Read Ebook: The Toxins and Venoms and Their Antibodies by Pozzi Escot M Emm Marius Emmanuel Cohn Alfred I Alfred Isaac Translator

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Rey-Pailhade, and later by Abelous and G?rard.

It must be remarked that the organs we have studied are essentially reducers, and that the more powerful reducers yield the most toxic extracts. We find here a confirmation of Armand Gautier's views regarding the anaerobic origin of the toxic substances formed within the organism.

Blood serum precipitated by alcohol affords products which possess very marked toxic power. It would appear that the toxic products we speak of here are thermogenic diastatic substances derived from the white blood corpuscles. In certain diseases the blood serum may acquire a high degree of toxicity. We will recur again presently to this property as a normal characteristic of the blood of various animal species, and will study it in greater detail in a future volume of this collection, devoted to the immunizing active principles.

The greater number of the other glands contain proteid matters and various peptones, more or less toxic, with amides and alkaloids.

Particular mention must be made of the thyroid gland, the secretions of which exercise a powerful action on the nervous centers and on nutrition. It appears reasonable to attribute to the secretions of this gland a very powerful antitoxic action, and the first proof of this fact is that the organisms deprived of this gland become the seat of serious derangements; the urines of such organisms become particularly toxic, while, on the other hand, the hypodermic injections of the aqueous extract of the gland, when the derangements spoken of exist, cause the immediate disappearance of the derangements caused by the excision of the gland.

The suprarenal capsules also possess properties that have often attracted the attention of physiologists during the last few years. They are considered as being, just like the thyroid gland, producers of antitoxins; they destroy, or seem to destroy, toxins that are artificially introduced into the circulation.

LANGLOIS: Th?se de doctorat en M?d., Paris, 1897.

The microbial toxins possess two essential properties; one the pyogenic property, thanks to which the toxins first attract, then destroy the white blood corpuscles or leucocytes, and transform them into pus, and the other the pyretogenic property, which appears to belong only quite indirectly to the pyogenic substance. The toxins in general retard the heart action.

We will not speak of the distinctions it has been sought to establish between the substances which possess these different properties, but will at once take up the discussion of several of the microbial toxins.

The cultures of the bacillus are made in Liebig's bouillon, to which has been added 0.1% of fibrin, the whole being carefully sterilized for a long time at 110? C. The cultures medium is inoculated with a drop of blood taken from the heart or spleen of an animal that has died of anthrax. At the end of a week, the culture is filtered, and the filtrate acidulated with a little acetic acid and precipitated by adding powdered ammonium sulphate. The flocculent precipitate is collected, washed, dissolved in distilled water, and dialyzed. The dialyzed solution is concentrated in vacuo at 40-45? C., and precipitated by adding to it alcohol. The precipitate formed is then collected and dried.

In this manner there is obtained a grayish-white substance which is soluble in water, and which is fatal in large doses, but which, given in repeated small doses, confers immunity against anthrax.

According to Hankin, it seems that the toxic property of this toxin is due to an albumose.

Marchoux has been able to confer immunity upon sheep by injecting first small quantities of the filtered culture of the anthrax bacilli, and then the virulent anthrax itself.

The animals thus rendered immune yield a serum which may be used as a vaccin against anthrax, and which even possesses curative properties under certain conditions.

In every case the acquired immunity is only temporary. We will recall to recollection the method employed by Pasteur for vaccinating against anthrax, using attenuated cultures, a method which is practiced daily at the present time.

From the cultures of symptomatic anthrax Chauv?e extracted a very active toxin which can withstand without impairment a temperature of 110?C. Roux has shown that the serum of animals that have succumbed to the symptomatic anthrax is capable of vaccinating against this disease; we have here a new proof that the antitoxin is in fact a product of the defense of the cells of the organism, and the author mentioned has been able to vaccinate guinea-pigs by injecting into the peritoneum culture bouillon sterilized by heating to 115? C. or by filtering through porcelain.

Prolonged boiling on the water-bath completely destroys the activity of this tuberculin, which moreover hardly ever keeps longer than three weeks. It has been found possible to preserve it for an indefinite period, however, by adding to it 30 to 40 per cent. of glycerin. It possesses all the general reactions of albuminoids.

Tuberculin is not toxic in the proper sense of the word. Injected in small quantities into the healthy human being and into healthy animals, it exerts no effect; on the other hand, however, in tubercular organisms, even in incipient stages of the disease, even where it is almost impossible to make a clinical diagnosis, the injection of very small quantities develops a lively and characteristic reaction.

Grasset and Vedel consider the tuberculin as an excellent means of diagnosing tuberculosis in man, but in such a case it is necessary to operate with the greatest caution, with very small quantities of the tuberculin, and to feel, in some sort, the sensitiveness of the patient, particularly in the case of children.

