Toxins

TOXINS. The concept of "toxin" entered immunobiology at the end of the 19th century, when substances were discovered in animals, plants, and bacteria that possessed the following main properties: 1) When introduced into an animal organism, they cause symptoms of poisoning and sometimes death of animals. 2) Upon repeated introduction, starting with non-lethal doses, they cause the appearance in the animal organism of antitoxins capable of neutralizing their poisonous effect. 3) They exhibit strict specificity, combining only with their corresponding antitoxin. Toxins are found in animals, plants, and bacteria. Representatives of animal toxins include snake venom, scorpion venom, and the venom of some spiders (for example, Theraphosa) of tropical countries. Representatives of plant toxins are ricin, abrin, and crotonin. These poisons can be characterized as nerve and blood poisons. Ehrlich, who studied them, obtained sera capable of neutralizing their poisonous effect. Even greater importance is attached to bacterial toxins. From pathogenic bacteria, when grown on artificial nutrient media, substances were obtained that act poisonously in small doses. A feature of the first toxins obtained from microbes (diphtheria and tetanus) was that, like the microbes themselves, they caused characteristic symptoms of disease after a certain latent (incubation) period of action. The incubation period distinguishes toxins from simple chemical poisons. There are two main hypotheses to explain the formation of bacterial toxins: 1) the toxin is formed intracellularly in the microbes, 2) the toxin is formed extracellularly in the environment surrounding the microbe. The first hypothesis in turn is divided into two opinions, according to which: 1) the toxin is secreted by the living bacterial cell, 2) the toxin appears in the environment after the death of the cell and its destruction. The hypothesis of extracellular formation of toxins (Walbum's theory) for some time predominated in immunobiology. According to this theory, it was assumed that the toxin is present in the cell in the form of "protoxin" (a non-poisonous substance). In this form, it enters the surrounding microbe environment, where, combining with the products of protein breakdown, mainly with the albumoses of the medium, it acquires its poisonous properties. Walbum's arguments were as follows: 1) the presence of albumoses and peptones in the medium is necessary for toxin formation, 2) the artificial addition of these substances to the toxin enhances its effect. However, Walbum's experiments were not confirmed by Prigge, who proved that fluctuations in the strength of the toxin's effect depend on the individuality of the animal. The presence of albumoses and peptones in the medium for obtaining toxins affects the improvement of the vital activity of microbes, which facilitates the obtaining of strong toxins. Albumoses and other relatively large fragments of broken-down protein do not lead to the obtaining of strong toxins in all species of microbes. For example, the tetanus bacillus requires for enhanced toxin formation a more complete breakdown of these substances. Thus, the most probable assumption is that toxins are formed inside the microbial cell and then, during the life of the cell or after its death, pass into the medium. The large size of the toxin molecule, which with difficulty passes through an animal membrane during dialysis, suggests that the transition of the toxin into the medium during the life of the microbe is difficult. Observing toxin formation in various pathogenic microbes during their growth on liquid nutrient media, one can observe that some species give a significant concentration of toxin in the medium, while others rather firmly retain these toxic substances in the body of the cell, and they enter the medium in small quantities. On this basis, as well as based on various properties of toxins, Pfeiffer gave a classification, according to which toxins are divided into exo- and endotoxins. Based on the study of the diphtheria toxin discovered by Roux and Yersin and studied by Behring and Ehrlich, and the tetanus toxin by Kitasato, the following characteristics were given to exotoxins, otherwise called true toxins. 1. The toxin acts in infinitesimally small quantities. 2. It does not act immediately, but after a certain incubation period, which noticeably decreases with an increase in dose. 3. These toxins are very labile under the action of elevated temperature, light, and other physicochemical factors. 