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Objective: Multiple sclerosis (MS) is a multifactorial chronic disease that affects the central nervous system (CNS). Toll-like receptors (TLRs) play a central role in cytokine production after pathogen- and danger-associated molecular patterns (PAMPs and DAMPs) and contribute to CNS damage in MS patients. Here, we evaluated the effects of interferon (IFN)-β treatment in TLR2 and TLR4-dependent cytokine production and mRNA expression in whole-blood cell cultures from MS patients., Methods: We evaluated cytokine production by ELISA from whole-blood cell culture supernatants and mRNA expression by real-time polymerase chain reaction in peripheral blood mononuclear cells (PBMCs)., Results: In patients treated with IFN-β, tumor necrosis factor (TNF)-α production after exposure to TLR2 agonist (Pam3Cys) was lower than in healthy controls and untreated MS patients. However, IFN-β treatment had no significant effect on TNF-α production after TLR4 agonist (LPS) stimulation. On the other hand, interleukin (IL)-10 production was increased in TLR4- but not in TLR2-stimulated whole-blood cell culture from MS patients under IFN-β treatment when compared to the controls. No differences in TNF-α or IL-10 mRNA expression in PBMCs from healthy controls and untreated or treated MS patients were detected, although PBMCs from treated patients presented higher levels of IL-32γ mRNA than those from controls., Conclusions: Our data suggest that IFN-β treatment alters the TLR-dependent immune response of PBMCs from MS patients. This may contribute to the beneficial effects of IFN-β treatment., (© 2019 S. Karger AG, Basel.)

Human leishmaniasis caused by Leishmania (Viannia) braziliensis can be presented as localized cutaneous leishmaniasis (LCL) or mucosal leishmaniasis (ML). Macrophages kill parasites using nitric oxide (NO) and reactive oxygen species (ROS). The aim of this study was to evaluate the ability of parasites obtained from patients with LCL or ML to produce and resist NO or ROS. Promastigotes and amastigotes from LCL or ML isolates produced similar amounts of NO in culture. Promastigotes from ML isolates were more resistant to NO and H 2 O 2 than LCL parasites in a stationary phase, whereas amastigotes from LCL isolates were more resistant to NO. In addition, in the stationary phase, promastigote isolates from patients with ML expressed more thiol-specific antioxidant protein (TSA) than LCL isolates. Therefore it is suggested that infective promastigotes from ML isolates are more resistant to microbicidal mechanisms in the initial phase of infection. Subsequently, amastigotes lose this resistance. This behavior of ML parasites can decrease the number of parasites capable of stimulating the host immune response shortly after the infection establishment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The disseminated form of leishmaniasis is a serious and rare disease, being diagnosed in 2% of the cutaneous cases registered per year in Brazil. The main characteristic is the appearance of multiple pleomorphic lesions on the cutaneous surface. A 68-year-old male from the rural area of Tocantins, Brazil, presented atypical disseminated cutaneous leishmaniasis (ACL). The clinical course and histopathological and immunological findings presented a mixed pattern that hindered diagnosis and therapeutic management. Molecular typing revealed a mixed infection with Leishmania (V.) guyanensis and Leishmania (L.) amazonensis . Molecular identification of the agents responsible for ACL is important for adequate therapeutic planning, minimizing the possibility of sequellae that impact the quality of life of the patient.

Interleukin 32 (IL-32) is an intracellular cytokine produced by immune and non immune cells after different stimuli. It contributes to inflammation and control of intracellular pathogens mainly by inducing proinflammatory cytokines and microbicidal molecules. Evidence is rising showing that IL-32 can be considered an endogenous danger signal after tissue injury, amplifying the inflammatory process and acquired immune responses. It seems to be a master regulator of intracellular infectious diseases. In this review, first the general properties of IL-32 are described followed by its role in the immunopathogenesis of inflammatory and infectious diseases. Roles of IL-32 in the control of infectious diseases caused by intracellular pathogens are reported, and later a focus on IL-32 in leishmaniases, diseases caused by an intracellular protozoan, is presented., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Leishmania (Viannia) guyanensis is a causal agent of American tegumentary leishmaniasis (ATL). This protozoan has been poorly investigated; however, it can cause different clinical forms of ATL, ranging from a single cutaneous lesion to severe lesions that can lead to destruction of the nasopharyngeal mucosa. L. (V.) guyanensis and the disease caused by this species can present unique aspects revealing the need to better characterize this parasite species to improve our knowledge of the immunopathological mechanisms and treatment options for ATL. The mechanisms by which some patients develop a more severe form of ATL remain unclear. It is known that the host immune profile and parasite factors may influence the clinical manifestations of the disease. Besides intrinsic parasite factors, Leishmaniavirus RNA 1 (LRV1) infecting L. guyanensis can contribute to ATL immunopathogenesis. In this review, general aspects of L. guyanensis infection in humans and mouse models are presented.

