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Abstract

Leishmaniasis include a spectrum of human infectious disease, ranging from self-healing cutaneous ulceration to a progressive and lethal visceral infection. The disease is estimated to affect about 12 millions of people, with 400.000 new cases worldwide per year. The first-line drugs for Leishmaniasis treatment, remains on pentavalent antimonial, since their discover in the beginning of the century. Despite of the high toxicity and the cumbersome schedule, many reports discusses clinical resistance and treatment failures. Venoms of numerous land snakes posses cytotoxic or lytic effects on tumor cells in vitro. Growth inhibition of Trypanosomatidae by snake venoms was described, but no attempt to purify the active fraction was done. In this work, we demonstrate that Bothrops moojeni venom has a killing effect in vitro against Leishmania spp promastigotes, determined by cellular viability assay, using oxidative conversion of MTT. This activity was sequentially purified from crude venom by molecular exclusion and ion exchange chromatography. When eluted fractions were tested, anti-Leishmania and L-amino acid oxidase (L-AAO) activities coeluted in the same fractions. The molecular weight of the enzyme was estimated to be 140kDa by molecular exclusion chromatography, and 69kDa, by SDS-PAGE, migrating as a single band, with an isoeletric point of 4.8, determined by isoelectric focusing. This 135-fold purified L-AAO of B.moojeni venom is an acid enzyme with homodimeric constitution, active against Leishmania spp promastigotes from New World, with an efficient concen-Ultrastructural studies of promastigotes affected by L-AAO, demonstrated cytoplasmic death, with edema in several organelles, as mitochondria and nuclear membrane, before cell disruption. The action of L-AAO was demonstrated to be hydrogen peroxide-dependent, by abolishing the killing effect by catalase, absence of substrate or by showing similar effects by added hydrogen peroxide. These data could help in the development of alternative therapeutic approaches in leishmaniasis treatment.Financial Support: CNPq, LIMHCFMUSP(49). We observed previously that the aqueous extract of Kalanchoe pinnata (Kp) administrated by oral route controlled lesion of Balb/c mice infected with Leishmania amazonensis (Da-Silva et al, Acta Tropica, 60: 201-210, 1995). The protective effect of Kp in leishmaniasis was related with immunossupression (Rossi-Bergmann et al, Phytoterapy Research, 8: 399-402, 1994 ; Da-Silva et al, Acta Tropica, 60: 201-210, 1995) and nitric oxide production (Da-Silva et al, Parasitology 118: 575-582, 1999.). In the present work we investigated whether Kp treatment can modulate production of cytokines in normal and infected mice. We also investigated the effect of palmitic acid, a major fatty acid present in the most immunossupressive fraction of Kp, on cytokine production by normal mice. QM-2 -AQUEOUS EXTRACT OFNormal mice (4-5/group) were orally treated with 8mg of Kp extract or palmitic acid for 3 consecutive days, and then sacrificed. Alternativel...