Treatment of leishmaniasis is a challenging subject. Although available, chemotherapy is limited, presenting toxicity and adverse effects. New drugs with antileishmanial activity are being investigated, such as antiparasitic compounds derived from plants. In this work, we investigated the antileishmanial activity of the biflavonoid amentoflavone on the protozoanLeishmania amazonensis. Although the antileishmanial activity of amentoflavone has already been reportedin vitro, the mechanisms involved in the parasite death, as well as its actionin vivo, remain unknown. Amentoflavone demonstrated activity on intracellular amastigotes in macrophages obtained from BALB/c mice (IC₅₀2.3 ± 0.93 μM). No cytotoxicity was observed and the selectivity index was estimated as greater than 10. Using BALB/c mice infected withL. amazonensiswe verified the effect of an intralesional treatment with amentoflavone (0.05 mg/kg/dose, in a total of 5 doses every 4 days). Parasite quantification demonstrated that amentoflavone reduced the parasite load in treated footpads (46.3% reduction by limiting dilution assay and 56.5% reduction by Real Time Polymerase Chain Reaction). Amentoflavone decreased the nitric oxide production in peritoneal macrophages obtained from treated animals. The treatment also increased the expression of ferritin and decreased iNOS expression at the site of infection. Furthemore, it increased the production of ROS in peritoneal macrophages infectedin vitro. The increase of ROSin vitro, associated with the reduction of NO and iNOS expressionin vivo, points to the antioxidant/prooxidant potential of amentoflavone, which may play an important role in the balance between inflammatory and anti-inflammatory patterns at the infection site. Taken together these results suggest that amentoflavone has the potential to be used in the treatment of cutaneous leishmaniasis, working as an ally in the control and development of the lesion.

The Vero cell line, a non-phagocytic cell, has supported the intracellular mechanism of Leishmania (L.) chagasi. This strain (MHOM/BR/501/MS00) was isolated from a human case of visceral leishmaniasis in Mato Grosso do Sul, Brazil and cultivated in Schneider's Drosophila medium with 20 % of heat inactivated fetal calf serum. It was allowed to infect the Vero cells at a ratio of 10 to 20 promastigotes per cell. Within six hours of incubation, promastigote forms were found attached to Vero cells without any particular orientation. The number of amastigotes per cell increased during the incubation period. Results showed that promastigotes of L. (L..) chagasi could interact, transform to amastigote forms and multiply in non-phagocytic cells, demonstrating a new model to study the intracellular cycle of this protozoan. La lignée de cellule Vero, qui n'est pas une cellule phagocytaire professionnelle, a supporté la croissance de Leishmania (L.) chagasi. Le parasite (MHOM/BR/501/MS00) a été isolé d'un patient souffrant de leishmaniose viscérale au Mato Grosso du Sud, Brésil. Les parasites ont été cultivés sur un milieu de culture de Schneider supplémenté avec 20 % de sérum fœtal bovin inactivé par la chaleur; ils ont été mis en contact avec les cellules à raison de 10 à 20 promastigotes par cellule. Après six heures d'incubation, les promastigotes sont retrouvés attachés aux cellules, sans orientation particulière. Le nombre de parasites par cellule augmente pendant la période d'incubation. Les résultats montrent que les promastigotes de L. (L.) chagasi peuvent interagir, se transformer en amastigotes et se multiplier dans des cellules qui ne sont pas des phagocytes professionnels. Ces résultats caractérisent ainsi un nouveau modèle pour étudier le cycle intracellulaire de ce parasite.

Leishmaniasis represents a complex of diseases with a broad clinical spectrum and epidemiological diversity, considered a major public health problem. Although there is treatment, there are still no vaccines for cutaneous leishmaniasis. Because Leishmania spp. is an intracellular protozoan with several escape mechanisms, a vaccine must provoke cellular and humoral immune responses. Previously, we identified the Leishmania homolog of receptors for activated C kinase (LACK) and phosphoenolpyruvate carboxykinase (PEPCK) proteins as strong immunogens and candidates for the development of a vaccine strategy. The present work focuses on the in silico prediction and characterization of antigenic epitopes that might interact with mice or human major histocompatibility complex class I. After immunogenicity prediction on the Immune Epitope Database (IEDB) and the Database of MHC Ligands and Peptide Motifs (SYFPEITHI), 26 peptides were selected for interaction assays with infected mouse lymphocytes by flow cytometry and ELISpot. This strategy identified nine antigenic peptides (pL1-H2, pPL3-H2, pL10-HLA, pP13-H2, pP14-H2, pP15-H2, pP16-H2, pP17-H2, pP18-H2, pP26-HLA), which are strong candidates for developing a peptide vaccine against leishmaniasis.

In the search for new compounds with antileishmanial activity, we synthesized a triazole hybrid analogue of the neolignans grandisin and machilin G (LASQUIM 25), which was previously found highly active against both promastigotes and intracellular amastigote forms of Leishmania amazonensis. In this work, we investigated the leishmanicidal effects of LASQUIM 25 to identify the mechanisms involved in the cell death of L. amazonensis promastigotes. Transmission electron microscopy (TEM) analysis showed marked effects of LASQUIM 25 (IC₅₀ = 7.2 µM) on the morphology of promastigote forms, notably on mitochondria. The direct action of the triazole derivative on the parasite was noticed over time from 2 h to 48 h, and cells displayed several ultrastructural alterations characteristic of apoptotic cells. Also, flow cytometric analysis (FACS) after TMRE staining detected changes in mitochondrial membrane potential after LASQUIM 25 treatment (64.83% labeling versus 83.38% labeling in nontreated cells). On the other hand, FACS after PI staining in 24 h-treatment showed a slight alteration in the integrity of the cell membrane, a necrotic event (16.76% necrotic cells versus 3.19% staining in live parasites). An abnormal secretion of lipids was observed, suggesting an exocytic activity. Another striking finding was the presence of autophagy-related lysosome-like vacuoles, suggesting an autophagic cell death that may arise as consequence of mitochondrial stress. Taken together, these results suggest that LASQUIM 25 leishmanicidal mechanisms involve some degree of mitochondrial dysregulation, already evidenced by the treatment with the IC₅₀ of this compound. This effect may be due to the presence of a methylenedioxy group originated from machilin G, whose toxicity has been associated with the capacity to generate electrophilic intermediates.