, which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on eryt... more , which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on erythrocytes (RBC) from different species despite the fact that enzymatically active CGMT-I was able to hydrolyze RBC membrane phospholipids and disrupt liposomes prepared from RBC lipids. Human RBC did not become susceptible to the toxins after treatment with neuraminidase or after altering membrane fluidity with cholesterol or sublytic concentrations of detergent. Unlike normal RBC, significant hemolysis was induced by CGMT-II and another similar Lys-49 isoform, B. asper MT-II (BAMT-II), in RBC enriched with phosphatidylserine (PS). Hemolysis was greater in RBC preincubated with pyridyldithioethylamine (PDA), a potent inhibitor of aminophospholipid transport. RBC enriched with phosphatidic acid (PA) also became susceptible to the myotoxins but was unaffected by PDA. Cells enriched with phosphatidylcholine (PC) remained resistant to the action of the toxins. BAMT-II also induced damage in black lipid membranes prepared with PS but not PC alone. When RBC binding of BAMT-II was measured by enzyme-linked immunosorbent assay, it was observed that PS-and PA-enriched erythrocytes were always able to capture more toxin than normal and PC-enriched RBC. This effect was significantly improved by PDA (in the case of PS) and it was observed either in the presence or in the absence of calcium in the medium. These data suggest that negatively charged lipids in the outer leaflet of cell membranes constitute myotoxic PLA2 binding sites. The scarcity of anionic phospholipids in the outer leaflet of RBC could explain their resistance to the action of these PLA2s.
Caprylic acid purification of IgG, currently used in the manufacture of horse-derived antivenoms,... more Caprylic acid purification of IgG, currently used in the manufacture of horse-derived antivenoms, was successfully adapted for the preparation of sheep and camel IgG. Sheep IgG had a molecular mass of w150 kDa, whereas camel IgG presented two bands of molecular masses of w160 and 100 kDa, the latter corresponding to heavy-chain IgG, which is devoid of light chains. Horse, sheep and camel IgGs were compared by several parameters aiming at predicting their potential for induction of early and late adverse reactions. Horse and sheep IgGs showed a higher anticomplementary activity than camel IgG, and also elicited a higher anti-IgG response than camel IgG, when injected in mice. Horse IgG agglutinated human type OC erythrocytes, whereas no such reactivity was observed in sheep and camel IgG preparations. A novel procedure was used for the detection of antibodies in human serum against animal IgGs. It was found that a pool of human sera collected in Costa Rica had a higher titer of antibodies directed against horse and sheep IgGs than against camel IgG. Overall, camel IgG showed the lowest potential for the induction of adverse reactions among the three IgGs tested. q
Interest in studies on the neutralization of snake venoms and toxins by diverse types of inhibito... more Interest in studies on the neutralization of snake venoms and toxins by diverse types of inhibitors is two-fold. From an applied perspective, results enclose the potential to be translated into useful therapeutic products or procedures, to benefit patients suffering from envenomings. From a basic point of view, on the other hand, neutralizing agents may be used as powerful dissecting tools to determine the relative role of toxins within the context of the overall pathology induced by a venom, or to increase our understanding on the molecular mechanisms by which toxins exert their harmful actions upon particular targets. The venom of the snake Bothrops asper has been the subject of a number of experimental studies addressing its neutralization by antibodies, as well as by nonimmunologic inhibitors, including natural products derived from plants or animals, or synthetic drugs. As summarized in the present review, neutralization studies on this venom and some of its isolated toxins have contributed to a better understanding of envenomings by this species, and their treatment. In addition, such studies have provided valuable knowledge on the mechanisms of action and the relative functional importance of particular toxins of this venom, especially in the case of its myotoxic phospholipases A 2 and hemorrhagic metalloproteinases.
The ability of the chelating agent CaNa2EDTA to inhibit local tissue damage induced by Bothrops a... more The ability of the chelating agent CaNa2EDTA to inhibit local tissue damage induced by Bothrops asper venom was studied in mice and in horses used for polyvalent (Crotalinae) antivenom production. CaNa2EDTA was devoid of toxicity when injected i.m. or s.c. inducing only a mild edema. Preincubation of B. asper venom with CaNa2EDTA inhibited hemorrhagic and dermonecrotic activities, but did not reduce edema-forming and myotoxic effects. A group of horses initially immunized with native venoms developed less severe local tissue reactions when injected with booster doses of venom and CaNa2EDTA than when receiving booster injections of venom alone, although they showed a similar antivenom response. Moreover, antivenoms produced from plasmas of horses that received booster injections of either venom alone or venom plus CaNa2EDTA had similar neutralizing activity against lethal, hemorrhagic and coagulant effects induced by B. asper venom. The similar antibody response was corroborated by Western blotting using crude venom and by an ELISA that estimates anti-myotoxin titer. It is concluded that the chelating agent CaNa2EDTA reduces the extent of local tissue damage induced by B. asper venom, without affecting the immune response of horses against pharmacologically-relevant venom components.
