Use of a fluorescent stain for evaluating in vitro infection with Leishmania panamensis.

Leishmaniasis is a group of endemic diseases produced by infection with Leishmania parasites and affects tropical and subtropical regions of the world. Due to the severe problems related to the treatment of this condition (resistance and toxicity), further studies are needed to evaluate new antileishmanial compounds. The activity of antileishmanial prototypes should be analyzed in models that allow a better interpretation of the findings with respect to natural infection. In this sense, the use of an infection model with macrophages and dendritic cells is a better than using promastigotes alone, in order to establish the potential leishmanicidal activity of a prototype compound. For infection analysis, staining with polychromatic dyes such as Giemsa plus microscopic examination is the gold standard. However, it is common to find problems associated with color uniformity, expertise of the observer, sensitivity and specificity of the technique. For this reason, it's necessary to develop tools and protocols to overcome such limitations. This study assessed the utility of the SYBR® Safe fluorescent dye, considering its affinity for nucleic acids as a useful property for staining the nucleus and kinetoplast of Leishmania parasites within an infected cell. Infection (and subsequent treatment) assays were performed in dendritic cells and macrophages infected with Leishmania panamensis parasites to compare SYBR® Safe and Giemsa stain for the same assay. Correlation coefficients were found to be above 0.9 for both techniques; however, unlike Giemsa, SYBR® Safe staining was easier and provided a clearer observation of internalized parasites. These results support the use of SYBR® Safe as a promising tool for evaluating potential antileishmanials given its advantages over the traditional technique.

Leishmanicidal activity of synthetic antimicrobial peptides in an infection model with human dendritic cells.

Different species of Leishmania are responsible for cutaneous, mucocutaneous or visceral leishmaniasis infections in millions of people around the world [14]. The adverse reactions caused by antileishmanial drugs, emergence of resistance and lack of a vaccine have motivated the search for new therapeutic options to control this disease. Different sources of antimicrobial molecules are under study as antileishmanial agents, including peptides with antimicrobial and/or immunomodulatory activity, which have been considered to be potentially active against diverse species of Leishmania[7,39]. This study evaluated the cytotoxicity on dendritic cells, hemolytic activity, leishmanicidal properties on Leishmania panamensis and Leishmania major promastigotes and effectiveness on parasite intracellular forms (dendritic cells infected with L. panamensis and L. major promastigotes), when each parasite in culture was exposed to different concentrations of a group of synthetic peptides with previously reported antimicrobial properties, which were synthesized based on their naturally occurring reported sequences. Dermaseptin, Pr-2 and Pr-3 showed inhibitory activity on the growth of L. panamensis promastigotes, while Andropin and Cecropin A (with a selectivity index of 4 and 40, respectively) showed specific activity against intracellular forms of this species. The activities of Andropin and Cecropin A were exclusively against the intracellular forms of the parasite, therefore indicating the relevance of these two peptides as potential antileishmanial agents. In the case of L. major promastigotes, Melittin and Dermaseptin showed inhibitory activity, the latter also showed a selectivity index of 8 against intracellular forms. These findings suggest Andropin, Cecropin A and Dermaseptin as potential therapeutic tools to treat New and Old World cutaneous leishmaniasis.