone, he maintains, is a product of the diphtheria bacillus itself,
but the toxoids (protoxoids and syntoxoids) represent the toxin modified
without further aid from the bacillus. The toxoids, though not toxic,
retain all their avidity for antitoxin. According to Ehrlich’s
conception, the molecule of toxin, under the influence of various
factors, readily loses its toxic or _toxophore_ group, capable of
poisoning the animal, whilst still retaining its _haptophore_ group, the
group that combines with the antitoxin. The toxoids then would represent
this haptophore group of the diphtheria toxin. Without being injurious
to animals, the toxoids are capable of neutralising the antitoxin and of
setting up in the animal the formation of this antibody. In the
experiments carried out by the method of Babes and of the Russian
authors we have just mentioned, there would be, according to the view
held by Ehrlich and his school, an immunisation by the toxoids.
[Sidenote: [368]]
The toxones, however, are also capable of vaccinating against the toxin
and the toxone and of giving rise to the production of a diphtheria
antitoxin, active against these two poisons. This is what is affirmed by
Madsen[546] and by Dreyer[547], according to a communication made by the
latter to the International Congress of Medicine held at Paris.
[Sidenote: [369]]
By means of the various methods briefly described above, is obtained a
real acquired immunity against the various bacterial and vegetable
poisons and the venoms. On the other hand, with the methods of
vaccination mentioned in the eighth chapter, which confer a substantial
immunity against micro-organisms, we cannot demonstrate, in the
vaccinated animals, a resistance against the corresponding toxins
greater than in the unvaccinated control animals. The animals, so
thoroughly vaccinated against certain micro-organisms that they
withstood enormous doses of culture, did not become capable of resisting
the minimal lethal dose of the poison. We are led to conclude,
therefore, that immunity can only be obtained against certain of the
toxins. For this reason we must regard the attempt made by von Behring
to obtain a real immunisation against the toxin of cholera as an
important forward step. Before von Behring’s attempt, various species of
animals had been frequently and very substantially vaccinated against
the cholera vibrio, but these animals, even when most thoroughly
vaccinated, were completely non-resistant to the cholera toxin. Von
Behring suggested to his pupil Ransom[548] the idea of immunising
guinea-pigs, not with microbial cultures living or dead, as had usually
been done previously, but exclusively with the fluids of the cultures,
deprived of the vibrios by filtration. In order, however, to attain the
desired object, it was necessary to prepare fluids sufficiently active
to poison the unvaccinated control guinea-pigs with certainty. The
results of these investigations confirmed his anticipation, and Ransom
soon found himself in possession of guinea-pigs well vaccinated against
the cholera poison. He was mistaken, however, in supposing that, in all
cases of immunity acquired against Koch’s vibrio, we have to do, in the
main, with a purely antitoxic immunity. An investigation carried out in
the Pasteur Institute[549], whilst confirming the facts discovered by
Ransom, lead to different results as regards their interpretation. It
was demonstrated that the immunity against the vibrio is in no way
founded on a resistance against its toxin and that we have to do with
two very different acquired immunities. The vaccination obtained with
the bodies of the micro-organisms induced a refractory condition against
infection by the living vibrio, but not the slightest resistance against
the toxin. The immunity, on the other hand, which is conferred by the
injection of soluble products, deprived of the micro-organisms, is
effective not only against the toxin of cholera, but also against
infection by the vibrio. When an animal is vaccinated with cultures, or
even with the bodies only of the vibrios, cholera toxin is introduced,
but the toxin, under these conditions, is incapable of setting up
antitoxic immunity. It would appear that the presence of the vibrios may
constitute some obstacle to the production of this immunity.
Soon afterwards, Wassermann[550] pointed out that the same rule applies
in the case of the _Bacillus pyocyaneus_. With whole cultures of this
bacillus he obtained in guinea-pigs an immunity exclusively against
infection, whilst with cultures in a fluid medium, deprived of the
bacilli, he was able to vaccinate his animals both against the pyocyanic
toxin and against the infective peritonitis produced by the living
micro-organism. The same double immunity could also be obtained in
laboratory animals against the typhoid bacillus and several other
bacteria.
When animals were subjected to different methods of vaccination against
toxins, the manifestation of certain phenomena more or less constant was
observed; amongst these must be pointed out especially the rise of
temperature, a local reaction and certain modifications in the body
fluids.
Fever is a very general symptom in the course of the vaccination of
mammals. A rise of temperature is almost always observed as a result of
the injection of toxins. It is very variable, both as regards duration
and intensity, and cannot serve as an indicator of the result of the
vaccination. In this respect, such great differences have been observed
that the attempt to establish any general laws has had to be abandoned.
[Sidenote: [370]]
[Sidenote: [371]]
Local reaction is also a phenomenon which is very frequently observed
during vaccination; to this von Behring[551] paid great attention. He
and his collaborators found that normal horses when injected
subcutaneously with small or large doses of tetanus toxin did not
present any exudation at the seat of inoculation. The horses which died
as the result of a tetanus intoxication and those which got better
behaved from this point of view in much the same fashion. In horses,
however, which are being vaccinated and which are periodically subjected
to gradually increasing doses of toxin, tumefaction at the seat of
injection is never absent. Von Behring attributes this difference to the
primordial insusceptibility of the living elements which govern
exudation in the subcutaneous tissue to tetanus poison. It is only
during the process of vaccination that these cells become susceptible
and capable of manifesting a visible reaction. I consider that this
difference is due more probably to a change in the chemiotaxis of the
various elements which contribute to the inflammatory exudation
reaction, from a negative to positive type. The cells do not react at
the commencement, not because they are not susceptible to the toxin, but
rather because their susceptibility is too great. During the course of
vaccination they become sufficiently adapted to the poison to be able to
manifest their normal inflammatory reaction. This explanation certainly
harmonises with the fact that during the period of vaccinations in
general and of vaccination against toxins in particular, the blood
usually presents a more or less distinct hyperleucocytosis. Now, as is
well known, this phenomenon of hyperleucocytosis is one of the most
striking manifestations of a positive chemiotaxis in white corpuscles.
