Gustavo Rojas - Academia.edu (original) (raw)

Papers by Gustavo Rojas

Research paper thumbnail of COMPARATIVE STUDY ON THE ABILITY OF IgG AND F(ab 9)2 ANTIVENOMS TO NEUTRALIZE LETHAL AND MYOTOXIC EFFECTS INDUCED BY MICRURUS NIGROCINCTUS(CORAL SNAKE) VENOM

A comparative study was performed on the ability of IgG and F(abЈ) 2 antivenoms to neutralize let... more A comparative study was performed on the ability of IgG and F(abЈ) 2 antivenoms to neutralize lethal and myotoxic activities of Micrurus nigrocinctus venom. Both antivenoms were adjusted to a similar neutralizing potency in experiments where venom and antivenoms were preincubated prior to injection. No significant differences were observed between IgG and F(abЈ) 2 antivenoms concerning neutralization of lethal effect in rescue experiments, i.e., when antivenom was administered intravenously after envenomation. However, F(abЈ) 2 antivenom was more effective in prolonging the time of death when subneutralizing doses were administered immediately after venom injection. Both products partially reversed the binding of M. nigrocinctus ␣-neurotoxins to acetylcholine receptor in vitro. The IgG and F(abЈ) 2 antivenoms effectively neutralized venom-induced myotoxicity when administered intravenously immediately after envenomation, although neutralization was poor if antivenom injections were delayed. Intramuscular injection of venom promoted diffusion of antivenom antibodies throughout muscle tissue, and F(abЈ) 2 diffused to a higher extent than IgG molecules. Thus, despite the observation that F(abЈ) 2 antivenom was more effective than IgG antivenom in prolonging the time of death when subneutralizing doses were administered immediately after envenomation, no major differences were observed in antivenom neutralization of lethal and myotoxic effects or in their capacity to reverse neurotoxin binding to the acetylcholine receptor.

Research paper thumbnail of Immunoglobulin G and F(ab′) 2 polyvalent antivenoms do not differ in their ability to neutralize hemorrhage, edema and myonecrosis induced by Bothrops asper (terciopelo) snake venom

Research paper thumbnail of Preclinical assessment of the ability of polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced in Costa Rica to neutralize the venoms of North American snakes

Toxicon, 2003

Polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced by Instituto Clodomiro Picad... more Polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced by Instituto Clodomiro Picado, Costa Rica, were assessed for their ability to neutralize various toxic activities of the venoms of North American snakes of the genera Crotalus, Agkistrodon and Micrurus, in assays involving preincubation of venom and antivenom. When the intraperitoneal route of injection was utilized, polyvalent (Crotalinae) antivenom was effective in the neutralization of the venoms of Crotalus atrox, Crotalus adamanteus, Crotalus viridis viridis, Crotalus horridus atricaudatus, Agkistrodon contortrix contortrix and Agkistrodon piscivorus piscivorus, whereas the venom of Crotalus scutulatus was not neutralized. When the intravenous route was used, results differed depending on the 'challenge dose' of venom employed. Polyvalent antivenom neutralized all venoms when mice were challenged with 2 LD 50 s of venom. When 5 LD 50 s were used, antivenom neutralized the venoms of C. atrox, C. adamanteus, C. v. viridis and C. h. atricaudatus, being ineffective in the neutralization of C. scutulatus, A. c. contortrix and A. p. piscivorus. Polyvalent antivenom was effective in the neutralization of hemorrhagic and myotoxic activities of all venoms studied. It also neutralized coagulant activity of C. adamanteus venom, whereas most of the venoms were devoid of clotting activity on plasma in vitro. Moreover, it neutralized defibrinating activity of the only three venoms that induced this effect (i.e. C. adamanteus, A. c. contortrix and A. p. piscivorus). Anticoral (Elapidae) antivenom neutralized lethality induced by the venom of Micrurus fulvius, using either the intravenous or the intraperitoneal routes of injection. Moreover, it neutralized myotoxic effect of this venom as well. It is concluded that polyvalent antivenom neutralizes lethality and other activities of most of the crotaline venoms tested. However, since it is ineffective in neutralizing the lethal effect of C. scutulatus venom, it is suggested that a venom containing presynaptically-active neurotoxic phospholipases A 2 related to 'mojave toxin' needs to be introduced in the immunizing mixture in order to increase the neutralizing scope of this product in North America. Anticoral antivenom is highly effective in the neutralization of the venom of M. fulvius. q

