Evaluation of antileishmanial drugs activities in an ex vivo model of leishmaniasis.

Leishmaniasis is a poverty-related disease, the chemotherapy of which is based on few drugs. The in vitro macrophage-amastigote model using mouse peritoneal cells, human-monocyte transformed macrophages and immortalized cell lines have been used to test new and safe antileishmanial drugs. Considering the differences for drug sensitivities between these Leishmania infected cells, the efficacy of amphotericin B, pentavalent antimonial, miltefosine and resveratrol was evaluated in a recently developed ex vivo culture of macrophages isolated from mouse lesion induced by L. amazonensis (CD11b+F4/80+CD68+CD14+) compared with infected peritoneal macrophages (CD11b+F4/80+CD68+CD14+). The results show that IC50 values of amphotericin B, miltefosine and pentavalent antimonial for parasites in lesional and peritoneal macrophages were similar, although high doses of these compounds did not result in total clearance of parasites in lesional cells (amphotericin B), peritoneal cells (miltefosine) and both cell cultures (pentavalent antimonial). Amastigotes infecting lesional macrophages were more resistant to resveratrol as compared to parasites in peritoneal macrophages. The cytoxicity of miltefosine and resveratrol was higher in infected peritoneal macrophages than in lesional cells. These data suggest that the antileishmanial effect and citotoxicity of some anti leishmanial compounds are dependent of macrophage source and mouse peritoneal macrophages loaded with amastigotes do not represent the lesion cell.

Long-term cell culture isolated from lesions of mice infected with Leishmania amazonensis: a new approach to study mononuclear phagocyte subpopulations during the infection.

Leishmanioses are neglected diseases and the parasite Leishmania survives and proliferates within mononuclear phagocytes, particularly macrophages. In vitro studies of the immunology and cell biology of leishmaniosis are performed in murine peritoneum and bone marrow macrophages and immortalized cell lines despite the normal and injured tissue-specific heterogeneity of macrophages. In this work, we established an ex vivo methodology to culture lesional cells from BALB/c mice infected with Leishmania amazonensis. The cells were successfully isolated from footpad skin lesions and those exhibiting macrophage morphology were maintained in long-term culture (12 days), while the small number of lymphocytes, polymorphonuclear and unidentified cells died after 1 day of culture. The frequency of infected cells decreased over 2 days. Most lesional cells cultivated ex vivo were myeloid CD11b+ CD14+ F4/80+ CD68+ cells. Low levels of IFN-γ and IL-4, IL-10 production and low arginase and phagocytic activities were detected in ex vivo lesional cell cultures. The ex vivo model developed in this study open perspectives for studying the biology of leishmanial lesions in cellular subpopulations and at the single-cell level.