Toxicity of two North American Loxosceles (brown recluse spiders) venoms and their neutralization by antivenoms

The toxic, biochemical, and immunological characteristics of L. boneti and L. reclusa venoms and its neutralization by anti-L. boneti and anti-L. reclusa antivenoms were studied. The electrophoretic profile showed very similar patterns and the toxic activities were very close. Immunological studies showed cross-reactivity among L. boneti and L. reclusa venoms, with L. boneti and L. reclusa experimental antivenoms, and anti-L. gaucho and anti-L. laeta antivenoms. The venom of L. laeta showed low immunological reactivity with the North American Loxosceles antivenoms. Experimental anti-North American Loxosceles antivenoms protected mice of the systemic toxicity and were able to prevent necrosis in rabbit skin after the injection of the venom. Both antivenoms displayed cross neutralization. The results showed that both Loxosceles venoms have very close toxic, biochemical, and immunological characteristics, and that either monospecific antivenoms or an antivenom raised with L. boneti and L. reclusa venoms as immunogens could be useful for treating bites by North American Loxosceles spiders.     

STUDIES ON THE BIOLOGIC RELATIONSHIP OF ENDOTOXIN AND OTHER TOXIC PROTEINS : II. ENHANCEMENT OF SUSCEPTIBILITY TO SNAKE VENOM BY ENDOTOXIN

1. Injections of sublethal quantities of Agkistrodon piscivorus venom into endotoxin-treated rabbits produces a consistent early death. 2. The endotoxin-induced hypersusceptibility state (EIHS) to venom is produced by intravenous, intradermal, and intraperitoneal administration of endotoxin. The latency and duration of the EIHS vary with the route of administration. 3. EIHS is induced by as little as 1 gamma of endotoxin administered intravenously. Although the degree of susceptibility was no greater with a 100 gamma dose than with 1 gamma, 1 mg of endotoxin made the rabbits susceptible to smaller venom doses. 4. EIHS was demonstrated with intravenous, intradermal and intraperitoneal injection of Agkistrodon piscivorus venom. Endotoxin-pretreated animals were not as susceptible to venom given intramuscularly. 5. Normal rabbits are very resistant to venom given intradermally and intraperitoneally. 6. Enhanced susceptibility to intravenous endotoxin was demonstrated in animals pretreated with sublethal doses of moccasin venom. It differed from EIHS in its short duration and its outcome: a late, slowly progressive death. 7. EIHS was demonstrated with other venoms: Crotalus adamanteus and Vipera russellii, and in a modified form (resulting in late death) with Notechis scutatus. Endotoxin pretreatment had no effect on susceptibility to Naja flava, Bungarus candidus, or Crotalus durissus terrificus venoms. 8. Major hypotheses regarding the nature of endotoxin-induced alterations in non-specific resistance were considered in relation to EIHS to venom.     

