Poincianella pluviosa is a tree whose stem bark is reputed to have antidiarrheal, wound healing, antitumoral and antimalarial properties. Considering that the leaves of the tree are a more abundant and renewable source than the stem bark, we chose to study the antimalarial potential of this organ part. The aqueous leaf extract exhibited a rich phenolic profile. It was active (IC50= 6.88 ± 1.64 µg mL-1) against chloroquine resistant Plasmodium falciparum (W2) without toxicity to HepG2 A16 cells. The organic fraction was more active (IC50= 1.83 µg mL-1) than the aqueous leaf extract itself. Ten phenolic compounds were obtained from the latter. This is the first report on the phenolic composition of P. pluviosa leaves. Ellagic acid (IC50= 0.215 ± 0.007 µg mL-1) proved to be 32 fold more active than the aqueous leaf extract itself. Considering their renewable aspect, leaves of P. pluviosa appear to be a more interesting source of antimalarial compounds than stem bark.

Chemical profiles of extracts of four species from Laurencia complex (Ceramiales, Rhodophyta) from different populations collected along Southeast Brazilian coast were assessed by High Performance Liquid Chromatography coupled with a Diode Array Detector in order to observe geographic chemical variability. Aiming to evaluate the impact of chemical diversity on potential pharmaceutical uses, the extracts were tested against the promastigote form of Leishmania amazonensis. The most active extracts were submitted to anti-amastigote and cytotoxicity assays. Principal Component Analysis of the chromatograms resulted in four major groups of chemical profiles according to the presence of leishmanicidal chamigranes (-)-elatol and obtusol. The existence of chemotypes, displaying variable pharmacological action, is proposed for the differences observed in L. dendroidea samples. Although all extracts were found active against promastigote form of L. amazonensis, their efficacy was remarkably different and not related to the variation of (-)-elatol and obtusol, which indicates the presence of additional compounds with antileishmanial activity. Moreover, the active extracts also displayed anti-amastigote activity and none of them were considered cytotoxic. The results highlight that the knowledge of chemical geographic variability can be valuable in the search of new antileishmanial compounds from marine sources.

Ketoconazole is a clinically safe antifungal agent that also inhibits the growth of Leishmania spp. A study was undertaken to determine whether Leishmania parasites are prone to becoming resistant to ketoconazole by upregulating C14-demethylase after stepwise pharmacological pressure. Leishmania amazonensis promastigotes [inhibitory concentration (IC)50 = 2 µM] were subjected to stepwise selection with ketoconazole and two resistant lines were obtained, La8 (IC50 = 8 µM) and La10 (IC50 = 10 µM). As a result, we found that the resistance level was directly proportional to the C14-demethylase mRNA expression level; we also observed that expression levels were six and 12 times higher in La8 and La10, respectively. This is the first demonstration that L. amazonensis can up-regulate C14-demethylase in response to drug pressure and this report contributes to the understanding of the mechanisms of parasite resistance.

As pterocarpanquinonas 8a-c, previamente sintetizadas em nosso laboratório, e uma série homóloga de derivados, substâncias 9a-c preparadas neste trabalho, foram avaliadas em células de câncer de mama (MCF-7) e em cultura dos parasitas Leishmania amazonensis e Plasmodium falciparum. As substâncias 8a-c foram mais potentes que 9a-c nas células tumorais e em Leishmania amazonensis. Por outro lado, 9a-c mostraram ser as mais ativas sobre o Plasmodium falciparum. Todas as substâncias estudadas foram biosseletivas, apresentando baixa citotoxicidade para linfócitos murinos frescos e linfócitos humanos ativados pelo mitógeno fitoemoaglutinina (PHA).

Pterocarpanquinones 8a-c, previously synthesized in our laboratory, and an homologous series of derivatives, compounds 9a-c prepared in this work, were evaluated on breast cancer cells (MCF-7) and on the parasites Leishmania amazonensis and Plasmodium falciparum, in culture. Compounds 8a-c were more potent than 9a-c on tumor cells and Leishmania amazonensis. On the other hand, 9a-c showed to be more active on Plasmodium falciparum. All the compounds studied were bioselective, presenting negligible cytotoxicity against fresh murine lymphocytes and human lymphocytes activated by the mitogen phytohemaglutinin (PHA).

The critical role of interferon-gamma (IFN-g) in the resistance of C57Bl/6 mice to Leishmania major is widely established but its role in the relative resistance of these animals to L. amazonensis infection is still not clear. In this work we use C57Bl/6 mice congenitally deficient in the IFN-g gene (IFN-g KO) to address this issue. We found that IFN-g KO mice were as resistant as their wild-type (WT) counterparts at least during the first two months of infection. Afterwards, whereas WT mice maintained lesion growth under control, IFN-g KO mice developed devastating lesions. At day 97 of infection, their lesions were 9-fold larger than WT controls, concomitant with an increased parasite burden. At this stage, lesion-draining cells from IFN-g KO mice had impaired capacity to produce interleukin-12 (IL-12) and tumour necrosis factor-a in response to parasite antigens whereas IL-4 was slightly increased in comparison to infected WT mice. Together, these results show that IFN-g is not critical for the initial control of L. amazonensis infection in C57Bl/6 mice, but is essencial for the developmente of a protective Th1 type immune response in the later stages.

A Leishmania amazonensis é o principal agente etiológico da leishmaniose cutânea difusa, uma doença associada a respostas imunes anérgicas. Neste estudo nós mostramos que o extrato bruto de promastigotas de Leishmania amazonensis (LaAg), mas não de L. braziliensis (LbAg), contém substâncias que suprimem respostas proliferativas, espontâneas e mitogênicas, de células T. As substâncias supressoras no LaAg são termo-resistentes (100°C/1h) e parcialmente dependentes da atividade de proteases. A anergia de células T não foi devida à diminuição na produção de fatores de crescimento, uma vez que não foi revertida pela adição de: IL-2, IL-4, IFN-gama ou IL-12. O LaAg não inibiu a ativação de células T induzida por anti-CD3, sugerindo que a anergia é devida a um defeito na apresentação de antígenos. A anergia não foi devida à necrose celular, mas foi acompanhada de uma expressiva fragmentação de DNA nas células de linfonodos, indicativo de apoptose. Apesar da pré-incubação de macrófagos com LaAg ter inibido sua capacidade de apresentação de antigenos, este efeito não foi devido à apoptose dos primeiros. Estes resultados sugerem que a anergia de células T encontrada na leishmaniose difusa deve ser devida à apoptose dessas células que se segue à apresentação defeituosa de antígenos pelo antígeno do parasito.

Leishmania amazonensis is the main agent of diffuse cutaneous leishmaniasis, a disease associated with anergic immune responses. In this study we show that the crude antigen of Leishmania amazonensis (LaAg) but not L. braziliensis promastigotes (LbAg) contains substances that suppress mitogenic and spontaneous proliferative responses of T cells. The suppressive substances in LaAg are thermoresistant (100ºC/1h) and partially dependent on protease activity. T cell anergy was not due to a decreased production of growth factors as it was not reverted by addition of exogenous IL-2, IL-4, IFN-gamma or IL-12. LaAg did not inhibit anti-CD3-induced T cell activation, suggesting that anergy was due to a defect in antigen presentation. It was also not due to cell necrosis, but was accompanied by expressive DNA fragmentation in lymph node cells, indicative of apoptosis. Although pre-incubation of macrophages with LaAg prevented their capacity to present antigens, this effect was not due to apoptosis of the former. These results suggest that the T cell anergy found in diffuse leishmaniasis may be the result of parasite antigen-driven apoptosis of those cells following defective antigen presentation.