G.D. Laing

An affinity purified ovine antivenom for the treatment of Vipera berus envenoming

A novel antivenom for treating patients with Vipera berus bite has been developed. Sheep are immunised monthly with relatively small amounts of Vipera berus (common adder) venom and the resultant antisera pooled. The immunoglobulin fraction is precipitated with sodium sulphate then cleaved with papain to produce Fab fragments. Finally, those Fab fragments that are directed specifically against components in the venom are purified by affinity chromatography on columns comprising V. berus venom coupled to cyanogen bromide activated Sepharose 4B. The resultant product is some three times more effective than the non-purified Fab in protecting mice against the lethal venom effects.

Crotaline snake bite in the Ecuadorian Amazon: randomised double blind comparative trial of three South American polyspecific antivenoms

Abstract Objective To compare the efficacy and safety of three polyspecific antivenoms for bites by pit vipers. Design Randomised double blind comparative trial of three antivenoms. Setting Shell, Pastaza, southeastern Ecuador. Participants 210 patients with incoagulable blood were recruited from 221 consecutive patients admitted with snake bite between January 1997 and December 2001. Intervention One of three antivenoms manufactured in Brazil, Colombia, and Ecuador, chosen for their preclinical potency against Ecuadorian venoms. Main outcome measures Permanent restoration of blood coagulability after 6 and 24 hours. Results The snakes responsible for the bites were identified in 187 cases: 109 patients (58%) were bitten by Bothrops atrox, 68 (36%) by B bilineatus, and 10 (5%) by B taeniatus, B brazili, or Lachesis muta. Eighty seven patients (41%) received Colombian antivenom, 82 (39%) received Brazilian antivenom, but only 41 (20%) received Ecuadorian antivenom because the supply was exhausted. Two patients died, and 10 developed local necrosis. All antivenoms achieved the primary end point of permanently restoring blood coagulability by 6 or 24 hours after the start of treatment in > 40% of patients. Colombian antivenom, however, was the most effective after initial doses of 20 ml (two vials), < 70 ml, and any initial dose at both 6 and 24 hours. An initial dose of 20 ml of Colombian antivenom permanently restored blood coagulability in 64% (46/72) of patients after 6 hours (P = 0.054 compared with the other two antivenoms) and an initial dose of < 70 ml was effective at 6 hours (65%, P = 0.045) and 24 hours (99%, P = 0.06). Early anaphylactoid reactions were common (53%, 73%, and 19%, respectively, for Brazilian, Colombian, and Ecuadorian antivenoms, P < 0.0001) but only three reactions were severe and none was fatal. Conclusions All three antivenoms can be recommended for the treatment of snakebites in this region, though the reactogenicity of Brazilian and Colombian antivenoms is a cause for concern.

Inflammatory pathogenesis of snake venom metalloproteinase-induced skin necrosis

Local tissue damage, characterized by edema, hemorrhage and necrosis, is a common consequence of envenoming by many vipers. We have investigated the contribution of inflammatory responses induced by the venom metalloproteinase jararhagin (isolated from Bothrops jararaca venom) in the development of these lesions. Local venom effects (edema, hemorrhage and necrosis) were inducedexperimentally in knockout mice deficient in the TNF receptors TNFR1 or TNFR2, IL‐1βR, IL‐6 and iNOS. Jararhagin‐induced dermal necrosis was abolished in mice deficient in the TNF receptors TNFR1 and TNFR2, and the same activity was significantly reduced in IL‐6–/– mice. There was no significant difference in edema and hemorrhage activities following jararhagin insult between knockout and WT strains, indicating that these local venom metalloproteinase‐induced effects are independent of these pro‐inflammatory mediators. The contribution of both TNF receptors and IL‐6 in local tissue necrosis raises important therapeutic issues regarding the treatment of local envenoming.

The effect of jararhagin, a metalloproteinase from Bothrops jararaca venom, on pro-inflammatory cytokines released by murine peritoneal adherent cells.

The release of pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) from murine peritoneal adherent cells (MPAC) was studied after exposure to jararhagin, a metalloproteinase/disintegrin isolated from Bothrops jararaca venom. MPACs were treated with LPS (lipopolysaccharide), jararhagin, or EDTA-inactivated jararhagin for up to 24h. Following incubation, the culture supernatant was assayed by ELISA for the presence of cytokines, while the cells were analysed for viability and cytokine mRNA expression. The cells exposed to native jararhagin released TNF-alpha and IL-1beta after 4 and 24h respectively. When MPACs were exposed to Jararhagin treated with EDTA, TNF-alpha and IL-1beta production was sustained throughout the culture period and IL-6 production was observed. TNF-alpha, IL-6 and IL-1beta mRNA were detected 4h after stimulation with either native or EDTA-treated jararhagin. Addition of jararhagin to LPS stimulated cells resulted in a dramatic decrease in the release of IL-6 and TNF-alpha. RT-PCR showed that this inhibition does not occur at the transcriptional level and further experiments showed that jararhagin degraded soluble cytokines by proteolytic activity. This study suggests that jararhagin induces TNF-alpha, IL-1beta and IL-6 expression, which may be rapidly degraded by its proteolytic activity.