The ability of sheep antivenoms, consisting of whole IgG molecules or Fab fragments, to neutraliz... more 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 eects in assays where venom and antivenoms were incubated prior to injection. Thus, if dierences are observed in experiments involving independent injection of venom and antivenoms, they would depend on the pharmacokinetic pro®les of the products. Despite the observation that both antivenoms neutralized the three eects if preincubated with venom, neutralization was only partial when antivenoms were administered i.v. at various time intervals after envenomation. No signi®cant dierences were observed between IgG and Fab antivenoms concerning neutralization of hemorrhagic and edema-forming activities, whereas IgG antivenom was slightly more eective 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 eective than whole IgG molecules in the neutralization of locally-acting toxins from B. asper venom. #
Transactions of The Royal Society of Tropical Medicine and Hygiene, 2005
A polyspecific Pan-African antivenom has been produced from the plasma of horses immunized with a... more A polyspecific Pan-African antivenom has been produced from the plasma of horses immunized with a mixture of the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the three most medically important snakes in sub-Saharan Africa. The antivenom is a whole IgG preparation, obtained by caprylic acid precipitation of non-IgG plasma proteins. The antivenom effectively neutralizes the most important toxic activities of the three venoms used in the immunization in standard assays involving preincubation of venom and antivenom before testing. This antivenom compares favourably with other antivenoms designed for use in Africa with respect to neutralization of the toxins present in the venom of E. ocellatus. Caprylic acid fractionation of horse hyperimmune plasma is a simple, convenient and cheap protocol for the manufacture of high quality whole IgG antivenoms. It constitutes a potentially valuable technology for the alleviation of the critical shortage of antivenom in Africa.
Early adverse reactions occur in a number of patients treated with heterologous antivenoms and ha... more Early adverse reactions occur in a number of patients treated with heterologous antivenoms and have been associated with anticomplementary activity (ACA). In order to reduce the ACA of equine whole IgG antivenoms produced by caprylic acid fractionation, three different fractionation protocols were compared: (a) routine caprylic acid fractionation; (b) caprylic acid fractionation followed by b-propiolactone treatment; and (c) caprylic acid fractionation followed by ion-exchange chromatography using a quaternary ammonium membrane. The three protocols yielded products with similar physicochemical characteristics and anti-Bothrops asper venom antibody titers, except that ion-exchange purified antivenom had a lower protein concentration. Antivenoms fractionated by using b-propiolactone or filtration through quaternary ammonium membrane had a significantly reduced in vitro ACA. A preparation of caprylic acid-fractionated antivenom was heated in order to induce the formation of protein aggregates; however, its ACA was similar to non-heated antivenom. None of the antivenoms affected the hemolytic activity of serum complement in rabbits after a bolus intravenous administration. It is concluded that (a) b-propiolactone and quaternary ammonium membranes significantly reduce in vitro ACA of caprylic acid-fractionated equine antivenom, and (b) the validity of in vitro ACA as a predictor of EAR needs to be reexamined in clinical and experimental studies, since it may not adequately predict in vivo complement activation by antivenoms. q
Snakebite envenoming represents a neglected tropical disease that has a heavy public health impac... more Snakebite envenoming represents a neglected tropical disease that has a heavy public health impact, particularly in Asia, Africa and Latin America. A global initiative, aimed at increasing antivenom production and accessibility, is being promoted by the World Health Organization and others. This work discusses several aspects of antivenom manufacture and control in which the proteomic analysis of snake venoms, for which the term 'snake venomics' has been coined, might play a relevant supporting role. Snake venomics has already shown its usefulness for generating knowledge at different levels (ontogenetic, individual, and geographic) on inter-and intraspecies venom variability. This information has applications for the quality control of venom preparations used in antivenom manufacture. Moreover, the design of the best venom mixtures for immunization, aimed at increasing the effectiveness of antivenoms, may also be guided by venom proteome analysis, including molecular studies of the cross-reactivity of antivenoms and heterologous venoms through a recently developed methodological approach termed 'antivenomics'. Results generated by proteomic protocols should be complemented by preclinical testing of antivenom efficacy using functional neutralization assays. Snake venomics might be also helpful in designing alternative in vitro tests for the assessment of antivenom efficacy that would eventually substitute current in vivo tests.
