Severity of a Vipera palaestinae envenomation objective findings associated with a complicated hospitalization course following a Vipera palaestinae bite.

The most common snake in Israel, responsible for most snakebites is Vipera palaestinae (VP). Envenomation signs and symptoms vary from local manifestations to systemic reactions that may end with death. Antivenom treatment, given to high-risk patients, reduces complications and mortality but carries risks. As of now, there is no standardized protocol for adults bitten by VP based on objective clinical and laboratory findings. We conducted A retrospective analysis of 159 patients admitted to two large tertiary care institutions in the center (Hadassah University Medical Center) and south (Soroka University Medical Center) of Israel with Vipera palaestinae bites during 1990-2017. Epidemiological and clinical data were extracted, and the patients were divided into two groups based on hospitalization time (over or under 48 h). 159 patients were included in this study. The average hospitalization time was 66.1 h, with 49.7% of patients admitted over 48 h. The main factors that statistically correlated with a longer hospitalization time were: Male gender, lower extremity bite, platelets lower than 150 K at presentation, leukocyte count of over 10 K at presentation and elevated D-Dimer levels. This study provides factors which are associated with a severe VP envenomation. These clinical or laboratory findings (along with accompanying clinical symptoms) are associated with a higher risk of a prolonged hospitalization with more complications and may require a more intensive treatment and monitoring.

Adverse reactions to four types of monovalent antivenom used in the treatment of snakebite envenoming in South China.

To evaluate the adverse reactions associated with the four types of monovalent antivenom currently used in China, we retrospectively analysed the data of all patients admitted for snakebites who received antivenom treatment at the main institution for the treatment of venomous snakebites in Guangzhou from January 2013 to December 2021. A total of 1658 patients were analysed in our study, and 60.7% (n = 1007) of the snakebite patients received antivenom treatment. The incidence rate of adverse reactions that occurred after the administration of all types of monovalent antivenom was 4.9% (n = 49), and the incidence rate of acute adverse reactions was 2.7% (n = 27). The number of adverse reactions that occurred was 38/744 (5.1%) in patients who received prophylactic application of glucocorticoids alone and 10/217 (4.6%) in those who received a combination of antihistamines and glucocorticoids (P = 0.83). The average doses of the antivenoms used in patients exhibiting acute adverse reactions and serum sickness were 3.31 ± 0.75 vials and 2.36 ± 0.26 vials, respectively (P = 0.28). The antivenom skin test showed high specificity (98.3%, 95% CI: 97.24%-99.01%) but low sensitivity (14.3%, 95% CI: 6.41%-27.86%). Our results showed that the four types of monovalent antivenom were safe. No significant difference was observed between the use of glucocorticoids alone and the use of antihistamines combined with glucocorticoids as premedication for the prevention of adverse reactions. Reducing the dose of antivenoms or reducing the combination of antivenoms did not help to reduce the occurrence of adverse reactions. Skin testing should not be recommended due to its low sensitivity.

Uncommon defibrinogenation and coagulopathy caused by Trimeresurus stejnegeri stejnegeri envenomation in a patient with swelling above the ankle.

In Taiwan, Trimeresurus stejnegeri stejnegeri (Stejneger's Bamboo pitviper) is responsible for more than half of all venomous snakebites annually. This species often causes local envenoming characterized by tissue swelling and pain, occasional local ecchymosis, bullae and blister formation, and lymphangitis and lymphadenitis. The pathophysiology and treatment of potentially life-threatening coagulopathy and defibrinogenation induced by T. s. stejnegeri systemic envenoming have not been specifically addressed. Here, we describe the case of a man who was bitten by T. s. stejnegeri on his right first toe, which later developed into swelling above the ankle. It was found that there was severe hypofibrinogenemia, prolonged prothrombin time, and reduced activities of factors V and XI, plasminogen, and α2-antiplasmin. Even though a favorable outcome was achieved after repeatedly administering specific antivenom, fresh frozen plasma, and cryoprecipitate, probably low effectiveness of antivenom against the coagulopathy and prodigious amounts of replacement products were observed. To control coagulopathy early and avoid the needless replacement of coagulation factor, which are associated with inherent adverse reactions, more frequent serial blood assessment (e.g., every 6 h) and higher initial antivenom doses may be helpful. Knowledge of the specific coagulation factor deficiencies may improve our understanding of the relationship between hemotoxins and the resulting envenoming syndromes in this snakebite.

