BackgroundVisceral leishmaniasis (VL) is a severe disease caused by infection with protozoa of the genus Leishmania. Classic VL is characterized by a systemic infection of phagocytic cells and an intense activation of the inflammatory response. It is unclear why 90% of infected individuals do not develop the disease while a minority develop the classical form. Furthermore, among those that develop disease, a small group progresses to more severe form that is unresponsive to treatment. The presence of inflammatory mediators in serum could theoretically help to control the infection. However, there is also a release of anti-inflammatory mediators that could interfere with the control of parasite multiplication. In this study, we took advantage of the spectrum of outcomes to test the hypothesis that the immune profile of individuals infected with Leishmania (L.) infantum is associated with the development and severity of disease.Methodology/Principal FindingsSera from patients with confirmed diagnosis of VL were evaluated for the presence of numerous molecules, and levels compared with healthy control and asymptomatic infected individuals.Conclusions/Principal FindingsAlthough differences were not observed in LPS levels, higher levels of sCD14 were detected in VL patients. Our data suggest that L. infantum may activate the inflammatory response via CD14, stimulating a generalized inflammatory response with production of several cytokines and soluble molecules, including IFN-γ, IL-27, IL-10, IL-6 and sCD14. These molecules were strongly associated with hepatosplenomegaly, neutropenia and thrombocytopenia. We also observed that IL-6 levels greater than 200 pg/ml were strongly associated with death. Together our data reinforce the close relationship of IFN-γ, IL-10, IL-6, TNF-α and IL-27 in the immune dynamics of VL and suggest the direct participation of sCD14 in the activation of the immune response against L. infantum.

Development of immunoprotection against visceral leishmaniasis (VL) focused on the identification of antigens capable of inducing a Th1 immune response. Alternatively, antigens targeting the CD8 and T-regulatory responses are also relevant in VL pathogenesis and worthy of being included in a preventive human vaccine. We assessed in active and cured patients and VL asymptomatic subjects the clinical signs and cytokine responses to the Leishmania donovani nucleoside hydrolase NH36 antigen and its N-(F1), central (F2) and C-terminal (F3) domains. As markers of VL resistance, the F2 induced the highest levels of IFN-γ, IL-1β, and TNF-α and, together with F1, the strongest secretion of IL-17, IL-6, and IL-10 in DTH+ and cured subjects. F2 also promoted the highest frequencies of CD3+CD4+IL-2+TNF-α−IFN-γ−, CD3+CD4+IL-2+TNF-α+IFN-γ−, CD3+CD4+IL-2+TNF-α−IFN-γ+, and CD3+CD4+IL-2+TNF-α+IFN-γ+ T cells in cured and asymptomatic subjects. Consistent with this, the IFN-γ increase was correlated with decreased spleen (R = −0.428, P = 0.05) and liver sizes (R = −0.428, P = 0.05) and with increased hematocrit counts (R = 0.532, P = 0.015) in response to F1 domain, and with increased hematocrit (R = 0.512, P 0.02) and hemoglobin counts (R = 0.434, P = 0.05) in response to F2. Additionally, IL-17 increases were associated with decreased spleen and liver sizes in response to F1 (R = −0.595, P = 0.005) and F2 (R = −0.462, P = 0.04). Conversely, F1 and F3 increased the CD3+CD8+IL-2+TNF-α−IFN-γ−, CD3+CD8+IL-2+TNF-α+IFN-γ−, and CD3+CD8+IL-2+TNF-α+IFN-γ+ T cell frequencies of VL patients correlated with increased spleen and liver sizes and decreased hemoglobin and hematocrit values. Therefore, cure and acquired resistance to VL correlate with the CD4+-Th1 and Th-17 T-cell responses to F2 and F1 domains. Clinical VL outcomes, by contrast, correlate with CD8+ T-cell responses against F3 and F1, potentially involved in control of the early infection. The in silico-predicted NH36 epitopes are conserved and bind to many HL-DR and HLA and B allotypes. No human vaccine against Leishmania is available thus far. In this investigation, we identified the NH36 domains and epitopes that induce CD4+ and CD8+ T cell responses, which could be used to potentiate a human universal T-epitope vaccine against leishmaniasis.

