The control of human visceral leishmaniasis (VL) is hard since there are no vaccines available as well as the treatment is hampered by toxicity and resistant parasites. Furthermore, as human, and canine VL causes immunosuppression, the combination of drugs with immunostimulatory agents is interesting to upregulate the immunity, reducing side-effects, improving treatment approaches against disease. Herein, we assessed the immunochemotherapy using miltefosine along with a vaccine formulated by Leishmania braziliensis antigens + saponin + monophosphoryl lipid-A (LBSapMPL) in L. infantum-infected hamsters. Two months after infection, the animals received treatments, and after 15 days they were evaluated for the treatment effect. The potential anti-Leishmania effect of miltefosine + LBSapMPL-vaccine was revealed by a specific immune response activation reflecting in control of spleen parasitism using half the miltefosine treatment time. The treated animals also showed an increase of total and T-CD4 splenocytes producing IFN-γ and TNF-α and a decrease of interleukin-10 and anti-Leishmania circulating IgG. In addition, it was demonstrated that the control of spleen parasitism is related to the generation of a protective Th1 immune response. Hence, due to the combinatorial action of miltefosine with LBSapMPL-vaccine in immunostimulating and controlling parasitism, this immunochemotherapy protocol can be an important alternative option against canine and human VL.

Leishmania spp. parasites have a complex biological cycle presenting basically two different morphological stages, the amastigote and promastigote forms. In vitro cultivation allows a more complete study of the biological aspects of these parasites, indicating better conditions for infection, immunoassay tests, drug evaluations, and vaccines. Thus, we evaluated the three most used culture media for Leishmania spp., Grace's insect cell culture medium (Grace's), liver infusion tryptose (LIT), and Schneider's insect medium (Schneider's), without supplementation or supplemented with fetal calf serum (FCS) and bovine serum albumin (Albumin) to evaluate the growth, viability, and infectivity of the L. infantum promastigotes. It was observed that promastigote forms have a better growth in LIT and Schneider's with or without FCS when compared to that in Grace's. The supplementation with albumin promoted greater viability of the parasites independent of the medium. For in vitro infection of J774.A1 macrophages using light microscopy and flow cytometry analyses, FCS-supplemented LIT and Grace's promoted higher percentage of infected macrophages and parasite load compared with Schneider's media. Taken together, our results demonstrated that the supplementation of LIT culture medium with FCS is the most suitable strategy to cultivate Leishmania infantum parasites enabling the maintenance of growth and infective parasites for research uses.

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