José Roberto Marcelino

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

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 different 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 effective doses 50% (ED(50)) differed 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 effectively 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 effective in the neutralization of this activity. In conclusion, our results provide evidence of an extensive cross reactivity between these antivenoms and Central and South American crotaline snake venoms.

Production of the First Effective Hyperimmune Equine Serum Antivenom against Africanized Bees

Victims of massive bee attacks become extremely ill, presenting symptoms ranging from dizziness and headache to acute renal failure and multiple organ failure that can lead to death. Previous attempts to develop specific antivenom to treat these victims have been unsuccessful. We herein report a F(ab)´2-based antivenom raised in horse as a potential new treatment for victims of multiple bee stings. The final product contains high specific IgG titers and is effective in neutralizing toxic effects, such as hemolysis, cytotoxicity and myotoxicity. The assessment of neutralization was revised and hemolysis, the primary toxic effect of these stings, was fully neutralized in vivo for the first time.

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.

Characterization of anti-crotalic antibodies

Crotalus durissus terrificus, C. d. collilineatus, C. d. cascavella and C. d. marajoensis are responsible minor but severe snake bites in Brazil. The venoms of these snakes share the presence of crotoxin, a neurotoxin comprising of two associated components, crotapotin and phospholipase A2 (PLA2). Treatment of the victims with specific antiserum is the unique effective therapeutic measure. The ability of anti-Crotalus antisera produced by the routine using crude venom to immunize horses or purified crotoxin and PLA2 as individual immunogens was compared. Antisera obtained from horses immunized with C. durissus terrificus crude venom were able to recognize and neutralize not only the toxins presents in C. durissus terrificus, but also the ones present in the venoms from C. d. collilineatus, C. d. cascavella and C. d. marajoensis. Antisera from horses immunized with individual crotoxin or PLA2, although in lesser titers, were also able of recognizing the toxins in all four Crotalus species and neutralize the lethality of the C. d. terrificus venom.

Development of Equine IgG Antivenoms against Major Snake Groups in Mozambique

Background Snake envenoming is a significant public health problem in underdeveloped and developing countries. In sub-Saharan Africa, it is estimated that 90,000–400,000 envenomations occur each year, resulting in 3,500–32,000 deaths. Envenomings are caused by snakes from the Viperidae (Bitis spp. and Echis spp.) and Elapidae (Naja spp. and Dendroaspis spp.) families. The African continent has been suffering from a severe antivenom crisis and current antivenom production is only sufficient to treat 25% of snakebite cases. Our aim is to develop high-quality antivenoms against the main snake species found in Mozambique. Methods Adult horses primed with the indicated venoms were divided into 5 groups (B. arietans; B. nasicornis + B. rhinoceros; N. melanoleuca; N. mossambica; N. annulifera + D. polylepis + D. angusticeps) and reimmunized two times for antivenom production. Blood was collected, and plasma was separated and subjected to antibody purification using caprylic acid. Plasmas and antivenoms were subject to titration, affinity determination, cross-recognition assays and in vivo venom lethality neutralization. A commercial anti-Crotalic antivenom was used for comparison. Results The purified antivenoms exhibited high titers against B. arietans, B. nasicornis and B. rhinoceros (5.18 x 106, 3.60 x 106 and 3.50 x 106 U-E/mL, respectively) and N. melanoleuca, N. mossambica and N. annulifera (7.41 x 106, 3.07 x 106 and 2.60 x 106 U-E/mL, respectively), but lower titers against the D. angusticeps and D. polylepis (1.87 x 106 and 1.67 x 106 U-E/mL). All the groups, except anti-N. melanoleuca, showed significant differences from the anti-Crotalic antivenom (7.55 x 106 U-E/mL). The affinity index of all the groups was high, ranging from 31% to 45%. Cross-recognition assays showed the recognition of proteins with similar molecular weight in the venoms and may indicate the possibility of paraspecific neutralization. The three monospecific antivenoms were able to provide in vivo protection. Conclusion Our results indicate that the anti-Bitis and anti-Naja antivenoms developed would be useful for treating snakebite envenomations in Mozambique, although their effectiveness should to be increased. We propose instead the development of monospecific antivenoms, which would serve as the basis for two polyvalent antivenoms, the anti-Bitis and anti-Elapidae. Polyvalent antivenoms represent an increase in treatment quality, as they have a wider range of application and are easier to distribute and administer to snake envenoming victims.

