Gustavo Rojas

Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom

The polyvalent antivenom produced at the Instituto Clodomiro Picado, Costa Rica, was tested for its capacity to neutralize proteolytic and hemorrhagic activities of ten Costa Rican crotaline venoms. In experiments with preincubation of venom and antivenom, the latter efficiently neutralized proteolytic activities of nine venoms, with ED50 ranging from 50 to 300 microliters antivenom/mg venom. The venom of Bothrops nummifer was neutralized less efficiently (ED50 = 760 microliters/mg.) Antivenom was also very effective in neutralizing hemorrhagic activity, having its lowest neutralizing ability against the venom of B. picadoi (ED = 430 microliters/mg) and its highest towards the venom of B. asper (Pacific region) (ED50 = 47 microliters/mg). There was a significant correlation between the ability of antivenom to neutralize proteolytic and hemorrhagic effects. In spite of the ability of antivenom to neutralize hemorrhage when incubated with venom prior to injection, hemorrhage was only partially neutralized when antivenom was administered i.v. at different time periods after envenomation. This suggests that the rapid development of local hemorrhage, instead of the absence of antivenom antibodies, is the explanation for the poor neutralization observed in these types of experiments.

Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production

A simple methodology for hyperimmune horse plasma fractionation, based on caprylic acid precipitation, is described. Optimal conditions for fractionation were studied; the method gives best results when concentrated caprylic acid was added to plasma, whose pH had been adjusted to 5.8, until a final caprylic acid concentration of 5% was reached. The mixture was vigorously stirred during caprylic acid addition and then for 60 min; afterwards the mixture was filtered. Non-immunoglobulin proteins precipitated in these conditions, whereas a highly enriched immunoglobulin preparation was obtained in the filtrate, which was then dialysed to remove caprylic acid before the addition of NaCl and phenol. Thus, antivenom was produced after a single precipitation step followed by dialysis. In order to compare this methodology with that based on ammonium sulfate fractionation, a sample of hyperimmune plasma was divided into two aliquots which were fractionated in parallel by both methods. It was found that caprylic acid-fractionated antivenom was superior in terms of yield, production time, albumin/globulin ratio, turbidity, protein aggregates, electrophoretic pattern and neutralizing potency against several activities of Bothrops asper venom. Owing to its efficacy and simplicity, this method could be of great value in antivenom and antitoxin production laboratories.

Comparative study on coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of Costa Rican crotaline snake venoms and their neutralization by a polyvalent antivenom.

The coagulant, defibrinating, fibrino lytic and fibrinogenolytic activities of venoms from ten species of Costa Rican crotaline snakes were studied, together with the neutralization of these effects by a polyvalent antivenom. The venoms of Bothrops asper, B. schlegelii, B. nummifer, B. godmani, Lachesis muta and Crotalus durissus induced a coagulant effect in vitro, and all of them, with the exception of B. nummifer, also induced defibrination in vivo. The four non-coagulant venoms (B. lateralis, B. ophryomegas, B. nasuta and B. picadoi) induced a degradation of the alpha (A) chain of fibrinogen, thereby inhibiting coagulation. However, they did not induce defibrination upon i.v. injection. All of the venoms showed fibrinolytic activity in vitro. Polyvalent antivenom was effective in the neutralization of coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of these venoms, with the exception of coagulant effect induced by C. durissus venom. Since only three venoms are used in the immunization of horses, these results demonstrate the high degree of immunological cross reactivity between components affecting coagulation in Costa Rican crotaline snake venoms.

A randomized blinded clinical trial of two antivenoms, prepared by caprylic acid or ammonium sulphate fractionation of IgG, in Bothrops and Porthidium snake bites in Colombia: correlation between safety and biochemical characteristics of antivenoms.