ACTIVE IMMUNITY PRODUCED BY SO CALLED BALANCED OR NEUTRAL MIXTURES OF DIPHTHERIA TOXIN AND ANTITOXIN

The foregoing and earlier data taken together demonstrate that an active immunity lasting several years can be produced in guinea-pigs, by the injection of toxin-antitoxin mixtures which have no recognizable harmful effect either immediate or remote. They also show, what might have been anticipated, that under the same conditions mixtures which produce local lesions and which, therefore, contain an excess of toxin produce a much higher degree of immunity than the neutral mixtures, and that an excess of antitoxin reduces the possibility of producing an active immunity, and may extinguish it altogether. There is, therefore, a certain definite relation between the components of the mixture and the degree of immunity producible. Furthermore, toxin-antitoxin mixtures do not change materially within five days at room temperature. They are apparently more efficacious at the end of forty-eight hours than immediately after preparation. The experiments finally prove that a relatively high degree of active immunity can be induced by a harmless procedure, whereas the use of toxin alone leading to very severe local lesions is incapable of producing more than an insignificant protection. The method, therefore, invites further tests in regard to its ultimate applicability to the human being. Unless the subcutis of the guinea-pig reacts to toxin-antitoxin mixtures in a manner peculiar to itself, a practical, easily controlled method for active immunization can be worked out which should afford a larger protection than the serum alone and avoid the complications associated with horse serum. That proportion of toxin and antitoxin which would produce the highest desirable immunity consistent with the least discomfort would have to be carefully worked out for the human subject. From the nature of the immunity induced it is obvious, however, that such a method of immunization cannot take the place of a large dose of antitoxin in exposed individuals who must be protected at once. It would be applicable only as a general protective measure without reference to any immediate danger, since it would take several weeks, perhaps longer, to perfect the attainable immunity. Passing to the theoretical aspects of the facts observed, we find no publications bearing directly upon the subject before us. Madsen has, however, approached it very closely in his experiments on the immunization of animals with mixtures not fully balanced, or, in other words, in which the "toxones" were still free. He found that the injection of such mixtures in rabbits, goats and horses produces an active immunity. He makes the significant remark that perhaps in the immunizing capacity we may possess the keenest reagent for a poison which is not able to exert any toxic action in the body. This is fully borne out by the experiments described, for in these we pass beyond the visible spectrum, so to speak, of the toxin-antitoxin effects, and we are able to recognize toxic action only by the lasting immunizing effects. Another publication which touches upon some phases of the same problem is that of Morgenroth on the union between toxin and antitoxin. Morgenroth brought out the fact that a given toxin-antitoxin mixture is more toxic when injected directly into the circulation than when injected under the skin. Thus, an L₊ dose of 0.78 c.c. toxin + one unit antitoxin applied subcutaneously was of the same toxicity as 0.68 c.c. toxin + one unit antitoxin injected into the circulation. When the mixture had stood twenty-four hours this (L₊) dose was still 0.78 c.c. subcutaneously, but it had risen to 0.74 c.c. when introduced by the intracardiac route. The author makes two deductions from these results. He assumes that the velocity of reaction between toxin and antitoxin is slow, and that the union is not completed until the mixture has stood twenty-four hours. Hence, the L₊ dose of toxin injected into the blood is higher after twenty-four hours than immediately after mixing the toxin and antitoxin. He furthermore explains the fact that the subcutaneous L₊ dose remains the same whether the mixture is injected at once or after twenty-four hours, by assuming that in the subcutis of the guinea-pig there is a catalytic acceleration of the union of toxin and antitoxin. In view of the writer''s results it seems that not only immediately, but four to five days after the preparation of the mixture of toxin and antitoxin, there are still toxic substances available for the production of immunity in the body of the guinea-pig, when the dose of toxin in the mixture is far below the L₀ or neutral level. These toxins may be free, either because uncombined in vitro, or else because the mixture is partially dissociated in vivo, or there may be a third possibility. It is obvious that Morgenroth''s investigations, however extensive and thorough, have not exhausted the subject, for both these inferences are incompatible with his. Perhaps his recent important studies on the recovery of toxin from its combination with antitoxin with weak acids may throw more light on this subject. The only conclusion which we may safely draw at this time is that the toxin-antitoxin mixture produces two sets of effects, essentially identical, however. One is visible, as injury (œdema, loss of hair, superficial and deep necrosis of skin, paralysis and death), and corresponds to the toxin spectrum of Ehrlich. The other is invisible and manifests itself only in degrees of active immunity. At what ratio of toxin to antitoxin in the mixture active immunity is no longer produced will vary somewhat with the guinea-pig used, but it is evident that traces of immunity are still transmitted to the young when the amount of toxin approaches half the L₀ dose.     

A SPREADING FACTOR IN CERTAIN SNAKE VENOMS AND ITS RELATION TO THEIR MODE OF ACTION

The venom of several species of poisonous snakes acts to spread India ink through the skin as do the spreading factors procurable from certain tissues and elaborated by invasive bacteria. The factor is most abundant in the venom of the Viperidae (rattlesnake) family and relatively scant in the venom of Colubridae proteroglypha (cobra) family, and it is absent from toad venom. Extracts of the supralabial glands of harmless snakes contain only negligible amounts of the factor. Rattlesnake venom heated at 65° to 100° loses a large proportion of its toxicity but retains the ability to spread ink. Rattlesnake venom that has lost its toxicity on standing or on heating markedly enhances the infection produced by bacterial or virus suspension in the rabbit skin. Antivenine serum inactivates both the toxic and spreading factors of venom.     