Intravenous administration of antivenoms is associated with early adverse reactions in a number o... more Intravenous administration of antivenoms is associated with early adverse reactions in a number of cases, but the causes of this phenomenon are still unclear. The effect of preservatives (phenol and thimerosal) on IgG aggregate and dimer formation, in vitro complement-activating effect and hypotensive activity of a whole IgG horse liquid polyvalent antivenom, produced by caprylic acid fractionation, was assessed. These parameters were studied since they have been associated with the development of early adverse reactions to the administration of antivenoms and human immunoglobulins. After a three-year storage period at 4°C, antivenoms with preservatives had an increased content of IgG aggregates and dimers when compared with antivenom devoid of phenol and thimerosal. These observations correlate with a slight increment in the turbidity of preservative-containing antivenoms. The three antivenoms studied (formulation: no preservatives; with phenol and thimerosal; with thimerosal alone) activated human complement in vitro, with only minor quantitative differences among them. When antivenoms were administered as a bolus intravenous injection in rats, a rapid and prominent hypotension of short duration was observed after injection of phenol-containing antivenom, whereas such an effect was absent in antivenom free of preservative and in the one containing only thimerosal. Bolus injection of saline solution with phenol resulted in a similar hypotension, indicating that the effect is due to phenol. However, when phenol-containing antivenom was diluted 1:5 with saline solution before infusion, as occurs in the clinical use of this product, no hypotension was observed. Our results stress the need to evaluate the effects of preservatives on the physicochemical and pharmacological characteristics of antivenoms.
Whole IgG and F(ab')(2) equine-derived polyvalent (Crotalinae) antivenoms... more Whole IgG and F(ab')(2) equine-derived polyvalent (Crotalinae) antivenoms, prepared from the same batch of hyperimmune plasma, were compared in terms of neutralization of the lethal and defibrinating activities induced by Bothrops asper venom, their ability to reach the muscle tissue compartment in envenomated mice, and their potential for the induction of adverse reactions. Both preparations were adjusted to the same potency against the lethal effect of B. asper venom in experiments involving preincubation of venom and antivenom. Then, "rescue" experiments were performed, i.e. antivenom was administered either intravenously or intramuscularly at various times after envenomation. IgG and F(ab')(2) antivenoms were equally effective in the neutralization of lethality, both being more effective when administered i.v. than after i.m. injection. Neutralization decreased as the time lapse between envenomation and treatment increased. No significant differences were observed in the ability of antivenoms to neutralize defibrinating activity of B. asper venom in experiments involving independent injection of venom and antivenoms. There was a much higher accumulation of equine antibodies in muscle tissue that had been injected with B. asper venom than in non-envenomated tissue, indicating that venom-induced microvessel damage probably favors a prominent and similar extravasation of both IgG and F(ab')(2) antibodies. This may explain the similar effectiveness of both types of antivenom in previously reported studies on the neutralization of venom-induced local tissue damage. Both IgG and F(ab')(2) antivenoms activate human complement in vitro and induce an anti-equine immunoglobulin response in mice, indicating that Fc removal per se does not eliminate the potential for inducing adverse reactions. However, IgG antivenom had higher anticomplementary activity and induced a stronger anti-immunoglobulin response than F(ab')(2) antivenom.
, which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on eryt... more , which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on erythrocytes (RBC) from different species despite the fact that enzymatically active CGMT-I was able to hydrolyze RBC membrane phospholipids and disrupt liposomes prepared from RBC lipids. Human RBC did not become susceptible to the toxins after treatment with neuraminidase or after altering membrane fluidity with cholesterol or sublytic concentrations of detergent. Unlike normal RBC, significant hemolysis was induced by CGMT-II and another similar Lys-49 isoform, B. asper MT-II (BAMT-II), in RBC enriched with phosphatidylserine (PS). Hemolysis was greater in RBC preincubated with pyridyldithioethylamine (PDA), a potent inhibitor of aminophospholipid transport. RBC enriched with phosphatidic acid (PA) also became susceptible to the myotoxins but was unaffected by PDA. Cells enriched with phosphatidylcholine (PC) remained resistant to the action of the toxins. BAMT-II also induced damage in black lipid membranes prepared with PS but not PC alone. When RBC binding of BAMT-II was measured by enzyme-linked immunosorbent assay, it was observed that PS-and PA-enriched erythrocytes were always able to capture more toxin than normal and PC-enriched RBC. This effect was significantly improved by PDA (in the case of PS) and it was observed either in the presence or in the absence of calcium in the medium. These data suggest that negatively charged lipids in the outer leaflet of cell membranes constitute myotoxic PLA2 binding sites. The scarcity of anionic phospholipids in the outer leaflet of RBC could explain their resistance to the action of these PLA2s.
