ERIKA GRACIELLE PINTO
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- Antileishmanial and antitrypanosomal activity of the cutaneous secretion of Siphonops annulatus(2014) PINTO, Erika Gracielle; ANTONIAZZI, Marta Maria; JARED, Carlos; TEMPONE, Andre GustavoBackground: Among the tropical parasitic diseases, those caused by protozoans are considered a challenge to public health, being represented by leishmaniasis and Chagas disease. In view of the low effectiveness and toxicity of the current therapy, animal venoms such as amphibian secretions have been used as a promising source of new drug prototypes. The present work aimed to achieve bioguided fractionation of metabolites present in a cutaneous secretion of the caecilian Siphonops annulatus (Amphibia: Gymnophiona: Siphonopidae) with antileishmanial and antitrypanosomal activity. Methods: Through liquid-liquid partition and chromatographic techniques, the secretion was fractionated using bioguided assays. The 50% inhibitory concentration (IC50) of the main fraction (SaFr1) was studied against Leishmania (L.) infantum promastigotes and intracellular amastigotes, trypomastigotes of Trypanosoma cruzi and mammalian cells; viability was detected by the colorimetric MTT assay. By using a spectrofluorimetric assay with the probe SYTOX (R) Green and transmission electron microscopy (TEM), we also investigated the potential damage caused by SaFr1 in the plasma membrane and mitochondria of Leishmania. Results: The bioguided assay enabled isolation of a highly purified fraction (SaFr1) with an IC50 of 0.065 mu g/mL against promastigotes and 2.75 mu g/mL against trypomastigotes. Due to its high toxicity to peritoneal macrophages, SaFr1 showed no selectivity towards the intracellular forms of Leishmania. Ultrastructural studies with Leishmania demonstrated severe mitochondrial damage and the formation of large cytoplasmic vacuoles, leading to the parasite's death within a few hours. Nevertheless, it caused no alteration in the plasma membrane permeability as detected by the fluorescent probe and TEM. Conclusions: The present study demonstrated for the first time the antiparasitic activity of the skin secretion of the caecilian S. annulatus against Leishmania and T. cruzi, confirming that skin secretions of these amphibians, similarly to those of anurans and salamanders, are also potential tools for the development of new drug candidates against neglected diseases.
- Melittin induces in vitro death of Leishmania (Leishmania) infantum by triggering the cellular innate immune response(2016) PEREIRA, Andreia Vieira; BARROS, Gustavo de; PINTO, Erika Gracielle; TEMPONE, Andre Gustavo; ORSI, Ricardo de Oliveira; SANTOS, Lucilene Delazari dos; CALVI, Sueli; FERREIRA JR., Rui Seabra; PIMENTA, Daniel Carvalho; BARRAVIERA, BeneditoBackground: Apis mellifera venom, which has already been recommended as an alternative anti-inflammatory treatment, may be also considered an important source of candidate molecules for biotechnological and biomedical uses, such as the treatment of parasitic diseases. Methods: Africanized honeybee venom from Apis mellifera was fractionated by RP-C18-HPLC and the obtained melittin was incubated with promastigotes and intracellular amastigotes of Leishmania (L.) infantum. Cytotoxicity to mice peritoneal macrophages was evaluated through mitochondrial oxidative activity. The production of anti-and pro-inflammatory cytokines, NO and H2O2 by macrophages was determined. Results: Promastigotes and intracellular amastigotes were susceptible to melittin (IC50 28.3 mu g.mL(-1) and 1.4 mu g.mL(-1), respectively), but also showed mammalian cell cytotoxicity with an IC50 value of 5.7 mu g.mL(-1). Uninfected macrophages treated with melittin increased the production of IL-10, TNF-alpha, NO and H2O2. Infected melittin-treated macrophages increased IL-12 production, but decreased the levels of IL-10, TNF-alpha, NO and H2O2. Conclusions: The results showed that melittin acts in vitro against promastigotes and intracellular amastigotes of Leishmania (L.) infantum. Furthermore, they can act indirectly on intracellular amastigotes through a macrophage immunomodulatory effect.