Visceral leishmaniasis (VL) is a chronic parasitic disease caused by Leishmania infantum in the Americas. During VL, several proinflammatory cytokines are produced in spleen, liver, and bone marrow. However, the role of interleukin-32 (IL-32) has not been explored in this disease. IL-32 can induce production of proinflammatory cytokines in innate immune cells and polarize the adaptive immune response. Herein, we discovered that L. infantum antigens induced expression of mRNA mainly for the IL-32γ isoform but also induced low levels of the IL-32β transcript in human peripheral blood mononuclear cells. Furthermore, infection of human IL-32γ transgenic mice (IL-32γTg mice) with L. infantum promastigote forms increased IL-32γ expression in the spleen and liver. Interestingly, IL-32γTg mice harbored less parasitism in the spleen and liver than wild-type (WT) mice. In addition, IL-32γTg mice showed increased granuloma formation in the liver compared to WT mice. The protection against VL was associated with increased production of nitric oxide (NO), interferon gamma (IFN-γ), IL-17A, and tumor necrosis factor alpha by splenic cells restimulated ex vivo with L. infantum antigens. In parallel, there was an increase in the number of Th1 and Th17 T cells in the spleens of IL-32γTg mice infected with L. infantum IL-32γ induction of IFN-γ and IL-17A expression was found to be essential for NO production by splenic cells of infected animals. These data indicate that IL-32γ potentiates the Th1/Th17 immune response during experimental VL, thus contributing to the control of L. infantum infection., (Copyright © 2018 American Society for Microbiology.)

Cryptococcal species vary in capsule and cell size, thermotolerance, geographic distribution, and affected populations. Cryptococcus gattii sensu stricto and C. deuterogattii affect mainly immunocompetent hosts; however, C. bacillisporus , C. decagattii , and C. tetragattii cause infections mainly in immunocompromised hosts. This study aimed to compare the capacities of different species of the C. gattii species complex to induce cytokines and antimicrobial molecules in human peripheral blood mononuclear cells (PBMCs). Cryptococcus bacillisporus and C. deuterogattii induced the lowest levels of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 among the five species of the C. gattii complex. Cryptococcus deuterogattii induced higher levels of IL-22 than those induced by C. tetragattii and the environmental species C. flavescens In addition, C. bacillisporus and C. gattii sensu stricto proliferated inside human monocyte-derived macrophages after 24 h of infection. All Cryptococcus species were able to generate reactive oxygen species (ROS) in human PBMCs, with C. bacillisporus and C. deuterogattii being more efficient than the other species. In conclusion, C. bacillisporus and C. deuterogattii induce lower levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 and higher ROS levels than those induced by the other species. Species of the Cryptococcus gattii complex have different abilities to induce cytokine and ROS production by human PBMCs., (Copyright © 2018 American Society for Microbiology.)

Because of visceral leishmaniasis (VL) urbanization and spreading of the human immunodeficiency virus (HIV) infection to rural areas, coinfection has become more common. Here, we compared the accuracy of Kalazar Detect® (KD), an rK39-based immunochromatographic (IC) test, and OrangeLife® (OL), an rK39 + rK28 IC test, for diagnosing VL in patients coinfected with HIV in an endemic area in Brazil. Seventy-six VL patients and 40 patients with other diseases, of which 31 and 21 patients, respectively, were infected with HIV, were examined. The sensitivity of OL and KD tests was 88.89 and 95.45%, respectively, in patients without HIV. The sensitivity dropped to 67.74 and 61.29%, respectively, in coinfected patients. The decrease in sensitivity was not related to a decrease in the production of Leishmania-specific IgG. Because of the low sensitivity of rk39 test in HIV-infected patients, we suggest that patients with negative rK39 results should undergo further investigation with additional serological tests that are not based only on the rK39 antigen and examination of bone marrow aspirates.

Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by loss of dopaminergic neurons associated with neuroinflammation. Toll-like receptors (TLRs) are expressed in peripheral blood leukocytes and also in neurons and glial cells mediating inflammation. This study aimed to investigate the peripheral blood leukocyte response to TLR2 and TLR4 agonists in young and elderly PD patients. Two groups of patients with PD were evaluated (≤ 55 years old and ≥ 65 years old), age-matched with healthy controls (n = 26). Severity of PD was evaluated by Unified Parkinson's Disease Rating Scale (UPDRS). Whole blood cultures were stimulated with lipopolysaccharide (LPS), a TLR4 agonist or Pam 3 Cys (Pam), a TLR2 agonist. Tumor necrosis factor alpha (TNFα) and interleukin 10 (IL-10) were measured by immunoenzimatic assay. 6 h-TNFα production was increased after TLR4 stimulation, mainly in young PD patients, whereas TLR2-induced TNFα and IL-10 levels were decreased in PD patients independent of age (p < 0.05). A reverse correlation between LPS-induced TNFα production and age was observed in PD patients and controls, but TNFα induced by TLR2 agonist was not associated with age of PD patients or controls. TNFα production induced by TLR4 but not by TLR2 was reversely associated with the age at PD onset and disease duration. No associations between UPDRS scores and cytokine levels were detected. In conclusion, TLR4 and TLR2 responses seem to be differentially affected during PD. Data suggest that TLR2 deficiency in periphery is independent of age of the patients, age at PD onset, or PD duration.

American Tegumentary Leishmaniasis is a chronic infection caused by Leishmania protozoan. It is not known whether genetic variances in NOD-like receptor (NLR) family members influence the immune response towards Leishmania parasites and modulate intracellular killing. Using functional genomics, we investigated whether genetic variants in NOD1 or NOD2 influence the production of cytokines by human PBMCs exposed to Leishmania. In addition, we examined whether recognition of Leishmania by NOD2 contributes to intracellular killing. Polymorphisms in the NOD2 gene decreased monocyte- and lymphocyte-derived cytokine production after stimulation with L. amazonensis or L. braziliensis compared to individuals with a functional NOD2 receptor. The phagolysosome formation is important for Leishmania-induced cytokine production and upregulation of NOD2 mRNA expression. NOD2 is crucial to control intracellular infection caused by Leishmania spp. NOD2 receptor is important for Leishmania recognition, the control of intracellular killing, and the induction of innate and adaptive immune responses.

Platelet-activating factor (PAF) is produced by macrophages during inflammation and infections. We evaluated whether PAF is able to modulate the infection of human macrophages by Leishmania braziliensis, the main Leishmania sp. in Brazil. Monocyte-derived macrophages were incubated with promastigote forms in absence or presence of exogenous PAF. We observed that the treatment of macrophages with low concentrations of PAF prior to infection increased the phagocytosis of L. braziliensis. More importantly, exogenous PAF reduced the parasitism when it was added before, during or after infection. In addition, treatment with a PAF antagonist (PCA 4248) resulted in a significant increase of macrophage infection in a concentration-dependent manner, suggesting that endogenous PAF is important to control L. braziliensis infection. Mechanistically, while exogenous PAF increased production of reactive oxygen species (ROS) treatment with PCA 4248 reduced oxidative burst during L. braziliensis infection. The microbicidal effects of exogenous PAF were abolished when macrophages were treated with apocynin, an NADPH oxidase inhibitor. The data show that PAF promotes the production of ROS induced by L. braziliensis, suggesting that this lipid mediator may be relevant to control L. braziliensis infection in human macrophages., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Interleukin 32 (IL-32) is a pro-inflammatory cytokine induced in patients with American tegumentary leishmaniasis (ATL) caused by Leishmania braziliensis. Here, we investigated whether IL-32 is also expressed in patient lesions caused by L. amazonensis. In addition, we evaluated experimental L. amazonensis and L. braziliensis infections in C57BL/6 transgenic mice for human IL-32γ (IL-32γTg) in comparison with wild-type (WT) mice that do not express the IL-32 gene., Results: Human cutaneous lesions caused by L. amazonensis express higher levels of IL-32 than healthy control skin. In mice, the presence of IL-32γ promoted the control of cutaneous lesions caused by L. braziliensis, but not lesions caused by L. amazonensis in an ear dermis infection model. In addition, IL-32γTg mice displayed less tissue parasitism and inflammation in IL-32γTg than WT mice during the healing phase of L. braziliensis infection. Production of antigen-specific pro-inflammatory cytokines was higher in IL-32γTg mice than in WT mice during L. braziliensis infection but not during L. amazonensis infection., Conclusions: Human cutaneous lesions caused by L. amazonensis express high levels of IL-32. In mice, the presence of IL-32γ contributes to the lesion healing caused by L. braziliensis but not by L. amazonensis. Data suggest that despite the ability for both species to induce IL-32 in humans, the connections between this cytokine and other immune players induced by related species of parasites can lead to distinct outcomes of the murine infections.

