Background: Leishmaniasis is a group of diseases with a variety of clinical manifestations. The form of the disease is highly dependent on the infectiveLeishmaniaspecies and the immunological status of the host. The infectivity of the parasite strain also plays an important role in the progression of the infection. The aim of this work is to understand the influence of the natural infectivity ofLeishmaniastrains in the outcome of visceral leishmaniasis.Methods: In this study we have characterized four strains ofL. infantumin terms of molecular typing,in vitrocultivation and differentiation. Two strains were isolated from HIV+ patients with visceral leishmaniasis (Bibiano and E390M), one strain was isolated from a cutaneous lesion in an immunocompetent patient (HL) and another internal reference strain causative of visceral leishmaniasis (ST) also from an immunocompetent patient was used for comparison. For this objective, we have compared their virulence byin vitroandin vivoinfectivity in a murine model of visceral leishmaniasis.Results: Molecular typing unraveled a newk26sequence attributed to MON-284 zymodeme and allowed the generation of a molecular signature for the identification of each strain.In vitrocultivation enabled the production of promastigotes with comparable growth curves and metacyclogenesis development. The HL strain was the most infective, showing the highest parasite loadsin vitrothat were corroborated with thein vivoassays, 6 weeks post-infection in BALB/c mice. The two strains isolated from HIV+ patients, both belonging to two different zymodemes, revealed different kinetics of infection.Conclusion: Differences ininvitro andin vivoinfectivity found in the murine model were then attributed to intrinsic characteristics of each strain. This work is supported by other studies that present the parasite’s inherent features as factors for the multiplicity of clinical manifestations and severity of leishmaniasis.

A differential behavior among infected and bystander dendritic cells (DCs) has been explored in different infection models. We have analyzed both populations sorted on contact with visceral Leishmania infantum on a susceptible mice model evaluating the subsequent repercussions on adaptive immune response. Our results demonstrate a clear dichotomy between the immunomodulatory abilities of bystander and infected DCs. The bystander population presents increased levels of IL-12p40 and costimulatory molecules being capable to induce CD4⁺ T cell activation with immune protective capabilities. In contrast, infected DCs, which express lower costimulatory molecules and higher levels of IL-10, promote the development of Leishmania Ag-specific, nonprotective T-bet⁺IFN-γ⁺IL-10⁺ CD4⁺ T cells with an effector phenotype. This specific polarization was found to be dependent on IL-12p70. Splenic infected DCs recovered from chronic infected animals are similarly capable to polarize ex vivo syngeneic naive CD4⁺ T cells toward a T-bet⁺IFN-γ⁺IL-10⁺ phenotype. Further analysis revealed that only MHC class II^high–infected DCs were responsible for this polarization. The adoptive transfer of such polarized CD4⁺ T cells facilitates visceral leishmaniasis in BALB/c mice in a clear contrast with their counterpart generated with bystander DCs that significantly potentiate protection. Further, we demonstrated that CD4⁺ T cells primed by infected DCs in an IL-10 free system, thus deprived of T-bet⁺IFN-γ⁺IL-10⁺ population, restore the immune response and reduce parasite load, supporting a deleterious role of IFN-γ⁺IL-10⁺ T cells in the maintenance of infection. Overall, our results highlight novel subversion mechanisms by which nonprotective T-bet⁺IFN-γ⁺IL-10⁺ T cells are associated with chronicity and prolonged parasite persistence.

SUMMARY: An ideal culture medium forLeishmaniapromastigotes should retain the basic characteristics of promastigotes found in sandflies (morphology and infectivity). Furthermore, the media should not create a bias in experimental settings, thus enabling the proper extrapolation of results. To assess this we studied several established media for promastigote growth. We analysed morphology, viability, cell cycle progression, metacyclic profile, capacity to differentiate into axenic amastigotes and infectivity. Furthermore, using a rational approach from the evaluated media we developed a simple serum-free medium (cRPMI). We report that parasites growing in different media present different biological characteristics and distinctin vitroandin vivoinfectivities. The developed medium, cRPMI, proved to be a less expensive substitute for traditional serum-supplemented media for thein vitromaintenance of promastigotes. In fact, cRPMI is ideal for the maintenance of parasites in the laboratory, diminishing the expected loss of virulence over time typical of the parasite cultivation. Ultimately this report is a clear warning that the normalization of culture media should be a real concern in the field as media-specific phenomena are sufficient to induce biological bias with consequences in infectivity and general parasite biology.

Leishmaniasis remains one of the world's most devastating neglected tropical diseases. It mainly affects developing countries, where it often co-exists with chronic malnutrition, one of the main risk factors for developing the disease. Few studies have been published, however, on the relationship between leishmaniasis progression and malnutrition. The present paper reports the influence of protein malnutrition on the immune response and visceral disease development in adult hamsters infected with Leishmania infantum fed either standard or low protein diets. The low protein diet induced severe malnutrition in these animals, and upon infection with L. infantum 33% had severe visceral leishmaniasis compared to only 8% of animals fed the standard diet. The infected, malnourished animals showed notable leukocyte depletion, mild specific antibody responses, impairment of lymphoproliferation, presence of parasites in blood (16.67% of the hamsters) and significant increase of the splenic parasite burden. Animals fed standard diet suffered agranulocytosis and monocytopenia, but showed stronger specific immune responses and had lower parasite loads than their malnourished counterparts. The present results show that protein malnutrition promotes visceral leishmaniasis and provide clues regarding the mechanisms underlying the impairment of the immune system.