4. They are active antigens, causing the formation of highly active serum that neutralizes the toxin. Ehrlich believed that the combination of toxin with antitoxin obeys the law of multiple proportions, similar to the combination of a strong acid with a base. Bordet and his followers dispute this (see Immunity). In contrast to "true" toxins, endotoxins, which are closely connected with the body of the microbe, are thermostable, weakly poisonous, and little antigenic. Typical representatives of microbes that produce endotoxins were considered the typhoid bacillus, the cholera vibrio, Pfeiffer's bacillus, the meningococcus, Bordet-Jangu's bacillus, the gonococcus, etc. The expansion of knowledge about the nature and properties of toxins has forced doubt to be cast on the correctness of dividing them into two opposite groups: exo- and endotoxins. Thus, among bacterial poisons classified as exotoxins, some were found that either did not correspond to the properties of true toxins or killed animals without a noticeable incubation period. As for the antigenic properties of these representatives, when animals were immunized with them, active sera were obtained. Endotoxins, as methods for obtaining them were improved, turned out to be more and more active and antigenic. Progress in this respect can be illustrated, for example, by the fact that the diphtheria toxin at one time killed animals only in a dose of 1 cm³, while at present it kills in doses of 0.001-0.0005 cm³. It is therefore obvious that Pfeiffer's old position about the differences between exo- and endotoxins does not reflect reality. Proof of this are the reports of various authors about obtaining free soluble toxins from microbes that were classified as producing only endotoxins. On the other hand, by obtaining extracts from microbial bodies, it is possible to prove the identity of exo- and endotoxins in microbes that were considered capable of producing only exotoxins, for example, in diphtheria (Prigge), dysentery (Kolle, Eisler), tetanus and botulinus (Kovacs). The methods for obtaining exo- and endotoxins are different. Exotoxins are obtained by growing microbes in a liquid medium for 1-8 days and then freeing this medium from microbial bodies by filtering through a paper filter (microbes growing in films), or through infusorial earth, or talc filter until complete transparency, followed by the addition of preservatives. For complete sterilization, the toxins are filtered through Chamberland and Berkefeld candles, which however significantly weakens their titers. The resulting filtrates in practice are called toxins. They are used for immunizing horses and people. To obtain high-quality toxins, certain conditions must be observed, selecting them individually, applicable to a given type of microbe. The selection concerns the strain, the medium, its pH, the duration of growth, temperature conditions, etc. Not all strains of a given type of microbe have the ability to produce a strong toxin. Thus, for diphtheria toxin, the Park-Williams diphtheria culture is used everywhere, for scarlet fever—the Dochez streptococcus strain, etc. Some microbes produce the most active toxin if the medium contains large derivatives of protein (albumoses, peptones), others—if the medium contains products of more complete breakdown of protein (amino acids). Temperature conditions are also not indifferent for toxin formation. Most microbes actively produce toxins at 37°, while for example the diphtheria toxin is obtained most actively at 33-35° (Madsen, Park and Williams). The hydrogen ion number is a very important factor in toxin formation. Thus, pH = 7.6 is most optimal for some microbes, for example diphtheria, while for others (meningococcus) a lower pH = 6.8-7.0 is necessary. Furthermore, the method of sterilizing the medium is also important, since it depends on the greater or lesser breakdown of protein derivatives, which reduces the nutritional value of the medium. To obtain endotoxins, various methods of extracting toxic substances from the microbial cell are used: 1) autolysis; prolonged cultivation in an incubator; 2) extraction from bacterial bodies with distilled water and physiological solution; 3) extraction at elevated temperature and with shaking; 4) the action of alkalis and acids, anti-formalin; 5) grinding bacteria in an agate mortar and subsequent extraction (Bezredka); 6) freezing and thawing; 7) dissolution by enzymes (pepsin, trypsin, lysozyme, bacteriophage). When choosing one method or another, it must be kept in mind that when using all the above-mentioned substances that destroy the cell, the toxin itself may also be destroyed, and therefore the most recommended methods are freezing and thawing and mechanical damage to the cell with subsequent extraction with physiological solution. As early as Ehrlich, observing the interaction of toxins and sera, noted the complexity of the structure of toxins.