Comparison of the potency of three Brazilian Bothrops antivenoms using in vivo rodent and in vitro assays

Three Brazilian polyspecific Bothrops antivenoms were compared using standard W.H.O. rodent in vivo and in vitro assays of their ability to neutralize the principal venom activities of pooled whole Bothrops jararaca venom. On a volume basis, the antivenoms were equally effective in neutralizing lethal activity in mice, and there were only minor differences in their ability to neutralize venom-induced haemorrhage, necrosis and procoagulant activity. Antivenom efficacy in neutralizing defibrinogenation varied. However, when equal amounts of antivenom IgG were compared, it was found that the FUNED antivenom best neutralized lethality, haemorrhage, necrosis and fibrinogen clotting activity. Vital Brazil and FUNED antivenoms were equally effective in neutralizing plasma coagulant activity but Vital Brazil antivenom was the more effective in neutralizing defibrinogenation.

Isolation and comparison of myotoxins isolated from venoms of different species of Bothrops snakes.

Venoms of nine different snake species of the genus Bothrops were fractionated using fast protein liquid chromatography (FPLC). Basic proteins with phospholipase A2 and/or myotoxic activities were isolated from venoms of B. jararacussu, B. moojeni, B. neuwiedi and B. pradoi. B. jararaca venom possessed very low concentrations of these proteins, which were undetectable in venoms of B. atrox, B. alternatus, B. cotiara and B. erythromelas. Basic proteins from B. moojeni and B. pradoi venoms were isolated in pure form. All active fractions possessed a common band of 15,000 mol. wt which caused a rise in serum creatine phosphokinase levels and histopathological changes in muscle cells following i.m. injection into mice. Levels of phospholipase A2 activity were variable. The implications of the possession of varying levels of myotoxins and phospholipase A2 in these venoms are discussed.

Collagen-induced secretion-dependent phase of platelet aggregation is inhibited by the snake venom metalloproteinase jararhagin

Jararhagin, a 52 kDa metalloproteinase from Bothrops jararaca snake venom, belongs to the family of enzymes with an N-terminal Zn2+-containing enzymatic domain, a disintegrin-like domain and a cysteine-rich C-terminal domain. Both jararhagin and jararhagin C, a 28 kDa-protein from the same venom identical to the disintegrin-like domain of jararhagin, inhibit collagen-induced platelet aggregation. In this study, jararhagin and synthetic linear peptides based on the disintegrin-like domain of jararhagin overlapping with the RGD sequence of venom disintegrins, were shown for the first time to inhibit the release of 5-hydroxytryptamine (5-HT) from platelets preloaded with [14C]5-HT and stimulated with collagen. The normal phosphorylation of the 21-kDa myosin light chain (p21) in response to the stimulation indicated that jararhagin and the peptides did not interfere with platelet shape change. The selective inhibition of the secretion-dependent phase of the platelet response to collagen by the enzyme and its peptides was confirmed by the defective phosphorylation of pleckstrin, a 47-kDa platelet protein (p47) involved in dense granule secretion.

Experimental assessment of a new, low-cost antivenom for treatment of carpet viper (Echis ocellatus) envenoming

Morbidity and mortality due to envenoming by the carpet viper (Echis ocellatus) in northern Nigeria remains unacceptably high and constitutes a severe economic and public health problem to the local farming community in particular. The only effective treatment of systemic envenoming is antivenom, but supplies are very limited as the little that is available is either too expensive, ineffective or both. Here, we describe a new ovine antivenom, designed both to be effective and to be available at low cost. The antivenom, a polyclonal ovine Fab preparation, provides superior protection, both in vivo and in vitro, to the best alternatives, the monospecific South African Institute of Medical Research antivenom and the polyspecific Pasteur Isper Africa antivenom. Fab fragments, which have the advantages of large volumes of distribution and, theoretically, low immuno-reactivity, are produced by a reusable solid-phase papain matrix which eliminates enzyme contamination of the product and reduces cost. The antivenom is lyophilised for increased stability and extended shelf-life in tropical climates where it is often impossible to keep such products cool.

Comparison of the purity and efficacy of affinity purified avian antivenoms with commercial equine crotalid antivenoms

Antivenoms were raised in laying hens by repeated immunizations with detoxified crotalid snake venoms and purified from egg yolks by affinity chromatography. While the affinity purified avian antivenoms were essentially pure IgG, commercial equine (Wyeth) and W.H.O. international reference antivenoms (Trimeresurus flavoviridis) contained several non-immunoglobulin contaminants. In standard mouse protection assays, the purified avian Crotalus atrox and T. flavoviridis antivenoms were 6.3 and 2.0 times as potent, respectively, as these equine antivenoms in neutralizing venom lethality. The purity, efficacy, and ease of manufacture of avian antivenoms, and their inability to fix mammalian complement, make them an attractive alternative to equine and other mammalian antivenoms.

A new antivenom to treat eastern coral snake (Micrurus fulvius fulvius) envenoming

An Fab based ovine antivenom has been prepared and compared both in vitro and in vivo with two commercial preparations. The product was found to be at least four times more effective on a weight basis. The increased potency, combined with the low incidence of side-effects associated with ovine Fab, should result in a safer, more effective antivenom.