Caprylic acid purification of IgG, currently used in the manufacture of horse-derived antivenoms,... more Caprylic acid purification of IgG, currently used in the manufacture of horse-derived antivenoms, was successfully adapted for the preparation of sheep and camel IgG. Sheep IgG had a molecular mass of w150 kDa, whereas camel IgG presented two bands of molecular masses of w160 and 100 kDa, the latter corresponding to heavy-chain IgG, which is devoid of light chains. Horse, sheep and camel IgGs were compared by several parameters aiming at predicting their potential for induction of early and late adverse reactions. Horse and sheep IgGs showed a higher anticomplementary activity than camel IgG, and also elicited a higher anti-IgG response than camel IgG, when injected in mice. Horse IgG agglutinated human type OC erythrocytes, whereas no such reactivity was observed in sheep and camel IgG preparations. A novel procedure was used for the detection of antibodies in human serum against animal IgGs. It was found that a pool of human sera collected in Costa Rica had a higher titer of antibodies directed against horse and sheep IgGs than against camel IgG. Overall, camel IgG showed the lowest potential for the induction of adverse reactions among the three IgGs tested. q
Interest in studies on the neutralization of snake venoms and toxins by diverse types of inhibito... more Interest in studies on the neutralization of snake venoms and toxins by diverse types of inhibitors is two-fold. From an applied perspective, results enclose the potential to be translated into useful therapeutic products or procedures, to benefit patients suffering from envenomings. From a basic point of view, on the other hand, neutralizing agents may be used as powerful dissecting tools to determine the relative role of toxins within the context of the overall pathology induced by a venom, or to increase our understanding on the molecular mechanisms by which toxins exert their harmful actions upon particular targets. The venom of the snake Bothrops asper has been the subject of a number of experimental studies addressing its neutralization by antibodies, as well as by nonimmunologic inhibitors, including natural products derived from plants or animals, or synthetic drugs. As summarized in the present review, neutralization studies on this venom and some of its isolated toxins have contributed to a better understanding of envenomings by this species, and their treatment. In addition, such studies have provided valuable knowledge on the mechanisms of action and the relative functional importance of particular toxins of this venom, especially in the case of its myotoxic phospholipases A 2 and hemorrhagic metalloproteinases.
The ability of the chelating agent CaNa2EDTA to inhibit local tissue damage induced by Bothrops a... more The ability of the chelating agent CaNa2EDTA to inhibit local tissue damage induced by Bothrops asper venom was studied in mice and in horses used for polyvalent (Crotalinae) antivenom production. CaNa2EDTA was devoid of toxicity when injected i.m. or s.c. inducing only a mild edema. Preincubation of B. asper venom with CaNa2EDTA inhibited hemorrhagic and dermonecrotic activities, but did not reduce edema-forming and myotoxic effects. A group of horses initially immunized with native venoms developed less severe local tissue reactions when injected with booster doses of venom and CaNa2EDTA than when receiving booster injections of venom alone, although they showed a similar antivenom response. Moreover, antivenoms produced from plasmas of horses that received booster injections of either venom alone or venom plus CaNa2EDTA had similar neutralizing activity against lethal, hemorrhagic and coagulant effects induced by B. asper venom. The similar antibody response was corroborated by Western blotting using crude venom and by an ELISA that estimates anti-myotoxin titer. It is concluded that the chelating agent CaNa2EDTA reduces the extent of local tissue damage induced by B. asper venom, without affecting the immune response of horses against pharmacologically-relevant venom components.
