NEUTRALIZATION OF LOCAL TISSUE DAMAGE

INDUCED BY BOTHROPS ASPER (TERCIOPELO)

SNAKE VENOM

JOSEÂ MARIÂ A GUTIEÂ RREZ,* GUILLERMO LEOÂ N,GUSTAVO ROJAS, BRUNO LOMONTE,

ALEXANDRA RUCAVADO and FERNANDO CHAVESInstituto Clodomiro Picado, Facultad de Microbiologõ a, Universidad de Costa Rica, San Jose ,

Costa Rica

(Received 9 December 1997; accepted 19 January 1998)

J. M. Gutie rrez, G. Leo n, G. Rojas, B. Lomonte, A. Rucavado and F.Chaves. Neutralization of local tissue damage induced by Bothrops asper(terciopelo) snake venom. Toxicon 36, 1529±1538, 1998.ÐLocal tissuedamage represents a serious consequence of Bothrops asper envenomations.It encompasses a complex series of alterations, including myonecrosis, der-monecrosis, hemorrhage and edema. Due to its rapid development it is di�-cult to neutralize by antivenoms, especially if there is a delay in serotherapy.Experimental studies with this venom and the polyvalent (Crotalinae) antive-nom produced in Costa Rica indicate that antivenom is e�ective in neutraliz-ing these toxic activities when incubated with the venom prior to injection.However, if venom and antivenom are injected independently in mice, neu-tralization of these e�ects is only partial. Moreover, neutralization is notcomplete even if homologous or heterologous antibodies are present in thecirculation before venom is injected. Despite di�erences in their pharmacoki-netic pro®les, equine whole IgG and F(ab')2 antivenoms show similar e�cacyin the neutralization of edema, hemorrhage and myonecrosis induced by B.asper venom, suggesting that the use of antivenoms made of antibody frag-ments may not improve neutralization of these e�ects. This is due, at least inpart, to the fact that microvessel disruption by venom components favors asimilar antibody concentration in the a�ected tissues. Recent advances in thedevelopment of neutralizing substances of rapid di�usion, that could beinjected locally in the ®eld, may contribute to the neutralization of metallo-proteinases and phospholipases A2. In addition, the rapid administration ofantivenoms with high antibody titers against locally-acting toxins is very im-portant in the treatment of these e�ects. # 1998 Elsevier Science Ltd. Allrights reserved

Toxicon Vol. 36, No. 11, pp. 1529±1538, 1998# 1998 Elsevier Science Ltd. All rights reserved

Printed in Great Britain0041-0101/98 $19.00+0.00PII: S0041-0101(98)00145-7

*Author to whom correspondence should be addressed.

1529

INTRODUCTION

Local tissue damage, i.e. hemorrhage, myonecrosis, dermonecrosis and edema, constitu-tes one of the most common and serious consequences of crotaline snake envenomation(Fan and Cardoso, 1995; Gutie rrez, 1995; Warrell, 1996). The pathogenesis of thesealterations is rather complex, as they are induced by di�erent toxins present in thevenoms, such as myotoxins (Mebs and Ownby, 1990; Gutie rrez and Lomonte, 1997),hemorrhagic metalloproteinases (Ownby, 1982; Bjarnason and Fox, 1994), and variousedema-inducing components (Bjarnason et al., 1983; Teng et al., 1989; Lomonte andGutie rrez, 1989). In addition, local tissue damage can be complicated by bacterial infec-tion and the development of compartmental syndrome (Gar®n et al., 1985; Cardoso etal., 1993; Warrell, 1996).

Neutralization of these e�ects by antivenoms is di�cult to achieve, not only due tothe variety of components inducing these alterations, but also to the rapid onset ofthese pathological changes. Thus, the delay in antivenom administration that occurs inmany rural areas of tropical countries hampers the success of serotherapy. Moreover,some aspects of local tissue damage are caused by venom-induced release of endogenousmediators, such as eicosanoids, vasoactive amines, kinins and cytokines (Rothschild andRothschild, 1979; Moura da Silva et al., 1996). It has been stated that the evaluation ofantivenoms should include not only the neutralization of lethality, but also of other rel-evant pathophysiological e�ects, such as those characteristic of local tissue damage(WHO, 1981; Gutie rrez et al., 1990; Theakston et al., 1995). Several studies have beenperformed in our laboratory in order to investigate the ability of the polyvalent(Crotalinae) antivenom produced in Costa Rica to neutralize local e�ects induced bythe venom of Bothrops asper, the most important poisonous snake in Central America.These results and their implications are discussed in this contribution.