AN EXPERIMENTAL STUDY OF THE LATE GLOMERULAR LESIONS CAUSED BY CROTALUS VENOM

The acute exudative glomerular lesion of the rabbit''s kidney caused by crotalus venom does not lead to a subacute or chronic glomerulonephritis. The hemorrhagic lesion of the glomerular tuft may show a process of repair characterized by the ingrowth, into the hemorrhagic masses, of endothelial cells from the uninjured part of the tuft. This process is, however, more analogous to the organization of a red thrombus than it is to any form of glomerular lesion known in man, and can hardly serve as an experimental demonstration of the mode of development of a subacute or chronic glomerular nephritis. On the other hand, crotalus venom causes a persistent albuminuria and extensive tubular degeneration and cast formation, with death, preceded by great emaciation, after five to six weeks.     

PRODUCTION OF A REFRACTORY STATE AS CONCERNS THE SHWARTZMAN PHENOMENON BY THE INJECTIONS OF VENOM OF THE MOCCASIN SNAKE (ANCISTRODON PISCIVORUS)

The majority of rabbits receiving intradermal, intraperitoneal or intravenous injections of moccasin venom became refractory to the development of the Shwartzman phenomenon. An incubation period of about 14 days was required for their resistance to develop. The incidence of refractory animals was inversely proportional within limits to the amount of toxin given intravenously to elicit the Shwartzman phenomenon. The intravenous route was the most efficacious in developing refractivity. The refractory state was still present 44 days after the primary injection of moccasin venom. Rattlesnake venom was not efficacious in inducing a refractory state. The refractory animals did not show a changed reaction to moccasin venom in the concentrations used. No circulating antibodies could be demonstrated to explain the refractory state. Antivenin had no effect on the course of the Shwartzman phenomenon.     

STUDIES ON THE MOLECULAR WEIGHT OF DIPHTHERIA TOXIN,ANTITOXIN, AND THEIR REACTION PRODUCTS

Purified diphtheria antitoxic horse pseudoglobulin has been prepared which is homogeneous by sedimentation, diffusion, and electrophoresis. Immunologically, however, the preparation contains only 43.5 per cent antitoxin specifically precipitable by toxin. The inactive pseudoglobulin remaining after specific precipitation was found to have the same physical and chemical properties as the original antitoxic pseudoglobulin. Although the molecular weight of antitoxin is the same as that of the normal horse serum globulins, the electrophoretic mobility does differ from those normally present. The molecular weight of diphtheria toxin is 70,000 and of antitoxin is 150,000. From ultracentrifuge studies on the two reactants and on mixtures of toxin and antitoxin in the soluble inhibition zones, the average molecular composition of the specific floccules at certain reference points throughout the equivalence zone and the maximum "valence" of toxin and antitoxin with respect to each other have been calculated. The significance of the results has been discussed in relation to antigen-antibody reactions in general and a possible explanation for the exceptional behavior of the toxin-antitoxin reaction in the region of excess antitoxin has been suggested.     