The ability of sheep antivenoms, consisting of whole IgG molecules or Fab fragments, to neutraliz... more 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 eects in assays where venom and antivenoms were incubated prior to injection. Thus, if dierences are observed in experiments involving independent injection of venom and antivenoms, they would depend on the pharmacokinetic pro®les of the products. Despite the observation that both antivenoms neutralized the three eects if preincubated with venom, neutralization was only partial when antivenoms were administered i.v. at various time intervals after envenomation. No signi®cant dierences were observed between IgG and Fab antivenoms concerning neutralization of hemorrhagic and edema-forming activities, whereas IgG antivenom was slightly more eective 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 eective than whole IgG molecules in the neutralization of locally-acting toxins from B. asper venom. #
, which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on eryt... more , which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on erythrocytes (RBC) from different species despite the fact that enzymatically active CGMT-I was able to hydrolyze RBC membrane phospholipids and disrupt liposomes prepared from RBC lipids. Human RBC did not become susceptible to the toxins after treatment with neuraminidase or after altering membrane fluidity with cholesterol or sublytic concentrations of detergent. Unlike normal RBC, significant hemolysis was induced by CGMT-II and another similar Lys-49 isoform, B. asper MT-II (BAMT-II), in RBC enriched with phosphatidylserine (PS). Hemolysis was greater in RBC preincubated with pyridyldithioethylamine (PDA), a potent inhibitor of aminophospholipid transport. RBC enriched with phosphatidic acid (PA) also became susceptible to the myotoxins but was unaffected by PDA. Cells enriched with phosphatidylcholine (PC) remained resistant to the action of the toxins. BAMT-II also induced damage in black lipid membranes prepared with PS but not PC alone. When RBC binding of BAMT-II was measured by enzyme-linked immunosorbent assay, it was observed that PS-and PA-enriched erythrocytes were always able to capture more toxin than normal and PC-enriched RBC. This effect was significantly improved by PDA (in the case of PS) and it was observed either in the presence or in the absence of calcium in the medium. These data suggest that negatively charged lipids in the outer leaflet of cell membranes constitute myotoxic PLA2 binding sites. The scarcity of anionic phospholipids in the outer leaflet of RBC could explain their resistance to the action of these PLA2s.
Caprylic acid purification of IgG, currently used in the manufacture of horse-derived antivenoms,... more Caprylic acid purification of IgG, currently used in the manufacture of horse-derived antivenoms, was successfully adapted for the preparation of sheep and camel IgG. Sheep IgG had a molecular mass of w150 kDa, whereas camel IgG presented two bands of molecular masses of w160 and 100 kDa, the latter corresponding to heavy-chain IgG, which is devoid of light chains. Horse, sheep and camel IgGs were compared by several parameters aiming at predicting their potential for induction of early and late adverse reactions. Horse and sheep IgGs showed a higher anticomplementary activity than camel IgG, and also elicited a higher anti-IgG response than camel IgG, when injected in mice. Horse IgG agglutinated human type OC erythrocytes, whereas no such reactivity was observed in sheep and camel IgG preparations. A novel procedure was used for the detection of antibodies in human serum against animal IgGs. It was found that a pool of human sera collected in Costa Rica had a higher titer of antibodies directed against horse and sheep IgGs than against camel IgG. Overall, camel IgG showed the lowest potential for the induction of adverse reactions among the three IgGs tested. q
Interest in studies on the neutralization of snake venoms and toxins by diverse types of inhibito... more Interest in studies on the neutralization of snake venoms and toxins by diverse types of inhibitors is two-fold. From an applied perspective, results enclose the potential to be translated into useful therapeutic products or procedures, to benefit patients suffering from envenomings. From a basic point of view, on the other hand, neutralizing agents may be used as powerful dissecting tools to determine the relative role of toxins within the context of the overall pathology induced by a venom, or to increase our understanding on the molecular mechanisms by which toxins exert their harmful actions upon particular targets. The venom of the snake Bothrops asper has been the subject of a number of experimental studies addressing its neutralization by antibodies, as well as by nonimmunologic inhibitors, including natural products derived from plants or animals, or synthetic drugs. As summarized in the present review, neutralization studies on this venom and some of its isolated toxins have contributed to a better understanding of envenomings by this species, and their treatment. In addition, such studies have provided valuable knowledge on the mechanisms of action and the relative functional importance of particular toxins of this venom, especially in the case of its myotoxic phospholipases A 2 and hemorrhagic metalloproteinases.
The ability of the chelating agent CaNa2EDTA to inhibit local tissue damage induced by Bothrops a... more The ability of the chelating agent CaNa2EDTA to inhibit local tissue damage induced by Bothrops asper venom was studied in mice and in horses used for polyvalent (Crotalinae) antivenom production. CaNa2EDTA was devoid of toxicity when injected i.m. or s.c. inducing only a mild edema. Preincubation of B. asper venom with CaNa2EDTA inhibited hemorrhagic and dermonecrotic activities, but did not reduce edema-forming and myotoxic effects. A group of horses initially immunized with native venoms developed less severe local tissue reactions when injected with booster doses of venom and CaNa2EDTA than when receiving booster injections of venom alone, although they showed a similar antivenom response. Moreover, antivenoms produced from plasmas of horses that received booster injections of either venom alone or venom plus CaNa2EDTA had similar neutralizing activity against lethal, hemorrhagic and coagulant effects induced by B. asper venom. The similar antibody response was corroborated by Western blotting using crude venom and by an ELISA that estimates anti-myotoxin titer. It is concluded that the chelating agent CaNa2EDTA reduces the extent of local tissue damage induced by B. asper venom, without affecting the immune response of horses against pharmacologically-relevant venom components.
