Inflammation induced by Bothrops asper venom

Inflammation is a major characteristic of envenomation by snakes from viperine and crotaline species. Bothrops asper snake venom elicits, among other alterations, a pronounced inflammatory response at the site of injection both in humans and experimental animals. This review describes the current status of our understanding of the inflammatory reaction, including pain, triggered by Bothrops asper venom. The experimental studies on the action of this venom as well as the complex network of chemical mediators involved are summarized. Moreover, aspects of the molecular mechanisms orchestrating this important response to envenomation by Bothrops asper are presented. Considering that isolated toxins are relevant tools for understanding the actions of the whole venom, studies dealing with the mechanisms of inflammatory and nociceptive properties of phospholipases A₂, a metalloproteinase and serine-proteases isolated from Bothrops asper venom are also described.     

Estudio ultraestructural de la mionecrosis inducida en raton por el veneno de terciopelo (Bothrops asper) de Costa Rica

Skeletal muscle from white mice was examined one and three hr after i.m. injection of 50 μg of Bothrops asper venom. At the light microscopic level we observed nectoric fibers with myolitic appearance, as early as the first hr and more marked by the third hr. At the ultrastructural level three kinds of fibers were observed: (a) fibers with normal morphology, (b) fibers with slight alteration at the sarcoplasmic reticulum and (c) fibers with strong alterations in several organelles; in these fibers, the main pathological feature was the disruption of myofibril architecture. This disruption started with the disappearance of A, H, I and M bands, followed by the alteration of Z line. At the end of this process an amorphous mass of proteins was observed. Shortly following envenomation, sarcoplasmic reticulum showed moderate dilatation and proliferation, but when degenerative changes were more evident, smaller and more abundant vacuoles were seen. Also, the plasma membrane was altered and disrupted in some fibers, whereas many mitochondria showed a swollen appearance and, in some cells, they were disrupted. An amorphous extracellular material was also observed as a consequence of envenomation. It is proposed that the ultrastructural basis of the muscular necrosis induced by terciopelo venom is the disruption and severe alteration of the myofibril architecture.     

Mionecrosis, hemorragia y edema inducidos por el veneno de Bothrops asper en ratón blanco

Pathogenesis of the myonecrotic, hemorrhagic and edema-forming effects in white mice inoculated with Bothrops asper venom was studied at different time intervals by means of light microscopy, determination of serum levels of the enzyme creatinephosphokinase (CPK), and evaluation of edema. Although the three effects were evident in the first hour, their development was relatively independent. Myonectoric activity reached its maximum levels in the first 3 hr, as judged by serum CPK. This indicates that myonecrotic toxins have a very rapid action on muscle fibers, after which CPK levels decrease to near normal values 24 hr after venom inoculation. Histological studies showed severe myonecrosis at 3, 6, 9, 12 and 24 hr, characterized by myolytic and coagulative necrotic fibers mixed with normal fibers. Maximum hemorrhagic effect was observed between 9 and 12 hr, whereas at 6, 9, 12 and 24 hr there was an intense polymorphonuclear leucocyte infiltration. On the other hand, 1 hr after venom injection there was a local edema of 44%, reaching a maximum value of 70% within the first 24 hr, decreasing afterward. This edema is more prolonged than that induced by Agkistrodon piscivorous or Trimeresurus elegans venoms. This could have important clinical implication since local swelling contributes to tissue compression and is related to other physiological alterations. One week after venom inoculation there were fibroblasts, fragments of necrotic muscle fibers, and a mononuclear cell exudate. At this time, we did not observe hemorrhagic areas. Determination of serum levels of CPK could be a useful and powerful technique in order to quantify muscle tissue destruction due to snake bite. Since only scanty hemorrhagic and necrotic areas were observed in heart muscle, the drastic and rapid increase in CPK levels mainly reflected skeletal muscle necrosis. CPK determination is a simple and quick laboratory technique that should be used to evaluate muscle necrosis and to improve treatment in snake venom poisoning.     