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

Toxicon, 2000

A study was performed on the ability of antivenoms, produced in Brazil and Costa Rica, to neutral... more 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 dierent 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 eective doses 50% (ED 50 ) diered 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 eectively 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 eective in the neutralization of this activity. In conclusion, our results provide evidence of an extensive

Research paper thumbnail of Ability of a polyvalent antivenom to neutralize the venom of Lachesis Muta Melanocephala, a new Costa Rican subspecies of the bushmaster

Toxicon, 1987

Acceptedfor publication 2 February 1987) J. M. GuTtÉRRP-z, G. RojAs and L. CP .atDAs . Ability of... more Acceptedfor publication 2 February 1987) J. M. GuTtÉRRP-z, G. RojAs and L. CP .atDAs . Ability of a polyvalent antivenom to neutralize the venom of Lachesis muta melanocephala, a new Costa Rican subspecies of the bushmaster . Toidcon 25, 713 -720, 1987 . -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. metanocephala venom.

Research paper thumbnail of Experimental myonecrosis induced by the venoms of South American Micrurus (coral snakes

Research paper thumbnail of Neutralization, by a monospecific Bothrops lanceolatus antivenom, of toxic activities induced by homologous and heterologous Bothı́rops snake venoms

Toxicon, 1999

A monospeci®c Bothrops lanceolatus antivenom, currently used in Martinique, was tested for its ec... more A monospeci®c Bothrops lanceolatus antivenom, currently used in Martinique, was tested for its ecacy 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 LD 50 of 12.8 mg/g. In addition, it induced local tissue damage (hemorrhage, edema and myotoxicity) and showed indirect hemolytic activity, but was devoid of coagulant eect on human plasma in vitro and of de®brinating activity in mice. Antivenom was fully eective in the neutralization of lethal, hemorrhagic, edema-forming, myotoxic and indirect hemolytic eects 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 eects, and was partially eective in neutralizing edema-forming activity. In contrast, the antivenom was ineective in the neutralization of in vitro coagulant and in vivo de®brinating eects induced by these two venoms. #

Research paper thumbnail of Standardization of assays for testing the neutralizing ability of antivenoms

Toxicon, 1990

1. Toxicon. 1990;28(10):1127-9; author reply 1129-32. Standardization of assays for testing the n... more 1. Toxicon. 1990;28(10):1127-9; author reply 1129-32. Standardization of assays for testing the neutralizing ability of antivenoms. Gutiérrez JM, Rojas G, Lomonte B, Gené JA, Chaves F, Alvarado J, Rojas E. PMID: 2264065 [PubMed - indexed for MEDLINE]. Publication Types: ...

Research paper thumbnail of Pan-African polyspecific antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa

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.

Research paper thumbnail of Effect of storage temperature on the stability of the liquid polyvalent antivenom produced in Costa Rica

Toxicon, 1990

The effect of storage temperature on the stability of the liquid polyvalent (crotaline) antivenom... more The effect of storage temperature on the stability of the liquid polyvalent (crotaline) antivenom produced at the Instituto Clodomiro Picado, Costa Rica, was studied during a twelve-month period. The following parameters were evaluated: neutralizing potency against lethal activity of Bothrops asper venom; protein and phenol concentrations; pH; turbidity; safety; and sterility. Analyses were performed each month on different samples of a batch, stored at 4, 23, 30 and 37 degrees C. No significant (P greater than 0.1) variations occurred in potency, protein and phenol concentrations, pH, sterility or safety, at any of the storage temperatures during the study period. However, visual inspection revealed a moderate increase in turbidity of the samples stored at 23, 30 and 37 degrees C, at nine, four and three months, respectively. Culture of samples excluded the possibility of microbial contamination of the product leading to turbidity. Chromatographic and electrophoretic analyses demonstrated that turbidity was caused by the formation of heterogeneous protein aggregates of high molecular weight. Present results support the conclusion that, although storage temperature (up to 37 degrees C for twelve months) does not alter antivenom potency, it significantly influences the formation of protein aggregates. This phenomenon can be prevented by recommending the storage of antivenom at refrigeration temperature.