Leishmania ( Viannia ) braziliensis is the main causative species of tegumentary leishmaniasis in Brazil. In this study, we evaluated the susceptibility of 16 clinical isolates of L. ( V. ) braziliensis from different regions of Brazil to miltefosine in vitro. Half-maximal inhibitory concentrations of miltefosine varied from 22.9 to 144.2 μM against promastigotes and from 0.3 to 4.2 μM against intracellular amastigotes. No significant differences were found between isolates of different geographical origins. A clear correlation between the EC 50 against promastigotes and amastigotes within each isolate was found. These findings contribute to the evaluation of miltefosine's potential and limitations for the treatment of tegumentary leishmaniasis in Brazil.

Background: Interleukin-32 (IL-32) is expressed in lesions of patients with American Tegumentary Leishmaniasis (ATL), but its precise role in the disease remains unknown., Methodology/principal Findings: In the present study, silencing and overexpression of IL-32 was performed in THP-1-derived macrophages infected with Leishmania (Viannia) braziliensis or L. (Leishmania) amazonensis to investigate the role of IL-32 in infection. We report that Leishmania species induces IL-32γ, and show that intracellular IL-32γ protein production is dependent on endogenous TNFα. Silencing or overexpression of IL-32 demonstrated that this cytokine is closely related to TNFα and IL-8. Remarkably, the infection index was augmented in the absence of IL-32 and decreased in cells overexpressing this cytokine. Mechanistically, these effects can be explained by nitric oxide cathelicidin and β-defensin 2 production regulated by IL-32., Conclusions: Thus, endogenous IL-32 is a crucial cytokine involved in the host defense against Leishmania parasites.

American tegumentary leishmaniasis (ATL) is a neglected disease widely distributed in Latin America. In Brazil, it is caused by different Leishmania species belonging to the Subgenera Viannia and Leishmania. ATL diagnosis is routinely based on clinical, epidemiological, parasitological and immunological (delayed-type hypersensitivity skin test-DTH) evidences. The main objective of this work was to determine the efficacy of a previous immunohistochemical (IHC) method developed by our group. Seventy eight skin biopsies from patients with different ATL clinical forms and origins were evaluated. The method was previously standardized in ATL patients from the municipality of Caratinga, Minas Gerais, Brazil, all infected with Leishmania (V.) braziliensis. Here, it is evaluated in patients from the North, Southeast and Midwest regions of Brazil. Clinical, parasitological (biopsy PCR) and immunological (Montenegro skin test-MST) diagnosis were performed prior to IHC procedure. The IHC procedure detected 70.5% of the cases having a high agreement with MST diagnosis (kappa=0.84). A distinguished contribution of this work is that IHC succeed in diagnosing some negative DTH patients. Those were infected with Leishmania (L.) amazonensis, commonly causing the anergic form of the disease. In conclusion, IHC succeed in detecting ATL caused by different Leishmania species from various geographic regions and clinical status. Although it was not able to detect ATL in all patients, it was better than MST providing an additional tool for the diagnosis of ATL patients. There was no significant correlation between clinical forms and histological features including the presence of necrosis., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Interleukin 32 (IL-32) is a proinflammatory cytokine, expressed as 9 distinct isoforms. The most active isoform is the predominantly intracellular-functioning IL-32γ. Involvement of IL-32 in infectious diseases is increasingly being appreciated. Production of IL-32 promotes pathways that serve to control bacterial infection, especially those caused by mycobacteria. A similar role for this cytokine is observed in the cellular response to viral infections. In addition to its protective effects against microorganisms, IL-32 is involved in immunopathogenesis of some infectious diseases. In parasitic diseases, it has been demonstrated that this cytokine is induced by Leishmania infection. In this review, we summarize the present data on the role of IL-32 in infectious diseases, highlighting this cytokine as new target for control of infections., (© Society for Leukocyte Biology.)