ASSAYS WITH PREINCUBATION AND ASSAYS WITH INDEPENDENT INJECTION OF VENOM

AND ANTIVENOM

A basic methodology to approach the study of neutralization of pharmacological ac-tivity of venoms by antivenoms was adapted in our laboratory (see Gutie rrez et al.,1990 and references therein). It includes two basic types of test: (a) assays with preincu-bation of venom and antivenom, and (b) assays with independent injection of venomand antivenom. In the ®rst type of assay, a ``challenge dose'' of venom is selected foreach e�ect to be evaluated, on the basis of dose±response studies. Then, a constantamount of venom is mixed with various dilutions of antivenom, in a constant ®nalvolume, and incubated for 30 min at 378C. Controls include venom alone, physiologicsaline solution alone and antivenom alone. After incubation, aliquots of the mixtures,containing the ``challenge dose'' of venom, are tested in the corresponding assay system.The neutralizing ability of antivenom is expressed as e�ective dose 50%, de®ned as theratio of ml antivenom/mg venom in which the e�ect of venom alone is neutralized by50%. On the other hand, assays with independent injection of venom and antivenomattempt to reproduce the natural course of envenomation. In these assays, the ``chal-lenge dose'' of venom is injected ®rst and then, at various time intervals, antivenom isadministered intravenously.

Several test systems are used for the study of local e�ects. Hemorrhagic activity isquanti®ed by the skin test (Kondo et al., 1960; Gutie rrez et al., 1985) and by the spec-trophotometric determination of hemoglobin in muscle (Ownby et al., 1984; Gutie rrez

J. M. GUTIERREZ et al.1530

et al., 1987). Edema-forming activity is evaluated in the rodent foot pad model eitherplethysmographically (Trebien and Calixto, 1989; Chaves et al., 1995) or by determiningthe increments in weight (Yamakawa et al., 1976; Gutie rrez et al., 1986) or in thickness(Lomonte et al., 1993a; Rucavado and Lomonte, 1996). Myonecrosis is quanti®ed eitherhistologically (Ownby et al., 1982; Preston et al., 1990; Lomonte et al., 1993b) or bymeasuring the creatine kinase activity in plasma (Gutie rrez et al., 1981) or in tissuehomogenates (Gutie rrez et al., 1987). Local necrotic activity is studied by a skin test inwhich necrotic lesions are observed 72 h after venom injection (Theakston and Reid,1983).

When these tests were applied to study the ability of polyvalent antivenom to neutral-ize local e�ects induced by B. asper venom, a consistent picture emerged: antivenomwas e�ective in experiments where venom and antivenom were incubated before injec-tion, but neutralization was only partial when antivenom was administered after enve-nomation (Fig. 1 and 2) (Gutie rrez et al., 1981, 1985). Similar results were obtainedwhen testing the neutralization of other Central American crotaline snake venoms(Gutie rrez et al., 1985, 1986, 1987; Rojas et al., 1987). Thus, the polyvalent antivenomproduced in Costa Rica contains antibodies capable of neutralizing locally-acting toxinsif incubated prior to injection. This conclusion has been strengthened by the determi-nation of antibody titres, by ELISA, against myotoxins present in Bothrops venoms(Lomonte et al., 1991). However, neutralization is not achieved if antivenom is injectedafter envenomation, probably due to the extremely rapid development of local tissuedamage. This hypothesis is supported by the studies on the pathogenesis of hemorrhage(Gutie rrez et al., 1984; Moreira et al., 1992), myonecrosis (Gutie rrez et al., 1984) andedema (Gutie rrez et al., 1980; Chaves et al., 1995) induced by B. asper venom in mice.Moreover, this has been corroborated in intravital microscopy studies after application

Fig. 1. Neutralization of hemorrhagic (Q), myotoxic (R) and edema-forming (.) activities ofBothrops asper venom by polyvalent (Crotalinae) antivenom in assays in which venom and anti-venom are incubated prior to injection. For each e�ect, a constant amount of venom was incu-bated with various dilutions of antivenom for 30 min at 378C. Then, aliquots of the mixtureswere injected in mice and the e�ects evaluated by standard laboratory assays. Results areexpressed as percentage of activity, taking as 100% the activity of samples in which venom was

incubated with saline solution instead of antivenom.

Neutralization of Tissue Damage Induced by Bothrops Venom 1531

of venom on the mouse cremaster muscle (Lomonte et al., 1994a). In the three cases thee�ects were prominent very soon after venom injection.

These methods have been also utilized in the evaluation of other antivenoms distribu-ted in Central America. In general, antivenoms have high antibody titers against hemor-rhagic toxins, as shown by the e�cient neutralization in preincubation-type assays(BogarõÂ n et al., 1995), although they are partially ine�ective in neutralizing hemorrhageinduced by B. asper venom in assays with independent injection of venom and antive-nom. On the other hand, drastic di�erences in antibody titers against myotoxins wereobserved in various commercial antivenoms, suggesting that some of them might beine�ective in the neutralization of B. asper-induced local myotoxicity (Lomonte et al.,1991; BogarõÂ n et al., 1995). Such disparity in antivenom e�cacy is probably based onthe antigenic variability of venom components between species and, sometimes, betweenpopulations of the same species (Warrell, 1997). Thus, since antivenoms are producedusing di�erent immunizing mixtures of venoms, an antivenom e�ective against a venomin a particular region might not be e�ective against a di�erent venom in anothercountry or region, as has been demonstrated in many instances (Theakston et al., 1995;Theakston, 1996). These observations stress the relevance of evaluating the neutralizinge�cacy of antivenoms against the most important venoms of each country or region.

