W.Dias da Silva

Comparison of the potency of three Brazilian Bothrops antivenoms using in vivo rodent and in vitro assays

Three Brazilian polyspecific Bothrops antivenoms were compared using standard W.H.O. rodent in vivo and in vitro assays of their ability to neutralize the principal venom activities of pooled whole Bothrops jararaca venom. On a volume basis, the antivenoms were equally effective in neutralizing lethal activity in mice, and there were only minor differences in their ability to neutralize venom-induced haemorrhage, necrosis and procoagulant activity. Antivenom efficacy in neutralizing defibrinogenation varied. However, when equal amounts of antivenom IgG were compared, it was found that the FUNED antivenom best neutralized lethality, haemorrhage, necrosis and fibrinogen clotting activity. Vital Brazil and FUNED antivenoms were equally effective in neutralizing plasma coagulant activity but Vital Brazil antivenom was the more effective in neutralizing defibrinogenation.

Endotoxemic-like shock induced by Loxosceles spider venoms: pathological changes and putative cytokine mediators

The systemic symptoms, tissue lesions and release of cytokines were analysed in four isogenic mouse strains with distinct haplotypes injected with various doses of Loxosceles intermedia spider venom. The estimated LD50 were 24.5 microg for C57Bl/6, 17.6 microg for BALB/c, 6.3 microg for C3H/HeJ and 4.6 microg for A/Sn mice. Prostration, acute cachexia, hypothermia, neurological disorders and hemoglobinuria were the signals preceding death. Accumulation of eosinophilic material inside the proximal and distal renal tubules and acute tubular necrosis were the most common histopathological findings. Death was prevented by previous treatment of venom with specific antivenom serum. The protein F35 purified from the whole venom retained the ability to induce the symptoms of the whole venom. The cytokines tumor necrosis factor (TNF), interleukins IL-6 and IL-10 and the radical nitric oxide were detected in serum at different levels after venom injection. These findings indicate that the state of shock produced in mice by whole endotoxin-free L. intermedia venom or by its purified fraction, protein F35, mimics the endotoxemic shock, that susceptibility to the systemic effects of the venom varies among mice of different haplotypes and that the pattern of in vivo cytokine release resembles that of endotoxemic shock.

Antigenic cross-reactivity among the venoms from several species of Brazilian scorpions

The venoms of seven species of scorpions living in different regions of Brazil were analysed with regard to their lethality, antigenic cross-reactivity and ability to induce antibody production. In mice, the tested scorpion venoms can be grouped as: (a) highly toxic: Tityus stigmurus Thorell (LD50 = 0.773 mg/kg), Tityus bahiensis (Perty) (LD50 = 1.062 mg/kg), Tityus serrulatus Lutz and Mello (LD50 = 1.160 mg/kg), and Tityus costatus (Karsch) (LD50 = 1.590 mg/kg); (b) moderately toxic: Tityus cambridgei Pocock (LD50 = 12.136 mg/kg); and (c) practically nontoxic: Rhopalurus agamemnon (Koch) (LD50 = 36.363 mg/kg), and Brotheas amazonicus Lourenço (LD50 = 90.909 mg/kg). On electrophoresis the venoms showed many protein bands displayed along the chromatogram, most of them cross-reacting in immunoelectrophoresis and immunoblotting using horse anti-T. serrulatus, anti-T. bahiensis or anti-T. serrulatus+T. bahiensis sera as probes. The antibodies present in these antivenoms combine with venom components as measured in vitro by the ELISA assay, and neutralize their lethal effects in vivo. These results indicate that horse anti-venoms against a mixture of T. serrulatus and T. bahiensis venoms or only against T. serrulatus venom yield an antibody population able to neutralize the toxic effects found in all venoms 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.

Caracterizacion de las actividades biologicas de los venenos ‘amarillo’ y ‘blanco’ de Crotalus durissus ruruima comparados con el veneno de Crotalus durissus terrificus. Poder neutralizante de los antivenenos frente a los venenos de Crotalus durissus ruruima

Abstract The biological activities of ‘yellow’ and ‘white’ venom of a rattlesnake Crotalus durissus ruruima Hoge, 1965, found in the savanna-like vegetation (cerrado) of northern Brazil (Roraima) and Venezuela have been studied, and compared to the reference Crotalus durissus terrificus venom. The lethal activity of venoms depended on the inoculation route. The most toxic venom was the white one. The venoms of C. d. terrificus and the yellow of C. d. ruruima had similar lethalities. The yellow venom of C. d. ruruima showed a caseinolytic activity three times higher than that obtained with either the venom of C. d. terrificus or the white one of the C. d. ruruima . Hemorrhagic and necrotic activities were found only in the yellow venom. White and yellow venoms from C. d. ruruima showed a similar action on fibrinogen; this thrombin-like action was greater with C. d. terrificus venom. On histopathological sections local and pulmonary hemorrhage was found only with the yellow venom, but myonecrotic activity was observed with both venoms of C. d. ruruima . Among all antivenoms studied, the anti-bothropic-crotalic was the best at neutralizing hemorrhagic and hemolytic activities. These results suggest that antivenom bothropic-crotalic should be used in the treatment of patients with snakebite by C. d. ruruima : besides its neutralization on lethal activity, it also neutralizes the hemorrhagic activity present in some venoms.