Utilization of gallic acid to inhibit some toxic activities caused by Bothrops jararaca or B. jararacussu snake venoms.

Snake bite envenoming is a serious public health issue, affecting thousands of people worldwide every year, especially in rural communities of tropical and subtropical countries. Injection of venom into victims may cause hemorrhaging, blood coagulation imbalance, inflammation, pain, edema, muscle necrosis, and eventually, death. The official validated treatment recommended by governments is the administration of antivenom that efficiently prevents morbidity and mortality. However, this therapy does not effectively neutralize the local effects of Viperidae venoms which constitute one of the leading causes of disability or amputation of the affected limb. Thus, bioprospecting studies seeking for alternative therapies to complement antivenom should be encouraged, especially those investigating the blockage of local venomic toxicity. Plants produce a great diversity of metabolites with a wide range of pharmacological and biological properties. Therefore, the objective of this study was to assess the utilization of gallic acid, which is widely found in plants, against some toxic in vitro (coagulation, proteolytic, and hemolytic) or in vivo (edematogenic, hemorrhagic, and lethal) activities of Bothrops jararaca or B. jararacussu venom. Gallic acid was incubated with B. jararaca or B. jararacussu venom (incubation protocol), after which, in vitro or in vivo assays were performed. Additionally, a gel containing gallic acid was developed and topically applied over the skin of mice after injection of B. jararaca or B. jararacussu venom (treatment protocol), and then, a hemorrhagic assay was carried out. As a result, gallic acid inhibited the toxic activities, with variable efficacy, and the gallic acid gel neutralized B. jararaca or B. jararacussu venom-induced hemorrhagic activity. Gallic acid was devoid of in vitro toxicity as shown through a hemocompatibility test. Thus, these findings demonstrate the potential of gallic acid in the development of an alternative agent to treat victims of snake bites inflicted by Bothrops species.

Assessment of quality, safety, and pre-clinical toxicity of an equine polyvalent anti-snake venom (Pan Africa): Determination of immunological cross-reactivity of antivenom against venom samples of Elapidae and Viperidae snakes of Africa.

Snakebite causes a large amount of morbidities and mortalities in Africa. The safety, efficacy, and homogeneity of anti-snake venoms are crucial for snakebite treatments to be effective with minimal adverse effects. We assessed the homogeneity of preparations of three different batches of Combipack snake venom antiserums (Pan Africa) [CSVAPA] by quantitatively analysing F(ab')2, IgG, and other contaminating proteins of plasma. LC-MS/MS analysis showed that approximately 92.4% of the proteins from the CSVAPA samples was IgG/F(ab')2 and the percent composition of contaminating proteins in CSVAPA varied from 0.07 to 4.6%. Batch 1 of the CSVAPA also contained a minor amount of undigested IgG and F(ab')2 aggregates. CSVAPA contained more than 60% venom-specific antibodies, showed moderate complement activation, no IgE contamination, safe level of endotoxin, and also showed pre-clinical safety. The immuno cross-reactivity of CSVAPA against 14 Viperidae and Elapidae snake venoms of Africa was tested by ELISA and immunoblotting, and the neutralization of major enzymatic venom activities, demonstrating that high molecular weight (>50 kDa) venom proteins are better recognized/neutralized compared to relatively low molecular weight (<20 kDa) venom proteins. CSVAPA at a dose of 3-12 times higher than the clinical dose did not cause deaths or adverse reaction of treated rabbits. The results suggest the satisfactory quality, safety, and efficacy of CSVAPA.