Efectos locales inducidos por el veneno de la serpiente coral Micrurus nigrocinctus en ratón blanco

The venom of the coral snake, Micrurus nigrocintus, from Costa Rica produces a myonecrotic effect in the white mouse. Experimental inoculation induces histological necrosis of the skeletal muscles, which becomes evident a few min after inoculation. This activity was confirmed by high levels of the enzyme creatinephosphokinase (CPK) which reach maximum levels 3 hr after inoculation, descending to near normal values at 24 hr. An infiltrate rich in polymorphonuclear neutrophils was present, reaching a maximum at 24–48 hr. One or two weeks after inoculation, the histological picture was of necrotic and regenerative tissue, fibroblasts and exudative cells of a mononuclear type. There were no vascular alterations nor hemorrhagic effects and the Kondo skin test in mice was negative. Furthermore, the venom produced little edema at the inoculation site and a weak proteolytic effect on casein.     

Resistencia inducida del conejo a dosis elevadas de veneno de Loxosceles laeta

Venom of the spider Loxosceles laeta was prepared from venom glands triturated in saline. Venom of female spiders had a minimum necrotizing dose of 1/16 gland and an ₅₀ of 1·5 glands on i.d. injection into rabbits. Venom of male spiders was much weaker and was not used in the experiments dealing with resistance. When increasing doses of venom are given to rabbits on alternate days, tolerance is rapidly established. After 8 injections, the animals can withstand a dose of 64 glands without ill effect other than the production of a local necrotic lesion. This resistance lasts at least 120 days after the last injection. Precipitins were demonstrated in the serum of the rabbits on the sixth day and reached a high titer by the fifteenth day. The resistance of the rabbits is believed to be due to specific antibody response plus increased ability of the animal to detoxify and eliminate the venom.     

Venom of Bothrops asper from Mexico and Costa Rica: Intraspecific variation and cross-neutralization by antivenoms

Bothrops asper is the species that induces the highest incidence of snakebite envenomation in southern Mexico, Central America and parts of northern South America. The intraspecies variability in HPLC profile and toxicological activities between the venoms from specimens collected in Mexico (Veracruz) and Costa Rica (Caribbean and Pacific populations) was investigated, as well as the cross-neutralization by antivenoms manufactured in these countries. Venoms differ in their HPLC profiles and in their toxicity, since venom from Mexican population showed higher lethal and defibrinogenating activities, whereas those from Costa Rica showed higher hemorrhagic and in vitro coagulant activities. In general, antivenoms were more effective in the neutralization of homologous venoms. Overall, both antivenoms effectively neutralized the various toxic effects of venoms from the two populations of B. asper. However, antivenom raised against venom from Costa Rican specimens showed a higher efficacy in the neutralization of defibrinogenating and coagulant activities, thus highlighting immunochemical differences in the toxins responsible for these effects associated with hemostatic disturbances in snakebite envenoming. These observations illustrate how intraspecies venom variation may influence antivenom neutralizing profile.     

Papel del farmacéutico de hospital en la prevención,identificación y manejo de los efectos adversos asociados al tratamiento antirretroviral

At present, the side effects associated with antiretroviral treatment are the main reasons for discontinuation of this kind of therapy, both in clinical trials and in regular clinical practise.On the other hand, due to the change of direction that our profession has suffered in recent years, we face the need to establish a different relationship with the patient, achieving direct and effective Pharmaceutical Care within a framework of shared responsibility for therapeutic results.Pharmacist interventions should be aimed at improving the quality of life of patients, which can only be achieved with a multidisciplinary approach and individualised and adjusted to new patterns of toxicity of the drugs currently used.The pharmacist who does this work must know how to interpret these side effects, giving accurate information to the patient about both pharmacological and non-pharmacological treatment and correct pharmaceutical follow-up which clearly sets forth the criteria for referral to medical appointments.The aim of this paper is to establish baselines so that the hospital pharmacist can perform clearly and uniformly in the prevention, identification and management of major side effects: gastrointestinal, cardiovascular, dermatological, at the central nervous system and kidney level, associated with antiretroviral therapy.     

