, 2004). C1P is implicated in the stimulation of cell proliferation via a pathway that involves inhibition of acid sphingomyelinase and the simultaneous blocking of ceramide synthesis ( Gomez-Munoz et al., 2004). LPA is known to induce various biological and pathological responses such
as platelet aggregation, endothelial Enzalutamide cell line hyperpermeability, and pro-inflammatory responses by signaling through three G-protein-coupled receptors ( Anliker and Chun, 2004; Moolenaar et al., 2004). In this study, we defined the antigenic/immunogenic potential of PLlv and BLlv by ELISA and immunoblotting. Immune sera anti-PLlv and anti-BLlv were produced in rabbits and their cross-reactivity against L. gaucho, L. intermedia, Phoneutria nigriventer venoms and Tityus click here serrulatus scorpion venom was evaluated. Fig. 4A and B show the ELISA reactivity (A492 nm) using different serum dilutions (1:100
to 1:62,500). As expected, each serum reacted strongly against its own venom antigens, and also with venoms from L. intermedia and L. gaucho. Notably, PLlv ( Fig. 4A) is moderately more immunogenic than BLlv ( Fig. 4B). None of the antivenoms reacted with P. nigriventer spider or T. serrulatus scorpion venoms. These observations suggest the presence of similar antigenic identities or common epitopes across the four Loxosceles spiders venoms studied. The antigen–antibody reactivity was also examined using Calpain western blotting and the cross-reactivity between Loxosceles venoms and anti-PLlv and anti-BLlv antivenom sera were confirmed ( Fig. 5A and B). A strong cross-reactivity with components ranging from 25 to 35 kDa was evident. Proteins with molecular masses between 25 and 35 kDa have been found to be the most immunogenic components of Loxosceles venoms ( Barbaro et al.,
1996). Antibodies against dermonecrotic toxins can be responsible for the strong cross-reactivity in the ELISA assay of the four spider venoms analyzed in this study ( Barbaro et al., 1994; Guilherme et al., 2001). The in vivo neutralizing activity in rabbits immunized with whole PLlv or BLlv venoms was studied by assaying protection against dermonecrosis, hemorrhage and edema. Ten days after the last immunization, rabbits were challenged by intradermal injection of 10 μg whole venoms (PLlv or BLlv), an amount equivalent to 1 MND/kg ( Felicori et al., 2006). Rabbits immunized with PLlv and challenged showed full protection against dermonecrosis and 80–90% protection against the hemorrhagic activity induced by both venoms ( Fig. 6A). Concerning the edematogenic activity, immunized rabbits afforded about 50% protection to BLlv, but lower protection against PLlv ( Fig. 6A). On the other hand, rabbits immunized with BLlv ( Fig. 6B) showed similar pattern of neutralization for dermonecrosis and edema, but close to 50% protection against the hemorrhagic-inducing activities by BLlv.