Assessment of the anti-snakebite properties of extracts of Aniba fragrans Ducke (Lauraceae) used in folk medicine as complementary treatment in cases of envenomation by Bothrops atrox.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of leaves and bark of Aniba fragrans are used as tea (decoction) to treat snakebites in communities in the Brazilian Amazon. The aqueous extract of the leaves of A. fragrans has been proven to be effective against Bothrops venom, but only when pre-incubated with the venom. This study sought to assess the potential of different types of extract of this species to inhibit the biological activities of Bothrops atrox venom (BaV) when used the same way as in folk medicine. The main classes of secondary metabolites and the concentrations of phenolics in the extracts were also determined. MATERIALS AND METHODS: Four types of extract of A. fragrans were prepared: aqueous extract of the leaf (AEL), aqueous extract of the bark (AEB), hydroalcoholic leaf extract (HLE) and extract of the residue from hydrodistillation of the leaf (ERHL). The phytochemical profiles of the aqueous extracts were determined using thin layer chromatography (TLC), and the concentrations of phenolics were measured by colorimetric assays. To investigate the potential of the extracts to inhibit the biological activities of BaV, in vitro tests for antiphospholipase and antifibrinolytic activities were performed. In vivo tests for antihemorrhagic and antidefibrinating activities were also carried out, as well as antimicrobial tests for activity against the main bacteria found in the oral cavity of snakes. Interaction between the extracts and the proteins in BaV was assessed by electrophoresis (SDS-PAGE) and Western blot (WB). The cytotoxicity of the extracts was assessed in a strain of MRC-5 human fibroblasts. RESULTS: Terpenoids, flavonoids and condensed and hydrolysable tannins were detected in all the extracts. Metabolites such as coumarins, fatty acids and alkaloids were present in some extracts but not in others, indicating different phytochemical profiles. Phenolics content varied between extracts, and there were more tannins in AEB and HLE. In the in vitro tests, the extracts inhibited the phospholipase and fibrinolytic activities of BaV in the two ratios of venom to extract used. HLE exhibited effective antimicrobial action as it inhibited growth of 11 of the 15 bacteria investigated, including Morganella morganii, the main bacteria described in the oral cavity of snakes. The extracts failed to inhibit the defibrinating activity of BaV, and only the Bothrops antivenom had a significant effect (96.1%) on this activity. BaV-induced hemorrhage was completely inhibited by AEL and AEB when the pre-incubation (venom:extract) protocol was used. When administered orally, as in folk medicine, both AEB and AEL produced significant inhibition of hemorrhagic activity (maximum inhibition 46.5% and 39.2%, respectively). SDS-PAGE and WB of the extracts pre-incubated with BaV showed that the main proteins in the venom had been precipitated by the extracts. None of the four extracts showed cytotoxic effects in the tests carried out with a human fibroblast cell line. CONCLUSION: In addition to being effective in reducing hemorrhage when administered orally, the extracts displayed a high antimicrobial potential against microorganisms involved in secondary infections at the site of the snakebite. Once the extracts have been tested in accordance with the appropriate regulations, this species could potentially be used to produce a phytomedicine for complementary treatment of the secondary infections due to bacteria that aggravate the local signs and symptoms after snakebite envenomation.

Anti-scorpion venom activity of Thapsia garganica methanolic extract: Histopathological and biochemical evidences.

ETHNOPHARMACOLOGICAL RELEVANCE: Thapsia garganica, is a herbal medicine traditionally used as diuretic, emetic and purgative. It is also used as anti-scorpion venom in Morocco; however, its protective effects against scorpion venom remain elusive. AIM OF THE STUDY: The present study was undertaken to evaluate anti-venom activity of T. garganica in vivo through histological and biochemical studies. MATERIALS AND METHODS: Methanolic leaves extract of T. garganica was evaluated for anti-venom activity against buthus. occitanus under in vivo conditions. Histopathological and biochemical changes in envenomed and treated mice were also examined. Phytochemical screening was conducted to estimate the major constituents whereas DPPH, ß -Carotene-linoleic acid and reducing power assays were performed to evaluate the anti-oxidant activity of T. garganica extract. RESULTS: Methanolic leaves extract of T. garganica (2g/kg) increased the survival time (> 18h) of mice injected with lethal doses of B. occitanus venom, with remarkable recovery of histology damage. Furthermore T. garganica induced a significant decreased of biochemical markers of kidney, liver and heart function. Phytochemistry screening revealed the presence of phenolic compounds, flavonoids, tannins and steroids/terpenoids, which might explain the bioactivity of the extract. It was also shown that the extract has an exceptionally high antioxidant activity compared to well-known antioxidants used as standards. CONCLUSION: The present study provides strong evidence that support the use of T. garganica as anti-scorpion venom in traditional medicine in Morocco. However, additional studies are required to isolate and identify the metabolites responsible for the activity.