It is true that, as to this reaction during the course of vaccination,
the views of observers are not unanimous. Besredka[552], as the outcome
of his work on this subject, expresses himself very distinctly. “During
the course of an immunisation against diphtheria toxin,” he writes, “one
always observes a marked reaction in the goat, either at the beginning
or at an advanced stage of the period of injections and especially in
the first few hours after injection” (p. 322). Nicolas and Courmont[553]
in their first memoir maintain that hyperleucocytosis “is not necessary
for immunisation.” Nevertheless, in the description of their
experiments, which were performed on horses vaccinated against
diphtheria, it is clear that the number of white corpuscles is often
markedly increased. Further, in several cases they describe the
formation of tumours at the point of inoculation, some of which end in
suppuration. Under these conditions, it is not possible to deny a
vaccinal reaction on the part of the leucocytes. Later, Nicolas,
Courmont and Prat[554] published a second memoir on the same subject, in
which they seek to confirm their view of the uselessness of
hyperleucocytosis in vaccination against the poison of diphtheria. They
give details of experiments on several species of animals and insist
specially on the conditions in which they have not observed
hyperleucocytosis. “The doses from the first have always been extremely
weak and with the addition of Lugol’s solution to attenuate them; only
very gradually have we reached stronger doses, as _that is one of the
indispensable conditions for the avoidance of leucocytic variations_,
whilst obtaining a good and rapid immunisation” (p. 974). These special
precautions to avoid hyperleucocytosis demonstrate clearly that this
phenomenon is usually produced during the course of vaccination. It is
quite natural that we should, by proceeding very slowly and with small
doses of toxin, succeed in diminishing or even suppressing the afflux of
leucocytes; but this fact cannot in any way minimise the importance of
the leucocytic reaction in vaccination. In these particular cases, this
reaction may take place without the number of leucocytes in the blood
being noticeably increased. In reading the details of the experiments
made by the Lyons observers, it will be seen that, in spite of all their
precautions, they were unable to prevent the production of
hyperleucocytosis. In all their cases, where they took the precaution to
count the leucocytes several times a day, there was an undoubted
increase of these cells. We may here recall Salomonsen and Madsen’s
account of the immunisation of a horse against diphtheria toxin, in
which they point out the frequency of tumefactions and even of
abscesses. In most cases the pus was sterile, which renders it probable
that the white corpuscles had accumulated at the seat of inoculation as
the result of some influence exerted by the diphtheria toxin.
[Sidenote: [372]]
By far the most important and remarkable change met with in animals
vaccinated against toxins and venoms, consists in the appearance of
antitoxic power in their blood and fluids in general. This fact was, as
already mentioned, first demonstrated by von Behring and Kitasato[555]
in the blood of rabbits immunised against tetanus. The blood itself, or
the blood serum, mixed with a quantity of tetanus toxin more than
sufficient to cause fatal poisoning, sets up no disease when injected
into animals. In their earliest researches, von Behring and Kitasato
kept the mixtures in contact _in vitro_ for 24 hours, before injecting
them into test animals. Later, they found that this prolonged contact
outside the body was unnecessary and that they could obtain successful
results by injecting the serum of vaccinated animals and the toxin
simultaneously, even at different points of the body. This discovery was
immediately afterwards applied by its authors to diphtheria and, in the
case of both intoxications, confirmed by numerous observers.
[Sidenote: [373]]
For some time we were satisfied with vaccinating small laboratory
animals and establishing the antitoxic power of their blood serum;
later, the vaccination of large animals, especially horses, was
commenced with the object of obtaining large quantities of antitetanus
and antidiphtheria serum for medical use. During the course of these
experiments the principal characters of the antitoxic fluids were
established. It was deemed desirable to isolate the antitoxic substance
from the blood serum in order to get rid of every unnecessary and
inactive admixture, so that the antitoxin might be used in as pure a
form as possible. This idea of isolating the antitoxic substance had,
however, soon to be abandoned as impossible of realisation. Antitoxin is
a non-crystallisable substance, of unknown chemical composition, which
adheres firmly to the albuminoid substances of the serum. It is usually
regarded as belonging to the same albuminoid group of substances, though
it is not possible to prove this satisfactorily. Von Behring[556],
however, who studied this question in collaboration with Knorr, denies
the albuminoid nature of tetanus antitoxin. After demonstrating that
this antitoxin, when the antitetanus serum is submitted to dialysis,
passes through the dialysing membrane, these observers found that they
could not obtain the characteristic reactions of albuminoids in the
dialysed fluid. It must be admitted, however, that this negative result
is not sufficient to justify a denial of the albuminoid nature of
antitoxin. When Nencki and Mme Sieber[557] sought to produce the
reactions of albuminoid substances with the digestive juice of
_Nepenthes_ (the well-known insectivorous plant) they got no result; but
after the concentration of the juice _in vacuo_, it at once gave the
characteristic reaction with nitric acid, and also with acetic acid,
potassium ferrocyanide and Millon’s reagent.
The antitoxins may be precipitated along with the globulins and are
distinguished, in general, by a fairly great resistance against physical
and chemical influences. In this respect they are allied to the
agglutinins, the fixatives and the precipitins, considered elsewhere,
and are sharply distinguished from the cytases. The antitoxins resist
temperatures which destroy the cytases and remain unaltered to beyond
60°–65° C. They are more stable than the delicate toxins of tetanus and
diphtheria, but they are more easily altered than the toxins of cholera,
of _Bacillus pyocyaneus_ and the venoms. When stored in a dry state in
the residue of evaporated serums and protected from light and air, the
antitoxins will keep for a very long time without showing any notable
attenuation. This property is very important in practice.
[Sidenote: [374]]
The antitoxins, in this respect also resembling the fixatives and the
agglutinins, are humoral substances in the strictest sense of the term.
They are found not only in prepared serums but abound also in the plasma
of the circulating blood, and in the plasmas of the lymph and of
exudations. Vaillard and Roux[558] have shown that the clear acellular
serous fluid of the oedema produced by the slowing of the circulation in
rabbits vaccinated against tetanus toxin, is as antitoxic as the blood
itself. Even the aqueous humour of a strongly immunised animal is
antitoxic, though to a less degree. On the other hand, the saliva and
urine exhibit very little antitoxic power, even when they are derived
from animals hyperimmunised against tetanus toxin. Milk, as first
demonstrated by Ehrlich[559], is fairly rich in antitoxin, although much
less so than the blood. According to the estimation of Ehrlich and
Wassermann[560], in the same immunised animal, milk contains
one-fifteenth to one-thirtieth of the amount of diphtheria or tetanus
antitoxin contained in the blood. Pus is always less antitoxic than
blood or blood serum. According to Roux and Vaillard (_l. c._, p. 82),
the pus of their rabbits vaccinated against tetanus toxin was only
one-sixth or one-eighth as antitoxic as the serum of the blood. In
Salomonsen and Madsen’s[561] antidiphtheritic horse the cellular
sediment of the pus was about one-half as antitoxic as the blood.
For the development of the antitoxic property in the fluids of the body,
it is not essential that animals should belong to species susceptible to
the corresponding toxin. Animals naturally most refractory against the
poisons of diphtheria and tetanus are also capable of producing
antitoxins. Vaillard[562] demonstrated this fact in the fowl. This bird,
which is naturally refractory against tetanus, usually acquires a very
marked antitetanic power in its blood after one or more injections of
tetanus toxin. He observed, however, that, in fowls thus treated, at a
stage when the fluids of the body are antitoxic, the albumen of the egg
is not so. The antitoxin, therefore, does not pass into this nutritive
secretion, as it does into the milk of mammals. On the other hand, as
has been demonstrated by F. Klemperer[563], the vitellus of the eggs of
fowls treated with tetanus toxin in time acquires an antitoxic property
of the most marked character.
