Evaluation of antivenoms in the neutralization of hyperalgesia and edema induced by Bothrops jararaca and Bothrops asper snake venoms

Neutralization of hyperalgesia induced by Bothrops jararaca and B. asper venoms was studied in rats using bothropic antivenom produced at Instituto Butantan (AVIB, 1 ml neutralizes 5 mg B. jararaca venom) and polyvalent antivenom produced at Instituto Clodomiro Picado (AVCP, 1 ml neutralizes 2.5 mg B. aspar venom). The intraplantar injection of B. jararaca and B. asper venoms caused hyperalgesia, which peaked 1 and 2 h after injection, respectively. Both venoms also induced edema with a similar time course. When neutralization assays involving the independent injection of venom and antivenom were performed, the hyperalgesia induced by B. jararaca venom was neutralized only when bothropic antivenom was administered iv 15 min before venom injection, whereas edema was neutralized when antivenom was injected 15 min or immediately before venom injection. On the other hand, polyvalent antivenom did not interfere with hyperalgesia or edema induced by B. asper venom, even when administered prior to envenomation. The lack of neutralization of hyperalgesia and edema induced by B. asper venom is not attributable to the absence of neutralizing antibodies in the antivenom, since neutralization was achieved in assays involving preincubation of venom and antivenom. Cross-neutralization of AVCP or AVIB against B. jararaca and B. asper venoms, respectively, was also evaluated. Only bothropic antivenom partially neutralized hyperalgesia induced by B. asper venom in preincubation experiments. The present data suggest that hyperalgesia and edema induced by Bothrops venoms are poorly neutralized by commercial antivenoms even when antibodies are administered immediately after envenomation.

Anti-inflammatory and antispasmodic activity of Ipomoea imperati (Vahl) Griseb (Convolvulaceae)

Ipomoea imperati (Convolvulaceae) lives on the sandy shores of the Brazilian coast and in other areas of the world. The anti-inflammatory activity of a methanol-water extract of the leaves of I. imperati was investigated in experimental models of acute and subchronic inflammation. Topical application of the extract (10 mg/ear) inhibited mouse ear edema induced by croton oil (89.0 ± 1.3% by the lipid fraction with an IC50 of 3.97 mg/ear and 57.0 ± 1.3% by the aqueous fraction with an IC50 of 3.5 mg/ear) and arachidonic acid (42.0 ± 2.0% with an IC50 of 4.98 mg/ear and 31.0 ± 2.0% with an IC50 of 4.72 mg/ear). Phospholipase A2, purified from Apis mellifera bee venom, was also inhibited by the extract (5.0 mg/ml lipid and aqueous fraction) in vitro in a dose-dependent manner (85% by the lipid fraction with an IC50 of 3.22 mg/ml and 25% by the aqueous fraction with an IC50 of 3.43 mg/ml). The methanol-water extract of I. imperati (1000 mg/kg) administered by the oral route also inhibited the formation of cotton pellet-induced granulomas (73.2 ± 1.2% by the lipid fraction and 56.14 ± 2.7% by the aqueous fraction) and did not cause gastric mucosal lesions. I. imperati extracts (10 mg/ml) also inhibited in a dose-dependent manner the muscle contractions of guinea pig ileum induced by acetylcholine and histamine (IC50 of 1.60 mg/ml for the lipid fraction and 4.12 mg/ml for the aqueous fraction). These results suggest the use of I. imperati as an anti-inflammatory and antispasmodic agent in traditional medicine.

Disintegrins: integrin selective ligands which activate integrin-coupled signaling and modulate leukocyte functions

Extracellular matrix proteins and cell adhesion receptors (integrins) play essential roles in the regulation of cell adhesion and migration. Interactions of integrins with the extracellular matrix proteins lead to phosphorylation of several intracellular proteins such as focal adhesion kinase, activating different signaling pathways responsible for the regulation of a variety of cell functions, including cytoskeleton mobilization. Once leukocytes are guided to sites of infection, inflammation, or antigen presentation, integrins can participate in the initiation, maintenance, or termination of the immune and inflammatory responses. The modulation of neutrophil activation through integrin-mediated pathways is important in the homeostatic control of the resolution of inflammatory states. In addition, during recirculation, T lymphocyte movement through distinct microenvironments is mediated by integrins, which are critical for cell cycle, differentiation and gene expression. Disintegrins are a family of low-molecular weight, cysteine-rich peptides first identified in snake venom, usually containing an RGD (Arg-Gly-Asp) motif, which confers the ability to selectively bind to integrins, inhibiting integrin-related functions in different cell systems. In this review we show that, depending on the cell type and the microenvironment, disintegrins are able to antagonize the effects of integrins or to act agonistically by activating integrin-mediated signaling. Disintegrins have proven useful as tools to improve the understanding of the molecular events regulated by integrin signaling in leukocytes and prototypes in order to design therapies able to interfere with integrin-mediated effects.

In vitro evaluation of the cytotoxic and trypanocidal activities of Ampelozizyphus amazonicus (Rhamnaceae)

Ampelozizyphus amazonicus Ducke is a tree commonly found in the Amazon region and an extract of its stem bark is popularly used as an antimalarial and anti-inflammatory agent and as an antidote to snake venom. Ursolic acid; five lupane type triterpenes: betulin, betulinic acid, lupenone, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid, and 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid, and three phytosteroids: stigmasterol, sitosterol and campesterol, have been isolated from stem extracts of A. amazonicus Ducke. Their structures were characterized by spectral data including COSY and HMQC. In an in vitro biological screening of the isolated compounds, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid was cytotoxic against the SKBR-3 human adenocarcinoma cell line (1 to 10 mg/mL), while 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid exhibited cytotoxicity against both SKBR-3 human adenocarcinoma and C-8161 human melanoma tumor cell lines (>0.1 mg/mL). In the present study, different extracts and some fractions of this plant were also investigated for trypanocidal activity due to the presence of pentacyclic triterpenes. The triterpene classes are potent against Trypanosoma cruzi. The bioassays were carried out using blood collected from Swiss albino mice by cardiac puncture during the parasitemic peak (7th day) after infection with the Y strain of T. cruzi. The results obtained showed that A. amazonicus is a potential source of bioactive compounds since its extracts and fractions isolated from it exhibited in vitro parasite lysis against trypomastigote forms of T. cruzi at concentrations >100 µg/mL. Fractions containing mainly betulin, lupenone, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid, and 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid showed more activity than crude extracts.