Research paper thumbnail of Caprylic acid fractionation of hyperimmune horse plasma: Description of a simple procedure for antivenom production

Toxicon, 1994

G. ROJAS, J. M. JIM~NEZ and J. M. GUTII~RREZ. Caprylic acid fractionation of hyperimmune horse pl... more G. ROJAS, J. M. JIM~NEZ and J. M. GUTII~RREZ. Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon 32, 351-363, 1994.--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 NaC1 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.

Research paper thumbnail of Turbidity of hyperimmune equine antivenom: The role of phenol and serum lipoproteins

Toxicon, 1993

of hyperimmune equine antivenom: the role of phenol and serum lipoproteins . Toxicon 31, 61-66, 1... more of hyperimmune equine antivenom: the role of phenol and serum lipoproteins . Toxicon 31, 61-66, 1993.-Twenty batches of polyvalent antivenom produced at the Instituto Clodomiro Picado were analyzed for turbidity, both before and after freezing-thawing and lyophilization . Eight batches became turbid upon freezing-thawing, and this change correlated with high levels of cholesterol, triglycerides and lipoproteins, especially ß-lipoprotein. Since normal horse serum does not become turbid after freezing-thawing, despite the fact that it has high lipoprotein levels, the possibility was raised that phenol, used as a preservative during serum fractionation, might affect lipoproteins, inducing the appearance of turbidity after freezing-thawing . This hypothesis was tested by fractionating a sample of hyperimmune serum either without phenol or using two different phenol concentrations (0.1 g/dl and 0.25 g/dl). Results showed that, although the three samples had the same cholesterol and triglyceride levels before fractionation, only the one having 0.25 g/dl phenol became turbid upon freezing-thawing, containing a diffuse lipoprotein band on electrophoresis. This finding suggests that turbidity in equine antivenoms depends on the interaction of at least three factors: (a) freezing, (b) high initial cholesterol and lipoprotein concentration in the serum, and (c) addition of phenol during fractionation of serum.

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

Toxicon, 1989

The coagulant, defibrinating, fibrino lytic and fibrinogenolytic activities of venoms from ten sp... more 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.

Research paper thumbnail of Neutralization of four Peruvian Bothrops sp. snake venoms by polyvalent antivenoms produced in Perú and Costa Rica: preclinical assessment

Acta Tropica, 2005

Envenomations after bites inflicted by snakes of the genus Bothrops constitute a public health ha... more Envenomations after bites inflicted by snakes of the genus Bothrops constitute a public health hazard in Perú, and the intravenous administration of equine-derived antivenoms represents the only scientifically validated treatment. This study presents a preclinical assessment of the efficacy of two whole IgG antivenoms, prepared in Perú and Costa Rica, to neutralize the most relevant toxic effects induced by the venoms of Bothrops atrox, B. brazili, B. barnetti and B. pictus from Perú. Peruvian antivenom is produced by immunizing horses with Bothrops sp. venoms from this country, whereas the production of Costa Rican antivenom involves immunization with venoms from Central American snakes. The neutralization of lethal, hemorrhagic, edema-forming, myotoxic, coagulant and defibrinating activities was evaluated in assays involving incubation of venom and antivenom prior to testing. Both antivenoms were effective in the neutralization of these effects, with quantitative variations in the values of effective dose 50% depending on the effects being studied. Peruvian antivenom was more effective in the neutralization of lethality induced by B. atrox and B. barnetti venoms. However, Peruvian antivenom failed to neutralize coagulant activity of B. barnetti venom and edema-forming activity of B. brazili venom, whereas neutralization was achieved by Costa Rican antivenom. It is concluded that an extensive immunological cross-reactivity exists between Bothrops sp. venoms from Perú and Costa Rica, and that both antivenoms are effective in the neutralization of these four venoms in a rodent model of envenoming.

Research paper thumbnail of Effect of Preservatives on IgG Aggregation, Complement-activating Effect and Hypotensive Activity of Horse Polyvalent Antivenom Used in Snakebite Envenomation

Biologicals, 2002

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.