The ability of sheep antivenoms, consisting of whole IgG molecules or Fab fragments, to neutraliz... more 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 eects in assays where venom and antivenoms were incubated prior to injection. Thus, if dierences are observed in experiments involving independent injection of venom and antivenoms, they would depend on the pharmacokinetic pro®les of the products. Despite the observation that both antivenoms neutralized the three eects if preincubated with venom, neutralization was only partial when antivenoms were administered i.v. at various time intervals after envenomation. No signi®cant dierences were observed between IgG and Fab antivenoms concerning neutralization of hemorrhagic and edema-forming activities, whereas IgG antivenom was slightly more eective 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 eective than whole IgG molecules in the neutralization of locally-acting toxins from B. asper venom. #
Transactions of The Royal Society of Tropical Medicine and Hygiene, 2005
A polyspecific Pan-African antivenom has been produced from the plasma of horses immunized with a... more A polyspecific Pan-African antivenom has been produced from the plasma of horses immunized with a mixture of the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the three most medically important snakes in sub-Saharan Africa. The antivenom is a whole IgG preparation, obtained by caprylic acid precipitation of non-IgG plasma proteins. The antivenom effectively neutralizes the most important toxic activities of the three venoms used in the immunization in standard assays involving preincubation of venom and antivenom before testing. This antivenom compares favourably with other antivenoms designed for use in Africa with respect to neutralization of the toxins present in the venom of E. ocellatus. Caprylic acid fractionation of horse hyperimmune plasma is a simple, convenient and cheap protocol for the manufacture of high quality whole IgG antivenoms. It constitutes a potentially valuable technology for the alleviation of the critical shortage of antivenom in Africa.
Early adverse reactions occur in a number of patients treated with heterologous antivenoms and ha... more Early adverse reactions occur in a number of patients treated with heterologous antivenoms and have been associated with anticomplementary activity (ACA). In order to reduce the ACA of equine whole IgG antivenoms produced by caprylic acid fractionation, three different fractionation protocols were compared: (a) routine caprylic acid fractionation; (b) caprylic acid fractionation followed by b-propiolactone treatment; and (c) caprylic acid fractionation followed by ion-exchange chromatography using a quaternary ammonium membrane. The three protocols yielded products with similar physicochemical characteristics and anti-Bothrops asper venom antibody titers, except that ion-exchange purified antivenom had a lower protein concentration. Antivenoms fractionated by using b-propiolactone or filtration through quaternary ammonium membrane had a significantly reduced in vitro ACA. A preparation of caprylic acid-fractionated antivenom was heated in order to induce the formation of protein aggregates; however, its ACA was similar to non-heated antivenom. None of the antivenoms affected the hemolytic activity of serum complement in rabbits after a bolus intravenous administration. It is concluded that (a) b-propiolactone and quaternary ammonium membranes significantly reduce in vitro ACA of caprylic acid-fractionated equine antivenom, and (b) the validity of in vitro ACA as a predictor of EAR needs to be reexamined in clinical and experimental studies, since it may not adequately predict in vivo complement activation by antivenoms. q
Snakebite envenoming represents a neglected tropical disease that has a heavy public health impac... more Snakebite envenoming represents a neglected tropical disease that has a heavy public health impact, particularly in Asia, Africa and Latin America. A global initiative, aimed at increasing antivenom production and accessibility, is being promoted by the World Health Organization and others. This work discusses several aspects of antivenom manufacture and control in which the proteomic analysis of snake venoms, for which the term 'snake venomics' has been coined, might play a relevant supporting role. Snake venomics has already shown its usefulness for generating knowledge at different levels (ontogenetic, individual, and geographic) on inter-and intraspecies venom variability. This information has applications for the quality control of venom preparations used in antivenom manufacture. Moreover, the design of the best venom mixtures for immunization, aimed at increasing the effectiveness of antivenoms, may also be guided by venom proteome analysis, including molecular studies of the cross-reactivity of antivenoms and heterologous venoms through a recently developed methodological approach termed 'antivenomics'. Results generated by proteomic protocols should be complemented by preclinical testing of antivenom efficacy using functional neutralization assays. Snake venomics might be also helpful in designing alternative in vitro tests for the assessment of antivenom efficacy that would eventually substitute current in vivo tests.