NEUTRALIZATION OF LOCALLY-ACTING TOXINS WHEN ANTIBODIES ARE INJECTED

BEFORE ENVENOMATION

Since experiments in which antivenom was administered after envenomation demon-strated only partial neutralization of local e�ects, a study was performed to determinethe success of neutralization if antibodies are present in experimental animals before

Fig. 2. Neutralization of hemorrhagic (q), myotoxic (diagonally lined) and edema-forming (hori-zontally lined) activities of Bothrops asper venom by the polyvalent (Crotalinae) antivenom inassays in which venom and antivenom are injected independently in mice. In these experiments,venom was injected intradermally, intramuscularly or subcutaneously, depending on the e�ect tobe studied, and then antivenom was administered intravenously, at di�erent time intervals (im-mediately, 15 and 30 min after envenomation). Results are expressed as percentage of activity,taking as 100% the e�ect induced by venom alone, i.e. in mice injected with venom and not

receiving antivenom.

J. M. GUTIERREZ et al.1532

venom injection (Rucavado and Lomonte, 1996). This was investigated in two exper-imental ways: (a) mice immunized with B. asper venom and then challenged withvenom, and (b) mice injected intravenously with antivenom and then receiving venom.In both cases it was demonstrated that the antibodies present in blood would neutralizethe venom injected in preincubation-type experiments.

When actively-immunized mice were challenged with B. asper venom, local tissuedamage was signi®cantly, but not totally, reduced. Acute edema, i.e. edema at 1 h, wasnot inhibited, but at subsequent time intervals it was signi®cantly neutralized when com-pared with mice injected with venom alone. Hemorrhage was reduced by about 70%and myonecrosis by 80% (Rucavado and Lomonte, 1996). On the other hand, hemor-rhage was almost completely neutralized in animals passively immunized with heter-ologous antibodies (equine polyvalent antivenom) either 5 or 120 min beforeenvenomation. Edema was not neutralized at the ®rst time intervals evaluated, butdecreased signi®cantly when compared to mice injected with venom and not pretreatedwith antivenom, at later time intervals. Myotoxicity was neutralized by about 65% inpassively immunized mice, with no signi®cant di�erence if antivenom was administeredeither 5 or 120 min before envenomation.

These results clearly show that, although a signi®cant reduction in local tissuedamage is achieved when antibodies are present in the bloodstream before venom injec-tion, such neutralization is not complete, and hemorrhage, edema and myonecrosisdevelop to some extent. It is likely that the antibody concentration in the tissues by thetime venom is injected is insu�cient to achieve neutralization of locally-acting toxins.These results are important not only to understand the basis of antibody neutralizationof locally-acting toxins, but also to simulate the e�ect of active immunization withvenoms as a future strategy to confront the problem of local tissue damage.

ARE F(ab')2 FRAGMENTS MORE EFFECTIVE THAN WHOLE IgE MOLECULES IN THE

NEUTRALIZATION OF LOCAL EFFECTS?

Neutralization of locally-acting toxins may depend on the concentration of antibodiesin the tissues at the time venom is injected. Based on this assumption, a study was per-formed to test the hypothesis that an antivenom made of F(ab')2 fragments would bemore e�ective than a whole IgG antivenom in the neutralization of myonecrosis, hemor-rhage and edema induced by B. asper venom. This hypothesis is supported by pharma-cokinetic studies which demonstrate that, due to their lower molecular weight, F(ab')2fragments have a larger volume of distribution and reach the tissues more rapidly thanwhole IgG antibodies (Ismail and Abd-Elsalam, 1996).

Both types of antivenoms were prepared from the same batch of hyperimmune equineplasma, and it was assured that they had the same neutralizing potency in experimentswith preincubation of venom and antivenom (Leo n et al., 1997). Therefore, any di�er-ence in neutralization would depend on the di�erent pharmacokinetic pro®le of the pro-ducts, and not on variations in antibody titers against locally-acting toxins. This isparticularly relevant since many studies have compared the neutralizing ability of anti-venoms having di�erent potencies. In these cases it is not possible to conclude with cer-tainty if the di�erences observed are due to the varying potencies or to thepharmacokinetic characteristics of the antivenoms. In the study performed by Leo n etal. (1997), B. asper venom was injected locally and a high dose of antivenom was admi-nistered intravenously at 0, 15 and 30 min after envenomation. In some experiments,antivenom was injected by other routes (intramuscular and intraperitoneal).