Rapid diagnosis of Naja atra snakebites

Background. The clinical diagnosis of snakebites is critical and necessary in many parts of the world, especially in Southeastern Asia, where venomous snakebites are a burden on public health. It is difficult to define or recognize the species of venomous snake because of the overlapping clinical manifestations of envenomations. A quick and reliable method for identifying the snake species is necessary. We designed and tested a strip of lateral flow system for the diagnosis of cobra snake bites in Taiwan. Methods. We developed a kit based on an immunochromatographic method for rapid detection of cobra (Naja atra) venom in human serum. The test and control lines composed of 1 mg/ml polyclonal duck antivenom and 0.5 mg/ml goat anti-rabbit immunoglobulin antibody solutions, respectively, were coated on nitrocellulose strips. Colloidal gold was conjugated with rabbit polyclonal anti-cobra venom antibodies. From July 2007 to December 2012, we used the kit to test serum from snakebite patients and to examine the agreement between our rapid test and the currently used sandwich enzyme-linked immunosorbent assay (ELISA). Results. Our kit was able to detect cobra venom in serum samples in 20 minutes with a detection limit of 5 ng/ml. An absence of cross-reactivity with other non-cobra venoms from Taiwan was noted in vitro. A total of 88 snakebite patients (34 cobra and 54 other non-cobra) were tested. The sensitivity of the strips based on the ELISA results was 83.3% and the specificity was 100%. There was a strong agreement between the results of the ELISA and immunochromatographic strips (κ = 0.868). Discussion and conclusions. This data indicates that an immunochromatographic strip might be suitable for cobra venom detection and could be used as a quick diagnostic tool in cases of N. atra snakebite.     

North American Coral Snake Antivenin for the Neutralization of Non-native Elapid Venoms in a Murine Model

Objectives: North American coral snake antivenin (CSAV; Wyeth Antivenin [Micrurus fulvius], equine origin) is approved for the treatment of coral snake envenomations in the United States. The coral snake is the only elapid that is native to North America, but envenomations from non‐native elapids are occurring more commonly in this country. This study was designed to evaluate the efficacy of CSAV in the neutralization of two exotic elapid envenomations: Naja naja (Indian cobra) and Dendroaspis polylepsis (black mamba). Methods: A randomized, blinded, placebo‐controlled murine model of intraperitoneal venom injection was employed. Venom potency was determined in preliminary dosing studies. Study animals then were divided into five groups: 1) N. naja venom + CSAV, 2) N. naja venom + 0.9% normal saline (NS), 3) D. polylepsis venom + CSAV, 4) D. polylepsis venom + NS, and 5) CSAV + NS. The venom dose was chosen to be twice the estimated LD ₅₀. The amount of CSAV injected was ten times the amount necessary for neutralization of a 2 × LD₅₀ dose of M. f. fulvius venom in a murine model. Statistical analysis included Fisher's exact and log‐rank testing to compare survival rates and times. Results: Preliminary studies estimated the venom LD₅₀ to be 2.58 mg/kg and 0.45 mg/kg, respectively, for the N. naja and D. polylepsis. A significant difference was shown in comparison of survival times between CSAV–venom groups and normal saline–venom groups despite all animals in both treatment and control arms dying. Animals receiving CSAV and N. naja venom survived (mean ± SD) 24.4 ± 3.0 minutes, versus 17.8 ± 1.3 minutes in the control group (p < 0.001), whereas those receiving CSAV and D. polylepsis venom survived 203.8 ± 37.0 minutes versus 130.0 ± 42.6 minutes in the control group (p < 0.001). All animals in the CSAV + NS group survived to the conclusion of the study. Conclusions: When premixed with venom, CSAV increased survival time in a murine model of intraperitoneal N. naja and D. polylepsis venom injection. The clinical implications of this are unclear, given unchanged mortality rates.     

