In vitro interaction between SURFACEN® and surfactant protein A against Leishmania amazonensis.

Leishmaniasis is caused by a parasite of the Leishmania genus, affecting more than 12 million people in 98 countries. The control of leishmaniasis remains a serious problem. There are currently no vaccines for leishmaniasis. The drugs available are toxic, expensive and frequently ineffective. The in vitro activity of SURFACEN® and SP-A against Leishmania amazonensis was evaluated. The combination of both products resulted in a synergic pharmacology effect, demonstrated by a fractional inhibitory concentration index <0.5. A more effective combination was a SURFACEN/SP-A ratio of 4:1, using a method of fixed ratio. The therapeutic effect of SURFACEN and SP-A as antileishmanial compounds was demonstrated, with a potentiation of activity when they were incubated in conjunction. Our results propose an exploration of these products in order to design new formulations against the Leishmania parasite.

Mitochondria and trypanosomatids: targets and drugs.

The family Trypanosomatidae, flagellated parasitic protozoa, is responsible for important infectious diseases in humans: sleeping sickness, Chagas diseases and leishmaniasis. Currently, development of effective vaccines against these parasites remains an unrealized goal, and clinical management is based on chemotherapeutics. Cost, toxicity and resistance problems of conventional drugs result in an urgent need to identify and develop new therapeutic alternatives. The sound understanding of parasites, biology is key for identifying novel lead structures and new drug targets. This article reviews current knowledge about mitochondrial drug targets and existing drugs against Trypanosoma and Leishmania. In the past, several targets in trypanosomatid mitochondria (electron transport chain, kDNA and topoisomerases, tRNA import and fatty acid synthesis) have been identified. It has been suggested that inhibition of certain targets is involved in triggering apoptosis by impairment of mitochondrial membrane potential and/or production of reactive oxygen species. The inhibitory mechanism of approved drugs, such as pentamidine, nifurtimox, artemisinin and atovaquone, is described in parallel with others products from preclinical studies. In spite of the large amount of genetic information, the analysis of the phenotype of the trypanosomatid mitochondrion in different life stages will remain a useful tool to design new active compounds with selective toxicity against these parasites.

Thiadiazine derivatives as antiprotozoal new drugs.

The 3,5-disubstituted tetrahydro-2H-1,3,5-thiadiazine-2-thione scaffold have found many applications in recent years. This review is aimed at highlighting the most important aspects about these compounds: synthesis, spectroscopic characterization and antiprotozoan activities. How the chemical nature of N-substituents influences the overall activity / cytotoxicity profile will also be discussed.

Leishmanicidal activity of Echinaster (Othilia) echinophorus crude extract.

In this study, a methanolic extract from Echinaster (Othilia) echinophorus was evaluated for activity against Leishmania amazonensis. The extract showed activity against the promastigote and amastigote forms with IC50 values of 62.9 and 37.5 microg.mL-1 respectively. This extract showed a moderate toxicity on macrophages from BALB/c mice. A dose of 100 mg/kg/day was effective when administered during 15 days by intraperitoneal route to BALB/c mice infected experimentally.

Leishmanicidal activity of Echinaster (Othilia) echinophorus crude extract / Atividade leishmanicida do extrato de Echinaster (Othilia) echinophorus

In this study, a methanolic extract from Echinaster (Othilia) echinophorus was evaluated for activity against Leishmania amazonensis. The extract showed activity against the promastigote and amastigote forms with IC50 values of 62.9 and 37.5 μg.mL-1 respectively. This extract showed a moderate toxicity on macrophages from BALB/c mice. A dose of 100 mg/kg/day was effective when administered during 15 days by intraperitoneal route to BALB/c mice infected experimentally.

Neste estudo descreve-se o efeito de um extrato metanólico de Echinaster echinophorus spp. no parasita Leishmania amazonensis. Em testes com as formas promastigotas e amastigotas, o IC50 do extrato foi 62,9 e 37,5 μg.mL-1, respectivamente. O extrato também tem toxicidade moderada em macrófagos de camundongos BALB/c. O tratamento de camundongos BALB/c infectados com L. amazonensis com doses diárias de 100 mg/kg/dia via intraperitoneal durante 15 dias mostrou-se relativamente efetivo no controle da infecção. Esta investigação confirma a importância de produtos naturais como fonte para a descoberta de fármacos com funções anti-Leishmania.

Differentiation of Leishmania (Viannia) panamensis and Leishmania (V.) guyanensis using BccI for hsp70 PCR-RFLP.

Leishmania panamensis and Leishmania guyanensis are two species of the subgenus Viannia that are genetically very similar. Both parasites are usually associated with cutaneous leishmaniasis, but also have the potential to cause the mucocutaneous form of the disease. In addition, the study of foci and consequently the identification of vectors and probable reservoirs involved in transmission require a correct differentiation between both species, which is important at epidemiological level. We explored the possibility of identifying these species by using restriction fragment length polymorphisms (RFLP) in the gene coding for heat-shock protein 70 (hsp70). Previously, an hsp70 PCR-RFLP assay proved to be very effective in differentiating other Leishmania species when HaeIII is used as restriction enzyme. Based on hsp70 sequences analysis, BccI was found to generate species-specific fragments that can easily be recognized by agarose gel electrophoresis. Using the analysis of biopsies, scrapings, and parasite isolates previously grouped in a cluster comprising both L. panamensis and L. guyanensis, we showed that our approach allowed differentiation of both entities. This offers the possibility not only for identification of parasites in biological samples, but also to apply molecular epidemiology in certain countries of the New World, where several Leishmania species could coexist.

[Immunization with Leishmania amazonensis subgenomic libraries protects BALB/c mice against the challenge]. / Inmunización con subgenoteca de leishmania amazonensis protege contra el reto a ratones BALB/c.

A genomic library of Leishmania amazonensis in expression vector of eukaryote cells (pEF1HisA, pEF1HisB, pEF1HisC) was prepared. Also two subgenomic libraries having each 500 clones approximately were created and BALB/c mice were immunized with 50 mg/0,1 mL of DNA from each. Two immunizations were administered intramuscularly at 15-day interval. Groups of control mice were immunized with DNA from empty plasmid pEF1His, with soluble parasite antigen (100 mg/0,1 mL) and saline solution. The size of lesions was measured for 12 weeks and at the end of the experiment, the parasite load at lesion sites was determined by plaque microtitration method. In mice immunized with subgenomic library DNAI and with soluble antigens,the size of lesions was controlled, which reached an statistical difference (p< 0,05) in relation to the rest of groups whose lesions increased. The parasite load found in lesion sites confirmed the previous results; the number of promastigots was significantly lower in those mice already protected. It was concluded that in subgenomic library DNA1 there should be genes or gene fragments whose in vivo expression induces protective immune response against the challenge in the murine model used.

Significant and transitory control of lesions development in mice immunized with a genomic library of Leishmania amazonensis

DNA from a genomic library of Leishmania amazonensis and from pcDNA3 plasmid were used to immunize BALB/c mice. Three immunizations at two weeks intervals were done, with 50µg/0.1ml of DNA. A control group was also injected with the same volume of saline solution. Afterward, all mice were challenged with infective promastigotes of the parasite, and the development of lesions was followed during 16 weeks by dorsoventral measures of the footpad. Mice previously immunized with the genomic library were capable of controlling lesions at a significant level, with major significance between 9 and 13 weeks post challenge.