Neutralization of Tissue Damage Induced by Bothrops Venom 1533

No signi®cant di�erences were observed in neutralization between these two types ofantivenom (Fig. 3), thus indicating that administration of antibody fragments of lowermolecular weight than whole IgG molecules does not improve neutralization of B.asper-induced local e�ects. In agreement with previous works cited above, neutralizationof local tissue damage was only partial even if antivenom was administered immediatelyafter envenomation. Myonecrosis was reduced by 80%, whereas hemorrhage and edemawere neutralized only by 25% and 30%, respectively (Leo n et al., 1997). Antivenomswere even less e�ective if the time lapse between envenomation and serotherapyincreased. Although these results cannot be simplistically extrapolated to other venomand antivenom systems, Lomonte et al. (1996) obtained similar results when comparingneutralization by F(ab')2 and Fab fragment antivenoms of hemorrhage induced byVipera berus venom. The e�cacy of an Fab antivenom to neutralize local e�ectsinduced by B. asper venom is currently under investigation.

Several hypotheses can be proposed to explain these ®ndings:(1) It might be that, despite a higher concentration of F(ab')2 fragments in the tissues,

their amount is still too low to e�ectively neutralize edema-forming, hemorrhagic andmyotoxic venom components. Moreover, since these e�ects develop so fast after venominjection, this would further hamper the possibility of neutralization.

(2) Alternatively, it might be that B. asper venom-induced microvessel damage, i.e.edema and hemorrhage, induces a similar extravasation of antibodies, independently oftheir molecular weight and of their normal pharmacokinetic behavior. This hypothesishas been recently demonstrated in our laboratory (unpublished results), since antibodyconcentration increased signi®cantly in envenomated muscle tissue, as compared withmuscle tissue of non-envenomated mice injected with antivenoms. Moreover, a similarconcentration of IgG and F(ab')2 was observed in the tissue. Thus, by disrupting theintegrity of the microvasculature, B. asper venom promotes a prominent, and similar,

Fig. 3. Neutralization of hemorrhagic and myotoxic activities of Bothrops asper venom by poly-valent antivenoms made of either whole IgG molecules or F(ab')2 fragments. Mice were injectedwith venom and then, at various time intervals (immediately, 15 and 30 min after envenomation),a constant volume of antivenom was administered intravenously. Results are expressed as per-centage of activity, taking as 100% the e�ect induced by venom alone, i.e. in mice injected withvenom and not receiving antivenom. Neutralization of hemorrhage: IgG antivenom (.); F(ab')2

antivenom (T). Neutralization of myotoxicity: IgG antivenom (Q); F(ab')2 antivenom (R).

J. M. GUTIERREZ et al.1534

extravasation of both IgG and F(ab')2 antibodies. These observations may be also rel-evant for other hemorrhagic venoms and corroborate that antibody pharmacokinetics isdi�erent in normal and in envenomated animals, thus stressing the need of comparativepharmacokinetic studies in both situations.

(3) Despite the demonstration of increased extravasation of antibodies in venom-damaged tissues, another possibility to interpret these results would be that neutraliz-ation of toxin occurs in the vascular compartment, and that formation of toxin±anti-body complexes causes a redistribution of toxins from tissues to blood, where they areneutralized. Such hypothesis is supported by pharmacokinetic studies showing anincrease in total venom concentration in blood after intravenous administration of anti-venom (Choumet et al., 1996; Riviere et al., 1997). Thus, it is important to maintainhigh antibody concentrations in blood during prolonged periods of time in order to pro-mote a complete redistribution of venom components to the circulation. Such neutraliz-ation mechanism would not be completely e�ective in neutralizing locally-acting toxins,since they would have already caused tissue damage by the time of redistribution.

IS THERE A SOLUTION? FUTURE TRENDS IN THE NEUTRALIZATION OF VENOM-INDUCED

LOCAL EFFECTS

Results obtained so far concerning neutralization of local e�ects induced by B. aspervenom are discouraging. However, it is expected that the knowledge being gained onthe biochemical nature, immunochemistry and mechanism of action of hemorrhagic,myotoxic and edema-forming toxins will be of value in the search for novel avenues toconfront this problem. It is necessary to search for novel neutralizing agents. Forinstance, several monoclonal antibodies were produced against B. asper myotoxin I(Lomonte and Kahan, 1988). In preincubation-type experiments, two of these antibodiescompletely neutralized local myotoxicity induced by crude venom and myotoxin I, andone of them neutralized myotoxin II, a lys-49 phospholipase A2 present in this venom(Lomonte et al., 1992). However, preliminary studies using independent injection ofvenom and antibodies revealed the same partial neutralization described for polyclonalequine antibodies (Lomonte, unpublished results).