While the role of Toll-like receptors (TLRs) has been investigated in murine models of tegumentary leishmaniasis caused by Leishmania (Viannia) braziliensis, the interaction between TLRs and Leishmania sp. has not been investigated in human cells. The aim of this study was to evaluate the involvement of TLR4 in cytokine production of human peripheral blood mononuclear cells (PBMCs) induced by L. braziliensis, and whether the parasite alters the expression of TLR4 on monocytes/macrophages. Amastigote forms were obtained from mice lesions and PBMCs were isolated from healthy donors. PBMCs were cultured in absence or presence of IFNγ, TLR4 neutralizing antibodies, natural antagonist of TLR4 (Bartonella LPS), TLR4 agonist (E. coli LPS), and amastigote forms. The concentrations of tumor necrosis factor (TNFα) and interleukin 10 (IL-10) were assayed by ELISA and TLR4 expression by flow cytometry. Amastigotes forms of L. braziliensis induced TNFα and IL-10 production only in IFNγ-primed PBMCs. The TNFα and IL-10 production was inhibited by TLR4 neutralization, both with anti-TLR4 antibodies and Bartonella LPS. Interestingly, addition of E. coli LPS further increased TNFα but not IL-10 production induced by L. braziliensis amastigotes. Amastigotes of L. braziliensis strongly reduced membrane TLR4 expression on monocytes/macrophages, apparently by internalization after the infection. The present study reveals that TLR4 drives the production of TNFα and IL-10 induced by L. braziliensis amastigotes and that the parasites decrease TLR4 expression on monocyte surface., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mucosal leishmaniasis (ML) caused by Leishmania (Vianna) braziliensis usually appears after the healing of the primary lesion when amastigotes disseminate from the infection site to the mucosal area. Here, we investigated murine infection with amastigotes obtained from patients with ML or localized cutaneous leishmaniasis (LCL). Amastigotes were used to infect wild type, IFN-γ KO and inducible nitric oxide synthase (iNOS) KO mice. Amastigotes from patients with LCL induced lesions that appeared earlier in IFN-γ KO than parasites from ML. The lesion after infection with ML appeared early in iNOS KO than in IFN-γ KO mice and in iNOS KO mice parasites from ML and LCL cause similar lesions at the initial phase of infection, while parasites from ML induced greater lesions than the ones from LCL at the late phase. A greater number of parasites were observed in spleen of IFN-γ KO and iNOS KO mice infected with amastigotes from patients with ML than those with LCL. Parasites from ML infect a lower percentage of macrophages and are killed independent on IFN-γ and dependent on NO. The data suggest that amastigotes responsible for mucosal lesion in humans develop slowly on the initial phase of infection due to high susceptibility to NO and they have an increased ability to disseminate., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Toll-like receptors (TLRs) are expressed in several immune cells including blood monocytes and resident macrophages, such as microglia in the central nervous system. TLRs recognize pathogen- or damage-associated molecular patterns, leading to the release of inflammatory and toxic molecules, which can contribute to neuroinflammation associated with Parkinson's disease (PD). The aim of this study was to compare the potential of peripheral blood cells from PD patients or healthy subjects to produce cytokines after exposure to TLR agonists, and to investigate TLR2 and TLR4 expression on monocyte subsets., Methods: Twenty-one patients and 21 healthy controls were recruited. Patients were evaluated according to the Unified Parkinson's Disease Rating Scale, and Hoehn and Yahr stage. Cytokines were measured in supernatants of whole blood cultures after incubation with TLR2, TLR4, or TLR7/8 agonists, by cytometric bead array. Expression of CD14, CD16, TLR2, and TLR4 was analyzed by cytometry., Results: Patient blood cells produced lower levels of cytokines in response to TLR2 and also after TLR7/8/R848 activation than controls. Percentages of CD14+CD16+ or CD14+CD16- monocytes and TLR2 and TLR4 expression were similar between patients and controls., Conclusions: Blood leukocyte TLR2 and TLR7/8 responses are impaired in PD. This was neither associated with imbalance in monocyte subsets nor with TLR2/TLR4 expression on these cells. The association between a decreased TLR response in periphery and damage of brain in PD must be further investigated., (© 2016 S. Karger AG, Basel.)