Antivenomic approach of different Crotalus durissus collilineatus venoms

Our group has previously performed a proteomic study verifying that individual variations can occur among Crotalus durissus collilineatus venoms. These variations may lead to differences in venom toxicity and may result in lack of neutralization of some components by antivenom. In this way, this study aimed to evaluate the Brazilian anticrotalic serum capacity in recognizing twenty-two Crotalus durissus collilineatus venoms, as well as their fractions. Methods: The indirect enzyme-linked immunosorbent assay (ELISA) was chosen to evaluate the efficacy of heterologous anticrotalic serum produced by Instituto Butantan (Brazil) in recognizing the twenty-two Crotalus durissus collilineatus venoms and the pool of them. Moreover, the venom pool was fractionated using reversed-phase fast protein liquid chromatography (RP-FPLC) and the obtained fractions were analyzed concerning antivenom recognition. Results: Evaluation of venom variability by ELISA showed that all venom samples were recognized by the Brazilian anticrotalic antivenom. However, some particular venom fractions were poorly recognized. Conclusion: This study demonstrated that the Brazilian anticrotalic serum recognizes all the different twenty-two venoms of C. d. collilineatus and their fractions, although in a quantitatively different way, which may impact the effectiveness of the antivenom therapy. These results confirm the need to use a pool of venoms with the greatest possible variability in the preparation of antivenoms, in order to improve their effectiveness.(AU)

Pharmacological characterization of cnidarian extracts from the Caribbean Sea: evaluation of anti-snake venom and antitumor properties

Cnidarians produce toxins, which are composed of different polypeptides that induce pharmacological effects of biotechnological interest, such as antitumor, antiophidic and anti-clotting activities. This study aimed to evaluate toxicological activities and potential as antitumor and antiophidic agents contained in total extracts from five cnidarians: Millepora alcicornis, Stichodactyla helianthus, Plexaura homomalla, Bartholomea annulata and Condylactis gigantea (total and body wall). Methods: The cnidarian extracts were evaluated by electrophoresis and for their phospholipase, proteolytic, hemorrhagic, coagulant, fibrinogenolytic, neuromuscular blocking, muscle-damaging, edema-inducing and cytotoxic activities. Results: All cnidarian extracts showed indirect hemolytic activity, but only S. helianthus induced direct hemolysis and neurotoxic effect. However, the hydrolysis of NBD-PC, a PLA2 substrate, was presented only by the C gigantea (body wall) and S. helianthus. The extracts from P. homomalla and S. helianthus induced edema, while only C gigantea and S. helianthus showed intensified myotoxic activity. The proteolytic activity upon casein and fibrinogen was presented mainly by B. annulata extract and all were unable to induce hemorrhage or fibrinogen coagulation. Cnidarian extracts were able to neutralize clotting induced by Bothrops jararacussu snake venom, except M. alcicornis. All cnidarian extracts were able to inhibit hemorrhagic activity induced by Bothrops moojeni venom. Only the C. gigantea (body wall) inhibited thrombin-induced coagulation. All cnidarian extracts showed antitumor effect against Jurkat cells, of which C. gigantea (body wall) and S. helianthus were the most active; however, only C. gigantea (body wall) and M. alcicornis were active against B16F10 cells. Conclusion: The cnidarian extracts analyzed showed relevant in vitro inhibitory potential over the activities induced by Bothrops venoms; these results may contribute to elucidate the possible mechanisms of interaction between cnidarian extracts and snake venoms.(AU)

Production of the first effective hyperimmune equine serum antivenom against Africanized bees.

Victims of massive bee attacks become extremely ill, presenting symptoms ranging from dizziness and headache to acute renal failure and multiple organ failure that can lead to death. Previous attempts to develop specific antivenom to treat these victims have been unsuccessful. We herein report a F(ab)(´)(2)-based antivenom raised in horse as a potential new treatment for victims of multiple bee stings. The final product contains high specific IgG titers and is effective in neutralizing toxic effects, such as hemolysis, cytotoxicity and myotoxicity. The assessment of neutralization was revised and hemolysis, the primary toxic effect of these stings, was fully neutralized in vivo for the first time.

Maintaining Coral Snakes (Micrurus nigrocinctus, Serpentes: Elapidae) for venom production on an alternative fish-based diet.