A variety of natural and synthetic molecules that inhibit metalloproteinases and phos-pholipases A2 have been characterized (see for example Xue et al. (1996) and Mihelichet al. (1997)) and some of them are being used in the treatment of in¯ammatory diseasesand other pathologies associated with these endogenous enzymes. Based on the simi-larities described between some venom enzymes and these endogenous metalloprotei-nases and phospholipases A2, it is likely that some of these inhibitors may be e�ectivein the neutralization of venom components. For instance, in the case of B. asper venom,a recent study demonstrated that a novel neutralizing mixture, composed by CaNa2EDTA, polyvalent antivenom and an inhibitor of hemorrhagic toxins isolated from theblood of B. asper was quite e�ective in the neutralization of hemorrhage induced bythis venom, even when the mixture was administered after envenomation (Borkow etal., 1997). CaNa2 EDTA is being used successfully in reducing the extent of local tissuedamage in horses immunized with venoms for the production of polyvalent antivenomin Costa Rica (Leo n et al., 1998).

Moreover, heparin binds to B. asper myotoxin II in a region located near the C-ter-minus of the molecule, neutralizing its myotoxicity (Lomonte et al., 1994b,c). Similarobservations have been performed by Melo et al. (1993) with heparin and B. jararacussuvenom. Nevertheless, when these encouraging results obtained in preincubation-type

Neutralization of Tissue Damage Induced by Bothrops Venom 1535

experiments were performed in experiments with independent injection of venom andheparin, neutralization was poor (Lomonte, unpublished results). More recently, Lizanoet al. (1997) characterized a plasma protein from B. asper which is highly e�ective, inpreincubation-type experiments, in neutralizing myotoxicity induced by the four myo-toxins isolated from B. asper venom. This natural antidote is currently being evaluatedin independent injection tests. Plant extracts constitute another rich source of substancesof potential use in venom neutralization (Mors et al., 1989; Melo et al., 1994). Thus, thesearch for natural and synthetic inhibitors of high di�usibility and devoid of toxicitythat could be applied locally at the site of venom injection is a relevant area ofresearch.

Finally, it is necessary to remark that, despite the partial ine�ectiveness of antivenomsto neutralize B. asper venom-induced local e�ects, it is very important to test the abilityof commercial antivenoms to neutralize myonecrosis, hemorrhage, dermonecrosis andedema in experiments with preincubation of venom and antivenom. This would assurethat antivenoms with high antibody titers are being used therapeutically. If these pro-ducts are administered in the clinics rapidly after envenomation, some degree of neutral-ization would be achieved. Antivenom producers should continue their e�orts toimprove the antibody titers against locally-acting toxins, and quality control labora-tories must guarantee that antivenoms being used in the clinics have high titers againstthese toxins. The combination of rapid administration of antivenoms having high anti-body titers, together with the local injection of e�ective toxin inhibitors, may greatlyimprove our ability to cope with this relevant medical problem.

AcknowledgementsÐThe authors thank the sta� of Instituto Clodomiro Picado for their permanent support inthis research throughout these years. These studies have been supported by Vicerrectorõ a de Investigacio n,Universidad de Costa Rica (project 741-89-057) and by the International Foundation for Science (projects F/0883-4 and F/1388-3F).

REFERENCES

Bjarnason, J. B. and Fox, J. W. (1994) Hemorrhagic metalloproteinases from snake venoms. Pharmacol. Ther.62, 325±372.

Bjarnason, J. B., Barish, A., Direnzo, G. S., Campbell, R. and Fox, J. W. (1983) Kallikrein-like enzymes fromCrotalus atrox venom. J. Biol. Chem. 258, 12566±12573.

Bogarõ n, G., Segura, E., Dura n, G., Lomonte, B., Rojas, G. and Gutie rrez, J. M. (1995) Evaluacio n de lacapacidad de cuatro antivenenos comerciales para neutralizar el veneno de la serpiente Bothrops asper (ter-ciopelo) de Costa Rica. Toxicon 33, 1242±1245.

Borkow, G., Gutie rrez, J. M. and Ovadia, M. (1997) Inhibition of the hemorrhagic activity of Bothrops aspervenom by a novel neutralizing mixture. Toxicon 35, 865±877.

Cardoso, J. L. C., Fan, H. W., Franca, F. O. S., Jorge, M. T., Leite, R. P., Nishioka, S. A., Avila, A., Sano-Martins, I. S., Tomy, S. C., Santoro, M. L., Chudzinski, A. M., Castro, S. C. B., Kamiguti, A. S., Kelen,E. M. A., Hirata, M. H., Mirandola, R. M. S., Theakston, R. D. G. and Warrell, D. A. (1993)Randomized comparative trial of three antivenoms in the treatment of envenoming by lance-headed vipers(Bothrops jararaca) in Sao Paulo, Brazil. Quart. J. Med. 86, 315±325.