[Sidenote: [375]]
[Sidenote: [376]]
The antitoxins, found especially in the fluids of the body but only
scantily in the cells, exert some action on the toxins. What is the
nature of this action? This question, much studied and discussed, is one
of very great importance in connection with the general problem of
acquired immunity against toxins. In his first memoir, written in
collaboration with Kitasato, von Behring (_Deutsche med. Wchnschr._,
Leipzig, 1890, S. 1113) formulates his first thesis as follows: “the
blood of a rabbit immunised against tetanus possesses the property of
destroying tetanus toxin.” This idea of destruction, which would remove
all toxic power from the poison, would naturally present itself to the
mind and was at once accepted by a great many observers, but the
numerous facts now accumulated on the subject will not allow us to
accept a real destruction of toxins by antitoxins. Tizzoni[564] was one
of the first to point out certain contradictions between the theory of
destruction and the phenomena produced in animals injected with tetanus
toxin and antitoxin. Buchner[565] also brought forward new facts which
led him to conclude that antitoxin, instead of acting directly on the
toxin, exerts its influence exclusively on the living elements, thus
protecting the animal against intoxication. Amongst the arguments
advanced by the Munich observer, the principal one is drawn from the
different action of mixtures of tetanus toxin and antitetanus serum on
various species of animals. It has been clearly shown that the
guinea-pig is more susceptible to tetanus than is the mouse. In
poisoning with tetanus toxin it requires an absolutely larger quantity
of toxin to kill the guinea-pig than to kill the mouse. But if we take
into account the weight of these animals, the conditions change
completely. Thus, to cause a fatal tetanus in a guinea-pig, which weighs
twenty times more than a mouse, we need only inject into the former a
dose at most ten times greater than that necessary to produce fatal
intoxication in the mouse. Buchner prepared a mixture of tetanus toxin
and antitetanus serum which, in the mouse, produces no tetanic
phenomenon or only sets up feeble and transient symptoms. According to
the theory of direct action, we must assume that in this mixture the
toxin is completely or almost completely neutralised by the antitoxin of
the serum. But when Buchner injected the same quantity of mixture into
guinea-pigs, without increasing it in proportion to the greater weight
of these animals, he produced a tetanus of the most marked character.
There has, consequently, remained in the mixture a sufficient amount of
free toxin, whose tetanigenic action is manifested in the guinea-pig, an
animal, as we have seen, more susceptible than the mouse. Buchner’s
experiment has been verified by several observers. Roux and
Vaillard[566] carried out others which afford similar evidence. The same
mixture of tetanus toxin and specific serum which is borne without the
least difficulty by normal guinea-pigs, causes typical tetanus in other
guinea-pigs of the same weight, and apparently in the best of health,
but which have been immunised some time before against the Massowah
vibrio. In another series of experiments, Roux and Vaillard injected
into guinea-pigs a very large amount of antitetanus serum “capable of
immunising them thousands of times,” and, shortly afterwards, a lethal
dose of tetanus toxin. The normal guinea-pigs were thoroughly resistant
to this test, whilst several guinea-pigs into which were also injected
the products of other micro-organisms, acquired tetanus. Analogous
results were obtained with mixtures of diphtheria toxin and
antidiphtheria serum. Roux concludes from these facts “that the
antitoxins act on the cells.” Against the theory of the destruction of
toxins by antitoxins, he invokes the influence of heat on mixtures of
these two substances. Calmette[567], under Roux’s inspiration and in his
laboratory, carried out various experiments on antivenomous serum. A
mixture of this with snake venom, in such proportion that the poison
became inactive, regained its toxicity after being heated for five
minutes at 68° C. A normal animal, injected with this mixture, succumbed
as if it had received pure venom. On being heated at 68° C. the serum
lost all its antitoxic power over the venom, and the latter, which only
becomes modified at a much higher temperature, remained intact. Later, a
similar result was obtained by Wassermann[568] in his experiments with
pyocyanic toxin. This poison is resistant at even higher temperatures
than is snake venom, whilst the antitoxin of the serum is destroyed
under the same conditions as are the other antitoxins. Taking advantage
of these peculiarities, Wassermann boiled the mixture of pyocyanic toxin
and antitoxin serum, being careful to dilute it with two volumes of
distilled water before doing so. This mixture which, before it was
heated, was quite innocuous for guinea-pigs, again became a fatal poison
after the destruction of the antitoxin.
[Sidenote: [377]]
These experiments prove clearly that, in the action of the antitoxin on
the toxin, there can no longer be any question of an actual destruction
of the latter, a view which has been accepted by both von Behring and
Ehrlich. But, as pointed out by Roux at the International Congress at
Budapest in 1894, the manifestation of the toxic action of the venom
after it has been heated along with antitoxin, may be reconciled with
the view that the combination between the two substances, if such take
place, must be very unstable. This same remark may be applied to
Wassermann’s experiment. Therefore the great majority of observers, if
not all, admit that the antitoxin combines with the toxin to form an
innocuous and unstable substance which can be decomposed by heat and by
other agents. The researches on the action of antitoxins _in vitro_ have
had a powerful influence in determining this view.
[Sidenote: [378]]
We have already in Denys and van de Velde’s[569] experiments an
indication of the direct action of certain antitoxins. These observers
showed that the serum of animals vaccinated against a Staphylococcus is
capable of neutralising _in vitro_ a particular toxin to which van de
Velde gave the name of _leucocidin_. When it was added to a drop of the
exudation from a rabbit, this leucocidin in a very short time destroyed
the white corpuscles, by dissolving the cell content but leaving the
nucleus untouched. When Denys and van de Velde prepared mixtures of
leucocytes, leucocidin and antileucocidic serum _in vitro_, the white
corpuscles retained their normal condition for a very long time. The
leucocidin was, therefore, rendered inactive by the direct influence of
the corresponding antitoxin. These facts have been confirmed by
Bail[570] and other observers and even extended to certain other
microbial toxins. Thus, the _Bacillus pyocyaneus_ produces a leucocidin
which kills the white corpuscles and dissolves their contents[571]. With
the object of facilitating experiments with these leucocytic poisons and
the corresponding antitoxic serums, Neisser and Wechsberg[572], of the
Institute of Experimental Therapeutics at Frankfort, invented a method
which allows us to observe the phenomena of the destruction of the
leucocytes and of the antitoxic power in test tubes, without having
recourse to a microscopical examination. They applied the fact,
discovered by Ehrlich, that living formed elements reduce methylene blue
and, depriving it of its oxygen, decolorise it. Leucocytes from aseptic
exudations are introduced into tubes and a weak solution (2%) of
methylene blue is poured on them. To prevent the re-oxidation of this
colouringmatter by the oxygen of the air, the surface of the fluid is
covered with a layer of liquid paraffin. If the leucocytes are living,
the lower blue layer becomes decolorised in a short time (in about two
hours); when the corpuscles are dead, decoloration does not take place.
By adding to the mixture of leucocytes and colouring matter some
leucocidin, alone or along with antileucocidic serum, it is possible not
only to observe with the naked eye the phenomena which take place in
these cases, but also to estimate to some extent the proportions of
poison and counterpoison.
All these researches make it clear that the antitoxin acts directly on
the leucocidin. Similar facts have been noted as regards certain other
organic poisons and their antitoxins. Shortly after the discovery of
antileucocidin by Denys and van de Velde, Kanthack made a communication
to the Physiological Society in 1896[573], exhibiting tubes in which the
coagulating action of Cobra venom on the blood had been prevented by the
addition of antivenomous serum. Of all the experiments, however, made to
prove the direct action of antitoxin on toxin, Ehrlich’s[574] have
played the most important part in the study of this question. Ehrlich
directed his attention to ricin which, as Kobert demonstrated, has the
property of agglutinating the red corpuscles of defibrinated blood. This
phenomenon can be easily observed _in vitro_. In tubes containing red
blood corpuscles, the addition of ricin causes these corpuscles to
agglutinate into clumps and to fall to the bottom of the tube, leaving a
clear supernatant fluid. After adding progressively increasing
quantities of antiricic serum to the tubes containing fluid blood and
ricin, Ehrlich was able to demonstrate that small quantities of
antiricin merely retarded the precipitation of the red corpuscles,
whilst larger doses completely prevented it. Having studied the
proportions of ricin and its antidote, necessary to retard and prevent
the fatal poisoning of animals, Ehrlich was struck by the parallelism
which is exhibited between the action of the antitoxin in the living
animal and that in the test tubes.