Blood Evaluation Of Cl and Na Concentration In Crioulo Breed Horses Using NAA: Comparison With Humans Levels

Neutron Activation Analysis was utilized for determining the concentration of chlorine and sodium in blood of Crioulo breed horses used for hyperimmune sera production (Bothrops, Diphtheria and Tetanus) at Butantan Institute (Sao Paulo city, Brasil). These data are an important support for a toxicological control of adverse reactions in patients who will receive the hyperimmune serum.

Phenol used as a preservative in Bothrops antivenom induces impairment in leukocyte–endothelial interactions☆

The effect of Bothrops antivenom on blocking the disturbances induced by Bothrops jararaca venom in leukocyte-endothelial interactions (LEI) at the microcirculation of the cremaster muscle in mice was evaluated using intravital microscopy. Our findings showed that an i.v. injection of Bothrops antivenom, per se, induced changes in LEI, similar to those induced by an s.c. injection of B. jararaca venom, and that Bothrops antivenom can also induce ephemeral symptoms, such as tremor and dyspnea in mice. These effects were mostly due to phenol used in Bothrops antivenom as a preservative, since animals injected i.v. with a phenol solution, but not with phenol-free Bothrops antivenom, presented those effects on LEI, and also tremor and dyspnea. In addition, phenol-free antivenom abrogated venom-induced changes in LEI parameters. The present data demonstrate that Bothrops antivenom contains antibodies that neutralize toxins of B. jararaca venom that impair LEI, and suggest that the phenol used as a preservative in it can originate some undesired effects.

Does the administration of pilocarpine prior to venom milking influence the composition of Micrurus corallinus venom

Considering that the scarcity of venom represents a huge challenge for biochemical and functional studies of Micrurus species (coral snakes), in this report we describe for the first time the influence of pilocarpine administration prior to venom milking on the yield and protein composition of Micrurus corallinus venom. The administration of pilocarpine resulted in an increase of about 127% in the volume of venom milked, with similar protein content. Venoms showed similar protein bands distribution and intensity by SDS-PAGE and equivalents RP-HPLC profiles. Our proteomic analysis showed that venoms milked in the presence and absence of pilocarpine presented comparable protein profiles, in terms of protein composition and relative abundance. The toxins identified were assigned to 13 protein families and represent the most complete M. corallinus venom proteome described so far, in terms of number of protein families identified. Our data indicate that the administration of pilocarpine prior to venom milking increases the venom yield and does not change significantly the venom composition of M. corallinus. The employment of pilocarpine represents a useful approach to increase the yield of venom not only for Micrurus species, but also for other genera of snakes with limitations regarding the amount of venom available. SIGNIFICANCE In this report, we evaluated the influence of pilocarpine administration prior to venom milking in the overall composition of M. corallinus venom. We showed that the use of pilocarpine 10min before M. corallinus venom milking increases venom yield by ~127%. Not only the volume of venom obtained is higher, but also the protein concentration of both venoms is similar, opposing the idea that a more diluted venom is obtained as a result of pilocarpine administration, observed in non-front-fanged snakes. Shotgun proteomics analysis revealed that venom milked with and without the use of this drug showed similar overall protein composition and relative abundances. In addition, our proteomic approach allowed the identification of 13 toxin families in M. corallinus venom, representing the most complete M. corallinus venom proteome described so far. Moreover, two of these toxin families were identified for the first time in the venom of this species. Thus, considering the scarcity of Micrurus venom for biochemical and functional studies, we highlighted the usefulness of pilocarpine administration prior to venom milking to increase the venom yield of these snakes.

Analysis of Cl and Na in Hyperimmune Sera by NAA

The Cl and Na concentration values in four types of hyperimmune sera (anti‐Bothrops, anti‐Diphtheria, anti‐Rabies and anti‐Tetanus) used for immunological therapy were determined using Neutron Activation Analysis (NAA). These data were compatible with the specifications established by the Word Health Organization (WHO‐OMS) and with the Brazilian Official Pharmacopea (Pharmaceutical Code Official of the Country). These data are an important support for quality control of hyperimmune sera production at Butantan Institute (Sao Paulo city, Brazil), responsible for supplying the Brazilian market.

Blood Levels of Zinc in Creole Horses Used in Sera Production

Using Neutron Activation Analysis Zn concentrations were determined in blood of horses (Creole breed). No significant difference was observed between male (0.0029±.0007 gL−1) and female (0.0031±.0011 gL−1) animals. These data are an important support to understand the physiological functions of Zn in blood during the process of sera production at Butantan Institute (Sao Paulo, Brazil) using horses.