L.G.D. Heneine

Inhibition of mutalysin II, a metalloproteinase from bushmaster snake venom by human α2-macroglobulin and rabbit immunoglobulin

Mutalysin II is a 22.5-kDa zinc endopeptidase isolated from Lachesis muta muta snake venom. In order to determine whether the inhibitors human alpha2-macroglobulin (alpha2-M) and rabbit antibody to mutalysin II share a common mechanism, we have investigated the inhibition of mutalysin II by these two different glycoproteins. The proteolytic activity of mutalysin II with dimethylcasein as substrate was completely inhibited by human alpha2-M and by a purified rabbit antibody to mutalysin II. The protection of fibrin(ogen) digestion by alpha2-M was slightly better than the protection offered by the antibody. In addition, the purified antibody reacted only with the metalloproteinase in bushmaster venom, as demonstrated by immunodiffusion. SDS-PAGE analysis of reduced samples showed that the interaction of mutalysin II with alpha2-M resulted in the formation of high molecular complex ( approximately 180000) and M(r) 90000 fragments generated by the venom enzyme. Also, fragments at 85 and 23 kDa were detected under non-reducing conditions after incubation of rabbit immunoglobulin with enzyme. Proteolysis of dimethylcasein as substrate revealed that the stoichiometry of inhibition was 1.0 mol of human alpha2-M and 1.5 mol of rabbit IgG antimutalysin II per mole of enzyme. Furthermore, dimethylcasein hydrolysis indicated that several viperid snake venoms, including Bothrops atrox, B. alternatus and Trimeresurus flavoviridis cross-reacted with the specific rabbit antibody to varying degrees.

Detoxification of the T2 fraction from a scorpion (Tityus serrulatus, Lutz and Mello) venom by iodination and some immunogenic properties of the derivatives

The iodination of the T2 fraction abolished its lethal capacity, and doses up to 30 times the LD50 were injected i.p. in mice without noticeable toxic effects. The modified fraction retained its immunological properties. Antibodies generated against the iodinated T2 fraction were also reactive toward the native T2 fraction, T1 fraction and the whole soluble venom.

The effect of stepwise iodination on biological properties of Bothrops jararaca venom

By titrating 5 mg of native venom with aliquots of a 2 x 10(-2) M iodine monochloride solution, neutralization of lethality by the incorporation of iodine was found with 200 +/- 5 microliters of solution, and above, up to 310 +/- 10 microliters, when saturation with iodine was attained. Doses up to 1500 micrograms (equivalent to 32 LD50 of native venom), where injected i.p. in mice without lethal effects. Proteolytic, phospholipase A2 and esterolytic activities were greatly reduced, but a low activity persisted even in fully iodinated samples. Direct hemolysis was markedly inhibited, and incapacity to coagulate fibrinogen and horse plasma was also observed in the iodinated samples. Hemorrhage and necrosis in rat skin, caused by 20 micrograms of iodinated venom were not elicited by doses up to 120 micrograms of iodinated anavenom. In mice, the myonecrosis that resulted from direct i.m. injection of native venom, and the massive hemorrhage caused by 5 LD50 doses injected i.p. were abolished by venom iodination. Blood congestion in liver, spleen, kidneys, and lungs, almost disappeared with iodination to the level of neutralization, and was barely seen with venom samples iodinated to saturation. The clinical signs of impaired physical activity, appearing in mice injected with 700 to 1500 micrograms of the iodinated anavenom were intensified by captopril and attenuated by epinephrine.

Effects induced by Apis mellifera venom and its components in experimental models of nociceptive and inflammatory pain