TOXIN AND ANTITOXIN OF AND PROTECTIVE INOCULATION AGAINST BACILLUS WELCHII

Five cultures of Bacillus welchii have been studied and compared Four came from infected wounds in the western theatre of war, and one was obtained from a personal article of clothing. Each culture possesses the essential characteristics ascribed to that group of bacteria. The infectious processes caused by the five cultures in rabbits, guinea pigs, and pigeons, are local in character; and very few or no bacilli enter or are found in the general blood stream during life or immediately after death. Glucose broth cultures, injected intravenously, are fatal to rabbits. Death occurs, almost immediately or after a few hours. Agglutinative bacterial emboli have been ruled out as the cause of death, as has been an acid intoxication. The fluid part of the culture acts in the same manner as the full culture and irrespective of neutralization with sodium hydroxide. The full cultures and supernatant fluid are hemolytic when injected directly into the circulation of rabbits and pigeons, and the acute death produced may be ascribed to a massive destruction of red corpuscles. The passage of the fluid portion of glucose broth cultures through Berkefeld filters reduces materially the hemolytic and poisonous effects. Cultures of the Welch bacilli in plain broth to which sterile pigeon or rabbit muscle is added are highly toxic, and the toxicity is not noticeably diminished by Berkefeld filtration. The filtrates are hemolytic when injected intravenously and inflaming and necrotizing when injected subcutaneously and intramuscularly. The local lesions produced in the breast muscles of the pigeon closely resemble those caused by infection with the bacilli. The toxicity of these filtrates is not affected by neutralization with sodium hydroxide, but is materially reduced by heating to 62°C. and entirely removed by heating to 70°C. for 30 minutes. Successive injections of carefully graded doses of this toxic filtrate in pigeons and rabbits give rise to active immunity. The blood taken from the immunized rabbits is capable of neutralizing the toxic filtrate in vivo and in vitro. The filtrate has therefore been designated as toxin and the immune serum as antitoxin. The antitoxin neutralizes the toxin in multiple proportions. Hence the latter would seem to possess the properties of an exotoxin. Moreover, it neutralizes the hemolytic as well as the locally .injurious toxic constituent. Antitoxic serum prepared from a given culture of Bacillus welchii is neutralizing for the toxins yielded by the other four cultures of that microorganism. The antitoxin is protective and curative against infection with the spore and the vegetative stages of Bacillus welchii in pigeons. The limits of the protective and curative action are now under investigation.     

Cross neutralization of coral snake venoms by commercial Australian snake antivenoms

Context: Although rare, coral snake envenomation is a serious health threat in Brazil, because of the highly neurotoxic venom and the scarcely available antivenom. The major bottleneck for antivenom production is the low availability of venom. Furthermore, the available serum is not effective against all coral snake species found in Brazil. An alternative to circumvent the lack of venom for serum production and the restricted protection of the actually available antivenom would be of great value. We compared the Brazilian coral snake and mono and polyvalent Australian antivenoms in terms of reactivity and protection.

Methods: The immunoreactivity of venoms from 9 coral snakes species were assayed by ELISA and western blot using the Brazilian Micrurus and the Australian pentavalent as well as monovalent anti-Notechis, Oxyuranus and Pseudechis antivenoms. Neutralization assays were performed in mice, using 3 LD₅₀ of the venoms, incubated for 30 minutes with 100?μL of antivenom/animal.

Discussion: All the venoms reacted against the autologous and heterologous antivenoms. Nevertheless, the neutralization assays showed that the coral snake antivenom was only effective against M. corallinus, M. frontalis, M. fulvius, M. nigrocinctus and M. pyrrhocryptus venoms. On the other hand, the Australian pentavalent antivenom neutralized all venoms except the one from M. spixii. A combination of anti-Oxyuranus and Pseudechis monovalent sera, extended the protection to M. altirostris and, partially, to M. ibiboboca. By adding Notechis antivenom to this mixture, we obtained full protection against M. ibiboboca and partial neutralization against M. lemniscatus venoms.

Conclusions: Our findings confirm the limited effectiveness of the Brazilian coral snake antivenom and indicate that antivenoms made from Australian snakes venoms are an effective alternative for coral snake bites in South America and also in the United States were coral snake antivenom production has been discontinued.     

Experimental study of diphtheritic polyneuritis in the rabbit and guinea pig. I. Immunologic and histopathologic observations