Chaves, F., Barboza, M. and Gutie rrez, J. M. (1995) Pharmacological study of edema induced by venom ofthe snake Bothrops asper (terciopelo) in mice. Toxicon 33, 31±39.

Choumet, V., Audebert, F., Riviere, G., Sorkine, M., Urtizberea, M., Sabouraud, A., Scherrmann, J. M. andBon, C. (1996) Toxicokinetics of Vipera aspis envenoming and antivenom therapy. In Envenomings andTheir Treatments, eds C. Bon and M. Goy�on, pp. 127±133. Editions Fondation Marcel Me rieux, Lyon.

Fan, H. W. and Cardoso, J. L. (1995) Clinical toxicology of snake bites in South America. In Handbook ofClinical Toxicology of Animal Venoms and Poisons, eds J. Meier and J. White, pp. 667±688. CRC Press,Boca Raton.

Gar®n, S. R., Castilonia, R. R., Mubarak, S. J., Hargens, A. R., Akeson, W. H. and Russell, F. E. (1985)The e�ect of antivenin on intramuscular pressure elevations induced by rattlesnake venom. Toxicon 23, 677±680.

J. M. GUTIERREZ et al.1536

Gutie rrez, J. M. (1995) Clinical toxicology of snakebite in Central America. In Handbook of ClinicalToxicology of Animal Venoms and Poisons, eds J. Meier and J. White, pp. 645±665. CRC Press, BocaRaton.

Gutie rrez, J. M. and Lomonte, B. (1997) Phospholipase A2 myotoxins from Bothrops snake venoms. In VenomPhospholipase A2 Enzymes. Structure, Function and Mechanism, ed. R. M. Kini, pp. 321±352. Wiley,Chichester.

Gutie rrez, J. M., Arroyo, O. and BolanÄ os, R. (1980) Mionecrosis, hemorragia y edema inducidos por elveneno de Bothrops asper en rato n blanco. Toxicon 18, 603±610.

Gutie rrez, J. M., Chaves, F., BolanÄ os, R., Cerdas, L., Rojas, E., Arroyo, O. and Portilla, E. (1981)Neutralizacio n de los efectos locales del veneno de Bothrops asper por un antiveneno polivalente. Toxicon19, 493±500.

Gutie rrez, J. M., Ownby, C. L. and Odell, G. V. (1984) Pathogenesis of myonecrosis induced by crude venomand a myotoxin of Bothrops asper. Exp. Mol. Pathol. 40, 367±379.

Gutie rrez, J. M., Gene , J. A., Rojas, G. and Cerdas, L. (1985) Neutralization of proteolytic and hemorrhagicactivities of Costa Rican snake venoms by a polyvalent antivenom. Toxicon 23, 887±893.

Gutie rrez, J. M., Rojas, G., Lomonte, B., Gene , J. A. and Cerdas, L. (1986) Comparative study of the edema-forming activity of Costa Rican snake venoms and its neutralization by a polyvalent antivenom. Comp.Biochem. Physiol. 85C, 171±175.

Gutie rrez, J. M., Rojas, G. and Cerdas, L. (1987) Ability of a polyvalent antivenom to neutralize the venomof Lachesis muta melanocephala, a new Costa Rican subspecies of the bushmaster. Toxicon 25, 713±720.

Gutie rrez, J. M., Rojas, G., Lomonte, B., Gene , J. A., Chaves, F., Alvarado, J. and Rojas, E. (1990)Standardization of assays for testing the neutralizing ability of antivenoms. Toxicon 28, 1127±1129.

Ismail, M. and Abd-Elsalam, M. A. (1996) Serotherapy of scorpion envenoming: Pharmacokinetics of antive-noms and a critical assessment of their usefulness. In: Envenomings and Their Treatments, eds C. Bon andM. Goy�on, pp. 135±153. Editions Fondation Marcel Me rieux, Lyon.

Kondo, H., Kondo, S., Ikezawa, H., Murata, R. and Ohsaka, A. (1960) Studies on the quantitative methodfor determination of hemorrhagic activity of Habu snake venom. Jpn. J. Med. Sci. Biol. 13, 43±51.

Leo n, G., Rojas, G., Lomonte, B. and Gutie rrez, J. M. (1997) IgG and F(ab')2 polyvalent antivenoms do notdi�er in their ability to neutralize hemorhage, edema and myonecrosis induced by Bothrops asper (tercio-pelo) snake venom. Toxicon 35, 1627±1637.

Leo n, G., Estrada, R., Chaves, F., Rojas, G., Ovadia, M. and Gutie rrez, J. M. (1998) Inhibition byCaNa2EDTA of local tissue damage induced by Bothrops asper venom: Application in horse immunizationfor antivenom production. Toxicon 36, 321±331.

Lizano, S., Lomonte, B., Fox, J. W. and Gutie rrez, J. M. (1997) Biochemical characterization and biochemicalproperties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper. Biochem.J. 326, 853±859.