He discovered that T. becomes less toxic with aging, as well as under the influence of certain chemical substances, for example, formalin, iodoform, while retaining its antigenic function. The addition of formalin and exposure to elevated temperature are widely used at present to obtain non-toxic antigen (Ramon's anatoxin). Studying various series of T., Ehrlich found that some T., equal in their effect on animals (toxic function), possess different binding antigenic properties. These observations led Ehrlich to assume two constituent parts of T.—haptophoric, or binding, and toxophoric (poisonous). The haptophoric part is more stable, the toxophoric part is a labile component of T. Moreover, Ehrlich noted that T. is not stable, and in it during storage a series of changes occur, affecting its properties. Ehrlich called these derivatives of T. toxoids, toxons, etc. 'Toxon' is a modification of T. in which the toxic effect is weakened and is expressed in necroses and late paralyses (after 2-4 months). The affinity for antitoxin compared to T. is reduced in toxon. 'Toxoid' was named T. that has lost its toxic ability while retaining antigenic properties. 60S toxins Ehrlich explained the not always identical ratios between Lt and the minimum lethal dose (Dim) in different series of T. by this mosaic of substances contained in the filtrates of poisons. Later researchers confirmed the complexity of the structure of T. and the different stability of its individual fractions. Unchanged T. is a complete antigen. At present, complete antigens are decomposed into so-called 'haptens' and 'semihaptens'. Haptens are called the part of the antigen that carries specific properties, enters into immune reactions, but does not possess antigenic action (polysaccharides—Landsteiner, Avery and Heidelberger). Hemihapten (Sachs, Klopstok), or semihapten, is called the part of the hapten that enters into specific reactions with antibodies without a visible phase of reaction. They can be detected only by the absence of a specific reaction upon subsequent addition to the mixture of hemihapten with serum of complete antigens. American researchers (Avery, Goebel) propose a model of antigen structure, and they have shown that antigenic properties are acquired by haptens when they are attached to protein. This question, however, is not yet finally clarified; the work of Zo-zaya showed the possibility of immunizing mice with pneumococcal hapten and protecting them against live pneumococcus culture by using extremely small doses of hapten—polysaccharides. He showed that the hapten possesses antigenic properties. The author believes that the failures of Avery and Heidelberger when immunizing animals with large doses were due to the fact that haptens in these doses behave as aggressins (see). T. are substances easily destroyed by the action of various physical and chemical factors. Thus, raising the temperature to 60° acts lethally on diphtheria and tetanus T.; but there is a whole series of thermostable T. that withstand even higher temperatures. The surrounding medium affects sensitivity to temperature; this explains some inconsistency in the results of experiments studying the effect of physical and chemical agents on T. Freezing and thawing T. does not noticeably affect its strength. Dried T. are well preserved, which is used for preserving many standard T. (tetanus, etc.). When the pH of T. changes, its strength changes. At pH below 5.5 and above 10, toxicity is completely destroyed. The best preservation of T., its stabilization, occurs at pH=7.2-7.6. Ru and Hep-son showed that the weakening of toxicity under the action of acids can be restored again by alkalinization. This is confirmed by Dernby and Valbum, but this restoration is not complete. According to observations by Bronfenbrenner and Schlesinger, T. of botulinus, conversely, acquires greater toxicity upon acidification. Proteolytic enzymes (pepsin, trypsin) lead to the destruction of T.; it is possible that here it is not the enzyme itself but the acid and alkali that are required for manifestation of their enzymatic action. The size of the T. molecule is measured by dialyzing it through various membranes, and it has been established that T. slowly diffuse through parchment and do not pass through collodion membranes. Sunlight destroys T., scattered daylight weakens it much more slowly. Particularly active are ultraviolet rays. According to some authors, X-rays destroy T., while others state that this action is not strong. Radium is not active with respect