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Visceral leishmaniasis (VL) is a tropical disease endemic to Brazil. The clinical manifestations of the infection range from asymptomatic to severe. In VL, changes in lipid metabolism, such as hypocholesterolemia and hypertriglyceridemia, occur that are believed to be related to its progression and severity. This study investigated the associations between serum levels of cholesterol, triglycerides, and lipoproteins (high-density lipoprotein, low-density lipoprotein, and very low-density lipoprotein) with clinical and hematological parameters that predict severity in a case series of 83 VL patients. Severely ill patients had higher mean serum triglyceride levels than non-severely ill patients. There was a significant positive correlation between disease severity score and serum triglyceride levels, very low-density lipoprotein, international normalized ratio for prothrombin time test, total bilirubin, and age. An inverse correlation was detected between the disease severity score and mean platelet and neutrophil counts. Hypertriglyceridemia can be a prognostic indicator of severity in patients diagnosed with VL.

Visceral leishmaniasis (VL) is a chronic and often fatal disease caused by protozoans of the genus Leishmania that affects millions of people worldwide. Patients with symptomatic VL have an impaired anti-Leishmania-specific CD4+ T-cell response, which is reversed after clinical cure. In contrast, the quality of the CD4+ and CD8+ T-cell responses involved in resistance and/or cure of VL relies on the capability of these cells to activate polyfunctional and memory responses, which are associated with the simultaneous production of three cytokines: IFN-γ, IL-2, and TNF-α. Models for the development of CD4 and CD8 T-cell quality in memory and protection to leishmaniasis have been described previously. We aimed to assess the functionality of the T cells involved in the recovery of the immune suppression throughout the VL treatment. Therefore, we cultured peripheral blood mononuclear cells (PBMCs) from VL patients and healthy controls in vitro with soluble Leishmania antigen (SLA). Cell surface markers and intracellular cytokine production were determined on days 7, 14, 21, 30, 60, 90, and 180 after the beginning of chemotherapy. We observed that the frequencies of CD4+TNF-α+IFN-γ+ and the multifunctional CD4+IL-2+TNF-α+IFN-γ+, together with CD4+TNF-α+ and CD4+IFN-γ+ T cells, increased throughout and at the end of the treatment, respectively. In addition, enhanced frequencies of CD8+IL-2+TNF-α+IFN-γ+ and CD8+TNF-α+IFN-γ T cells were also relevant in the healing process. Noteworthy, the frequencies of the CD4+ and CD8 central-memory T cells, which produce IL-2, TNF-α, and IFN-γ and ensure the memory response against parasite reinfection, are significantly enhanced in cured patients. In addition, the subset of the non-functional CD8Low population is predominant in VL untreated patients and decreases along the chemotherapy treatment. In contrast, a CD8High subset increased towards the cure. Furthermore, the cure due to treatment with meglumine antimoniate or with liposomal amphotericin B was associated with the recovery of the T-cell immune responses. We described the evolution and participation of functional T cells during the treatment of patients with VL. Our results disclosed that the clinical improvement of patients is significantly associated with the participation of the CD4+ and CD8+ cytokine-secreting T cells.

Leprosy is a chronic disease caused by M. leprae infection that can cause severe neurological complications and physical disabilities. A leprosy-specific vaccine would be an important component within control programs but is still lacking. Given that multifunctional CD4 T cells [i.e., those capable of simultaneously secreting combinations of interferon (IFN)-γ, interleukin (IL)-2, and tumor necrosis factor (TNF)] have now been implicated in the protective response to several infections, we tested the hypothesis if a recombinant M. leprae antigen-specific multifunctional T cells differed between leprosy patients and their healthy contacts. We used whole blood assays and peripheral blood mononuclear cells to characterize the antigen-specific T cell responses of 39 paucibacillary (PB) and 17 multibacillary (MB) leprosy patients and 31 healthy household contacts (HHC). Cells were incubated with either crude mycobacterial extracts (M. leprae cell sonicate–MLCS) and purified protein derivative (PPD) or recombinant ML2028 protein, the homolog of M. tuberculosis Ag85B. Multiplex assay revealed antigen-specific production of IFN-γ and IL-2 from cells of HHC and PB, confirming a Th1 bias within these individuals. Multiparameter flow cytometry then revealed that the population of multifunctional ML2028-specific T cells observed in HHC was larger than that observed in PB patients. Taken together, our data suggest that these multifunctional antigen-specific T cells provide a more effective response against M. leprae infection that prevents the development of leprosy. These data further our understanding of M. leprae infection/leprosy and are instructive for vaccine development.