A randomized blinded clinical trial was performed in 53 patients bitten by Bothrops sp. and Porthidium sp. in Antioquia and Chocó, Colombia, in order to compare the efficacy and safety of two antivenoms made of whole IgG obtained by either ammonium sulphate (monovalent anti-B. atrox) or caprylic acid (polyvalent) fractionation. Additionally, antivenoms were compared by electrophoretic and chromatographic analyses and anticomplementary activity in vitro. With a protocol of 2, 4 and 6 antivenom vials for the treatment of mild, moderate and severe envenomings, respectively, both antivenoms were equally efficient to neutralize the most relevant signs of envenoming and to clear serum venom levels in patients from the first hour and later on. Three patients with severe envenoming and initially treated with less than six vials on admission had persistent or recurrent venom antigenemia within 12-48 h. Monovalent antivenom fractionated by ammonium sulphate precipitation had higher amounts of protein aggregates and nonimmunoglobulin proteins than polyvalent antivenom fractionated by caprylic acid precipitation. Both antivenoms presented anticomplementary activity in vitro, being higher in the monovalent product. In agreement, monovalent antivenom induced a significantly higher incidence of early antivenom reactions (52%) than polyvalent antivenom (25%).

Neutralization of crotaline snake venoms from Central and South America by antivenoms produced in Brazil and Costa Rica.

A study was performed on the ability of antivenoms, produced in Brazil and Costa Rica, to neutralize lethal, hemorrhagic and coagulant activities of the venoms of 16 species of Central and South American snakes of the subfamily Crotalinae. Neutralization of lethality was studied by two different methods routinely used in the quality control of antivenoms at Instituto Butantan (IB) and Instituto Clodomiro Picado (ICP). Both antivenoms neutralized the majority of the venoms studied, but the values of effective doses 50% (ED(50)) differed markedly depending on the method used. In general, higher potencies were obtained with the method of ICP, where a challenge dose corresponding to 4 LD(50)s is used, than with the method of IB, where a challenge dose of 5 LD(50)s is employed. All venoms induced hemorrhagic activity in the mouse skin test, which was effectively neutralized by the two antivenoms. All venoms, except those of Porthidium nasutum and Bothriechis lateralis, induced coagulation of human plasma in vitro and both antivenoms were effective in the neutralization of this activity. In conclusion, our results provide evidence of an extensive cross reactivity between these antivenoms and Central and South American crotaline snake venoms.

Comparative study on the ability of IgG and Fab sheep antivenoms to neutralize local hemorrhage, edema and myonecrosis induced by Bothrops asper (terciopelo) snake venom

The ability of sheep antivenoms, consisting of whole IgG molecules or Fab fragments, to neutralize local hemorrhage, edema and myonecrosis induced by Bothrops asper venom was comparatively studied in mice. The two antivenoms were produced from the same batch of hyperimmune plasma and were adjusted to the same neutralizing potency against these effects in assays where venom and antivenoms were incubated prior to injection. Thus, if differences are observed in experiments involving independent injection of venom and antivenoms, they would depend on the pharmacokinetic profiles of the products. Despite the observation that both antivenoms neutralized the three effects if preincubated with venom, neutralization was only partial when antivenoms were administered i.v. at various time intervals after envenomation. No significant differences were observed between IgG and Fab antivenoms concerning neutralization of hemorrhagic and edema-forming activities, whereas IgG antivenom was slightly more effective in neutralizing myotoxic activity in experiments involving independent injection of venom and antivenom. These results do not support the hypothesis that Fab fragments are more effective than whole IgG molecules in the neutralization of locally-acting toxins from B. asper venom.

Comparative study of the edema-forming activity of costa rican snake venoms and its neutralization by a polyvalent antivenom

The edema-forming activity of eight Costa Rican crotaline snake venoms and its neutralization by a polyvalent antivenom were studied using the mouse footpad test. All of the venoms induced edema, the highest activity being present in the venoms of Bothrops lateralis and Bothrops picadoi. When experiments were performed with preincubation of venom and antivenom, neutralization of edema was poor. Moreover, it was observed that, with some venoms, edema increased when large doses of antivenom were used. This effect was also observed when some venoms were incubated with coral snake antivenom, suggesting that venoms may release some pharmacologically active component(s) from antivenom, since the latter contains traces of alpha-2 and beta globulins. Based on these findings, an alternative approach to the study of the neutralization of edema was used; in this new method, antivenom was injected i.v. before venom administration, thereby avoiding preincubation. With this technique, a much better neutralization of edema was observed, although with some venoms it was still poor. Venoms contain low molecular weight factors which induce edema, suggesting that lack of immunogenicity of some components may cause a poor neutralization. However, such components are responsible for only a minor portion of the edema induced by crude venoms. It is suggested that experiments in which venom and antivenom are preincubated preincubated in testing the neutralization of edema should be avoided, and that a more adequate approach may be an independent inoculation of venom and antivenom.