Intravenous administration of antivenoms is associated with early adverse reactions in a number o... more Intravenous administration of antivenoms is associated with early adverse reactions in a number of cases, but the causes of this phenomenon are still unclear. The effect of preservatives (phenol and thimerosal) on IgG aggregate and dimer formation, in vitro complement-activating effect and hypotensive activity of a whole IgG horse liquid polyvalent antivenom, produced by caprylic acid fractionation, was assessed. These parameters were studied since they have been associated with the development of early adverse reactions to the administration of antivenoms and human immunoglobulins. After a three-year storage period at 4°C, antivenoms with preservatives had an increased content of IgG aggregates and dimers when compared with antivenom devoid of phenol and thimerosal. These observations correlate with a slight increment in the turbidity of preservative-containing antivenoms. The three antivenoms studied (formulation: no preservatives; with phenol and thimerosal; with thimerosal alone) activated human complement in vitro, with only minor quantitative differences among them. When antivenoms were administered as a bolus intravenous injection in rats, a rapid and prominent hypotension of short duration was observed after injection of phenol-containing antivenom, whereas such an effect was absent in antivenom free of preservative and in the one containing only thimerosal. Bolus injection of saline solution with phenol resulted in a similar hypotension, indicating that the effect is due to phenol. However, when phenol-containing antivenom was diluted 1:5 with saline solution before infusion, as occurs in the clinical use of this product, no hypotension was observed. Our results stress the need to evaluate the effects of preservatives on the physicochemical and pharmacological characteristics of antivenoms.
Whole IgG and F(ab')(2) equine-derived polyvalent (Crotalinae) antivenoms... more Whole IgG and F(ab')(2) equine-derived polyvalent (Crotalinae) antivenoms, prepared from the same batch of hyperimmune plasma, were compared in terms of neutralization of the lethal and defibrinating activities induced by Bothrops asper venom, their ability to reach the muscle tissue compartment in envenomated mice, and their potential for the induction of adverse reactions. Both preparations were adjusted to the same potency against the lethal effect of B. asper venom in experiments involving preincubation of venom and antivenom. Then, "rescue" experiments were performed, i.e. antivenom was administered either intravenously or intramuscularly at various times after envenomation. IgG and F(ab')(2) antivenoms were equally effective in the neutralization of lethality, both being more effective when administered i.v. than after i.m. injection. Neutralization decreased as the time lapse between envenomation and treatment increased. No significant differences were observed in the ability of antivenoms to neutralize defibrinating activity of B. asper venom in experiments involving independent injection of venom and antivenoms. There was a much higher accumulation of equine antibodies in muscle tissue that had been injected with B. asper venom than in non-envenomated tissue, indicating that venom-induced microvessel damage probably favors a prominent and similar extravasation of both IgG and F(ab')(2) antibodies. This may explain the similar effectiveness of both types of antivenom in previously reported studies on the neutralization of venom-induced local tissue damage. Both IgG and F(ab')(2) antivenoms activate human complement in vitro and induce an anti-equine immunoglobulin response in mice, indicating that Fc removal per se does not eliminate the potential for inducing adverse reactions. However, IgG antivenom had higher anticomplementary activity and induced a stronger anti-immunoglobulin response than F(ab')(2) antivenom.
, which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on eryt... more , which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on erythrocytes (RBC) from different species despite the fact that enzymatically active CGMT-I was able to hydrolyze RBC membrane phospholipids and disrupt liposomes prepared from RBC lipids. Human RBC did not become susceptible to the toxins after treatment with neuraminidase or after altering membrane fluidity with cholesterol or sublytic concentrations of detergent. Unlike normal RBC, significant hemolysis was induced by CGMT-II and another similar Lys-49 isoform, B. asper MT-II (BAMT-II), in RBC enriched with phosphatidylserine (PS). Hemolysis was greater in RBC preincubated with pyridyldithioethylamine (PDA), a potent inhibitor of aminophospholipid transport. RBC enriched with phosphatidic acid (PA) also became susceptible to the myotoxins but was unaffected by PDA. Cells enriched with phosphatidylcholine (PC) remained resistant to the action of the toxins. BAMT-II also induced damage in black lipid membranes prepared with PS but not PC alone. When RBC binding of BAMT-II was measured by enzyme-linked immunosorbent assay, it was observed that PS-and PA-enriched erythrocytes were always able to capture more toxin than normal and PC-enriched RBC. This effect was significantly improved by PDA (in the case of PS) and it was observed either in the presence or in the absence of calcium in the medium. These data suggest that negatively charged lipids in the outer leaflet of cell membranes constitute myotoxic PLA2 binding sites. The scarcity of anionic phospholipids in the outer leaflet of RBC could explain their resistance to the action of these PLA2s.