Ecto-nucleotidase activity is involved in the infection process of Leishmania and various other parasites that enables modulation of host immune responses to promote disease progression. One of the enzymes responsible for this activity is the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase). The enzyme hydrolyzes nucleotides tri- and/or di-phosphate into monophosphate products, which are subsequently hydrolyzed into adenosine. These nucleotides can serve as purinergic signaling molecules involved in diverse cellular processes that govern immune responses. Given the importance of the extracellular metabolism of these nucleotides during intracellular pathogen infections, this study evaluates the role of ecto-nucleotidase activity during Leishmania infantum (L. infantum) infection in human macrophages. E-NTPDase protein expression and activity was evaluated in L. infantum during purine starvation, adenosine-enriched medium, or in the presence of an inhibitor of ecto-nucleotidases. Results show that E-NTPDase is expressed in L. infantum parasites, including on the cell membrane. Furthermore, functional activity of the enzyme was modulated according to the availability of adenosine in the medium. Purine starvation increased the hydrolytic capacity of nucleotides leading to higher infectivity, while growth in adenosine-enriched medium led to lower infectivity. Moreover, inhibiting E-NTPDase function decreased L. infantum infection in macrophages, suggesting the enzyme may serve as a ligand. Taken together, the ability of L. infantum to hydrolyze nucleotides is directly associated with increased infectivity in macrophages.

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. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) fellowships 300639/2003-1 to MP, 310977/2014-2 to CP-d-S, and grant 404400/2012-4 to CP-d-S, MP, PL, RA, JM, EC); Fundação Carlos Chagas de Amparo à Pesquisa do Estado de Rio de Janeiro (FAPERJ) (grant E-26-201.583/2014, E-26-102957/2011, and E-26/111.682/2013 to CP-d-S and fellowships E-26/102415/2010 and E-26/201747/2015 to DN); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (grant 23038.005304/2011-0 to MS); CNPQ-Fundação de Apoio à Pesquisa e a Inovação Tecnológica do Estado de Sergipe-PRONEX (12/2009); and FAPITEC CNPq-(PRONEX)(019.203.02712/2009-8) to RA. EC was supported by a research contract funded via VII PN I+D+I 2013–2016 and FEDER Funds (RICET RD12/0018/0003). Sí