Research paper thumbnail of NEUTRALIZATION OF LOCAL TISSUE DAMAGE INDUCED BY BOTHROPS ASPER (TERCIOPELO) SNAKE VENOM

Research paper thumbnail of 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

Toxicon, 2000

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. #

Research paper thumbnail of Quantitation by enzyme-immunoassay of antibodies against bothrops myotoxins in four commercially-available antivenoms

Toxicon, 1991

B. LOMONTE, J. M. Gu~REZ, G. Ralws and L. Cw1.D>at6N. Quantitation by enzyme-immunoassay of antib... more B. LOMONTE, J. M. Gu~REZ, G. Ralws and L. Cw1.D>at6N. Quantitation by enzyme-immunoassay of antibodies against Bothrops myotoxins in four commercially-available antivenoms . Toxicon 29, 69702, 1991 .-An enzymeimmunoassay (EIA) for the quantitation of antibodies against myotoxins present in the venoms of Bothrops riper (Costa Rica), B. atrox (Colombia) and B. moojeni (Brazil), was developed. This EIA was utilized for the evaluation of four antivenoms produced in Mexico (Laboratorios Myn; MYI~, Costa Rica (Instituto Clodomiro Picador ICP), Colombia (Instituto National de Salud; INS) and Brazil (Instituto Butantan ; IB). Antivenoms ICP, IB and INS

Research paper thumbnail of Isolation of a galactose-binding lectin from the venom of the snake Bothrops godmani (Godmann's pit viper

Toxicon, 1990

Isolation of a galactose-binding lectin from the venom of the snake Bothrops godrnani (Godmann's ... more Isolation of a galactose-binding lectin from the venom of the snake Bothrops godrnani (Godmann's pit viper) . Toxicon 28, 75-81, 1990.-A galactose-binding lectin, isolated from the venom of B. gotlmani by affinity chromatography . is an acidic protein (pI 4.9) with a subunit mol. wt of about 14,000, occurring mostly as a disulfide-linked dimer of 28,000 . A small proportion of lectin appears as a monomer and as a tetramer . The lectin agglutinates erythrocytes from mice, rabbit, cow and human (all ABO types, either Rh positive or negative), but does not agglutinate horse, sheep, goat and snake (Oxybelis aeneus, Colubridae) erythrocytes . The agglutinating activity is inhibited by 1 mM EDTA . The lectin is devoid of lethal, hemorrhagic, myotoxic, proteolytic and phospholipase A2 activities . It is not mitogenic for human peripheral blood mononuclear cells. The only effect observed was a moderate induction of edema in the footpad of mice, with a minimal edema-forming dose of 22 kg . This effect developed rapidly, and was significantly inhibited by i.p . administration of cyproheptadine, a histamine and serotonin antagonist, before injection of the lectin . Despite the edema-forming activity observed, the low concentration of lectin in crude venom, together with its relatively low potency, suggest that this lectin is not a key component in the development of edema following envenomations by B. godrnani.

Research paper thumbnail of Effects of a myotoxic phospholipase A2 isolated from Bothrops asper venom on skeletal muscle sarcoplasmic reticulum

Toxicon, 1987

J . M . GU719RREZ, G. RojAs, B . LomoNTE, J . A . GENé and L . CERDAs . Effects of a myotoxic pho... more J . M . GU719RREZ, G. RojAs, B . LomoNTE, J . A . GENé and L . CERDAs . Effects of a myotoxic phospholipase A2 isolated from Bothrops asper venom on skeletal muscle sarcoplasmic reticulum. Taxicon 25, 1244Taxicon 25, -1248Taxicon 25, , 1987 . -The myotoxin from B. asper snake venom inhibited Ca-ATPase activity of rabbit sareoplasmie retieulum after incubation in vitro . Inhibition was non-competitive and albumin enhanced the effect of the toxin . Furthermore, B. riper myotoxin hydrolyzed sarcoplasmic reticulum phospholipids and induced a dose-dependent release of horseradish perolddase that had been trapped in sarcoplasmic reticulum vesicles . Binding studies indicated that myotoxin does not bind to any particular protein of this membrane, suggesting that the toxin might interact with phospholipids. Inhibition of Ca-ATPase is probably a consequence of an alteration in sarcoplasmic reticulum phospholipids .