[Sidenote: [379]]
The study of anticytotoxins, discussed in the fifth chapter, has
furnished another opportunity of observing the action of antitoxins _in
vitro_. Camus and Gley and H. Kossel were the first to observe the
action _in vitro_ of antitoxic serum against the ichthyotoxin of eel’s
serum. Since this observation, this phenomenon has been repeatedly
studied in the antihaemolysins and antispermotoxins. The antidiastatic
serums also act _in vitro_ and, as their effect can be demonstrated on
soluble ferments placed in contact with unorganised bodies, such as
gelatine and casein, the purely chemical character of the reaction is
all the more strikingly shown. We are indebted to von Dungern, Briot and
Morgenroth for accurate observations on this subject.
Martin and Cherry[575] made use of a different method to demonstrate the
direct action of antitoxins on toxins which exhibit their toxic power on
the animal organism. They chose snake venom mixed with antivenomous
serum. The mixtures were filtered under great pressure [50 atmospheres]
through a film of gelatine, under the idea that, if the venom and
antitoxin were not chemically combined, the former alone, owing to its
much smaller molecules as compared with those of the antivenom, would
pass into the filtered fluid. This fluid should, under these conditions,
possess a toxic power for animals, when the mixture, used for
filtration, was deprived of the larger molecules. Martin and Cherry left
the venom and the antitoxic serum in contact for periods of varying
length, before filtering the mixtures. As the result of a series of such
experiments carried out according to this scheme, they found that the
product of the filtration made after some minutes’ contact between the
two substances, was distinctly toxic; whilst the filtrate obtained after
a contact of half-an-hour was absolutely innocuous. From their
observations these authors conclude that the antitoxin enters into
chemical combination with the venom, but that the combination does not
take place instantaneously, a certain amount of time being necessary for
its accomplishment.
[Sidenote: [380]]
In addition to the time factor others have an influence on the
combination between toxins and antitoxins, as is seen from
Ehrlich’s[576] and Knorr’s[577] investigations. Both observers have
shown that antitoxin neutralises the toxin more slowly in dilute
solutions than in more concentrated form. For this reason, when animals
are injected with very weak solutions, the toxin may manifest its action
before it can be neutralised by the antitoxin; this may lead to
erroneous conclusions. On the other hand, according to data furnished by
these authors, temperature also exerts an influence on the combination.
Lowering the temperature retards, whilst raising it accelerates the
neutralisation of the toxins by the antitoxins. Insisting on the purely
chemical character of the combination between these two substances,
Ehrlich and Knorr adduce the fact that this combination, in cases where
we have a complete neutralisation of the toxin, follows, most
rigorously, the law of multiple doses, that is to say, in order to
render innocuous a hundred doses of toxin we have only to take a hundred
times the quantity of antitoxin.
The series of facts summarised above demonstrate distinctly that
antitoxins act directly on toxins. But how can this result be reconciled
with the observations given above according to which must be admitted
the no less real influence of the organism of the living animal on
intoxication by mixtures of antitoxin with toxin? Knorr[578] sought at
first to minimise the importance of the facts brought forward by Buchner
and Roux. He failed to corroborate Buchner’s results and found that the
injection of mixtures, made with very large doses of tetanus toxin
(20,000 times the minimal lethal dose) and corresponding quantities of
antitetanus serum, brought about the same effect in guinea-pigs and
mice. By modifying the quantity of antitoxin, he rendered the mixture
equally innocuous or equally toxic for these two species. But the data
given by Knorr are quite sufficient to prevent us from accepting his
conclusion. In his experiments, as in those of Buchner, the guinea-pigs
manifested a greater susceptibility and died from mixtures which, in
mice, caused merely a tetanus of medium intensity.
[Sidenote: [381]]
Some have sought to explain Buchner’s experiment by assuming that the
mixtures, lethal for the guinea-pig and innocuous for the mouse, owed
their toxic action to the presence of the _tetanus toxone_ and not of
the true tetanus poison, the _tetanospasmin_. This hypothesis of
toxones, as stated above, was put forward by Ehrlich as the outcome of
his ingenious researches on the constitution of the diphtheria poison.
As, however, the toxones must act differently from the toxins, we can
only attribute to their action the results in those cases where the
guinea-pigs die without presenting typical symptoms of true tetanus,
that is to say without spasms. Now, in Buchner’s experiments, a much
larger proportion of these animals, injected with the same mixtures as
the mice, succumbed and exhibited the characteristic tetanic
convulsions. Even in those cases, however, where the death of the
guinea-pigs might be attributable to an intoxication by the toxone, the
general result could not be altered. The toxones are, according to
Ehrlich, manufactured by the micro-organisms in the culture media and
form an integral part of the natural microbial poisons. Again, they are
neutralised by antitoxic serums. If, therefore, in spite of there being
the same quantity of toxones and of antitoxin in the mixtures, these
mixtures become more toxic for the guinea-pig than for the mouse, we
have an indication that some special change must take place in the
animal to upset the conditions of toxicity.
Weigert[579] accepts the accuracy of Buchner’s experiment, which,
indeed, can no longer be denied, but explains it on the hypothesis that
there is some substance in the animal possessing a very great affinity
for the toxin. This substance is supposed to be capable of decomposing
the innocuous combination of the antitoxin with the toxin, just as heat
does in Calmette’s and Wassermann’s experiments, described above. In
both cases the toxin would be set free to exert its noxious action. Such
a hypothesis is very probable, because it agrees with direct
observation, but it compels us to accept some new phenomenon which is
produced not _in vitro_, but in the living animal, and which carries on
its work in a very different fashion in the guinea-pig and in the mouse.
In the present imperfect state of our knowledge it is very difficult to
form any idea of the precise conditions which must intervene in the
organism of the guinea-pig to cause the tetanus toxin to act in a
mixture with antitoxin which is much more innocuous for the mouse. In
order, however, to satisfy those who seek to understand these complex
phenomena, it may be useful to cite another example of antitoxic action
in which certain factors are distinguished by their simplicity.
[Sidenote: [382]]
Lang, Heymans and Masoin[580] have demonstrated that hyposulphite of
soda prevents poisoning by prussic acid. This terrible poison becomes
innocuous if we take care to introduce into the animal by any channel
whatever (subcutaneously, intravenously, or by the stomach) a sufficient
quantity of hyposulphite of soda. Under these conditions the sulphite is
substituted for the hydrogen of the prussic acid, transforming the
poison into sulphocyanic acid, which has no action on the organism. The
hyposulphite of soda, then, acts as the antitoxin of the prussic acid,
thanks to a chemical reaction of substitution between bodies of simple
composition. We have never yet succeeded in reproducing this reaction
_in vitro_, whilst in the animal body it is effected with very great
ease. Consequently, we are quite justified in invoking special
conditions in the body of the living animal; this, however, does not
preclude the possibility of a transformation of the toxic substance into
an innocuous substance through a chemical reaction. It is probable that
analogous phenomena may also be met with in the action of true
antitoxins on the microbial toxins or allied substances (venoms,
vegetable toxalbumins).