It is chiefly for the diagnosis of tuberculosis in cattle, however, that tuberculin is valuable. Thanks to Nocard, the procedure has to-day become a common practice. The injection of a fairly large dose, 0.3 to 0.4 Gm., according to the size of the animal, causes, in about ten hours or so, if the animal is tuberculous, a strong febrile reaction with an elevation of temperature of 1.5 to 3? C., whereas if the animal is not tuberculous no such reaction takes place.

Cases in which tuberculosis is far advanced, and in which the organism is impregnated with tuberculin, do not react after the injection of tuberculin.

Tuberculin does not confer immunity, and the bacillus retains all its virulence, even in injected tissues; nevertheless, the return to health of animals in which injections have been recently made may be due to the action of large doses of the serum; and on the other hand the tuberculin, in large quantities, may render the location unsuitable for the development of the tubercle bacilli.

Roux and Yersin were the first to affirm that diphtheria is an autointoxication caused by a very active poison formed by the microbe in the restricted locality where it develops. In order to obtain this toxin a culture of the bacillus is first made in a mutton bouillon made strongly alkaline with sodium carbonate , and with the addition of 2 per cent. of peptone. At the end of about one month, the culture being kept at about 37? C., the liquid is filtered through porcelain. It is indispensable to employ a very virulent bacillus; it is hence frequently advantageous to increase the virulence and toxigenic power of the bacilli it is desired to use.

The toxic liquid obtained is exceedingly powerful: 0.1 Cc. kills a rabbit in forty-eight hours. This toxin is very sensitive to the effects of heat. When heated to 65? C. it loses almost all its toxicity; at 70? C. it becomes innocuous; and it only requires to be heated to 100? C. for fifteen minutes in order to lose all immediate activity even in large doses. Nevertheless toxins thus weakened are capable of proving fatal to an animal even after five or six months.

Light, oxygen, ozone and all oxidizers destroy the active principle of the diphtheria toxin, which is, moreover, rendered almost inactive by organic acids.

This toxin is capable of diffusing through animal membranes, a fact that is in agreement with the toxic effect seen in a subject attacked with diphtheria, and due to the toxin passing through the mucosa. In spite of this property, however, the diphtheritic poison may be taken into the stomach without any pernicious results.

Roux and Yersin have shown that, like all the diastases, it may be precipitated from its solutions by the development, within these, of certain precipitates, particularly calcium phosphate. It is precipitated from its solutions by alcohol, as has been observed also in the case of diastatic solutions. All the toxic substance is contained in the albuminous precipitate thus obtained; but the prolonged action of alcohol, or repeated successive precipitations, alter it finally. Diphtheria toxin is likewise precipitated by the reagents for albumoses, particularly sodium sulphate in saturated solution. This procedure has been utilized by Brieger and Fraenkel for preparing the pure toxin, which they obtained in the form of very light, brilliant white, amorphous flocks, affording all the principal reactions of the soluble albumoses , and which they characterized as a toxalbumin.

On injecting into healthy animals this diphtheria toxin attenuated by sufficiently heating at 70? C, employing at first small doses, and gradually increasing, it is possible to immunize them against diphtheria, as was first demonstrated by Carl Fraenkel.

Roux and Martin, who have specially studied this procedure, have shown that a horse may be easily immunized by injecting into the animal the toxin diluted with a third of its volume of Gram's iodine solution, and in successively increasing doses. The initial dose is 0.25 Cc.; then, after two days, 0.5 Cc. of the same toxin is injected, and in like manner the dose is increased up to the eighteenth day, when the pure toxin is injected, at first in small doses, which are gradually increased so that at the end of two or three months injections of 80 Cc. of the pure toxin may be given without danger; the animal is then completely immunized.

The serum of an animal rendered immune in this manner contains a diphtheria antitoxin which possesses high power. A guinea-pig which has received an injection of 0.01 Cc. of the antitoxin is perfectly capable of withstanding a lethal dose of 0.5 Cc. of the toxin. The antidiphtheria serum thus obtained, and in almost limitless quantities, from an immunized animal, is capable of saturating the therapeutic diphtheritic toxin, and has to-day taken rank in therapeutics as the most efficacious remedy in diphtheria. Injected in varying doses, it confers a temporary but immediate immunity.

Nevertheless antidiphtheria serum must not be considered as an antidote; and in pathological diphtheria, the more serum is required the later it is used. In certain cases, if employed too late, it may prove ineffective.

The preventive action of the serum is remarkable. In 10 000 inoculated cases Behring and Ehrlich have had but 10 cases of diphtheria, and these were, moreover, of a benign character. The duration of the immunizing action appears to be from three weeks to two months.

This diphtheria antitoxin was first prepared by Gu?rin and Mac? by adding to the antidiphtheria serum a large volume of alcohol, washing the precipitate, and drying it in a vacuum. It is soluble in water, and loses its activity when heated to 65? C. Wassermann has proposed to extract it from the milk of immunized animals, by first coagulating the milk by rennet in the presence of sodium chloride, filtering, and removing the fat from the clear liquid by means of chloroform. After decanting, the clear solution obtained is precipitated by adding to it 30 to 33 per cent. of ammonium sulphate. The precipitate is dried in a vacuum on a polished porcelain slab after having first been strongly expressed. It is then dissolved in water.