Background There is insufficient evidence to support visceral leishmaniasis (VL) treatment recommendations in Brazil and an urgent need to improve current treatments. Drug combinations may be an option. Methods A multicenter, randomized, open label, controlled trial was conducted in five sites in Brazil to evaluate efficacy and safety of (i) amphotericin B deoxycholate (AmphoB) (1 mg/kg/day for 14 days), (ii) liposomal amphotericin B (LAMB) (3 mg/kg/day for 7 days) and (iii) a combination of LAMB (10 mg/kg single dose) plus meglumine antimoniate (MA) (20 mg Sb+5/kg/day for 10 days), compared to (iv) standard treatment with MA (20 mg Sb+5/kg/day for 20 days). Patients, aged 6 months to 50 years, with confirmed VL and without HIV infection were enrolled in the study. Primary efficacy endpoint was clinical cure at 6 months. A planned efficacy and safety interim analysis led to trial interruption. Results 378 patients were randomized to the four treatment arms: MA (n = 112), AmphoB (n = 45), LAMB (n = 109), or LAMB plus MA (n = 112). A high toxicity of AmphoB prompted an unplanned interim safety analysis and this treatment arm was dropped. Per intention-to-treat protocol final analyses of the remaining 332 patients show cure rates at 6 months of 77.5% for MA, 87.2% for LAMB, and 83.9% for LAMB plus MA, without statistically significant differences between the experimental arms and comparator (LAMB: 9.7%; CI95% -0.28 to 19.68, p = 0.06; LAMB plus MA: 6.4%; CI95% -3.93 to 16.73; p = 0.222). LAMB monotherapy was safer than MA regarding frequency of treatment-related adverse events (AE) (p = 0.045), proportion of patients presenting at least one severe AE (p = 0.029), and the proportion of AEs resulting in definitive treatment discontinuation (p = 0.003). Conclusions Due to lower toxicity and acceptable efficacy, LAMB would be a more suitable first line treatment for VL than standard treatment. ClinicalTrials.gov identification number: NCT01310738. Trial registration ClinicalTrials.gov NCT01310738, Author summary Visceral leishmaniasis remains a worldwide public health concern with high mortality even when proper treatment is instituted. There is a need to develop efficacious, safer and shorter treatment alternatives as the current options suffer from high toxicity and long treatment duration. Combination therapies emerge as an alternative, and WHO has encouraged the conduct of studies to evaluate drug combinations where evidence for current treatment regimens is not available. In Brazil, there is no local evidence to support the current treatment recommendations. Therefore, a clinical trial was conducted in five hospitals in Brazil to evaluate the efficacy and safety of the current available treatments—meglumine antimoniate, amphotericin B deoxycholate and liposomal amphotericin B—and of a combination of liposomal amphotericin B, single dose, and meglumine antimoniate, in a shorter administration regimen. Preliminary safety results led to the discontinuation of the amphotericin B deoxycholate treatment arm and a planned interim analysis resulted in the trial interruption. The final results, comparing liposomal amphotericin B and liposomal amphotericin B plus meglumine antimoniate to the standard meglumine antimoniate treatment, did not show a statistically significant difference in cure rates, though cure rate was higher in the liposomal amphotericin B group. Liposomal amphotericin B treatment showed a better safety profile compared to meglumine antimoniate. These results will support future changes in treatment protocols in Brazil and potentially in Latin America.

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. This work was supported by the following: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) (Fellowships 300639/2003-1 to MP, 310977/2014-2 to CP-d-S, 310797/2015-2 to AM, and grant 404400/2012-4 to CP-d-S, MP, PL, RA, JM, EC, and AM); by Fundação Carlos Chagas de Amparo à Pesquisa do Estado de Rio de Janeiro (FAPERJ) (grant E-26-201.583/2014, E-26-102957/2011, and E-26/111.682/2013 to CP-d-S, and fellowships E-26/102415/2010 and E-26/201747/2015 to DN); by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (grant 23038.005304/2011-0 to MS), by CNPQ-Fundação de Apoio à Pesquisa e a Inovação Tecnológica do Estado de Sergipe-PRONEX (12/2009); and by FAPITEC CNPq (PRONEX) (019.203.02712/2009-8) to RA. EC was supported by a research contract funded via VII PN I+D+I 2013-2016 and FEDER Funds (RICET RD12/0018/0003). Sí

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. According to official reports from 121 countries across five WHO regions, there were 213 899 newly diagnosed cases in 2014. Although leprosy affects the skin and peripheral nerves, it can present across a spectrum of clinical and histopathological forms that are strongly influenced by the immune response of the infected individuals. These forms comprise the extremes of tuberculoid leprosy (TT), with a M. leprae-specific Th1, but also a Th17, response that limits M. leprae multiplication, through to lepromatous leprosy (LL), with M. leprae-specific Th2 and T regulatory responses that do not control M. leprae replication but rather allow bacterial dissemination. The interpolar borderline clinical forms present with similar, but less extreme, immune biases. Acute inflammatory episodes, known as leprosy reactions, are complications that may occur before, during or after treatment, and cause further neurological damages that can cause irreversible chronic disabilities. This review discusses the innate and adaptive immune responses, and their interactions, that are known to affect pathogenesis and influence the clinical outcome of leprosy. Electronic supplementary material The online version of this article (doi:10.1186/s40249-016-0229-3) contains supplementary material, which is available to authorized users.