[Sidenote: [383]]
The case of the destruction of micro-organisms, which is now more easily
studied because it is possible to observe with the eye the fate of these
organisms in the animal, is a further source of valuable information.
The direct action of cytases on certain bacteria, such as the cholera
vibrio, can be just as easily demonstrated _in vitro_ as can the action
of antiricin on ricin. If we proceeded to argue from this, a perfectly
accurate observation, that the living animal plays no part in the
destruction of the micro-organisms and that this destruction takes place
always in a fashion analogous to Pfeiffer’s phenomenon _in vitro_, we
should undoubtedly arrive at an erroneous conclusion. We know already,
as has been indicated in previous chapters, that the granular
transformation of vibrios is only part of a whole series of phenomena of
destruction of micro-organisms, the great majority of which phenomena
require more or less active intervention of the animal organism. In
reality, matters usually go on in a very complicated fashion, in which
direct and indirect actions are blended in varied proportions. In the
examples described elsewhere, we see, alongside the granular
transformation, an agglutination into clumps and immobilisation, and an
ingestion and intracellular destruction of micro-organisms. The final
phase, no doubt, is always a chemical or physico-chemical action,
exerted against the micro-organism, but how varied are the means used to
bring about this result! We may surely be allowed to suppose that
analogous phenomena may take place in the action of antitoxins on the
toxins.
[Sidenote: [384]]
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]]
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.
[Sidenote: [408]]
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.
[Sidenote: [409]]
[Sidenote: [410]]
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.
[Sidenote: [411]]
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.
[Sidenote: [412]]
[Sidenote: [413]]
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.
[Sidenote: [414]]
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.
[Sidenote: [415]]
[Sidenote: [416]]
The absorption of the tetanus toxin becomes evident when we study in
detail the phenomena produced in the experiments carried out according
to Vaillard’s methods with tetanus spores and those of Wassermann and
Takaki with poison to which cerebral emulsion has been added, or
according to Stoudensky’s method with grains of carmine. When, however,
it is desired to bring forward rigorous proof of the presence of the
tetanus toxin inside the leucocytes charged with spores, with granules
of cerebral substance or with grains of carmine, very great difficulties
are encountered. How, indeed, is it possible to demonstrate this poison
fixed upon these various bodies, a poison, the presence of which cannot
be demonstrated except by its injection into the animal? For this, in
the study of the reaction of the organism of the animal against the
poisons, it is very important to have recourse to substances, whose
presence can be demonstrated more easily than can the microbial toxins.
We must first have recourse to the alkaloids, especially atropin, which,
in this respect, present numerous advantages. We know that rabbits
resist considerable doses of sulphate of atropin, even when this poison
is injected directly into the blood. On the other hand, when it is
introduced into the brain, according to Roux and Borrel’s method, even
small quantities are quite sufficient, as demonstrated by Calmette[642],
to produce a fatal poisoning. The intracerebral injection of the
one-hundredth part of a dose which, when introduced into the circulation
of the rabbit, produces no disturbance, in the same animal at the end of
a few minutes sets up an enormous pupillary dilatation with symptoms of
very lively excitation, increase of the reflexes, and general
anaesthesia. These phenomena are succeeded by paralysis and death, which
supervenes three or four hours after the injection. The natural immunity
of the rabbit against atropin falls therefore into the same category as
that against morphin. It is not due to the innate insusceptibility of
the nerve cells, but to something which prevents the alkaloid from
reaching these living elements. With the object of ascertaining the
mechanism of this immunity, Calmette injected into the veins of rabbits
a fairly large quantity of sulphate of atropin (0·2), he then bled these
animals and collected from their blood the plasma and the white
corpuscles, separating them by centrifugalisation. When injected into
the brain of other rabbits, these constituents of the blood did not act
in the same way. Whilst large doses of plasma set up merely a short
period of excitation and a very transitory pupillary dilatation,
corresponding quantities of leucocytes caused grave disturbances,
sometimes followed by death in from seven to twelve hours. Calmette
concludes from his researches that the atropin does not remain in the
fluid part of the blood, since mere traces of it are found in the serum,
but that it is seized and absorbed almost immediately by the
leucocytes[643]. This result has been confirmed by Lombard[644] by
another series of experiments. After injecting very large quantities of
sulphate of atropin into rabbits and guinea-pigs, he bled these animals
and separated out the elements of their blood. Instead of introducing
these elements into the brain of rabbits, he injected them into cats,
animals very sensitive to atropin. The cats which received the red
corpuscles and the plasma exhibited very insignificant symptoms of
poisoning. Those, on the other hand, which were injected with a
corresponding quantity of leucocytes, had much graver symptoms of
intoxication, such as photophobia with maximal pupillary dilatation,
dysphagia and persistent diarrhoea.
It is, therefore, to the absorption of the atropin by the leucocytes
that naturally refractory animals owe their immunity, an immunity which
is very marked in spite of the susceptibility of the nervous elements of
these animals. We have been able to obtain this result thanks to the
delicate physiological reactions obtained with certain alkaloids. As
regards arsenic the demonstration could be pushed even further, for the
absorption of this mineral poison by the leucocytes has been established
by chemical analysis.
[Sidenote: [417]]
When engaged in my researches on the leucocytic phenomena in
intoxications I succeeded[645] in showing that in rabbits subjected to
rapidly fatal doses of arsenious acid, there is a marked diminution in
the number of white corpuscles in the blood. On the other hand, in
rabbits habituated to arsenic, the same doses which brought about
hypoleucocytosis and death of the control rabbits, induced a
considerable rise in the number of leucocytes. Later, Besredka[646] made
continuous and detailed researches upon this subject and obtained most
interesting results. In order to simplify the conditions of experiment,
he studied the reaction of the organism of the animal after the
introduction of a red trisulphide of arsenic[647], a not very soluble
salt, easily recognisable by its colour and markedly toxic. When
non-lethal doses of this salt were injected into the peritoneal cavity
of the guinea-pig, there was, first a transitory fall in the number of
the white corpuscles in the peritoneal fluid, followed by a
hyperleucocytosis of the most marked character. Of the leucocytes
accumulated in the exudation the macrophages almost exclusively seized
the yellowish-red granules of the trisulphide of arsenic. Very shortly,
the whole of the salt injected was found within the peritoneal
leucocytes, and the animals in which this marked phagocytosis occurred
remained in good health. The ingested granules could be observed for
several days in the macrophages; but in course of time, these arsenical
particles were broken up into very small granules and ultimately
disappeared. Here, then, we have an intraphagocytic solution of the
trisulphide of arsenic and very probably a transformation of this salt
into some other arsenical combination, innocuous to the animal. This
soluble substance escapes from the macrophages and is finally excreted
by the urinary passages.