Research paper thumbnail of COMPARATIVE STUDY ON THE ABILITY OF IgG AND F(ab 9)2 ANTIVENOMS TO NEUTRALIZE LETHAL AND MYOTOXIC EFFECTS INDUCED BY MICRURUS NIGROCINCTUS(CORAL SNAKE) VENOM

A comparative study was performed on the ability of IgG and F(abЈ) 2 antivenoms to neutralize let... more A comparative study was performed on the ability of IgG and F(abЈ) 2 antivenoms to neutralize lethal and myotoxic activities of Micrurus nigrocinctus venom. Both antivenoms were adjusted to a similar neutralizing potency in experiments where venom and antivenoms were preincubated prior to injection. No significant differences were observed between IgG and F(abЈ) 2 antivenoms concerning neutralization of lethal effect in rescue experiments, i.e., when antivenom was administered intravenously after envenomation. However, F(abЈ) 2 antivenom was more effective in prolonging the time of death when subneutralizing doses were administered immediately after venom injection. Both products partially reversed the binding of M. nigrocinctus ␣-neurotoxins to acetylcholine receptor in vitro. The IgG and F(abЈ) 2 antivenoms effectively neutralized venom-induced myotoxicity when administered intravenously immediately after envenomation, although neutralization was poor if antivenom injections were delayed. Intramuscular injection of venom promoted diffusion of antivenom antibodies throughout muscle tissue, and F(abЈ) 2 diffused to a higher extent than IgG molecules. Thus, despite the observation that F(abЈ) 2 antivenom was more effective than IgG antivenom in prolonging the time of death when subneutralizing doses were administered immediately after envenomation, no major differences were observed in antivenom neutralization of lethal and myotoxic effects or in their capacity to reverse neurotoxin binding to the acetylcholine receptor.

Research paper thumbnail of Immunoglobulin G and F(ab′) 2 polyvalent antivenoms do not differ in their ability to neutralize hemorrhage, edema and myonecrosis induced by Bothrops asper (terciopelo) snake venom

Research paper thumbnail of Preclinical assessment of the ability of polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced in Costa Rica to neutralize the venoms of North American snakes

Toxicon, 2003

Polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced by Instituto Clodomiro Picad... more Polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced by Instituto Clodomiro Picado, Costa Rica, were assessed for their ability to neutralize various toxic activities of the venoms of North American snakes of the genera Crotalus, Agkistrodon and Micrurus, in assays involving preincubation of venom and antivenom. When the intraperitoneal route of injection was utilized, polyvalent (Crotalinae) antivenom was effective in the neutralization of the venoms of Crotalus atrox, Crotalus adamanteus, Crotalus viridis viridis, Crotalus horridus atricaudatus, Agkistrodon contortrix contortrix and Agkistrodon piscivorus piscivorus, whereas the venom of Crotalus scutulatus was not neutralized. When the intravenous route was used, results differed depending on the 'challenge dose' of venom employed. Polyvalent antivenom neutralized all venoms when mice were challenged with 2 LD 50 s of venom. When 5 LD 50 s were used, antivenom neutralized the venoms of C. atrox, C. adamanteus, C. v. viridis and C. h. atricaudatus, being ineffective in the neutralization of C. scutulatus, A. c. contortrix and A. p. piscivorus. Polyvalent antivenom was effective in the neutralization of hemorrhagic and myotoxic activities of all venoms studied. It also neutralized coagulant activity of C. adamanteus venom, whereas most of the venoms were devoid of clotting activity on plasma in vitro. Moreover, it neutralized defibrinating activity of the only three venoms that induced this effect (i.e. C. adamanteus, A. c. contortrix and A. p. piscivorus). Anticoral (Elapidae) antivenom neutralized lethality induced by the venom of Micrurus fulvius, using either the intravenous or the intraperitoneal routes of injection. Moreover, it neutralized myotoxic effect of this venom as well. It is concluded that polyvalent antivenom neutralizes lethality and other activities of most of the crotaline venoms tested. However, since it is ineffective in neutralizing the lethal effect of C. scutulatus venom, it is suggested that a venom containing presynaptically-active neurotoxic phospholipases A 2 related to 'mojave toxin' needs to be introduced in the immunizing mixture in order to increase the neutralizing scope of this product in North America. Anticoral antivenom is highly effective in the neutralization of the venom of M. fulvius. q