Caprylic acid purification of IgG, currently used in the manufacture of horse-derived antivenoms,... more Caprylic acid purification of IgG, currently used in the manufacture of horse-derived antivenoms, was successfully adapted for the preparation of sheep and camel IgG. Sheep IgG had a molecular mass of w150 kDa, whereas camel IgG presented two bands of molecular masses of w160 and 100 kDa, the latter corresponding to heavy-chain IgG, which is devoid of light chains. Horse, sheep and camel IgGs were compared by several parameters aiming at predicting their potential for induction of early and late adverse reactions. Horse and sheep IgGs showed a higher anticomplementary activity than camel IgG, and also elicited a higher anti-IgG response than camel IgG, when injected in mice. Horse IgG agglutinated human type OC erythrocytes, whereas no such reactivity was observed in sheep and camel IgG preparations. A novel procedure was used for the detection of antibodies in human serum against animal IgGs. It was found that a pool of human sera collected in Costa Rica had a higher titer of antibodies directed against horse and sheep IgGs than against camel IgG. Overall, camel IgG showed the lowest potential for the induction of adverse reactions among the three IgGs tested. q
Interest in studies on the neutralization of snake venoms and toxins by diverse types of inhibito... more Interest in studies on the neutralization of snake venoms and toxins by diverse types of inhibitors is two-fold. From an applied perspective, results enclose the potential to be translated into useful therapeutic products or procedures, to benefit patients suffering from envenomings. From a basic point of view, on the other hand, neutralizing agents may be used as powerful dissecting tools to determine the relative role of toxins within the context of the overall pathology induced by a venom, or to increase our understanding on the molecular mechanisms by which toxins exert their harmful actions upon particular targets. The venom of the snake Bothrops asper has been the subject of a number of experimental studies addressing its neutralization by antibodies, as well as by nonimmunologic inhibitors, including natural products derived from plants or animals, or synthetic drugs. As summarized in the present review, neutralization studies on this venom and some of its isolated toxins have contributed to a better understanding of envenomings by this species, and their treatment. In addition, such studies have provided valuable knowledge on the mechanisms of action and the relative functional importance of particular toxins of this venom, especially in the case of its myotoxic phospholipases A 2 and hemorrhagic metalloproteinases.
The ability of the chelating agent CaNa2EDTA to inhibit local tissue damage induced by Bothrops a... more The ability of the chelating agent CaNa2EDTA to inhibit local tissue damage induced by Bothrops asper venom was studied in mice and in horses used for polyvalent (Crotalinae) antivenom production. CaNa2EDTA was devoid of toxicity when injected i.m. or s.c. inducing only a mild edema. Preincubation of B. asper venom with CaNa2EDTA inhibited hemorrhagic and dermonecrotic activities, but did not reduce edema-forming and myotoxic effects. A group of horses initially immunized with native venoms developed less severe local tissue reactions when injected with booster doses of venom and CaNa2EDTA than when receiving booster injections of venom alone, although they showed a similar antivenom response. Moreover, antivenoms produced from plasmas of horses that received booster injections of either venom alone or venom plus CaNa2EDTA had similar neutralizing activity against lethal, hemorrhagic and coagulant effects induced by B. asper venom. The similar antibody response was corroborated by Western blotting using crude venom and by an ELISA that estimates anti-myotoxin titer. It is concluded that the chelating agent CaNa2EDTA reduces the extent of local tissue damage induced by B. asper venom, without affecting the immune response of horses against pharmacologically-relevant venom components.
The ability of sheep antivenoms, consisting of whole IgG molecules or Fab fragments, to neutraliz... more 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 eects in assays where venom and antivenoms were incubated prior to injection. Thus, if dierences are observed in experiments involving independent injection of venom and antivenoms, they would depend on the pharmacokinetic pro®les of the products. Despite the observation that both antivenoms neutralized the three eects if preincubated with venom, neutralization was only partial when antivenoms were administered i.v. at various time intervals after envenomation. No signi®cant dierences were observed between IgG and Fab antivenoms concerning neutralization of hemorrhagic and edema-forming activities, whereas IgG antivenom was slightly more eective 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 eective than whole IgG molecules in the neutralization of locally-acting toxins from B. asper venom. #
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Papers by Guillermo León