American Elapid snakes (Coral Snakes) comprise the genera Leptomicrurus, Micruroides and Micrurus, which form a vast taxonomic assembly of 330 species distributed from the South of United States to the southern region of South America. In order to obtain venom for animal immunizations aimed at antivenom production, Coral Snakes must be kept in captivity and submitted periodically to venom extraction procedures. Thus, to maintain a snake colony in good health for this purpose, a complete alternative diet utilizing an easily obtained prey animal is desirable. The development of a diet based on fish is compared to the wild diet based on colubrid snakes, and assessed in terms of gain in body weight rate (g/week), longevity (weeks), venom yield (mg/individual), venom median lethal dose (LD50) and venom chromatographic profiles. The animals fed with the fish-based diet gained more weight, lived longer, and produced similar amount of venom whose biological and biochemical characteristics were similar to those of venom collected from specimens fed with the wild diet. This fish-based diet appears to be suitable (and preferable to the wild diet) to supply the nutritional requirements of a Micrurus nigrocinctus snake collection for the production of antivenom.

Cross neutralization of Hypnale hypnale (hump-nosed pit viper) venom by polyvalent and monovalent Malayan pit viper antivenoms in vitro and in a rodent model.

Hypnale hypnale (hump-nosed pit viper) is a medically important venomous snake in Sri Lanka and Southwestern India. Bite of this snake may result in hemostatic dysfunction, acute kidney injury and death. Clinical studies indicated that the locally available polyvalent antivenoms produced in India are not effective against hump-nosed pit viper envenoming. Hence, there is an urgent need to search for effective antivenom. In this paper, we examined the ability of Calloselasma rhodostoma (Malayan pit viper) monovalent antivenom and the Hemato polyvalent antivenom (both produced by Thai Red Cross Society, TRCS) to neutralize the lethality and toxic effects of H. hypnale venom, as C. rhodostoma is considered a sister taxon of H. hypnale. In vitro neutralization studies showed that the Hemato polyvalent antivenom effectively neutralized the lethality of H. hypnale venom (1.52mgvenom/mL antivenom) as well as the hemorrhagic, procoagulant and necrotic activities of the venom. The monovalent C. rhodostoma antivenom could also neutralize the lethality and toxic activities of the venom, but the potency was lower. The Hemato polyvalent antivenom also effectively protected mice from the lethal and local effects of H. hypnale venom in an in vivo rodent model of envenoming. Furthermore, the polyvalent antivenom could also effectively neutralize the venom of Daboia russelii (2.50mgvenom/mL antivenom), another common cause of snake bites in Sri Lanka and South India. These findings suggested that the Hemato polyvalent antivenom may be beneficial in the antivenom treatment of H. hypnale envenoming.

Stability of equine IgG antivenoms obtained by caprylic acid precipitation: towards a liquid formulation stable at tropical room temperature.

Liquid formulations of antivenom require a cold chain for their distribution and storage, especially in tropical countries characterized by high temperature and humidity (climatic zone IV). Since cold chain is often deficient in many regions, there is a need to develop novel formulations of liquid antivenoms of higher stability at room temperatures. The effect of addition of the polyols mannitol and sorbitol on the thermal stability of caprylic acid-fractionated equine whole IgG antivenoms was assessed in preparations having different concentrations of protein and phenol. Results evidenced that: (1) turbidity increases proportionally to phenol and protein concentration. (2) After one year of storage at 25 degrees C, caprylic acid-purified antivenoms, formulated with or without polyols, did not show evidences of instability. (3) Formulation of antivenoms with 2.0 M sorbitol prevents the appearance of turbidity after one year storage at 37 degrees C; however, there was a partial loss in neutralizing potency in these conditions. Results suggest that formulation based on sorbitol is an option to obtain liquid whole IgG antivenoms of higher stability at tropical room temperatures.

Apparent marked reduction in early antivenom reactions compared to historical controls: was it prophylaxis or method of administration?