Lomonte, B. and Kahan, L. (1988) Production and partial characterization of monoclonal antibodies toBothrops asper (terciopelo) myotoxin. Toxicon 26, 675±689.

Lomonte, B. and Gutie rrez, J. M. (1989) A new muscle-damaging toxin, myotoxin II, from the venom of thesnake Bothrops asper (terciopelo). Toxicon 27, 725±733.

Lomonte, B., Gutie rrez, J. M., Rojas, G. and Caldero n, L. (1991) Quantitation by enzyme-immunoassay ofantibodies against Bothrops myotoxins in four commercially-available antivenoms. Toxicon 29, 695±702.

Lomonte, B., Gutie rrez, J. M., Ramõ rez, M. and Dõ az, C. (1992) Neutralization of myotoxic phospholipasesA2 from the venom of the snake Bothrops asper by monoclonal antibodies. Toxicon 30, 239±245.

Lomonte, B., Tarkowski, A. and Hanson, L. A. (1993a) Host response to Bothrops asper snake venom.Analysis of edema formation, in¯ammatory cells and cytokine release in a mouse model. In¯ammation 17,93±105.

Lomonte, B., Gutie rrez, J. M., Romero, M., Nu nÄ ez, J., Tarkowski, A. and Hanson, L. A. (1993b) An MTT-based method for the in vivo quanti®cation of myotoxic activity of snake venoms and its neutralization byantibodies. J. Immunol. Methods 161, 231±237.

Lomonte, B., Lundgren, J., Johansson, B. and Bagge, U. (1994a) The dynamics of local tissue damage inducedby Bothrops asper snake venom and myotoxin II on the mouse cremaster muscle: An intravital and electronmicroscopic study. Toxicon 32, 41±55.

Lomonte, B., Tarkowski, A., Bagge, U. and Hanson, L. A. (1994b) Neutralization of the cytolytic and myo-toxic activities of phospholipases A2 from Bothrops asper snake venom by glycosaminoglycans of theheparin/heparan sulfate family. Biochem. Pharmacol. 47, 1509±1518.

Lomonte, B., Moreno, E., Tarkowski, A., Hanson, L. A. and Maccarana, M. (1994c) Neutralizing interactionbetween heparins and myotoxin II, a lysine 49 phospholipase A2 from Bothrops asper snake venom.Identi®cation of a heparin-binding and cytolytic toxin region by the use of synthetic peptides and molecularmodeling. J. Biol. Chem. 269, 29867±29873.

Lomonte, B., Leo n, G. and Hanson, L. A. (1996) Similar e�ectiveness of Fab and F(ab')2 antivenoms in theneutralization of hemorrhagic activity of Vipera berus snake venom in mice. Toxicon 34, 1197±1202.

Mebs, D. and Ownby, C. L. (1990) Myotoxic components of snake venoms: Their biochemical and biologicalactivities. Pharmacol. Ther. 48, 223±236.

Neutralization of Tissue Damage Induced by Bothrops Venom 1537

Melo, P. A., Homsi-Brandeburgo, M. I., Giglio, J. R. and Sua rez Kurtz, G. (1993) Antagonism of the myo-toxic e�ect of Bothrops jararacussu venom and Bothropstoxin by polyanions. Toxicon 31, 285±291.

Melo, P. A., do Nascimento, M. C., Mors, W. B. and Sua rez-Kurtz, G. (1994) Inhibition of the myotoxic andhemorrhagic activities of crotalid venoms by Eclipta prostrata (Asteraceae) extracts and constituents.Toxicon 32, 595±603.

Mihelich, E. D., Carlson, D. G., Fox, N., Song, M., Schevitz, R. W. and Snyder, D. W. (1997) Structure-based design and therapeutic potential of phospholipase A2 inhibitors. In Phospholipase A2. Basic andClinical Aspects in In¯ammatory Diseases, eds W. Uhl, T. J. Nevalainen and M. W. Buchler, pp. 140±145.Karger, Basel.

Moreira, L., Gutie rrez, J. M., Borkow, G. and Ovadia, M. (1992) Ultrastructural alterations in mouse capil-lary blood vessels after experimental injection of venom from the snake Bothrops asper (terciopelo). Exp.Mol. Pathol. 57, 124±133.

Mors, W. B., Nascimento, M. C., Parente, J. P., Da Silva, M. H., Melo, P. A. and Sua rez Kurtz, G. (1989)Neutralization of lethal and myotoxic activities of South American rattlesnake venom by extracts and con-stituents of the plant Eclipta prostrata (Asteraceae). Toxicon 27, 1003±1009.

Moura da Silva, A. M., Laing, G. D., Paine, M. J. I., Dennison, J. M. T. J., Politi, V., Crampton, J. M. andTheakston, R. D. G. (1996) Processing of pro-tumor necrosis factor-a by venom metalloproteinases: A hy-pothesis explaining local tissue damage following snake bite. Eur. J. Immunol. 26, 2000±2005.

