I. Mota

The effect of jararhagin, a metalloproteinase from Bothrops jararaca venom, on pro-inflammatory cytokines released by murine peritoneal adherent cells.

The release of pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) from murine peritoneal adherent cells (MPAC) was studied after exposure to jararhagin, a metalloproteinase/disintegrin isolated from Bothrops jararaca venom. MPACs were treated with LPS (lipopolysaccharide), jararhagin, or EDTA-inactivated jararhagin for up to 24h. Following incubation, the culture supernatant was assayed by ELISA for the presence of cytokines, while the cells were analysed for viability and cytokine mRNA expression. The cells exposed to native jararhagin released TNF-alpha and IL-1beta after 4 and 24h respectively. When MPACs were exposed to Jararhagin treated with EDTA, TNF-alpha and IL-1beta production was sustained throughout the culture period and IL-6 production was observed. TNF-alpha, IL-6 and IL-1beta mRNA were detected 4h after stimulation with either native or EDTA-treated jararhagin. Addition of jararhagin to LPS stimulated cells resulted in a dramatic decrease in the release of IL-6 and TNF-alpha. RT-PCR showed that this inhibition does not occur at the transcriptional level and further experiments showed that jararhagin degraded soluble cytokines by proteolytic activity. This study suggests that jararhagin induces TNF-alpha, IL-1beta and IL-6 expression, which may be rapidly degraded by its proteolytic activity.

Thalassophryne nattereri fish venom: biological and biochemical characterization and serum neutralization of its toxic activities

Envenomation by Thalassophryne nattereri fishes are an important medical problem in northeast of Brazil, causing in human victims considerable pain and edema followed by necrosis. Venom obtained from fresh captured specimens of this fish was tested in vitro or in animal models for a better characterization of its toxic activities. Intradermal injection of the venom in the foot pad of mice induced local edema and hemorrhage followed a few hours later by necrosis. Subcutaneous injection of the venom induced systemic effects consisting in jerking motions, paralysis of hind limbs, erection of hair, rotational movements and violent convulsions followed by death. Dead animals showed hyperemia of the small intestine and lungs. The venom showed distinct edematous, necrotizing and hemolytic activities, a low level of hemorrhagic, myotoxic and proteolytic activities and no detectable phospholipase A2 activity. SDS-PAGE analysis of the crude venom showed at least 17 components with the major band located around Mw = 19,000. Almost all proteins stained by amido black were also revealed by Western blotting with antibodies to T. nattereri venom. Fractionation of the venom by either gel filtration or cation exchange chromatography resulted in a few distinct peaks but in both situations the biological activities were located in only one of the peaks which corresponded to basic proteins with approximately Mw = 47,000. Heating of the venom at 56 degrees C for 60 min completely destroyed its biological activities. All venom toxic activities except edema were completely neutralized after in vitro incubation with anti-T. nattereri serum.

Toxic activities of venoms from nine Bothrops species and their correlation with lethality and necrosis.

The comparison of seven toxic activities contained in venoms from nine different species of Bothrops and the correlation of each activity with lethality and necrosis was the subject of this study. The haemorrhagic, coagulant, necrotizing, myotoxic, proteolytic and phospholipase activities were not equally distributed among the venoms studied except for the oedema-inducing activity which was almost equally distributed among them. The correlation coefficient was estimated for each activity in relation to lethality and necrosis induced by the venom. Lethality was significantly related to haemorrhagic and oedema-inducing activities, whereas the necrotizing activity showed significant correlation with phospholipase and coagulant activities. Proteolytic activity presented a significant inverse correlation with lethality.

Dermonecrotic and lethal components of Loxosceles gaucho spider venom

Loxosceles gaucho spider venom causes a typical dermonecrotic lesion in bitten patients and rarely causes lethal systemic effects. Gel filtration on Sephadex G 100 of L. gaucho spider venom resulted in three fractions: fraction A, containing the higher mol. wt components (approximately 35,000); fraction B, containing lower mol. wt components (approximately 15,000); and fraction C, containing very low mol. wt components (probably small peptides). The dermonecrotic and lethal activities were detected exclusively in fraction A. The venom and fraction A produced large dermonecrotic lesions in rabbits with necrosis spreading by gravity to the skin of the lateral body wall. Analysis by SDS-PAGE showed that the proteins contained in fraction A are approximately 35,000 and 33,000 mol. wt. Immunoblotting analysis showed that the proteins responsible for the dermonecrotic and lethal activity are very immunogenic and the first to be detected by antibodies during the course of immunization.

Compared chemical properties of dermonecrotic and lethal toxins from spiders of the genus Loxosceles (Araneae)

Loxosceles spider venom usually causes a typical dermonecrotic lesion in bitten patients, but it may also cause systemic effects that may be lethal. Gel filtration on Sephadex G-100 ofLoxosceles gaucho, L. laeta, orL. intermedia spider venoms resulted in three fractions (A, containing higher molecular mass components, B containing intermediate molecular mass components, and C with lower molecular mass components). The dermonecrotic and lethal activities were detected exclusively in fraction A of all three species. Analysis by SDS-PAGE showed that the major protein contained in fraction A has molecular weight approximately 35 kDa inL. gaucho andL. intermedia, but 32 kDa inL. laeta venom. These toxins were isolated from venoms ofL. gaucho, L. laeta, andL. intermedia by SDS-PAGE followed by blotting to PVDF membrane and sequencing. A database search showed a high level of identity between each toxin and a fragment of theL. reclusa (North American spider) toxin. A multiple sequence alignment of theLoxosceles toxins showed many common identical residues in their N-terminal sequences. Identities ranged from 50.0% (L. gaucho andL. reclusa) to 61.1% (L. intermedia andL. reclusa). The purified toxins were also submitted to capillary electrophoresis peptide mapping afterin situ partial hydrolysis of the blotted samples. The results obtained suggest thatL. intermedia protein is more similar toL. laeta toxin thanL. gaucho toxin and revealed a smaller homology betweenL. intermedia andL gaucho. Altogether these findings suggest that the toxins responsible for most important activities of venoms ofLoxosceles species have a molecular mass of 32–35 kDa and are probably homologous proteins.