The aim of this study was to characterize clinical field isolates of Leishmania spp. obtained from patients with American Tegumentary Leishmaniasis (ATL) who live in Goiás state, Brazil. The presumed areas of infection were in Goiás, Tocantins, and Pará states. Three isolates of parasites were identified as L. (Viannia) braziliensis and one as L. (V.) guyanensis. The in vitro growth profiles were found to be similar for all parasites. Nevertheless, in C57BL/6 mice, L. (V.) guyanensis infection was better controlled than L. (V.) braziliensis. Yet in C57BL/6 mice deficient in interferon gamma, L. (V.) guyanensis lesions developed faster than those caused by L. (V.) braziliensis isolates. In BALB/c mice, the development of lesions was similar for isolates from both species; however, on the 11th week of infection, amastigotes could not be observed in macrophages from L. (V.) guyanensis-infected mice. Thus, L. (V.) guyanensis can be circulating in Goiás, a state where autochthonous cases of this species had not yet been reported. Considering the difficulties to differentiate L. (V.) guyanensis from L. (V.) braziliensis at the molecular, morphological, and clinical (human and murine models) levels, the presence of L. (V.) guyanensis infections is possibly underestimated in several regions of Brazil.

This study was designed to assess in vitro metacyclogenesis of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis clinical field isolates obtained from patient lesions (L. braziliensis IMG3 and PPS6m; L. amazonensis MAB6). Metacyclogenesis was evaluated by different criteria, namely, promastigote size (morphometric analysis and flow cytometry), surface modifications (loss of lectin or monoclonal antibody (mAb) binding, complement resistance), and infectivity to human macrophages. Growth curves were similar for all parasites evaluated. The various features analyzed were expressed in a high percentage of promastigotes at 6th and 10th days of culture and a low percentage at the 2nd day. However, in most isolates, these features, considered as markers of metacyclogenesis, seemed to develop with different time courses, since the percentages of metacyclic forms detected with each technique were usually different. Parasites from 6th or 10th day and those negatively selected with lectin or mAb similarly infected human macrophages. From all isolates analyzed, L. amazonensis PH8 and MAB6 showed the highest and the lowest levels of susceptibility, respectively, to leishmanicidal activity of IFN-γ/LPS-activated macrophages. Our results showed that by using different techniques to evaluate different aspects of metacyclogenesis (morphological and biochemical modifications) different percentages of metacyclic promastigotes can be detected in each isolate culture.