[Sidenote: [418]]
Since the phagocytes ingest the trisulphide of arsenic and render it
innocuous, it was to be anticipated that the elimination of these
protective cells would lead to a fatal poisoning by doses which, under
normal conditions, are readily withstood by guinea-pigs. When Besredka
used sacs of reed pith containing non-fatal quantities of the red
trisulphide and introduced them into the peritoneal cavity of
guinea-pigs these animals were not long in exhibiting symptoms of
poisoning and died at the end of a longer or shorter period, this
varying with the amount of poison introduced. Even when the phagocytic
reaction had been impaired as the result of a previous injection of
carmine powder, the guinea-pigs died after doses of trisulphide of
arsenic which, under ordinary conditions, did not kill them. The
phagocytes in this experiment devoured numerous grains of carmine and
were rendered incapable of ingesting enough of the trisulphide of
arsenic to save the animal. On the other hand, when Besredka set up a
previous accumulation of macrophages in the peritoneal cavity of his
guinea-pigs, he succeeded in rendering these animals resistant to doses
of trisulphide of arsenic that, under normal conditions, were fatal. The
whole of these facts converge to show that the phagocytes, thanks to
their power of seizing the trisulphide of arsenic and of modifying it
within them, exercise a beneficent and immunising action on the organism
of the animal. The analogy of the main facts concerning this protective
influence with that observed in the immunity against infective
micro-organisms is indeed very considerable.
Having determined the part played by the macrophages in the resistance
of the organism of the animal against a not very soluble salt of
arsenic, Besredka proceeded to study the leucocytic phenomena in
poisoning by soluble arsenical compounds. In his experiments he made use
of potassium arsenite and he found that when lethal doses were injected
the guinea-pigs showed a diminution of leucocytes in the blood in less
than 24 hours, whilst with non-lethal doses, he produced a marked
hyperleucocytosis. When he injected lethal doses into rabbits accustomed
to arsenic, these animals manifested an increase of white corpuscles,
just as in animals injected with non-lethal doses. These oscillations in
the number of leucocytes, like those which have been observed after
poisoning by trisulphide of arsenic, certainly indicate that the
organism and its defensive cells behave in the same way to both slightly
soluble and very soluble salts of arsenic. In the first case it was easy
to demonstrate that the accumulation of leucocytes in the blood and in
the peritoneal exudation terminated in the ingestion of the granules of
trisulphide. With potassium arsenite, it was not so easy to prove the
point; a chemical analysis of the elements of the blood, however, has
given a decisive answer. After injecting the lethal dose of this soluble
salt into rabbits accustomed to arsenic, Besredka bled them in order to
separate the plasma, leucocytes and red corpuscles. Several experiments
made on these rabbits gave a concordant result which this observer sums
up thus: “Although the bulk of plasma and of red corpuscles was much
greater than that of the leucocytes, it was in the latter only that
arsenic was found” by chemical analysis. It was only in those cases
where the animals survived, and manifested hyperleucocytosis, that
Besredka succeeded in demonstrating the presence of arsenic in the white
corpuscles.
[Sidenote: [419]]
These experiments, excluding any doubt as to the protective part played
by the leucocytes against arsenical intoxication, of course suggested
the idea of investigating whether the nerve elements, submitted to the
direct influence of potassium arsenite, exhibit any real susceptibility
to this poison. The injection of solutions of this salt into the brain
demonstrated that the one-hundredth part of an ordinary lethal
subcutaneous dose was sufficient to cause fatal poisoning. This fact,
then, falls into line with other facts, already numerous, as to the
susceptibility of the nerve centres to microbial toxins, alkaloids and
other poisons. But in the case of potassium arsenite, it was even more
easily demonstrated than in the other cases that immunity natural or
acquired, is connected with the absorption of the poison by the
leucocytes. These cells, themselves much less susceptible to the toxic
action than are the nerve elements, protect them from contact with the
poison.
It is manifest that arsenic is not the only mineral substance capable of
being absorbed by the phagocytes, and there are already on record well
established facts in support of this thesis. Some time previous to the
researches on arsenical poisoning just summarised, Kobert, then in
Dorpat, set his pupils, Stender, Samoïloff, Lipsky and others[648] to
make systematic researches on the fate of iron in the animal organism.
For this purpose these observers made use of a very soluble preparation
of iron—or better expressed, as soluble as possible—Dr Hornemann’s
_ferrum oxydatum saccharatum solubile_, which does not precipitate in
alkaline media. They proved that a small quantity of the iron introduced
into the animal is eliminated by the kidneys and the wall of the
intestine, but that the greater part of the metal is arrested in the
organs, especially the liver, spleen and bone marrow. The iron is there
absorbed by the leucocytes which hold it for some time and then throw it
into the intestine.
[Sidenote: [420]]
I have had the opportunity of observing this circulation of Dr
Hornemann’s soluble salt in the organism of several species of
vertebrates. Some time after its introduction into the organism by the
blood vessels, peritoneally or subcutaneously, the iron may be found (by
means of the microchemical reaction with potassium ferrocyanide)
accumulated in the various phagocytes, especially the leucocytes, the
stellate Kupffer’s cells of the liver and the macrophages of the splenic
pulp. The non-phagocytic cells, as, for example, Ehrlich’s basophile
leucocytes, so abundant in the lymph of rats, take up very little of
this iron, although the macrophages and microphages are full of it[649].
Against these facts Weigert[650] has advanced the objection that the
leucocytes absorb only the iron precipitated in the form of granules,
but my own researches allow of no doubt that not only granular but
dissolved iron is absorbed. This discussion, however, loses much of its
importance in view of the results obtained with potassium arsenite.
According to Samoïloff[651], soluble salts of silver in the animal
organism undergo a fate similar to that of Hornemann’s soluble iron salt
and are absorbed by the phagocytic elements. It must be noted, further,
that according to the experiments of Arnozan and Montel[652], the
leucocytes absorb such drugs as calomel and salicylate of soda.