Neutralization of local effects of the terciopelo (Bothrops asper) venom by blood serum of the colubrid snake Clelia clelia

The neutralizing capacity of the blood serum of the non-venomous snake Clelia clelia against the hemorrhagic, edematous and myonecrotic effects of Bothrops asper venom in white mice was tested using in vitro preincubation experiments. Untreated serum neutralized to a different extent all local effects evaluated, but showed toxicity to mice, evidenced by edema and myonecrosis. Serum heated at 56°C for 30 min lost its toxic properties but also its neutralizing capacity against hemorrhagic and edematous effects of the venom. The myonecrotic effect was neutralized by large quantities of heated serum. The serum of C. clelia formed a single precipitation arc in immunoelectrophoretic slides against B. asper venom, corresponding to the slow anodic proteins of the serum.     

Immunochemical and biological characterization of monoclonal antibodies against BaP1, a metalloproteinase from Bothrops asper snake venom

BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (K_d), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The K_ds of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure-function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.     

Skeletal muscle regeneration after myonecrosis induced by crude venom and a myotoxin from the snake Bothrops asper (Fer-de-Lance)

Skeletal muscle regeneration was studied following injections of Bothrops asper venom and a myotoxin isolated from the crude venom. In toxin-injected muscle regeneration proceeded normally. By 4 days there were myotubes and small regenerating cells. The size of the cells increased by 1 and 2 weeks, and by 4 weeks regenerating cells were fully developed. The regenerated cells retained centrally located nuclei. The regenerative process in venom-injected muscle was not completely normal — by 1 and 2 weeks four main areas, based on the predominant cell type present, were observed in the tissue: (a) necrotic muscle cells; (b) regenerating muscle cells; (c) fibroblasts and collagen; (d) adipocytes. Furthermore, some nerve fibers were demyelinated. Samples obtained 4 weeks after venom injection showed an almost complete regeneration in many areas, whereas in other areas nests of small regenerating cells were surrounded by portions of adipose tissue and collagen. At four weeks regenerating cells in venom-injected muscle were significantly smaller than cells in toxin-injected and saline-injected muscles. There was a significant reduction in capillary/muscle cell ratio in areas of the muscle where hemorrhage and myonecrosis were present 30 min after injection of B. asper venom. Since B. asper venom drastically affects the microvasculature, it is proposed that impairment of regeneration after injection of crude venom is a consequence of diminished blood supply to some areas of the muscle.     

Identification of linear B-cell epitopes on myotoxin II, a Lys49 phospholipase A2 homologue from Bothrops asper snake venom

Knowledge on toxin immunogenicity at the molecular level can provide valuable information for the improvement of antivenoms, as well as for understanding toxin structure-function relationships. The aims of this study are two-fold: first, to identify the linear B-cell epitopes of myotoxin II from Bothrops asper snake venom, a Lys49 phospholipase A₂ homologue; and second, to use antibodies specifically directed against an epitope having functional relevance in its toxicity, to probe the dimeric assembly mode of this protein in solution. Linear B-cell epitopes were identified using a library of overlapping synthetic peptides spanning its complete sequence. Epitopes recognized by a rabbit antiserum to purified myotoxin II, and by three batches of a polyvalent (Crotalidae) therapeutic antivenom (prepared in horses immunized with a mixture of B. asper, Crotalus simus, and Lachesis stenophrys venoms) were mapped using an enzyme-immunoassay based on the capture of biotinylated peptides by immobilized streptavidin. Some of the epitopes identified were shared between the two species, whereas others were unique. Differences in epitope recognition were observed not only between the two species, but also within the three batches of equine antivenom. Epitope V, located at the C-terminal region of this protein, is known to be relevant for toxicity and neutralization. Affinity-purified rabbit antibodies specific for this site were able to immunoprecipitate myotoxin II, suggesting that the two copies of epitope V are simultaneously available to antibody binding, which would be compatible with the mode of dimerization known as “conventional” dimer.     