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

Toxicon, 2000

A study was performed on the ability of antivenoms, produced in Brazil and Costa Rica, to neutral... more 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 dierent 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 eective doses 50% (ED 50 ) diered 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 eectively 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 eective in the neutralization of this activity. In conclusion, our results provide evidence of an extensive

Research paper thumbnail of Ability of a polyvalent antivenom to neutralize the venom of Lachesis Muta Melanocephala, a new Costa Rican subspecies of the bushmaster

Toxicon, 1987

Acceptedfor publication 2 February 1987) J. M. GuTtÉRRP-z, G. RojAs and L. CP .atDAs . Ability of... more Acceptedfor publication 2 February 1987) J. M. GuTtÉRRP-z, G. RojAs and L. CP .atDAs . Ability of a polyvalent antivenom to neutralize the venom of Lachesis muta melanocephala, a new Costa Rican subspecies of the bushmaster . Toidcon 25, 713 -720, 1987 . -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. metanocephala venom.

Research paper thumbnail of Experimental myonecrosis induced by the venoms of South American Micrurus (coral snakes

Research paper thumbnail of Neutralization, by a monospecific Bothrops lanceolatus antivenom, of toxic activities induced by homologous and heterologous Bothı́rops snake venoms

Toxicon, 1999

A monospeci®c Bothrops lanceolatus antivenom, currently used in Martinique, was tested for its ec... more A monospeci®c Bothrops lanceolatus antivenom, currently used in Martinique, was tested for its ecacy 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 LD 50 of 12.8 mg/g. In addition, it induced local tissue damage (hemorrhage, edema and myotoxicity) and showed indirect hemolytic activity, but was devoid of coagulant eect on human plasma in vitro and of de®brinating activity in mice. Antivenom was fully eective in the neutralization of lethal, hemorrhagic, edema-forming, myotoxic and indirect hemolytic eects 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 eects, and was partially eective in neutralizing edema-forming activity. In contrast, the antivenom was ineective in the neutralization of in vitro coagulant and in vivo de®brinating eects induced by these two venoms. #

Research paper thumbnail of Standardization of assays for testing the neutralizing ability of antivenoms

Toxicon, 1990

1. Toxicon. 1990;28(10):1127-9; author reply 1129-32. Standardization of assays for testing the n... more 1. Toxicon. 1990;28(10):1127-9; author reply 1129-32. Standardization of assays for testing the neutralizing ability of antivenoms. Gutiérrez JM, Rojas G, Lomonte B, Gené JA, Chaves F, Alvarado J, Rojas E. PMID: 2264065 [PubMed - indexed for MEDLINE]. Publication Types: ...

Research paper thumbnail of Pan-African polyspecific antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa

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.

Research paper thumbnail of Effect of storage temperature on the stability of the liquid polyvalent antivenom produced in Costa Rica

Toxicon, 1990

The effect of storage temperature on the stability of the liquid polyvalent (crotaline) antivenom... more The effect of storage temperature on the stability of the liquid polyvalent (crotaline) antivenom produced at the Instituto Clodomiro Picado, Costa Rica, was studied during a twelve-month period. The following parameters were evaluated: neutralizing potency against lethal activity of Bothrops asper venom; protein and phenol concentrations; pH; turbidity; safety; and sterility. Analyses were performed each month on different samples of a batch, stored at 4, 23, 30 and 37 degrees C. No significant (P greater than 0.1) variations occurred in potency, protein and phenol concentrations, pH, sterility or safety, at any of the storage temperatures during the study period. However, visual inspection revealed a moderate increase in turbidity of the samples stored at 23, 30 and 37 degrees C, at nine, four and three months, respectively. Culture of samples excluded the possibility of microbial contamination of the product leading to turbidity. Chromatographic and electrophoretic analyses demonstrated that turbidity was caused by the formation of heterogeneous protein aggregates of high molecular weight. Present results support the conclusion that, although storage temperature (up to 37 degrees C for twelve months) does not alter antivenom potency, it significantly influences the formation of protein aggregates. This phenomenon can be prevented by recommending the storage of antivenom at refrigeration temperature.

