ABSTRACT
Therapeutic options for the treatment of leishmaniasis are insufficient and need improvements owing to their low efficiency and high toxicity as well as the emergence of resistant strains. The limited number of new drugs for neglected diseases and lack of innovation in your development are still challenges. In this context, the process of discovery and development of biological assays play a pivotal role for the identification of bioactive compounds. The assays currently used for screening of drugs with cytotoxic activity against Leishmania parasites, include different processes that utilize intact parasite (free or intracellular) or specific enzymes of metabolism as a target cell. These assays allow the screening of large numbers of samples followed by more detailed secondary confirmatory assays to confirm the observed activity and assess their toxicity. In the present study, we described the development of a new functional and more complete assay that enables simultaneous assessment of potential anti-Leishmania compounds through evaluation of internalization of fluorescein-labeled L. braziliensis promastigotes by human peripheral blood monocytes and their cytotoxicity by flow cytometry. We standardized the conditions for parasite labeling to achieve better phagocytosis analysis by setting the ratio of number of parasites per cell as 1 to 2, at incubation time of 6h. The cytotoxicity assessment was performed by the quantification of cells undergoing early/late apoptosis and necrosis using a double labelling platform employing 7AAD for late apoptosis and necrosis analysis and Annexin-V for early apoptosis evaluation. Hemolysis analysis was an additional parameter to test cytotoxicity. Two drugs used on clinic (Amphotericin B and Glucantime®) were used to validate the proposed methodology, and the assay was able to detect their known leishmanicidal activity and immunotoxicity properties. This new predictive assay will contribute to the development of translational medicine strategies in drug discovery for neglected diseases such as leishmaniasis.
Subject(s)
Animal Testing Alternatives/methods , Antiprotozoal Agents/toxicity , Flow Cytometry/methods , Leishmania/drug effects , Neglected Diseases/drug therapy , Adult , Amphotericin B/pharmacology , Amphotericin B/toxicity , Animals , Antiprotozoal Agents/pharmacology , Antiprotozoal Agents/therapeutic use , Drug Discovery/methods , Drug Evaluation, Preclinical/methods , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Humans , Leishmania braziliensis/drug effects , Leishmaniasis/drug therapy , Leukocytes/drug effects , Leukocytes/parasitology , Meglumine Antimoniate/pharmacology , Meglumine Antimoniate/therapeutic use , Meglumine Antimoniate/toxicity , Microscopy, Confocal , Middle Aged , Monocytes/drug effects , Monocytes/parasitology , Time Factors , Young AdultABSTRACT
Leishmaniasis is one of the most important parasitic diseases after malaria. The standard treatment of leishmaniasis includes pentavalent antimonials (SbV); however, these drugs are associated with serious adverse effects. There have been very few studies pertaining to their side effects and mechanism of action in the fetus. This investigation examines the effects of meglumine antimoniate (MA) on the survival rate, angiogenesis and cellular apoptosis in the human umbilical vein endothelial cells (HUVECs). HUVECs were treated with varying doses of MA (100-800⯵g/ml) for 24, 48 and 72â¯h and the survival rate was studied by colorimetric assay, flow cytometry, immunocytochemistry, migration (scratch) assay and tube formation assay. The results of quantitative real-time PCR (qPCR) studies indicated that the most important genes involved in presenting angiogenesis included VEGF and its receptors (Kdr and Flt-1), NP1 and Hif-1α genes including the anti-apoptotic gene of Bcl2, were significantly reduced compared to the control group (pâ¯<â¯0.05). In contrast, the most leading genes involved in the phenomenon of apoptosis were P53, Bax, Bak, Apaf-1 and caspases 3, 8 and 9, which were significantly up regulated compared to the control group (pâ¯<â¯0.05).
Subject(s)
Antiprotozoal Agents/toxicity , Human Umbilical Vein Endothelial Cells/drug effects , Meglumine Antimoniate/toxicity , Apoptosis/drug effects , Apoptosis Regulatory Proteins/genetics , C-Reactive Protein/genetics , Cell Movement/drug effects , Cells, Cultured , Human Umbilical Vein Endothelial Cells/physiology , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Neovascularization, Physiologic/drug effects , Nerve Tissue Proteins/genetics , Tumor Suppressor Protein p53/genetics , Vascular Endothelial Growth Factor A/geneticsABSTRACT
The present gold standard of the treatment of cutaneous leishmaniasis (CL) is pentavalent antimonials either sodium stibogluconate (Pentostam) or meglumine antimoniate (Glucantime), These drugs are quite toxic. They are given by injection and usually administered intramuscularly or intravenously for three weeks or intralesionally for seven or more weeks. That is why the successful introduction of radiofrequency-induced heat therapy using a Thermomed™ 1.8 instrument administered in a single application, with minimal toxic effects, is so important for the treatment of CL.