[Sidenote: [421]]
These observations all clearly show that the phagocytes must not be
looked upon as cells capable of seizing merely the dead bodies of
micro-organisms and of animal cells, always fearing and avoiding poisons
and only able to come forward when protected by some other antitoxic
function. The phagocytes no doubt often exhibit a negative
susceptibility for many poisons, when these are introduced into the
animal organism in too large a quantity. But these cells are most
resistant to toxic substances and protect the higher elements from the
poison. Under these conditions, it is quite natural to assign to the
phagocytes the rôle of the fighting agents of the animal organism
against poisons and we may even enquire whether these elements do not
produce the antitoxins. It has been pointed out that it is very
difficult to attribute this function to the cells susceptible to the
toxic action,—the spermatozoa in the production of antispermotoxin, the
red blood corpuscles in the development of antihaemotoxin, or the nerve
cells in the production of tetanus antitoxin. Moreover since, according
to Ehrlich’s theory, it is only the haptophore group which excites the
formation of antitoxins on the part of the elements which possess the
corresponding receptors, it is quite possible that the phagocytes,
thanks to the facility with which they absorb the poisons, occupy an
important place as producers of antitoxins. I have already formulated
this hypothesis, and several investigators, amongst whom may be cited
Gautier[653] and Courmont[654], have received it favourably, though in
the imperfect state of our knowledge, it cannot, as yet, be fully
demonstrated. It might perhaps be objected against this hypothesis that
in many instances, after the injection of micro-organisms living or
dead, in spite of a vigorous leucocytic reaction the organism of the
animal does not produce any antitoxin. In such cases, there is clearly a
development of antibodies, such as the fixatives, whose phagocytic
origin may reasonably be claimed, but no true antitoxins. It must not be
forgotten, however, that the various kinds of phagocytes present,
amongst themselves, great differences, and that perhaps certain only of
these elements are capable of producing antitoxins. When
micro-organisms, living or dead, are introduced into an animal it is
found that antitoxins do not as a rule appear in the fluids; in these
cases the reaction is set up mainly by the microphages. The macrophages
represent the principal source of antitoxins. In cases where these
phagocytes ingest the micro-organism the blood exhibits an undoubted
antitoxic power. Such is the case with bubonic plague in the human
subject, where the micro-organism is readily ingested by the
macrophages. Here we obtain antitoxic serums even after the introduction
of living or dead organisms into the animal, a fact observed by Roux and
his collaborators. Another fact in favour of the hypothesis I am
defending is furnished to us by the cayman. As noted above, this
reptile, of all known animals, supplies antitoxins most quickly and
easily. In the cayman the leucocytic system is composed of eosinophile
microphages filled with granules, and of macrophages. As the eosinophile
cells are only very weakly phagocytic, it is the macrophages almost
exclusively which intervene in the reaction against the micro-organisms.
It is probable, then, that in the cayman and in animals inoculated with
the plague bacillus the exclusion of the microphages from the struggle
constitutes a factor favourable to the production of antitoxins and at
the same time favourable to the manifestation of the activity of the
macrophages.
[Sidenote: [422]]
If these latter phagocytes play the primary rôle in the excretion of
antitoxins in the fluids of the body we should expect to find this
function exercised not only by the motile macrophages of the blood and
lymph, but also by the fixed macrophages, so widely diffused through
almost all the organs.
I advance this hypothesis for what it is worth, simply as a guiding idea
for new researches in this field, of which so much is still
unknown[655]. The brief account of the actual state of the question of
artificial immunity against toxins, has indicated to us that this is a
problem far more difficult of solution than is that of acquired immunity
against micro-organisms. The mere fact that these latter can still be
found some hours or even days after their entry into the refractory
animal, affords a great advantage in these researches as compared with
those on toxins which are lost, often almost immediately, after their
injection. Consequently our knowledge of antimicrobial immunity is more
advanced than is that on immunity against the soluble products of
micro-organisms.
The facts narrated in this chapter support the thesis I have defended on
the subject of immunity against micro-organisms—that antimicrobial
immunity in no way depends on a previous resistance against the toxins.
As a general rule the immunity against micro-organisms is developed more
readily than the immunity against their toxic products and at an earlier
stage.
Although much still remains to be done in the elucidation of the
mechanism of antitoxic immunity, the principal data acquired on the
subject of this immunity have undoubtedly led to applications of the
highest importance, as will be set forth in one of the following
chapters.
CHAPTER XIII
IMMUNITY OF THE SKIN AND MUCOUS MEMBRANES
Protective function of the skin.—Exfoliation of the epidermis as a
means of ridding the animal of micro-organisms.—Localisation and
arrest of micro-organisms in the dermis.—Intervention of
phagocytes in the defence of the skin.
Elimination of micro-organisms by the conjunctiva.—Microbicidal
function of the tears.—Absorption of toxins by the
conjunctiva.—Protection of the cornea.—Elimination of
micro-organisms by the nasal mucosa.—Protection by the respiratory
channels.—Dust cells.—Absorption of poisons by the respiratory
channels.
Alleged microbicidal property of the saliva.—Part played by microbial
products in the protection of the buccal cavity.—Antitoxic
function of the saliva.
Antiseptic action of the gastric juice.—Antitoxic function of pepsin.
Protective function of the alimentary canal.—Absence of microbicidal
power from the intestinal ferments.—Protective function of the
bile.—Antitoxic rôle of the digestive ferments.—Favouring and
retarding functions of the intestinal micro-organisms.—Destruction
of toxins by these micro-organisms.
Defensive rôle of the liver. Protective function of the lymphoid
organs of the alimentary canal.
Protective function of the mucous membrane of the genital
organs.—Autopurification of the vagina.
[Sidenote: [423]]
In the preceding chapters the phenomena of immunity which are exhibited
within the animal body in which the portals were open for the
penetration of the micro-organisms and their poisons have been studied.
We had to deal almost exclusively with experimental immunity, the study
of which constitutes the basis of our present knowledge concerning the
general problem of immunity. In natural immunity, however, things do not
follow the same course. The micro-organisms and their toxins are not
introduced directly into the tissues and blood by means of a syringe or
other instrument. The micro-organisms have to make their own way through
the skin and the mucosae, tissues which offer a resistance more or less
serious and effective; or they may have to take up their abode in the
cavities of the animal organism, in order that they may be able to
inundate it with their poisons. We must here review briefly these
natural barriers to microbial invasion.
[Sidenote: [424]]
The skin constitutes a protective covering of great importance in
connection with the preservation against microbial invasion of the
delicate parts of an animal. In many of the lower and higher animals,
and even in man himself, the skin becomes the seat of a microbial flora,
often very abundant, in which may be found, in addition to certain
inoffensive organisms, other minute parasites more or less harmful. The
pyogenic cocci, staphylococci and streptococci, are constantly found on
the human skin, most frequently hidden in the depths of the canals of
the hair follicles. These micro-organisms seize every favourable
opportunity to attack the organism, producing such local lesions of the
skin as acne, pimples, boils, and erysipelas, or even becoming
generalised in the blood and tissues, as in the septicaemias and
pyaemias. To the skin, therefore, must be assigned a very important
function in the prevention of the invasion of micro-organisms which are
found constantly on the surface of the body or which, along with all
kinds of dirt, are brought there accidentally.
[Sidenote: [425]]
The skin is able to fulfil this protective function from the fact that,
in most animals, it is covered with a not very permeable layer of some
considerable thickness. In the majority of the Invertebrata, of all
classes, the surface of the body is clothed with a chitinous layer,
sometimes very thin and capable of folding and following all the
movements of the body; or again it may be impregnated with calcareous
salts and very hard, as in the case of the integument of Insects and
Crustacea, and the shell of the Mollusca. In all cases this cutaneous
sheath constitutes a formidable obstacle to the entry of
micro-organisms. Even in animals of very small size the thin cuticle is
effective in preventing any invasion by these parasites. Thus the
_Saprolegniae_, fungi so fatal to many aquatic animals, are often quite
unable to pass through this cuticular layer. In order to pass this
obstacle their germs must take advantage of some fissure or wound,
produced by other causes. _Daphniae_, too, may often be observed to
succeed in ridding themselves of the _Monospora_ with its needle-like
spores by means of a mechanism which we have already described in
chapter VI. The white corpuscles of the blood surround the spores of
this parasite and transform them into an innocuous detritus. Sometimes,
however, a number of these fine spores manage to perforate the cutaneous
investment of the small crustacean; quite a small opening is made in the
chitinous wall, which in itself is a source of no danger. As soon,
however, as a spore of the _Saprolegnia_ approaches this opening, it
immediately begins to thrust a process through the small lesion, and
from that moment the fate of the _Daphnia_ is sealed. Incapable of
opposing the slightest phagocytic resistance to the filaments of the
fungus, it is invaded throughout by the mycelium and soon dies.