Research paper thumbnail of Caprylic acid fractionation of hyperimmune horse plasma: Description of a simple procedure for antivenom production

Toxicon, 1994

G. ROJAS, J. M. JIM~NEZ and J. M. GUTII~RREZ. Caprylic acid fractionation of hyperimmune horse pl... more G. ROJAS, J. M. JIM~NEZ and J. M. GUTII~RREZ. Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon 32, 351-363, 1994.--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 NaC1 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.

Research paper thumbnail of Turbidity of hyperimmune equine antivenom: The role of phenol and serum lipoproteins

Toxicon, 1993

of hyperimmune equine antivenom: the role of phenol and serum lipoproteins . Toxicon 31, 61-66, 1... more of hyperimmune equine antivenom: the role of phenol and serum lipoproteins . Toxicon 31, 61-66, 1993.-Twenty batches of polyvalent antivenom produced at the Instituto Clodomiro Picado were analyzed for turbidity, both before and after freezing-thawing and lyophilization . Eight batches became turbid upon freezing-thawing, and this change correlated with high levels of cholesterol, triglycerides and lipoproteins, especially ß-lipoprotein. Since normal horse serum does not become turbid after freezing-thawing, despite the fact that it has high lipoprotein levels, the possibility was raised that phenol, used as a preservative during serum fractionation, might affect lipoproteins, inducing the appearance of turbidity after freezing-thawing . This hypothesis was tested by fractionating a sample of hyperimmune serum either without phenol or using two different phenol concentrations (0.1 g/dl and 0.25 g/dl). Results showed that, although the three samples had the same cholesterol and triglyceride levels before fractionation, only the one having 0.25 g/dl phenol became turbid upon freezing-thawing, containing a diffuse lipoprotein band on electrophoresis. This finding suggests that turbidity in equine antivenoms depends on the interaction of at least three factors: (a) freezing, (b) high initial cholesterol and lipoprotein concentration in the serum, and (c) addition of phenol during fractionation of serum.

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

Toxicon, 1989

The coagulant, defibrinating, fibrino lytic and fibrinogenolytic activities of venoms from ten sp... more 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.

Research paper thumbnail of Neutralization of four Peruvian Bothrops sp. snake venoms by polyvalent antivenoms produced in Perú and Costa Rica: preclinical assessment

Acta Tropica, 2005

Envenomations after bites inflicted by snakes of the genus Bothrops constitute a public health ha... more Envenomations after bites inflicted by snakes of the genus Bothrops constitute a public health hazard in Perú, and the intravenous administration of equine-derived antivenoms represents the only scientifically validated treatment. This study presents a preclinical assessment of the efficacy of two whole IgG antivenoms, prepared in Perú and Costa Rica, to neutralize the most relevant toxic effects induced by the venoms of Bothrops atrox, B. brazili, B. barnetti and B. pictus from Perú. Peruvian antivenom is produced by immunizing horses with Bothrops sp. venoms from this country, whereas the production of Costa Rican antivenom involves immunization with venoms from Central American snakes. The neutralization of lethal, hemorrhagic, edema-forming, myotoxic, coagulant and defibrinating activities was evaluated in assays involving incubation of venom and antivenom prior to testing. Both antivenoms were effective in the neutralization of these effects, with quantitative variations in the values of effective dose 50% depending on the effects being studied. Peruvian antivenom was more effective in the neutralization of lethality induced by B. atrox and B. barnetti venoms. However, Peruvian antivenom failed to neutralize coagulant activity of B. barnetti venom and edema-forming activity of B. brazili venom, whereas neutralization was achieved by Costa Rican antivenom. It is concluded that an extensive immunological cross-reactivity exists between Bothrops sp. venoms from Perú and Costa Rica, and that both antivenoms are effective in the neutralization of these four venoms in a rodent model of envenoming.

Research paper thumbnail of Effect of Preservatives on IgG Aggregation, Complement-activating Effect and Hypotensive Activity of Horse Polyvalent Antivenom Used in Snakebite Envenomation

Biologicals, 2002

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.