Diphtheritic neuritis was produced in rabbits and guinea pigs by injection of underneutralized toxin-antitoxin mixtures. The disease is a progressive, ataxic paresis related in severity to the dose of injected toxin-antitoxin. Cerebrospinal fluids of rabbits with this disease showed "albuminocytologic dissociation" similar to that found in the same disease in man. Histologically the disease is a progressive demyelination of the peripheral nervous system, un-accompanied by axis cylinder damage or inflammation. It involves the spinal roots and sensory ganglia in the rabbit, the peripheral nerves in the guinea pig. Diphtheritic neuritis can be distinguished from experimental allergic neuritis in the rabbit and guinea pig on both clinical and histological grounds. The rabbit disease is however an excellent model of diphtheritic neuritis in man. In the animals with diphtheritic neuritis, studied in the present work, there was vigorous production of circulating antitoxin and infrequently complement-fixing antibody to horse serum proteins. No antibody against rabbit spinal cord or sciatic nerve was demonstrated. Skin reactivity to both rabbit nerve suspension and diphtheria toxoid was present at 1 to 2 weeks following inoculation and reached a maximum well before the peak of antitoxin response. These reactions did not seem to be typical delayed reactions. No correlation existed between the development of diphtheritic polyneuritis and any of these immunologic events or the circulating complement level, either in time or in degree. Treatment of rabbits with 400 r whole body irradiation 48 hours before inoculation resulted in severe leukopenia lasting about 3 weeks, delay of antitoxin formation with considerable reduction of peak titers, and some decreased skin reactivity to toxoid. It had no effect on the disease process. It is concluded that diphtheritic polyneuritis is produced by a non-immunologic mechanism, e.g., direct toxicity.     

Protein content of antivenoms and relationship with their immunochemical reactivity and neutralization assays

Context. Therapy for snakebites relies on the application of antivenoms, which may be produced with different immunogenic mixtures of venom and possess different pharmaceutical characteristics. For these reasons, immunological cross-reactivity and heterologous neutralization were analyzed relative to the protein content of three antivenoms used in the Americas. Methods. The antivenoms studied were composed of equine F(ab’)₂ fragments from animals immunized with Crotalinae venoms. The antivenoms were tested against venoms of seven pit viper species from Argentina, seven from Mexico, one from Costa Rica, and one from Colombia. Results. Immunoblotting showed high cross-reactivity of all major protein bands with all the antivenoms tested. ELISA results also showed high cross-reactivity among the different venoms and antivenoms, and a high heterologous neutralization was observed. The results can be interpreted in different ways depending on whether the reactivity is considered in terms of the volume of antivenom used or by the amount of protein contained in this volume of antivenom. The antivenoms with high immunochemical reactivity and neutralizing capacity were those with higher protein content per vial; but when doses were adjusted by protein content, antivenoms of apparently lower neutralizing capacity and immunochemical reactivity showed at least similar potency and reactivity although volumetrically at higher doses. Conclusion. Protein content relative to neutralization potency of different products must be taken into account when antivenoms are compared, in addition to the volume required for therapeutic effect. These results show the importance of obtaining high-affinity and high-avidity antibodies to achieve good neutralization using low protein concentration and low-volume antivenoms.     

A QUANTITATIVE STUDY OF THE SCARLET FEVER TOXIN-ANTITOXIN FLOCCULATION REACTION

1. Highly purified scarlet fever toxin has been prepared from culture filtrates of two scarlatinal strains (NY5 and 594B) of hemolytic streptococcus grown on a medium of defined composition. 2. The flocculation reaction of Rane and Wyman has been studied quantitatively and has been shown specific for scarlet fever toxin and antitoxin. 3. Scarlet fever toxin from strains NY5 and 594B are quantitatively identical in their immunological behavior. 4. Pure scarlet fever toxin contains 0.00023 mg. nitrogen per flocculating unit and close to 1.3 x 10⁸ skin test doses per mg. nitrogen as calculated from the immunological data. Both the immunological and the analytical data suggest that scarlet fever toxin is a protein. Similar calculations indicate that scarlet fever antitoxin contains 0.00093 mg. nitrogen per unit. 5. The scarlet fever toxin-antitoxin complex is readily dissociated in dilute solutions. In this respect the scarlet fever toxin-antitoxin system contrasts sharply with the diphtheria toxin-antitoxin system. 6. The scarlet fever toxin-antitoxin reaction is discussed in relation to other flocculation reactions.     