OBJECTIVE: Serious morbidity and mortality following snakebite injuries are common in tropical regions of the world. Although antivenom administration is clinically effective, it carries an important risk of early anaphylactic reactions, ranging from relatively benign nausea, vomiting, and urticaria to life-threatening angioedema, bronchospasm and hypotension. Currently, no adequately powered study has demonstrated significant benefit from the use of any prophylactic drug. A high rate of anaphylactic reactions observed during a trial of three different antivenoms in Ecuador prompted adoption of premedication with intravenous (i.v.) hydrocortisone and diphenhydramine together with dilution and slower administration of antivenom. DESIGN: In a rural mission hospital in Eastern Ecuador, 53 consecutive snakebite victims received a new antivenom regimen in 2004-2006, comprising prophylactic drugs and i.v. infusion of diluted antivenom over 60 min. They were compared to an historical control cohort of 76 patients treated in 1997-2002 without prophylactic drugs and with i.v. "push" injection of undiluted antivenom over 10 min. All these patients had incoagulable blood on admission and all were treated with Brazilian Instituto Butantan polyspecific antivenom. RESULTS: Baseline characteristics of the historical control and premedicated groups were broadly similar. In the historical group, early reaction rates were as follows: 51% of patients had no reaction; 35% had mild reactions; 6% moderate; and 6% severe. In the premedicated/slow i.v. group, 98% of patients had no reaction; 0 mild; 0 moderate; and 2% severe. The difference in reaction rates was statistically significant (p<0.001). CONCLUSIONS: Premedication with intravenous hydrocortisone and diphenhydramine together with dilution of antivenom and its administration by i.v. infusion over 60 min appeared to reduce both the frequency and severity of anaphylactic reactions. A randomized blinded controlled trial is needed to confirm these encouraging preliminary findings.

Snake venomics of the Lesser Antillean pit vipers Bothrops caribbaeus and Bothrops lanceolatus: correlation with toxicological activities and immunoreactivity of a heterologous antivenom.

The venom proteomes of the snakes Bothrops caribbaeus and Bothrops lanceolatus, endemic to the Lesser Antillean islands of Saint Lucia and Martinique, respectively, were characterized by reverse-phase HPLC fractionation, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venoms contain proteins belonging to seven ( B. caribbaeus) and five ( B. lanceolatus) types of toxins. B. caribbaeus and B. lanceolatus venoms contain phospholipases A 2, serine proteinases, l-amino acid oxidases and zinc-dependent metalloproteinases, whereas a long disintegrin, DC-fragments and a CRISP molecule were present only in the venom of B. caribbaeus, and a C-type lectin-like molecule was characterized in the venom of B. lanceolatus. Compositional differences between venoms among closely related species from different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. The venoms of these two species differed in the composition and the relative abundance of their component toxins, but they exhibited similar toxicological and enzymatic profiles in mice, characterized by lethal, hemorrhagic, edema-forming, phospholipase A 2 and proteolytic activities. The venoms of B. caribbaeus and B. lanceolatus are devoid of coagulant and defibrinogenating effects and induce only mild local myotoxicity in mice. The characteristic thrombotic effect described in human envenomings by these species was not reproduced in the mouse model. The toxicological profile observed is consistent with the abundance of metalloproteinases, PLA 2s and serine proteinases in the venoms. A polyvalent (Crotalinae) antivenom produced in Costa Rica was able to immunodeplete approximately 80% of the proteins from both B. caribbaeus and B. lanceolatus venoms, and was effective in neutralizing the lethal, hemorrhagic, phospholipase A 2 and proteolytic activities of these venoms.

Inhibitory effects on phospholipase A2 and antivenin activity of melanin extracted from Thea sinensis Linn.

Antivenin activity of melanin extracted from black tea (MEBT) was reported for the first time. The antagonistic effect of MEBT was evaluated for Agkistrodon contortrix laticinctus (broadbanded copperhead), Agkistrodon halys blomhoffii (Japanese mamushi), and Crotalus atrox (western diamondback rattlesnake) snake venoms administered i.p. to ICR mice. MEBT was injected i.p. immediately after the venom administration in dose of 3 mg per mouse in the same place of venom injection. MEBT demonstrated neutralization effect against all venoms tested. The greatest antivenin effect of MEBT was found against Japanese mamushi snake venom. In this case, half the mice died within 2.5 +/- 0.7 h after injection of 0.9 mg/kg of venom. An immediate injection of MEBT substantially reduced the toxic effect of venom and extended time at the 50% level of survival up to 52.3 +/- 2.3 h. The antivenin activity of MEBT is due to chelating of Ca++ and non-specific binding of phospholipase A2. The inhibitory effect of MEBT on phospholipase A2 assessed for different venoms was similar to that obtained with pure enzyme. Low toxicity of MEBT in combination with its antagonistic activity against different venoms may allow effective life-saving treatment against snakebites. Such application of MEBT is important when identification of the snake is impossible or if specific treatment is unavailable.