The integrity of the skin being so important for the preservation of
life, a fairly perfect mechanism has been elaborated for the maintenance
of this integrity. All animals, no matter what their position in the
animal scale, are liable to lesions and wounds of the surface of their
bodies. In the _Daphniae_ I have often[656] observed wounds produced by
the bites of other aquatic animals. The surface of these wounds soon
becomes covered with a rich microbial vegetation. The leucocytes are
brought up to the injured point and there produce a protective layer;
but, at the same time, a rapid proliferation of the neighbouring cells
of the epidermis takes place; this closes the wound and separates the
skin, so reconstituted, from the micro-organisms. Everything resumes its
original order and the leucocytes soon disperse, regaining the blood
stream.
These phenomena, which can be readily observed under the microscope in
such small and transparent animals as the _Daphniae_, may serve as the
prototype of those of a number of analogous processes in the animal
kingdom. The thicker and more solid the cuticular investment, the more
fully it guarantees the animal against the penetration of
micro-organisms. Cuénot[657] made the observation that Crustacea,
furnished with such a hard envelope as is the carapace of the Decapods,
are completely defenceless from the moment parasitic micro-organisms
make their way into their bodies. These intruders quietly instal
themselves in the tissues, without causing the slightest phagocytic
reaction, and thus bring about the inevitable death of the host. The
protection of the animal in this case is, so to speak, associated with
the resistance offered by the carapace.
[Sidenote: [426]]
Again, in many of the Vertebrata, the skin has a hard, thick sheath,
e.g., the scales of fishes and of reptiles. Man, with his supple and not
very thick skin, is less well endowed; this, however, does not prevent
him from defending himself against the entry of micro-organisms by the
cutaneous path. Sabouraud[658], a well-known dermatologist, has given a
very concise and at the same time very complete sketch of the part
played by the skin in the protection of the body against
micro-organisms; from this author the following data are borrowed.
The epidermic layer sets up a defence by the production and expulsion of
corneal cells. In the normal course of the life of the epidermis, the
cells of the deeper layers, coming to the surface, become exfoliated and
are thrown off. “There is thus produced, a continual exfoliation of the
dead layers, and a continual eviction of such micro-organisms as are
living on them. The epidermis is dense and its cells have a hard
envelope; the micro-organism is not endowed with motion, or at least not
with sufficient to be of service in effecting an entrance. It can only
penetrate the epidermis by multiplication _in situ_, a micro-organism
originates alongside another, another in front of it, and in front of
this again others. In this way they burrow between the apposed cells
just as a root penetrates into the ground; so great is the resistance of
the horny cells that we never find any micro-organisms within them, but
between them only” (p. 734). The epidermic cells, containing
micro-organisms, exfoliate, and the skin is thus ridded of them.
Frequently the process, as it goes on constantly and slowly, is
invisible; but often, on the other hand, it becomes exaggerated and
manifests itself in the form of a cuticular desquamation which leads to
the elimination of a large number of micro-organisms. The patient may
retain “such pellicles for ten years, and even longer, without
presenting anything else but these, and there are many other chronic
squamous infections in which the course is uncomplicated by even an
erosion or the slightest wound.”
The connective tissue of the human skin is also fully able to defend
itself; it is extremely vigorous and represents a real obstructing and
resisting tissue. The penetration of parasites sets up in it a
thickening of the fibrous tissue; this effects a localisation of the
microbial focus. To appreciate the effectiveness of this dermic defence,
we have only to compare the slow growth of lupus, a form of cutaneous
tuberculosis, with that of tuberculosis of the lungs and other viscera,
or the slow evolution of farcy, or cutaneous glanders, with that of the
visceral form of the disease.
[Sidenote: [427]]
If we examine more closely the process by which the dermis surrounds the
intruders with a fibrous capsule, we readily recognise in it a reaction
of the macrophages of the skin. In lupus these phagocytes seize the
tubercle bacilli, combining to form giant cells and giving rise to an
exaggerated development of the connective tissue fibres. Moreover, when
the skin is menaced with a microbial invasion, not only the local
macrophages but the leucocytes are mobilised. The migratory white
corpuscles travel through the epidermis and the connective tissue layer.
In spite of the absence of a lymphatic circulation in the epidermis, the
leucocytes penetrate into this layer “and, in a section through the
normal epidermis, it is very rare not to find here and there some
deformed and flattened leucocyte, surprised just as it was creeping
between the cells of the _rete mucosa_ or of the _stratum granulosum_.”
Immediately that the epidermis or the dermis finds itself menaced with a
microbial invasion, an accumulation of leucocytes of all kinds is
produced at once; this may remain microscopic or it may assume
proportions visible to the naked eye. Frequently the subjacent
epithelium throws off epidermic scales which are filled with leucocytes;
often also the leucocytic foci in the dermis become emptied, the
micro-organisms being expelled along with their enemies the phagocytes.
The tissues of the skin proper defend themselves against micro-organisms
as well as they are able; but so soon as the danger becomes serious
there is sent to their succour a whole army of mobile phagocytes. This
example of the defence made by the cutaneous investment may serve as a
prototype of that of every other region of the body. Alongside a local
action, there is always an intervention of mobile phagocytes; but when
this action becomes insufficient, a much more abundant accumulation of
leucocytes than is found in ordinary cases is immediately produced.
Like the skin, the mucous membranes are invested with an epithelial
layer, which serves as a barrier to the entry of micro-organisms. But
whilst the surface of the normal skin is dry or barely moistened by the
secretory products of the cutaneous glands, the mucous membranes are
always humid, a condition favourable to the multiplication of
micro-organisms. Hence the mucous membranes which are most exposed to
contact with the air and with external objects, always contain a larger
or smaller number of organisms, amongst which the pathogenic species,
notably staphylococci, pneumococci and streptococci, are the most
common. The part played by the animal organism in getting rid of these
micro-organisms becomes more complicated than in the case of the defence
made by the skin.
[Sidenote: [428]]
The first of the mucous membranes to be exposed to contamination by
micro-organisms is the conjunctiva of the eye. At the moment of birth it
is in contact with the vaginal mucous membrane and acquires from it some
of its micro-organisms, both innocuous and pathogenic. Tears fulfil the
function of averting the danger resulting from this proximity and from
the presence of micro-organisms in the conjunctival sac in general.
Ophthalmologists have shown that these tears transport the organisms
into the nasal cavity by means of the lachrymal canal. To determine this
point Bach[659] introduced a number of Kiel water bacilli along with
pyogenic staphylococci into the conjunctival sac of various individuals.
Seedings made with the tears showed a very rapid disappearance of the
two organisms, which passed into the nose where their presence could be
demonstrated by making plate cultures of the nasal mucus. Enormous
numbers of the Kiel bacilli, introduced into the conjunctival sac, were
all transferred to the nasal cavity, on the average, by the end of
half-an-hour. The pyogenic staphylococci persisted on the surface of the
conjunctiva for a longer period, but they also passed in large numbers
through the lachrymal canal into the nose.
[Sidenote: [429]]