This study aimed to evaluate the cytotoxicity of intracanal medications on L929 fibroblast cells at different periods of observation. The following experimental groups were studied: calcium hydroxide with camphorated paramonochlorophenol and glycerin (CPG); iodoform with glycerin (IG); calcium hydroxide with iodoform and distilled water (CIW); iodoform with distilled water (IW); calcium hydroxide with distilled water (CW); Otosporin® (OT); and a control group composed of cells and culture medium. Eluates were prepared from each group and placed in contact with 1 x 105 cells/well for periods of 30 minutes, 12, 24, 48 and 72 hours, 5 and 7 days. After each experimental period, a cytotoxicity test was performed using methyltetrazolium (MTT) and a spectrophotometer at an optical density of 570 nm to analyze cell viability. The ANOVA and Tukey test with a significance level of 5% was used to analyze the data. At 30 minutes and at 12 hours, all groups were equal to the control group. At 24 hours, there was greater cytotoxicity in the IG group than in the control group (P

Visceral leishmaniasis (VL) is a chronic and fatal disease caused byLeishmania infantumin Brazil. Leukocyte recruitment to infected tissue is a crucial event for the control of infections such as VL. Leucotriens are lipid mediators synthesized by 5-lipoxygenase (5-LO) and they display a protective role against protozoan parasites by inducing several functions in leucocytes. We determined the role of 5-LO activity in parasite control, focusing on the inflammatory immune response againstLeishmania infantuminfection. LTB4is released duringin vitroinfection. The genetic ablation of 5-LO promoted susceptibility in highly resistant mice strains, harboring more parasites into target organs. The susceptibility was related to the failure of neutrophil migration to the infectious foci. Investigating the neutrophil failure, there was a reduction of proinflammatory cytokines involved in the related Th17 axis released into the organs. Genetic ablation of 5-LO reduced the CD4+T cells producing IL-17, without interfering in Th1 subset.L. infantumfailed to activate DC from5-LO-/-, showing reduced surface costimulatory molecule expression and proinflammatory cytokines involved in Th17 differentiation. BLT1blockage with selective antagonist interferes with DC maturation and proinflammatory cytokines release. Thus, 5-LO activation coordinates the inflammatory immune response involved in the control of VL.

Background Soluble CD40 ligand (sCD40L) and matrix metalloproteinase 9 (MMP-9) are inflammation markers and have been poorly described in infectious disease. In this prospective study, we describe the sera kinetics of these two molecules in the course of treatment follow up in human visceral leishmaniasis (VL). Methods Sera from VL patients were collected before and during follow up of regular Antimony treatment. sCD40L and MMP-9 were measured by Luminex assay. Paired analysis by Wilcoxon signed test was used for comparison of values of the same subjects before and after initiation of treatment. Correlations between clinical data and parasite load with the serum levels of sCD40L and MMP-9 were performed by Spearman test. Tests were considered statistically significant if the probability of a type I error was less than 5% (p-value Results While sCD40L and MMP-9 were not observed in sera from non endemic controls which are at low risk of Leishmania chagasi infection, elevated levels were observed in sera from VL patients, and an increase in sCD40L and MMP-9 levels were detectable during the follow-up of VL patients undergoing antimony treatment. sCD40L levels were also high in individuals living in endemic settings at high risk of infection (endemic controls). Additionally, negative correlations were found between spleen sizes and MMP-9 before treatment and sCD40L at day 15 of treatment. Negative correlations were also found between parasite load with both sCD40L and MMP-9. Conclusion Serum sCD40L and MMP-9 are identified as new and simple biomarkers in two situations: (i) monitoring the success of therapy and (ii) predicting favorable clinical outcome of human VL.