Ownby, C. L. (1982) Pathology of rattlesnake envenomation. In Rattlesnake Venoms. Their Actions andTreatment, ed. A. T. Tu, pp. 163±209. Marcel Dekker, New York.

Ownby, C. L., Colberg, T. R. and Odell, G. V. (1984) A new method for quantitating hemorrhage induced byrattlesnake venoms: Ability of polyvalent antivenom to neutralize hemorrhagic activity. Toxicon 22, 227±233.

Ownby, C. L., Gutie rrez, J. M., Colberg, T. and Odell, G. V. (1982) Quantitation of myonecrosis induced bymyotoxin a from prairie rattlesnake (Crotalus viridis viridis) venom. Toxicon 20, 877±885.

Preston, S., Davis, C. E. and Harris, J. B. (1990) The assessment of muscle ®bre loss after the injection of thevenom of Notechis scutatus (Australian tiger snake). Toxicon 28, 201±214.

Riviere, G., Choumet, V., Audebert, F., Sabouraud, A., Debray, M., Scherrmann, J. M. and Bon, C. (1997)E�ect of antivenom on venom pharmacokinetics in experimentally envenomed rabbits: Toward an optimiz-ation of antivenom therapy. J. Pharmacol. Exp. Ther. 281, 1±8.

Rojas, G., Gutie rrez, J. M., Gene , J. A., Go mez, M. and Cerdas, L. (1987) Neutralizacio n de las actividadesto xicas y enzima ticas de cuatro venenos de serpientes de Guatemala y Honduras por el antiveneno poliva-lente producido en Costa Rica. Rev. Biol. Trop. 35, 59±67.

Rothschild, A. M. and Rothschild, Z. (1979) Liberation of pharmacologically-active substances by snakevenoms. In Snake Venoms, ed. C. Y. Lee, pp. 591±628. Springer Verlag, Berlin.

Rucavado, A. and Lomonte, B. (1996) Neutralization of myonecrosis, hemorrhage and edema induced byBothrops asper snake venom by homologous and heterologous pre-existing antibodies in mice. Toxicon 34,567±577.

Teng, C. M., Wang, J. P., Peng, H. C. and Ouyang, C. (1989) Edema-producing proteins isolated fromTrimeresurus mucrosquamatus snake venom. Toxicon 27, 899±905.

Theakston, R. D. G. (1996) Snake bite: The kinetics of envenoming and therapy. In Envenomings and TheirTreatments, eds C. Bon and M. Goy�on, pp. 117±126. Editions Fondation Marcel Me rieux, Lyon.

Theakston, R. D. G. and Reid, H. A. (1983) Development of simple standard assay procedures for the charac-terization of snake venoms. Bull. W.H.O. 61, 949±956.

Theakston, R. D. G., Laing, G. D., Fielding, C. M., Freire Lascano, A., Touzet, J. M., Vallejo, F., Guderian,R. H., Nelson, S. J., Wuster, W., Richards, A. M., Rumbea Guzma n, J. and Warrell, D. A. (1995)Treatment of snake bites by Bothrops species and Lachesis muta in Ecuador: Laboratory screening of candi-date antivenoms. Trans. Royal Soc. Trop. Med. Hyg. 89, 550±554.

Trebien, H. A. and Calixto, J. B. (1989) Pharmacological evaluation of rat paw edema induced by Bothropsjararaca venom. Agents Actions 26, 292±300.

Warrell, D. A. (1996) Clinical features of envenoming from snake bites. In Envenomings and Their Treatment,eds C. Bon and M. Goy�on, pp. 63±76. Editions Fondation Marcel Me rieux, Lyon.

Warrell, D. A. (1997) Geographical and intraspecies variation in the clinical manifestations of envenoming bysnakes. In: Venomous Snakes. Ecology, Evoilution and Snakebite, eds R. S. Thorpe, W. Wuster and A.Malhorta, pp. 189±203. Clarendon Press, New York.

World Health Organization (1981) Progress in the characterization of venoms and standardization of antivenoms.WHO O�set Publication No. 58, Geneva.

Xue, C. B., He, X., Roderick, J., DeGrado, W. F., Decicco, C. and Copeland, R. A. (1996) Potent matrixmetalloproteinase inhibitors: Amino-carboxylate compounds containing modi®cations of the P1 residue.Bioorg. Med. Chem. Lett. 4, 379±384.

Yamakawa, M., Nozaki, M. and Hokama, Z. (1976) Fractionation of Sakishima-habu (Trimeresurus elegans)venom and lethal, hemorrhagic and edema-forming activities of the fractions. In Animal, Plant andMicrobial Toxins, eds A. Ohsaka, K. Hayashi and Y. Sawai, Vol I, pp. 97±109. Plenum Press, New York.

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