Encapsulation of native crotoxin in liposomes: a safe approach for the production of antivenom and vaccination against Crotalus durissus terrificus venom.

Crotoxin, the neurotoxic component of Crotalus durissus terrificus (Cdt) venom that displays phospholipase A2 activity, was successfully encapsulated into dehydration-rehydration vesicles (DRV/crotoxin) and reverse-phase evaporation vesicles (REV/crotoxin) made from sphingomyelin and cholesterol. The encapsulation efficiency of native crotoxin was higher in DRV/crotoxin than in REV/crotoxin. DRV/crotoxin was not toxic when i.v. inoculated in mice at a dose of crotoxin as high as 91 times its L.D50 or when s.c. inoculated at 42 times its LD50. On the other hand, crotoxin released from DRV/crotoxin retained its original toxicity. REV/crotoxin was found to be at least 1.9 times more toxic than DRV/crotoxin. The fact that DRV/crotoxin retained crotoxin more efficiently than REV/crotoxin may account for the difference in acute toxicity between the two preparations. DRV/crotoxin, when s.c. inoculated in mice, induced anti-crotoxin antibodies that protected animals against the lethal effect of Cdt venom. Following immunization with three doses of DRV/crotoxin (3 x 20 micrograms of crotoxin/mouse) and challenge with 8 x LD50 of Cdt venom, 75% of mice were protected. The DRV/crotoxin preparation was compared to crotoxin emulsified in Freund's adjuvant (FCA/crotoxin). DRV/crotoxin was found to be less toxic than FCA/crotoxin, and to induce lower levels of anti-crotoxin antibodies but similar levels of protection when inoculated at high doses (20 or 70 micrograms crotoxin/mouse). When DRV/crotoxin was adsorbed to alum at the time of immunization, it induced antibody and protection levels comparable to those produced by FCA/crotoxin.

Dano da radiaçäo gama em crotamina (Toxina da cascavel brasileira) / Damage of gamma radiation in crotamine (Toxin of brazilian rattlesnake)

Radiaçöes ionizantes afetam a estrutura das moléculas devido à destruiçäo das suas ligaçöes químicas. Estas alteraçöes químicas poderäo levar a uma mudança nas propriedades biológicas das mesmas, como tem sido demosntrado na literatura. A Crotamina foi obtida a partir de um "pool" do veneno de Crotalus durissus terrificus através de cromatografia de exclusäo molecular, sendo posteriormente irradiada em soluçäo numa concentraçäo de 2 mg/ml de NaCl 0,85% com radiaçäo gama produzida por uma fonte de 60Co. Foram adotadas as doses de 100 Gy, 250 Gy, 500 Gy, 1000 Gy e 2000 Gy (taxa de dose = 1,19. 10**3 Gy/h). Realizou-se os seguintes ensaios: presença de grupos SH livres, determinaçäo do número de bandas na SDS-PAGE (sugerindo a formaçäo de agregados protéicos) que foi proporcional ao aumento das doses. Pela imunodifusäo näo houve perda da atividade imunoquímica quando testadas contra o antisoro produzido pelo Instituto Butantan

LD50 determination: objections to the method of Beccari as modified by Molinengo.

The efficiency of the method of Beccari as modified by Molinengo is compared with the sequential technique of Dixon and Mood. It is concluded that the sequential technique is less constrained, gives estimates that are less disperse and is simpler to analyze statistically than the regression of Beccari-Molinengo. Both techniques are comparable in the number of animals required, as well as in the time invested in each assay.

Preparation of highly potent Naja naja atra (Formosan cobra) antivenin.

As the preparation of high titer antiserum against Naja naja atra venom is a time-consuming process, attempts were made to develop a immunization procedure for producing highly potent antiserums within a short period. Rabbits were immunized for 12 weeks with (1) whole venom as used routinely in our Institute, (II) whole venom adsorbed on carboxymethyl-cellulose (CMC) and mixed with Freund's complete adjuvant and (III) neurotoxin adsorbed on CMC and adjuvant mixture followed by CMC-whole venom-adjuvant mixture. The results showed that one ml of the antiserum prepared by method (III) could neutralize 445 LD50 of whole venom, in other words, its potency was 4 and 40 times higher than those prepared by methods (II) and (I), respectively.