Experiment has shown that the culture bouillon thus obtained contains two kinds of toxic substances--highly toxic alkaloidal bases , and a true toxin, possessing diastatic properties, and of almost incredible toxic power.

This toxin had already been isolated by Kitasato. It is a toxalbumin, and is very sensitive to the action of heat. A temperature of 65? C., maintained for 30 minutes, renders it quite inactive; and it becomes oxidized and is destroyed by the action of the air in the presence of light.

Brieger and Boer, by precipitating with zinc chloride the filtered culture bouillon, obtained a pure, amorphous tetanus toxin, which they also considered as a toxalbumin, and which possesses exceedingly toxic properties.

If a precipitate be caused to form in these toxic solutions, as, for instance, a precipitate of calcium phosphate, this carries down with it all the toxin present in the liquid. 0.0005 Gm. of this precipitate is surely fatal to a guinea-pig.

Dozon and Cournemont have observed that even in doses of 300 to 400 Gm. of the filtered culture liquid, this toxin is not immediately toxic to a horse, but kills the animal only after a period of incubation of at least twenty-four hours. The blood of such an animal, however, is immediately and directly fatal to animals into which it is injected.

The immunized animals yield a serum which, mixed with tetanus cultures, renders these innocuous, and which enjoys an antitoxic power that borders on the marvelous. A quintillionth of a cubic centimeter of the serum per gramme weight of a live mouse suffices to protect the animal from an otherwise fatal quantity of tetanus toxin.

This serum is nevertheless powerless to preserve man in cases of acute tetanus; it confers an immediate, but only transitory, immunity.

As to its mode of action, it appears to cause a permanent condition of excitation or of nutritive reaction of the cells, which makes these resistant to the poison. As in the case of the other toxins, the quantity of antitoxin necessary to protect an organism is so much greater the later the treatment is applied.

This solution keeps well when kept from air, light, and heat. In practice it is employed in 10-per cent. solution in phenolated water . The mallein may be precipitated from the crude solution by the addition of alcohol, as recommended by Foth. Foth's mallein occurs as a white, light powder, very easily soluble in water.

Mallein enjoys a very important r?le in veterinary therapeutics, a r?le analogous to that of tuberculin, permitting the diagnosis of incipient glanders.

Experience has shown that in animals already attacked by glanders, even if ever so slightly, the thermic reaction never fails when 0.25 Cc. of the mallein solution is injected. In healthy animals, however, the injection of mallein, even in much larger quantities, causes no apparent effect. In animals attacked by glanders the reaction attains its maximum in twelve hours, and several days are required for the temperature to return to normal.

According to Nocard, mallein possesses no immunizing properties whatever.

The same author, in collaboration with Fraenkel, later on isolated a toxalbumin from the culture bouillon of the typhoid bacillus. Sanarelli obtained an active toxin by macerating for several days at 60? C. a month-old culture of the typhoid bacillus made with a 2-per cent. glycerin-bouillon. Chantemesse has also published a process which yields a highly virulent toxin.

Chantemesse and Widal have shown that on injecting into an organism increasing quantities of the sterilized cultures of Eberth's Bacillus, it is possible to fully immunize an animal against the bacillus itself, and even also against the Bacillus coli communis. The operation, however, is tedious and painful. The serum of immunized animals possesses preventive and curative properties respecting the effects of typhoid bacilli.

A dose of the filtered culture, which is fatal to a guinea-pig, becomes innocuous when mixed with 0.5 Cc. of the serum of a vaccinated guinea-pig; 6 Cc. of the serum injected six hours after an injection of the virulent culture, hence when this is in full action, suffice to save the animal. So far as the human being is concerned, the results obtained have not been sufficiently satisfactory.

The culture bouillon of the Bacillus coli communis, which is closely allied to Eberth's bacillus, also contains soluble toxic substances which have been named coli-bacillus toxin. This substance, which is produced only in small quantity by the microbe, is fatal only in very large doses.

Villiers found in it a liquid ptomaine; Klebs found another and crystallizable ptomaine; while Pitai discovered in it a toxin unalterable by heat, and which he considered as a toxopeptone. According to Gamaleia there is present a true toxin, alterable by heat, and the reactions of which entitle it to be considered as a nucleo-albumin; he has also found in it a toxic nuclein.

These toxic substances are found, according to Gamaleia, Pfeiffer, and Sanarelli, confined during the life of the microbe within its cellular envelope, and does not diffuse through this. Metchnikoff and Roux are of the contrary opinion, however, and they have prepared a toxin almost insensitive to a temperature of 100? C., and precipitable from its solutions by ammonium sulphate or strong alcohol; the toxin is a toxalbumin. This toxin is quite toxic; one-third of a cubic centimeter suffices to kill 100 Gm. of guinea-pig in 18 hours; with larger doses, death is almost immediate.

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