Effect of Crotalus venom on the humoral and cellular immune response

The effect of venom of South American rattlesnake Crotalus durissus terrificus (cdt) on the humoral and cellular immune response was studied in BALB/c mice that were immunized with soluble antigens [human serum albumin (HSA) or chicken ovoalbumin (OVA)] or sensitized to DNFB 1 hr after venom injection. Pretreatment of the animals with cdt venom induced a significant reduction in the level of anti-OVA and anti-HSA IgG antibodies. The effect of crotoxin, a major neurotoxic component of cdt venom, its acidic non-toxic subunit (CA) and its basic phospholipase A2 subunit (CB) was also studied. The whole crotoxin molecule was as able as cdt venom to induce a significant decrease in the level of anti-OVA and anti-HSA IgG antibodies. However, the CA and CB subunits of crotoxin did not change the antibody level to either antigen, suggesting that the suppressive effect of crotoxin requires the intact molecule. Both cdt venom and the whole crotoxin molecule were able to induce a significant decrease in the level of anti-HSA IgG1 antibodies. The levels of other IgG isotypes and IgE were barely detectable and could not be estimated. In spite of their suppressive effect on the humoral immune response neither cdt venom nor crotoxin had any effect on the cellular immune response as estimated by contact sensitivity reaction to DNFB. It is suggested that cdt venom and its crotoxin component have an inhibitory effect on the humoral but not on the cellular immune response.

Neutralization of different activities of venoms from nine species of Bothrops snakes by Bothrops jararaca antivenom

Antivenoms are the usual treatment in cases of systemic envenoming by Bothrops snakes. However, the neutralization of each venom component by the antivenom is not well established. Bothrops jararaca antivenom, produced in rabbits, recognizes the venoms of nine different Bothrops species with high ELISA antibody titres. Western blot analysis showed that almost all antigens present on both homologous and heterologous venoms are recognized. Neutralization tests were performed using whole antivenom or its IgG fraction. The antivenom was able to neutralize the haemorrhagic, coagulant and necrotizing activities of the heterologous venoms in the same antivenom/venom proportion as for the homologous venom. Myotoxic activity was only partially neutralized. Neutralization of the proteolytic activity of heterologous venoms required higher amounts of antivenom than for the homologous venom. Phospholipase and oedema-inducing activities were completely neutralized only in the homologous system.

Antigenic cross-reactivity of venoms from medically important Loxosceles (Araneae) species in Brazil

Antigenic cross-reactivity between the components of venoms from three spiders of the genus Loxosceles, L. gaucho, L. laeta and L. intermedia, was studied. Species-specific antisera were prepared by immunization of rabbits with each venom. Anti-L. gaucho horse hyperimmune serum provided by the Butantan Institute for treatment of accidents with these spiders was also used. Separation by SDS-PAGE showed the existence of many common components in the three antigens. No individual antigen was observed. Analysis of the antisera by ELISA and Western blotting showed cross-reactivity as well as several common bands between the three venoms. The horse anti-L. gaucho venom serum recognized many common proteins when antigens of the other two species were used. Antigens in the range of 33,000-35,000 mol. wt showed most cross-reactivity. Both horse and rabbit anti-venom sera contained antibodies able to neutralize the lethal and dermonecrotic activities of the venom of the three species studied.

Antigenic cross-reactivity of venoms obtained from snakes of genus bothrops

Antigenic cross-reactivity was studied among the components of venoms from nine species of the genus Bothrops using species-specific antivenoms. Sera titration by DOT-ELISA detected similar levels of antibody when either homologous or heterologous antigens were used. Transblotted antigens, after SDS-PAGE fractionation, were also revealed by homologous and heterologous antivenoms. Antigens with mol. wt greater than 30,000 seemed to be the most cross-reactive. Antigens of about 24,000 mol. wt were poorly immunogenic. Antigens between 14-18,000 mol. wt cross-reacted only with B. moojeni, B. jararacussu, B. neuwiedi and B. pradoi venoms. Neutralization of the lethality of B. jararaca venom was observed by homologous and heterologous antivenoms.

Isolation of Ascaris suum components which suppress IgE antibody responses

An extract from adult Ascaris suum was fractionated on a Sephadex G-200 column and individual elution fractions assessed for their ability to induce or suppress an IgE antibody response in mice immunized simultaneously with a heterologous antigen (ovalbumin). The results showed that, whilst fractions eluted in the first peak suppressed the anti-ovalbumin IgE antibody production, those eluted in the third peak induced a significant anti-Ascaris IgE response. The mixture of the two kinds of fractions resulted in suppression of anti-Ascaris IgE antibodies. The apparent molecular weight (MW) of the first and third peaks was 530,000 and 29,000 daltons, respectively. After delipidation, the extract still retained most of its biological activities. SDS-PAGE of this material showed selective loss of high-MW components when compared to the whole extract. No bands were observed in SDS-PAGE of peak I in contrast to peak III, which displayed several bands. Immunoblots of all these samples showed at least 3 bands above MW 150,000, which reacted with anti-peak I antiserum, in the unfractionated extracts and in peak I, but not in peak III. When the extract was fractionated by affinity chromatography using anti-peak I antibodies as ligands, the immunogenic components were present in the effluent, whereas the suppressive components were recovered in the eluate.