Visceral leishmaniasis is a life-threatening disease characterized by intense parasitism of the spleen, liver, and bone marrow. Antimonials have served as front-line antileishmanial therapeutics for decades, but the increasing failure rates under antimonial treatment have challenged the continued use of these drugs. Pentavalent antimonials are known to reinforce the killing mechanisms of macrophages, although the associated mechanism remains unclear. Here, for the first time, we determined whether Leishmania infantum strains isolated from patients refractory to antimony treatment (relapse cases) were cross-resistant to antimonials, liposomal amphotericin B, and/or nitric oxide, and also whether these strains modulate macrophage infection. We selected four clinical isolates from relapse cases and two clinical isolates from antimony-responsive patients (control group) for the present study. The L. infantum promastigotes from all four relapse cases were resistant to trivalent antimonial treatment and nitric oxide, while only one isolate was resistant to liposomal amphotericin B. We evaluated whether the resistant strains from relapse cases showed enhanced infectivity and amastigote survival in macrophages, or macrophage-killing mechanisms in macrophages activated by lipopolysaccharide plus interferon gamma. Infection indexes calculated using macrophages infected with isolates from relapse were higher than those observed with control strains that were stimulated independently. Macrophage infection was higher with L. infantum isolates from relapse cases and correlated with enhanced interleukin 1-β production but showed similar nitrite production. Our results demonstrate that L. infantum field isolates from relapse cases were resistant to antimonials and nitric oxide and that these parasites stimulated inflammatory cytokines and were resistant to macrophage-killing mechanisms, factors that may contribute to disease severity.

The Leishmania (Leishmania) donovani nucleoside hydrolase NH36 is the main antigen of the Leishmune® vaccine and one of the promising candidates for vaccination against visceral leishmaniasis. The antigenicity of the N-terminal (F1), the central (F2), or the C-terminal recombinant domain (F3) of NH36 was evaluated using peripheral blood mononuclear cells (PBMC) from individuals infected with L. (L.) infantum from an endemic area of visceral leishmaniasis of Spain. Both NH36 and F1 domains significantly increased the PBMC proliferation stimulation index of cured patients and infected asymptomatic individuals compared to healthy controls. Moreover, F1 induced a 19% higher proliferative response than NH36 in asymptomatic exposed subjects. In addition, in patients cured from visceral leishmaniasis, proliferation in response to NH36 and F1 was accompanied by a significant increase of IFN-γ and TNF-α secretion, which was 42–43% higher, in response to F1 than to NH36. The interleukin 17 (IL-17) secretion was stronger in asymptomatic subjects, in response to F1, as well as in cured cutaneous leishmaniasis after NH36 stimulation. While no IL-10 secretion was determined by F1, a granzyme B increase was detected in supernatants from cured patients after stimulation with either NH36 or F1. These data demonstrate that F1 is the domain of NH36 that induces a recall cellular response in individuals with acquired resistance to the infection by L. (L.) infantum. In addition, F1 and NH36 discriminated the IgG3 humoral response in patients with active visceral leishmaniasis due to L. (L.) donovani (Ethiopia) and L. (L.) infantum (Spain) from that of endemic and non-endemic area controls. NH36 showed higher reactivity with sera from L. (L.) donovani-infected individuals, indicating species specificity. We conclude that the F1 domain, previously characterized as an inducer of the Th1 and Th17 responses in cured/exposed patients infected with L. (L.) infantum chagasi, may also be involved in the generation of a protective response against L. (L.) infantum and represents a potential vaccine candidate for the control of human leishmaniasis alone, or in combination with other HLA epitopes/antigens.

Fatty acids, especially those from phospholipids (PLFA), are essential membrane components that are present in relatively constant proportions in biological membranes under natural conditions. However, under harmful growth conditions, such as diseases, environmental changes, and chemical exposure, the fatty acid proportions might vary. If such changes could be identified and revealed to be specific for adverse situations, they could be used as biomarkers. Such biomarkers could facilitate the identification of virulence and resistance mechanisms to particular chemotherapeutic agents. Therefore, specific biomarkers could lead to better therapeutic decisions that would, in turn, enhance treatment effectiveness. The objective of this study was to compare the fatty acid profiles of trivalent antimony and nitric oxide (NO)-resistant and -sensitive Leishmania chagasi and Leishmania amazonensis isolates. Fatty acid methyl esters (FAMEs) were obtained from total lipids (MIDI), ester-linked lipids (ELFA), and ester-linked phospholipids (PLFA). FAMEs were analyzed by chromatography and mass spectrometry. Species- or resistance-associated differences in FAME profiles were assessed by nonmetric multidimensional scaling, multiresponse permutation procedures, and indicator species analyses. The isolate groups had different MIDI-FAME profiles. However, neither the ELFA nor PLFA profiles differed between the sensitive and resistant isolates. Levels of the fatty acid 18:1 Δ9c were increased in sensitive isolates (p < 0,001), whereas the fatty acid 20:4 Δ5,8,11,14 showed the opposite trend (p < 0.01). We conclude that these two fatty acids are potential biomarkers for NO and antimony resistance in L. chagasi and L. amazonensis and that they could be helpful in therapeutic diagnoses.