Melittin induces in vitro death of Leishmania (Leishmania) infantum by triggering the cellular innate immune response

Abstract Background 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 μg.mL−1 and 1.4 μg.mL−1, respectively), but also showed mammalian cell cytotoxicity with an IC50 value of 5.7 μg.mL−1. Uninfected macrophages treated with melittin increased the production of IL-10, TNF-α, NO and H2O2. Infected melittin-treated macrophages increased IL-12 production, but decreased the levels of IL-10, TNF-α, 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.

Antileishmanial and antitrypanosomal activity of the cutaneous secretion of Siphonops annulatus

Background 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.) infantumpromastigotes and intracellular amastigotes, trypomastigotes ofTrypanosoma cruzi and mammalian cells; viability was detected by the colorimetric MTT assay. By using a spectrofluorimetric assay with the probe SYTOX® 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 g/mL against promastigotes and 2.75 g/mL against trypomastigotes. Due to its high toxicity to peritoneal macrophages, SaFr1 showed no selectivity towards the intracellular forms ofLeishmania. Ultrastructural studies withLeishmania demonstrated severe mitochondrial damage and the formation of large cytoplasmic vacuoles, leading to the parasites death within a few hours...

Antileishmanial and antitrypanosomal activity of the cutaneous secretion of Siphonops annulatus

Background 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.) infantumpromastigotes and intracellular amastigotes, trypomastigotes ofTrypanosoma cruzi and mammalian cells; viability was detected by the colorimetric MTT assay. By using a spectrofluorimetric assay with the probe SYTOX® 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 μg/mL against promastigotes and 2.75 μg/mL against trypomastigotes. Due to its high toxicity to peritoneal macrophages, SaFr1 showed no selectivity towards the intracellular forms ofLeishmania. Ultrastructural studies withLeishmania 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 againstLeishmania 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.(AU)

Investigação da atividade antimicrobiana do veneno de Rhinella icterica (Amphibia, Anura) / Research of the antimicrobial activity of the poison from (Amphibia, Anura)

Os anfíbios apresentam dois tipos de glândulas cutâneas: as mucosas e as granulosas. As secreções produzidas nas suas glândulas de sua pele apresentam componentes químicos diversos que têm sido estudados com relação as suas atividades biológicas, com efeito anestésico, alucinógeno e até antimicrobiano. Devido à diversidade de espécies no Brasil e ainda poucos estudos dessa natureza, o presente estudo objetivou investigar a atividade antimicrobiana do veneno das glândulas parotóides do sapo Rhinella icterica, procedentes do Distrito de Rubião Junior, Botucatu, estado de São Paulo. Foi avaliado o efeito de diferentes concentrações do veneno sobre colônias de bactérias Escherichia coli e Staphylococcusaureus, bem como o tempo necessário para a ação antimicrobiana. Observou-se que o veneno extraído apresentou atividade antimicrobiana leve para as duas bactérias estudadas, porém com maior ação para S. aureus. O veneno agiu somente em concentrações maiores de 50 mg/mL, com maior eficiência na concentração de 100 mg/mL, em tempo igualou superior a 30 minutos para S. aureus e a partir de 15 minutos para E. coli. Estes dados poderão servir de base para estudos futuros envolvendo o isolamento das substâncias do veneno que apresentaram atividade antimicrobiana e as concentrações mínimas necessárias para a referida ação.