Biochemical and pharmacological similarities between the venoms of newborn Crotalus durissus durissus and adult crotalus durissus terrificus rattlesnakes

A comparative study was performed with the venoms of newborn Crotalus durissus durissus, adult Crotalus durissus terrificus and adult Crotalus durissus durissus snakes. Venom of newborn specimens of C.d. durissus is very similar to that of adult specimens of C.d. terrificus, since they have strong lethal and myotoxic activities, and weak proteolytic, hemorrhagic and edema-forming effects, in contrast to venom of adult specimens of C.d. durissus. In addition, the two former venoms have high amounts of the neurotoxic complex crotoxin, whereas venom from adult C.d. durissus has a low concentration of crotoxin. Electrophoretic analysis corroborates the strong similarities between the former two venoms. It is concluded that venom of newborn C.d. durissus contains high concentrations of crotoxin and low amounts of hemorrhagic and proteolytic components, and that a drastic ontogenetic change takes place in the venom composition of this subspecies.

Ability of a polyvalent antivenom to neutralize the venom of Lachesis muta melanocephala, a new Costa Rican subspecies of the bushmaster

Several toxic and enzymatic activities of the venom of L. m. melanocephala were studied. This venom has many similarities with that of L. m. stenophrys, although there are quantitative differences in venom activities, as well as in the immunodiffusion patterns of these venoms when reacted against polyvalent antivenom. This antivenom was tested for its ability to neutralize a series of toxic and enzymatic effects of L. m. melanocephala venom. A new method to study myonecrosis, based on the quantitation of residual creatine kinase in injected muscle, was used. Antivenom was highly effective in neutralizing lethal, hemorrhagic, myotoxic, edema-forming, defibrinating, caseinolytic and fibrinolytic activities when venom and antivenom were incubated prior to the test or, in the case of edema-forming activity, when antivenom was administered before venom injection. On the other hand, when antivenom was injected i.v. at different time intervals after venom injection neutralization of lethality was good, although neutralization of local effects, i.e. hemorrhage and edema, was poor. These results indicate that polyvalent antivenom contains antibodies capable of neutralizing toxic and enzymatic activities of L. m. melanocephala venom. Moreover, the partial inability of antivenom to neutralize local effects when administered after venom injection is probably due to the rapid development of these effects once venom is injected.

Neutralization, by a monospecific Bothrops lanceolatus antivenom, of toxic activities induced by homologous and heterologous Bothírops snake venoms.

A monospecific Bothrops lanceolatus antivenom, currently used in Martinique, was tested for its efficacy in the neutralization of several toxic and enzymatic activities of the venoms of B. lanceolatus, B. atrox and B. asper. When tested by the i.p. route in mice, B. lanceolatus venom had an LD50 of 12.8 microg/g. In addition, it induced local tissue damage (hemorrhage, edema and myotoxicity) and showed indirect hemolytic activity, but was devoid of coagulant effect on human plasma in vitro and of defibrinating activity in mice. Antivenom was fully effective in the neutralization of lethal, hemorrhagic, edema-forming, myotoxic and indirect hemolytic effects of B. lanceolatus venom in assays involving preincubation of venom and antivenom. When tested against the venoms of B. asper and B. atrox, the antivenom completely neutralized the lethal, hemorrhagic, myotoxic and indirect hemolytic effects, and was partially effective in neutralizing edema-forming activity. In contrast, the antivenom was ineffective in the neutralization of in vitro coagulant and in vivo defibrinating effects induced by these two venoms.