The effects induced by Apis mellifera venom (AMV), melittin-free AMV, fraction with molecular mass < 10 kDa (F<₁₀) or melittin in nociceptive and inflammatory pain models in mice were investigated. Subcutaneous administration of AMV (2, 4 or 6 mg/kg) or melittin-free AMV (1, 2 or 4 mg/kg) into the dorsum of mice inhibited both phases of formaldehyde-induced nociception. However, F<₁₀ (2, 4 or 6 mg/kg) or melittin (2 or 3 mg/kg) inhibited only the second phase. AMV (4 or 6 mg/kg), but not F<₁₀, melittin-free AMV or melittin, induced antinociception in the hot-plate model. Paw injection of AMV (0.05 or 0.10 mg), F<₁₀ (0.05 or 0.1 mg) or melittin (0.025 or 0.050 mg) induced a nociceptive response. In spite of inducing nociception after paw injection, scorpion (Tityus serrulatus) or snake (Bothrops jararaca) venom injected into the dorsum of mice did not inhibit formaldehyde-induced nociception. In addition, AMV (6 mg/kg), but not F<₁₀ (6 mg/kg) or melittin (3 mg/kg), inhibited formaldehyde paw oedema. Concluding, AMV, F<₁₀ and melittin induce two contrasting effects: nociception and antinociception. AMV antinociception involves the action of different components and does not result from non-specific activation of endogenous antinociceptive mechanisms activated by exposure to noxious stimuli.

MODIFICATION OF BIOLOGICAL PROPERTIES OF PROTEIN TOXINS BY STEPWISE IODINATION

By gradual incorporation of stable iodine into toxins and whole venoms it is possible to abolish completely the physiological, lesion and lethal properties of the native components. The properties of iodinated antigens and from antibodies generated by these detoxified derivatives, are presented. The hapten is incorporated into tyrosyl and histidyl residues. The derivatives can be obtained in less than one hour. Within the same batch of protein, there is a determinable stoichiometric ratio hapten/protein to achieve the desired modified properties of the derivative. The iodinating solutions are easy to prepare, can be accurately standardized and have unlimited shelf lives. The cost of the whole procedure is very low. No side-effects, local or systemic were observed, even with prolonged use of the derivatives. The method was applied to toxic components and whole venom of the scorpion Tityus serrulatus, and the hypertensive, bradipneic, oliguric, lesional, lethal and cytotoxic effects were completely abolished. Polyclonal antibodies generated by these iodinated antigens neutralized the virulent effects of native components and reversed the α effects of the whole venom in frog sciatic nerves. They conferred active immunization in mice, rats, guinea pigs, goats, horses and pigeons. Crotoxin and whole venom of Crotalus durrissus terrificus lost the lesional and lethal activity, but conserving the immunogenic capacity. They produced antibodies against the native components, giving also vaccinal protection. While the virulent crotalic antigens had a cytotoxic activity, the iodinated antigens were highly mitogenic with human white cells. Five bothropic venoms were neutralized in the hemorrhagic, tissue lesion and lethal capacity, the derivatives were immunogenic. Repetitive sublethal doses of scorpionic, crotalic and bothropic venoms lead invariably to an amyloid-like deposit in tissues, whereas the iodinated samples were ineffective. Allergenic extracts of Schistosoma mansoni can be transformed into anallergic derivatives that retains antigenic properties. Violently allergenic extracts of Ascaris lumbricoides suum can be completely deactivated with iodination, but conserved immunological competence. Cholera, tetanus and botulinum toxins, as iodinated toxoids, had its lesional and lethal capacity completely avoided. Physiological proteins with strong biological activity can also be rendered innocuous. Iodinated insulin lost its capacity to lower blood glucose levels, but induced high avidity antibodies in guinea-pigs and rabbits. By iodination, kallikrein can be turned unable to contract rat uterus, and to liberate kinins from kinninogen. Modified tonin do not increase the blood pressure in rats. Aqueous extracts of Leptospira canis and L. icterohaemorrhagiae after iodination, were innocuous to hatched eggs, and immunogenic in mice and rabbits. A lectin from Macrotylema axillare, lost the hemaglutination capacity with only 75% of iodine saturation. The derivative was highly immunogenic in rabbits. Heavy iodination can transform self-antigens in non-self, generating antibodies in same species animals. All derivatives obtained were stable, did not show any reversion to toxicity, generated antibodies against the native antigens and gave active protection when injected in animals. The injections were also apparently painless. The time gap between the accident and the administration of antibodies is discussed for systemic and local effects. A new schedule for immunization, only feasible with toxoided venoms is presented. It is based on a clonal expansion induced by a small dose, followed by an exponential saturation dose of the same toxoid. The attainment of higher levels of protecting antibodies against the native antigen in the generated sera, is unmatched by other procedures. Data for practical use of iodination is presented.