In vitro sensitivity of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Brazilian isolates to meglumine antimoniate and amphotericin B Leishmania (Viannia) braziliensis is the most common species in the Americas and the most important causative agent of cutaneous and mucocutaneous leishmaniasis in Brazil, while Leishmania (Leishmania) amazonensis is the primary etiologic agent of the diffuse cutaneous form of the disease (Lainson & Shaw 1998).The therapeutic arsenal routinely employed to treat patients with leishmaniasis is limited and unsatisfactory. Successful leishmaniasis treatment depends on several aspects such as the immune status of the host, clinical manifestations of the disease and the sensitivity of the causative Leishmania. Widespread resistance to pentavalent antimonials has been identified for Leishmania (L.) donovani in some regions of India and Nepal (Sundar et al. 2000;Rijal et al. 2003). However, only a few studies correlated clinical response with in vitro antimony Tropical Medicine and International Health

Diffuse cutaneous leishmaniasis (DCL) is characterized by disseminated lesions and the absence of a specific cellular immune response. Here, the immunochemotherapy outcome of a patient with DCL from Amazonian Brazil infected with Leishmania (Leishmania) amazonensis is presented. After several unsuccessful chemotherapy treatment regimens and many relapses, a monthly immunotherapy scheme of L. amazonensis PH8 plus L. (Viannia) braziliensis M2903 monovalent vaccines associated with Bacillus Calmette-Guerin (BCG) was established, one round of which also included an M2903 vaccine associated with intermittent antimonial treatment. Temporary healing of all lesions was achieved, although Leishmania skin tests were negative and interferon gamma was not detected in mononuclear cell cultures stimulated with Leishmania antigens. The frequencies of CD16 (+)CD56(+) NK cells (approximately 2x) and CD14 (+)CD16(+) proinflammatory monocytes (approximately 8x) increased in peripheral blood, and CD56 (+) lymphocytes were found infiltrating the lesions. An association between the increase of the frequency of innate immune system cells and the healing of lesions is shown, suggesting that this protocol of immunotherapy reduced the parasite load and activated NK cells and monocytes.

Leishmania (Viannia) braziliensis causes cutaneous and mucosal leishmaniasis in several countries in Latin America. In mammals, the parasites live as amastigotes, interacting with host immune cells and stimulating cytokine production that will drive the type of the specific immune responses. Generation of Th17 lymphocytes is associated with tissue destruction and depends on IL-1β, IL-6, TGF-β and IL-23 production, whereas IL-10 and TGF-β are associated with tissue protection. Here, we evaluate whether amastigotes stimulate peripheral blood mononuclear cells (PBMCs) from healthy donors to produce the major cytokines responsible for the generation of Th17. Seven L. (V.) braziliensis isolates from patients with different clinical forms of leishmaniasis were expanded in interferon-γ knockout mice to obtain amastigotes and in culture to get promastigotes. The parasites were used to stimulate PBMCs from healthy donors, and cytokine production was evaluated by ELISA or qPCR. Amastigotes and promastigotes induced IL-10 production in PBMCs; however, only amastigotes induced IL-1β, IL-6 and TGF-β. These data demonstrate for the first time that L. (V.) braziliensis amastigotes directly stimulate production of a unique pattern of cytokines that could contribute to the generation of Th17.

BackgroundParasites of the Leishmania genus alternate between the flagellated extracellular promastigote stage and intracellular amastigotes. Here we report the characterization of a Leishmania isolate, obtained from a cutaneous leishmaniasis patient, which presents peculiar morphological features.MethodsThe parasite was cultured in vitro and characterized morphologically using optical and electron microscopy. Identification was performed based on monoclonal antibodies and internal ribosomal spacer typing. In vitro macrophage cultures, murine experimental models and sand fly infections were used to evaluate infectivity in vitro and in vivo.ResultsThe isolate was identified as Leishmania (Viannia) braziliensis. In the atypical promastigotes grown in culture, a short flagellum surrounded or interrupted by a protuberance of disorganized material was observed. A normal axoneme was present close to the basal body but without elongation much further outside the flagellar pocket. A disorganized swelling at the precocious end of the axoneme coincided with the lack of a paraflagellar rod structure. The isolate was able to infect macrophages in vitro, induce lesions in BALB/c mice and infect Lutzomyia longipalpis.ConclusionsNotwithstanding the lack of an extracellular flagellum, this isolate infects macrophages in vitro and produces lesions when inoculated into mice. Moreover, it is able to colonize phlebotomine sand flies. Considering the importance attributed to the flagellum in the successful infection and survival of Leishmania in the insect midgut and in the invasion of macrophages, these findings may bring new light into the infectious mechanisms of L. (V.) braziliensis.