to T. Oxidizers extremely affect T. Passing ozone, KMnO₄ causes strong destruction of it. Prolonged passage of atmospheric air has no noticeable effect on it. Metals (copper, silver) produce in vitro destruction not only of the toxophoric but also of the haptophoric group of T. (in vivo metals are inactive). The best preserving substances are carbolic acid and chloroform. The chemical nature of bacterial T. is still little studied. The former view that T. is a protein body is now increasingly being questioned. A number of researchers succeed in obtaining T. in such a purified state that its solutions no longer give protein reactions. Supporters of the protein nature of T. explain the inability to find protein in these solutions by the limited sensitivity of chemical reactions and, as proof of the presence of protein, cite the possibility of causing anaphylaxis with purified T. However, there are indisputable proofs that anaphylaxis can be caused by non-protein substances, for example, polysaccharides completely devoid of nitrogen. Attempts to purify T. lead mainly to weakening of its toxic function, and in this respect T. are compared with enzymes. As for the nature of the toxic action, they have great similarity with alkaloids. Hosoya and Miyata proposed a purification method that allows obtaining T. in a protein-free state, for example, diphtheria, tetanus, botulinus, and dysentery. Preparations by them have not yet been chemically studied. They possess toxic and antigenic properties without the ability to give precipitation and complement-binding reactions. By immunizing children, the authors succeeded in converting the Schick reaction to negative in 98% of cases. Moreover, scarlatinal, erysipelas, and meningococcal toxins were obtained in a protein-free state by Krestovnikova and Ryakhina, and both their chemical and immunobiological properties were studied. The composition of T. includes a carbohydrate core with an attached reducing group and a nitrogen-containing group with an amino group. Specific properties are associated with the carbohydrate group, as follows from the observations of American authors. When T. is mixed with serum, reactions occur manifested in the neutralization of T., the visible phase of which is the precipitation (precipitation, flocculation). The nature of their interaction is not yet studied, and in this respect various hypotheses have been expressed: chemical—Ehrlich's, physicochemical—Arrhenius', adsorption—Bordet's, etc. (see Immunity). The latter hypothesis is based on observations of the reversibility of the reaction between T. and antitoxin. At present it is known that the mixture of T. and antitoxin can be decomposed only at first, later a stable connection between them occurs. T. cause in animals and humans phenomena of general intoxication, and sometimes they reproduce characteristic symptoms. Intoxication is expressed in general weakness, dizziness, cerebral phenomena (vomiting, convulsions, paralyses), phenomena from the intestines. 5 fill At a certain dose, death occurs. When small doses are administered subcutaneously, an infiltrate forms, turning into necrosis with ulcer formation and subsequent scarring. Small doses of T. cause in sensitive animals and people transient reactions expressed in slight redness at the site of administration. Such a reaction is used to determine the degree of sensitivity of people to a given infection (Schick, Dick reactions, etc.). From the above, the importance of T. in the infectious process becomes apparent. Many infections proceed as intoxications (diphtheria, tetanus, botulinus), but in others the role of toxins in the pathogenesis of the disease is also very great. On the other hand, all phenomena of intoxication in infections cannot be reduced to the action of bacterial T.: toxic products arising in the body itself in the process of disturbance of tissue metabolism are also of great importance. For the role of T. in food poisonings—see Food poisonings, infections. Botulism.—The property of T. to cause the formation of antibodies in the body of animals and humans is widely used in the fight against infectious diseases. Antitoxic therapeutic sera are used successfully for therapeutic and prophylactic purposes in many infections (see Sera). In addition, there are diagnostic sera that allow determining the type of microbe being excreted. Bacterial T. and their preparations serve for active immunization of animals and humans in diphtheria, scarlet fever, etc. Individual representatives of bacterial toxins.