A single strain of Mycobacterium bovis bacillus Calmette-Guérin (BCG) grown in two different media evokes distinct humoral immune responses in mice

Two attenuated bacillus Calmette-Guérin (BCG) preparations derived from the same Moreau strain, Copenhagen but grown in Sauton medium containing starch and bacto-peptone (onco BCG, O-BCG), or asparagine (intradermal BCG, ID-BCG), exhibited indistinguishable DNA sequences and bacterial morphology. The number of viable bacilli recovered from spleen, liver and lungs was approximately the same in mice inoculated with the vaccines and was similarly reduced (over 90%) in mice previously immunized with either BCG vaccine. The humoral immune response evoked by the vaccines was, however, distinct. Spleen cell proliferation accompanying the growth of bacilli in tissue was significantly higher in mice inoculated with O-BCG. These cells proliferated in vitro upon challenge with the corresponding BCG extract. Previous cell treatment with mAb anti-CD4 T cells abolished this effect. Anti-BCG antibodies, as assayed either in serum by ELISA or by determining the number of antibody-producing spleen cells by the spot-ELISA method, were significantly higher in mice inoculated with ID-BCG. Anti-BCG antibodies were detected in all immunoglobulin classes, but they were more prevalent in IgG with the following distribution among its isotypes: IgG1>(IgG2a = IgG2b)>IgG3. When some well-characterized Mycobacterium tuberculosis antigens were used as substitutes for BCG extracts in ELISA, although antibodies against the 65-kDa and 96-kDa proteins were detected significantly, antibodies against the 71-kDa, 38-kDa proteins and lipoarabinomannan were only barely detected or even absent. These results indicate that BCG bacilli cultured in Sauton-asparagine medium permitted the multiplication of bacilli, tending to induce a stronger humoral immune response as compared with bacilli grown in Sauton-starch/bacto-peptone-enriched medium.

Activation of human complement system in paracoccidioidomycosis

Plasma samples of 14 patients with paracoccidioidomycosis were analysed for components that represent activation of the complement system. Most patients (12/13) showed significant titres of complement-fixing antibodies and 14/14 had increased C4d/C4 ratios. There was no conclusive correlation between these two immunological indices, however. Factor B values of patients were similar to normal donors and fragment Ba was not detected in any of the patients. These results indicate a classical complement pathway activation in the plasma of patients with paracoccidioidomycosis.

Complement Activation by Animal Venoms

AbstractDuring activation of complement (C) system, C4b and C3b attach covalently to targets such as cell membranes. Deposited C4b serve both as the focus for the assembly of the C3 convertases and as ligands for C3b receptors or inactivators. C4a, C3a and C5a, mediators of the early events of inflammation, are released into the fluid phase while C5b serves to organize the cytolytical complex C5b-C9. Among the Arthropoda the venom from some spiders (Loxosceles) contain activators of the mammalian C system, although the exact mechanism and point of the activation cascade in where they act were not yet elucidated. In the insect venoms as honeybee, yellow jacket, yellow hornet, white-facet hornet, caterpillars and wasp there are some components capable of reducing total haemolytic and serum levels. Interestingly, mellitin, a soluble haemolytic peptide present in bee venom shares structural homology with human C9. Scorpion venom apparently are not active on the C system. Snakes, belonging to ELAPIDAE, CROTALI...

Activation of human complement system Paracoccidioides brasiliensis and its deposition on the yeast form cell surface

The yeast form of Paracoccidioides brasiliensis strain Pb18 was able to activate C3 of normal human serum diluted in phosphate-buffered saline or EGTA-MgCl2 in vitro. C3 convertase was also permissive when Pb18 cells were pre-treated with a pool of immune serum from patients with paracoccidioidomycosis and incubated in serum diluted in EDTA-CaCl2. The components C3, and fragments C3c, C3d, C3g, factor H, factor B, C4 and C5b-9 were demonstrated on the Pb18 cell surface by immunofluorescence although no effect was seen on fungal viability.

A SENSITIVE AND SPECIFIC IMMUNOASSAY FOR THE MEASUREMENT OF THE ANTIBODIES PRESENT IN HORSE ANTIVENOMS ENDOWED WITH THE CAPACITY TO BLOCK THE PHOSPHOLIPASE A2-DEPENDENT HEMOLYSIS INDUCED BY SNAKE VENOMS

Phospholipase A2 (PLA2), a component of most snake venom toxins, cleaves 3-sn-phosphoglycerides releasing lysophosphatidyl-choline. The indirect quantitative assay method for PLA2 was standardized for specific antivenom titration in a fast and sensitive assay by the similarity with the hemolysis induced by PLA2 and by complement system in sheep erythrocytes. The curves obtained by plotting the degree of hemolysis against the doses of snake venom are concave to the abscissa axis following an equation similar to that previously described for the hemolysis induced by the C system. We observed that venoms of some Bothrops, Crotalus and Micrurus species contained around 1 x 10 to 10 Z/mg of venom, while the venom of Naja contained over one million Z/mg. Antibodies against PLA2 were titrated by incubating amounts of venom predetermined to give 1 to 5 Z with various dilutions of the antivenoms, and the remaining active PLA2 was determined in the hemolytic assay. We observed the following: a) the antivenoms contained specific antibodies against the PLA2 present in the corresponding venoms; b) cross-reactivity was not detected among PLA2 epitopes from venoms and nonspecific antivenoms; and c) the assay quantitatively performed determined the specific antibodies directed to epitopes on the molecule of PLA2. The method described in this paper is highly specific, sensitive and reproducible, besides being fast and inexpensive.