Research paper thumbnail of NEUTRALIZATION OF LOCAL TISSUE DAMAGE INDUCED BY BOTHROPS ASPER (TERCIOPELO) SNAKE VENOM

Research paper thumbnail of 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

Toxicon, 2000

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. #

Research paper thumbnail of Quantitation by enzyme-immunoassay of antibodies against bothrops myotoxins in four commercially-available antivenoms

Toxicon, 1991

B. LOMONTE, J. M. Gu~REZ, G. Ralws and L. Cw1.D>at6N. Quantitation by enzyme-immunoassay of antib... more B. LOMONTE, J. M. Gu~REZ, G. Ralws and L. Cw1.D>at6N. Quantitation by enzyme-immunoassay of antibodies against Bothrops myotoxins in four commercially-available antivenoms . Toxicon 29, 69702, 1991 .-An enzymeimmunoassay (EIA) for the quantitation of antibodies against myotoxins present in the venoms of Bothrops riper (Costa Rica), B. atrox (Colombia) and B. moojeni (Brazil), was developed. This EIA was utilized for the evaluation of four antivenoms produced in Mexico (Laboratorios Myn; MYI~, Costa Rica (Instituto Clodomiro Picador ICP), Colombia (Instituto National de Salud; INS) and Brazil (Instituto Butantan ; IB). Antivenoms ICP, IB and INS

Research paper thumbnail of Isolation of a galactose-binding lectin from the venom of the snake Bothrops godmani (Godmann's pit viper

Toxicon, 1990

Isolation of a galactose-binding lectin from the venom of the snake Bothrops godrnani (Godmann's ... more Isolation of a galactose-binding lectin from the venom of the snake Bothrops godrnani (Godmann's pit viper) . Toxicon 28, 75-81, 1990.-A galactose-binding lectin, isolated from the venom of B. gotlmani by affinity chromatography . is an acidic protein (pI 4.9) with a subunit mol. wt of about 14,000, occurring mostly as a disulfide-linked dimer of 28,000 . A small proportion of lectin appears as a monomer and as a tetramer . The lectin agglutinates erythrocytes from mice, rabbit, cow and human (all ABO types, either Rh positive or negative), but does not agglutinate horse, sheep, goat and snake (Oxybelis aeneus, Colubridae) erythrocytes . The agglutinating activity is inhibited by 1 mM EDTA . The lectin is devoid of lethal, hemorrhagic, myotoxic, proteolytic and phospholipase A2 activities . It is not mitogenic for human peripheral blood mononuclear cells. The only effect observed was a moderate induction of edema in the footpad of mice, with a minimal edema-forming dose of 22 kg . This effect developed rapidly, and was significantly inhibited by i.p . administration of cyproheptadine, a histamine and serotonin antagonist, before injection of the lectin . Despite the edema-forming activity observed, the low concentration of lectin in crude venom, together with its relatively low potency, suggest that this lectin is not a key component in the development of edema following envenomations by B. godrnani.

Research paper thumbnail of Effects of a myotoxic phospholipase A2 isolated from Bothrops asper venom on skeletal muscle sarcoplasmic reticulum

Toxicon, 1987

J . M . GU719RREZ, G. RojAs, B . LomoNTE, J . A . GENé and L . CERDAs . Effects of a myotoxic pho... more J . M . GU719RREZ, G. RojAs, B . LomoNTE, J . A . GENé and L . CERDAs . Effects of a myotoxic phospholipase A2 isolated from Bothrops asper venom on skeletal muscle sarcoplasmic reticulum. Taxicon 25, 1244Taxicon 25, -1248Taxicon 25, , 1987 . -The myotoxin from B. asper snake venom inhibited Ca-ATPase activity of rabbit sareoplasmie retieulum after incubation in vitro . Inhibition was non-competitive and albumin enhanced the effect of the toxin . Furthermore, B. riper myotoxin hydrolyzed sarcoplasmic reticulum phospholipids and induced a dose-dependent release of horseradish perolddase that had been trapped in sarcoplasmic reticulum vesicles . Binding studies indicated that myotoxin does not bind to any particular protein of this membrane, suggesting that the toxin might interact with phospholipids. Inhibition of Ca-ATPase is probably a consequence of an alteration in sarcoplasmic reticulum phospholipids .