Diphtherial toxin is obtained by growing the Park-Williams strain № 8 on Marten's broth at pH=7.6. The cultivation temperature is 35-36° for 7-8 days. Testing of the toxin's readiness is done approximately on a guinea pig: a dose of 0.005 cm³ should cause the death of the animal within a day. Antigenic properties are determined by the Rayon method (flocculation) and the Kraus method (on binding capacity). The 'yield of toxin' is 0.001-0.0005 cm³. The toxin is weakened at 50° and is rapidly destroyed at 80°. It is sensitive to the action of light. After a certain period of reduced toxicity, it stabilizes. As a standard toxin, one not less than 6 months old is taken. The toxin is preserved with phenol. To obtain anatoxin, formalin is added to the toxin.--Tetanus toxin is obtained by cultivation for 6-7 days on Marten's broth with the digestion of peptone at 45°. 'The broth is poured in a high layer, before seeding the broth is heated, pH=7.2. The toxin is labile, stored for the purpose of serum standardization in dry form. The toxin is destroyed at 60° for 20 minutes, at 65°-in 5 minutes. The toxin is highly poisonous for animals, the minimum lethal dose for a guinea pig weighing 250 g is 0.000001-0.0000001 g. Intoxication is expressed in characteristic convulsions, which are identical with the picture of tetanus disease.-Botulinum toxin was first discovered in meat preserves and is one of the causes of meat and fish poisoning. At present, it has been proven that it can also be found in vegetable preserves. This poison is very active, kills a white mouse in a dose of 0.0001. The toxin is quite resistant to various influences. Thus, t° of 60-80° destroys it, while type C toxins are destroyed only by boiling for 1 hour. The toxin is poisonous when taken per os. Dysentery toxin is obtained on a medium with 'Shapoto' peptone, cultivation for 15 days at 37°. This toxin is more heat-resistant, is destroyed only at 78-80° for 1 hour.-Streptococcal toxin is obtained by cultivating toxigenic strains on Marten's broth at pH=7.6 for 6-7 days. The toxin is measured in human skin doses, as animals are very little sensitive to it. At present, the strength of the toxin is calculated at 100,000-120,000 skin doses. This toxin is heat-resistant, is inactivated only by boiling for 1 hour.-Typhoid toxin is obtained by cultivating toxic strains on spleen (Derkach) or on Marten's broth at pH=7.6 (E. B. Ginzburg) for 6-7 days. The effect on animals is expressed by increasing phenomena of general intoxication.-Paratyphoid toxin is more active. The lethal dose for mice is 0.5-1 cm³, for a rabbit 3-5 cm³. The toxin is extremely resistant, is destroyed only in an autoclave at 120°.-Meningococcal toxin is obtained by cultivation on hormonal broth with 2% peptone at pH=6.8 for 6-7 days (Ferry, Norton and Steele) or on Marten's broth, pH=6.8-7.0, with the digestion of peptone at 45° (Krestovnikova, Belkina, Doser). Only specially selected toxic strains are capable of toxin formation in the medium. The toxin is quite stable, is inactivated by boiling for 1½ hours. Its effect on animals is expressed by increasing general intoxication, diarrhea. The lethal dose for mice is 1-2 cm³, for a rabbit 3-5 cm³ per 1 kg of weight.-Toxin of Pfeiffer's bacillus (influenza). The toxin is obtained on calf broth with the addition of 5% defibrinated rabbit serum, by cultivation for 18-24 hours at 37° (Parker) or on placental broth at pH=7.6 with the addition of 2½% defibrinated sheep blood, by cultivation for 7-8 days. The broth after the addition of blood is brought to boiling for 5 minutes, then sterile-filtered through a pleated filter and tested for sterility (L. I. Falkovich). The toxin is labile, is destroyed at 60° for 1 hour or by 10 minutes of boiling. The effect on animals occurs quickly after its introduction into the vein. It is characterized by rapid breathing, opisthotonus, paralysis of the limbs and rapidly developing diarrhea. Before death, strong excitement, convulsions appear. Death occurs either after 1-1½ hours or within the next few days. The lethal dose for a rabbit is 3-8 cm³. Standardization of toxins--cm³. Standardization, standardization of bacterial preparations.