Pediatric Acetaminophen Overdose: Assessing the Risk

Micrurus snakes (coral snakes) may produce severe envenomation that can lead to death by peripheral respiratory paralysis. Only few laboratories produce specific antivenoms, and despite the cross‐reactivity found in some Micrurus species venoms, the treatment is not always effective. To test two therapeutic antivenoms against the venom of four species of Micrurus from Southern America, North of South America, Central America, and North America, the determination of the lethal potency of the venoms, the study of some biochemical and immunochemical characteristics, and the determination of the neutralizing activity of both antivenoms were studied. North American and South American antivenoms neutralized well venoms from Micrurus species of the corresponding hemisphere but displayed lower effectiveness against venoms of species from different hemispheres. It was concluded that the neutralization of Micrurus venoms by regional antivenoms could be useful to treat the envenomation by some Micrurus snakes but is necessary to evaluate carefully the antivenoms to be used with the venoms from the snakes of the region. Also, considering the difficulties for coral snake antivenom production, the development of a polyvalent antivenom is useful to treat the envenomation by coral snakes from different regions is necessary.     

THE COAGULATION OF BLOOD BY SNAKE VENOMS AND ITS PHYSIOLOGIC SIGNIFICANCE

Nine of the 17 venoms here tested were found capable of coagulating citrated blood or plasma. As has been believed by most workers in the field, 7 of these 9 coagulant venoms convert fibrinogen to an insoluble modification resembling fibrin (Bothrops atrox, Bothrops jararaca, Bothrops nummifera, Crotalus adamanteus, Crotalus horridus, Crotalus terrificus basiliscus, Crotalus terrificus terrificus). The optimum pH for this coagulation was determined for 3 of these, and was found in each case to be approximately pH 6.5, the same as that for the action of thrombin on fibrinogen. Unlike thrombin, however, the fibrinogen-coagulating activity of the venoms was unaffected by the antithrombin elaborated in the course of anaphylactic shock. In addition to coagulating fibrinogen directly, 3 of these venoms (Bothrops atrox, Bothrops jararaca, and to a less extent, Crotalus terrificus basiliscus) acted on prothrombin to convert it to thrombin, without the necessary intervention of either calcium or platelets. Finally, 2 venoms (Notechis scutatus, and to a slight extent, a mixed Micrurus venom), which had no demonstrable effect on purified fibrinogen, nevertheless converted prothrombin to thrombin. Unlike the reaction between the venoms and fibrinogen, this activation of prothrombin has no definite pH optimum, but takes place over a wide zone (pH 5.6–8.3). In the case of Bothrops atrox, there was some indication that the initial velocity of the reaction increased with increasing alkalinity, but that the amount of thrombin ultimately formed decreased. Extraordinarily minute quantities of some of these venoms sufficed to produce a demonstrable activation of prothrombin. Thus, the fer de lance (Bothrops atrox) venom was active in a 1:25,000,000 dilution, and that of the Australian tiger snake (Notechis scutatus) was active in a 1:4,000,000 dilution. The thrombin formed was indistinguishable from that produced by the action of calcium + platelets on prothrombin. Like the latter type of thrombin, and unlike venoms which act directly on fibrinogen, thrombin formed from prothrombin by venom was inhibited by antithrombin. Every one of the 9 non-coagulant venoms in this series destroyed prothrombin; and 5 of these destroyed fibrinogen as well. As is discussed in the text, there is reason to believe that these several properties of the venoms (coagulation and destruction of fibrinogen; activation and destruction of prothrombin) depend on the proteolytic enzymes which they were found to contain. These observations lend further support to the thesis that, in the course of physiological coagulation, (a) calcium plus platelets (or tissue derivative) constitute an enzyme system which reacts with prothrombin to form thrombin, and which is thus analogous to trypsin and to several of the proteolytic venoms here discussed, and (b) the thrombin so formed is itself a proteolytic enzyme which, like papain and the majority of the coagulant and proteolytic snake venoms here studied, reacts with fibrinogen to form a fibrillar gel, fibrin.     