Comparison of the effect of Crotalus simus and Crotalus durissus ruruima venoms on the equine antibody response towards Bothrops asper venom: Implications for the production of polyspecific snake antivenoms

Antivenoms are preparations of immunoglobulins purified from the plasma of animals immunized with snake venoms. Depending on the number of venoms used during the immunization, antivenoms can be monospecific (if venom from a single species is used) or polyspecific (if venoms from several species are used). In turn, polyspecific antivenoms can be prepared by purifying antibodies from the plasma of animals immunized with a mixture of venoms, or by mixing antibodies purified from the plasma of animals immunized separately with single venom. The suitability of these strategies to produce polyspecific antibothropic-crotalic antivenoms was assessed using as models the venoms of Bothrops asper, Crotalus simus and Crotalus durissus ruruima. It was demonstrated that, when used as co-immunogen, C. simus and C. durissus ruruima venoms exert a deleterious effect on the antibody response towards different components of B. asper venom and in the neutralization of hemorrhagic and coagulant effect of this venom when compared with a monospecific B. asper antivenom. Polyspecific antivenoms produced by purifying immunoglobulins from the plasma of animals immunized with venom mixtures showed higher antibody titers and neutralizing capacity than those produced by mixing antibodies purified from the plasma of animals immunized separately with single venom. Thus, despite the deleterious effect of Crotalus sp venoms on the immune response against B. asper venom, the use of venom mixtures is more effective than the immunization with separate venoms for the preparation of polyspecific bothropic-crotalic antivenoms.     

A phospholipase A2 from Bothrops asper snake venom activates neutrophils in culture: Expression of cyclooxygenase-2 and PGE2 biosynthesis

In this study, the production of prostaglandin E₂ (PGE₂) and up-regulation in cyclooxygenase (COX) pathway induced by a phospholipase A₂ (PLA₂), myotoxin-III (MT-III), purified from Bothrops asper snake venom, in isolated neutrophils were investigated. The arachidonic acid (AA) production and the participation of intracellular PLA₂s (cytosolic PLA₂ and Ca²⁺-independent PLA₂) in these events were also evaluated. MT-III induced COX-2, but not COX-1 gene and protein expression in neutrophils and increased PGE₂ levels. Pretreatment of neutrophils with COX-2 and COX-1 inhibitors reduced PGE₂ production induced by MT-III. Arachidonyl trifluoromethyl ketone (AACOCF₃), an intracellular PLA₂ inhibitor, but not bromoenol lactone (BEL), an iPLA₂ inhibitor, suppressed the MT-III-induced AA and PGE₂ release. In conclusion, MT-III directly stimulates neutrophils inducing COX-2 mRNA and protein expression followed by production of PGE₂. COX-2 isoform is preeminent over COX-1 for production of PGE₂ stimulated by MT-III. PGE₂ and AA release by MT-III probably is related to cPLA₂ activation.     

Estudio comparativo de los venenos de serpiente Cascabel (Crotalus durissus durissus) de ejemplares adultos y recien nacidos

B. Lomonte, J. A. Gené, J. M. Gutiérrez and L. Cerdas. Estudio comparativo de los venenos de serpiente Cascabel (Crotalus durissus durissus) de ejemplares adultos y recién nacidos. Toxicon21, 379 – 384, 1983. — Venoms from adult and newborn Central American rattlesnakes (Crotalus durissus durissus) were compared for lethal, proteolytic, hemorrhagic, myonecrotic, edematigenous and in vitro hemolytic activities. Electrophoretic and immunoelectrophoretic patterns showed some differences between these venoms. Venom from newborn snakes was devoid of hemorrhagic and edematigenous activities, whereas the venom from adult specimens induced these effects. On the other hand, newborn snake venom showed higher lethality and indirect hemolytic activity, and lower proteolytic activity, than venom from adult specimens. Both types of venoms induced only slight myonecrosis in mice, as judged by histological observation. The ed₅₀ of an antivenom, in terms of absolute weight neutralized per ml of serum, was lower for the newborn specimens venom than for adult's venom, however, for each venom the number of ld₅₀ neutralized was similar.