Mast cell chymase reduces the toxicity of Gila monster venom, scorpion venom, and vasoactive intestinal polypeptide in mice

Mast cell degranulation is important in the pathogenesis of anaphylaxis and allergic disorders. Many animal venoms contain components that can induce mast cell degranulation, and this has been thought to contribute to the pathology and mortality caused by envenomation. However, we recently reported evidence that mast cells can enhance the resistance of mice to the venoms of certain snakes and that mouse mast cell-derived carboxypeptidase A3 (CPA3) can contribute to this effect. Here, we investigated whether mast cells can enhance resistance to the venom of the Gila monster, a toxic component of that venom (helodermin), and the structurally similar mammalian peptide, vasoactive intestinal polypeptide (VIP). Using 2 types of mast cell-deficient mice, as well as mice selectively lacking CPA3 activity or the chymase mouse mast cell protease-4 (MCPT4), we found that mast cells and MCPT4, which can degrade helodermin, can enhance host resistance to the toxicity of Gila monster venom. Mast cells and MCPT4 also can limit the toxicity associated with high concentrations of VIP and can reduce the morbidity and mortality induced by venoms from 2 species of scorpions. Our findings support the notion that mast cells can enhance innate defense by degradation of diverse animal toxins and that release of MCPT4, in addition to CPA3, can contribute to this mast cell function.     

THE R?LE OF THE RETICULO-ENDOTHELIAL SYSTEM IN IMMUNITY : V. PHENOMENA OF PASSIVE IMMUNITY IN BLOCKED AND SPLENECTOMIZED MICE.

1. Blockade of the reticulo-endothelial system by means of one intravenous injection of India ink as well as splenectomy did not alter the course of either Pneumococcus Type I infection or tetanus intoxication in mice. 2. The protective action of Antipneumococcus Type I serum against the corresponding infection, as determined by the injection of in vitro prepared mixtures of serum and culture, was definitely lower in mice which had received one blocking injection of India ink shortly before the test. 3. Titration of tetanus toxin and antitoxin in blocked and splenectomized mice gave results identical with those obtained in normal mice, if in vitro prepared and incubated toxin-antitoxin mixtures were injected. The degree of protection, however, conferred by a preceding dose of antitoxin against subsequent intoxication, was markedly lower in blocked mice than in normal control animals, this difference becoming more pronounced with the increase of the time interval.     

A STUDY OF THE COMPETITION OF LECITHIN AND ANTITOXIN FOR CL. WELCHII LECITHINASE

Lecithin has been found to interfere with the combining reaction of Cl. welchii alpha toxin (lecithinase) and its antitoxin. If the lecithinase is first brought into contact with lecithin, and the antitoxin is then added, the antitoxin fails to stop the enzymatic reaction, but gradually decelerates it. If the lecithinase is brought into contact with both lecithin and antitoxin at the same instant, it appears to combine in part with each, and the enzymatic process takes place at a reduced rate, which gradually declines further. If the lecithinase is first brought into contact with antitoxin, before the lecithin is added, the enzymatic reaction is completely inhibited. This ability of lecithin to inhibit the antitoxin-toxin combination cannot be explained adequately as a non-specific coating of the toxin-enzyme by the lecithin. It is rather suggested that lecithin and antitoxin compete specifically for combination with the same regions on the enzyme molecule. Lecithin has similarly been found to interfere with the combination of Crotalus terrificus venom and its antiserum. The above findings provide a partial explanation for the lack of effectiveness of antitoxin when given late in the course of Cl. welchii infection.     

ANTIBODY FORMATION : I. THE SUPPRESSION OF ANTIBODY FORMATION BY PASSIVELY ADMINISTERED ANTIBODY

The suppression of antibody formation by passively administered antibody is influenced by the dose and nature of the antigen, type of immunization procedure, ratio of antibody to antigen, species origin and characteristics of the antiserum used, as well as the species selected for immunization. In guinea pigs, diphtheria antitoxin formation can be effectively suppressed by an intravenous injection of excess homologous or heterologous antitoxin as long as 5 days after toxoid immunization and after delayed-type hypersensitivity to toxoid has developed. Following the period of antibody suppression which lasts 2 to 7 weeks, serum antibody can usually be demonstrated. It is proposed that this delayed immunization results from dissociation of antigen, since diphtheritic paralysis and death can be produced in guinea pigs and rabbits by the intravenous injection of toxin-antitoxin precipitates formed in antitoxin excess. This syndrome is prevented by injection of excess horse antitoxin 1 hour after injection of the toxin-antitoxin complexes.