Although Leishmania (Viannia) braziliensis is the most prevalent species that cause American tegumentary leishmaniasis (ATL), the immune response against this parasite has been poorly investigated. Upon activation, macrophages produce a series of pro-inflammatory molecules, including the lipid mediator leukotriene B4 (LTB4). LTB4 has been shown to enhance several macrophage functions, but its role in human macrophages is less known. Here, we investigated the role of LTB4 on human monocyte-derived macrophages infected with human isolate of L. (V.) braziliensis (IMG3). It was found that human macrophages produce LTB4 upon infection with Leishmania, which by autocrine or paracrine activation of its high affinity receptor BLT1, potentiates macrophage leishmanicidal activity. This LTB4 effect is mediated by increased secretion of reactive oxygen species (ROS). Moreover, Leishmania infection decreased the expression of BLT1, leading to the speculation that this could represent a parasite escape mechanism to establish a chronic inflammatory infection. Therefore, our data suggest that LTB4 could be used in therapeutic strategies to control Leishmania infection., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Background: The interleukin 32 (IL-32) is a proinflammatory cytokine produced by immune and non-immune cells. It can be induced during bacterial and viral infections, but its production was never investigated in protozoan infections. American Tegumentary Leishmaniasis (ATL) is caused by Leishmania protozoan leading to cutaneous, nasal or oral lesions. The aim of this study was to evaluate the expression of IL-32 in cutaneous and mucosal lesions as well as in peripheral blood mononuclear cells (PBMC) exposed to Leishmania (Viannia) braziliensis., Methods: IL-32, tumour necrosis factor (TNF) and IL-10 protein expression was evaluated by immunohistochemistry in cutaneous, mucosal lesions and compared to healthy specimens. The isoforms of IL-32α, β, δ, γ mRNA, TNF mRNA and IL-10 mRNA were assessed by qPCR in tissue biopsies of lesions and healthy skin and mucosa. In addition, PBMC from healthy donors were cultured with amastigotes of L. (V.) braziliensis. In lesions, the parasite subgenus was identified by PCR-RFLP., Results: We showed that the mRNA expression of IL-32, in particular IL-32γ was similarly up-regulated in lesions of cutaneous (CL) or mucosal (ML) leishmaniasis patients. IL-32 protein was produced by epithelial, endothelial, mononuclear cells and giant cells. The IL-32 protein expression was associated with TNF in ML but not in CL. IL-32 was not associated with IL-10 in both CL and ML. Expression of TNF mRNA was higher in ML than in CL lesions, however levels of IL-10 mRNA were similar in both clinical forms. In all lesions in which the parasite was detected, L. (Viannia) subgenus was identified. Interestingly, L. (V.) braziliensis induced only IL-32γ mRNA expression in PBMC from healthy individuals., Conclusions: These data suggest that IL-32 plays a major role in the inflammatory process caused by L. (Viannia) sp or that IL-32 is crucial for controlling the L. (Viannia) sp infection.

Tegumentary leishmaniasis is an endemic protozoan disease that, in Brazil, is caused by parasites from Viannia or Leishmania complex. The clinical forms of cutaneous disease comprise localized, disseminated, mucosal or mucocutaneous, and diffuse leishmaniasis. Viannia complex parasites are not easy to isolate from patient lesions, especially from mucosal lesions, and they are difficult to culture. The aim of the present study was to compare the efficiency of ex vivo (culture) and in vivo (IFNγ-deficient mice) parasite isolation methods to improve the isolation rate and storage of stocks of New World Leishmania sp that cause cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). Biopsy fragments from cutaneous or mucosal lesions were inoculated into culture medium or mouse footpads. We evaluated 114 samples (86 CL, 28 ML) using both methods independently. Samples from CL patients had a higher isolation rate in ex vivo cultures than in mice (34.1% vs. 18.7%, P<0.05). Nevertheless, almost twice the number of isolates from ML lesions was isolated using the mouse model compared to ex vivo cultures (mouse, 6/25; culture, 3/27). The overall rates of isolation were 40.2% for CL samples and 29.6% for ML samples. Of the 43 isolations, we successfully stocked 35 isolates (81.4%; 27 CL, 8 ML). Contaminations were more frequently detected in cultures of ML than CL lesions. For comparison, the use of both methods simultaneously was performed in 74 samples of CL and 25 samples of ML, and similar results were obtained. Of the eight ML isolates, five were isolated only in mice, indicating the advantage of using the in vivo method to obtain ML parasites. All parasites obtained from in vivo isolation were cryopreserved, whereas only 68% of ex vivo isolations from CL lesions were stocked. In conclusion, the use of genetically modified mice can improve the isolation of parasites from ML. Isolation and stocking of New World Leishmania parasites, especially those from ML that are almost absent in laboratory stocks, are critical for evaluating parasite genetic diversity as well as studying host-parasite interactions to identify biological markers of Leishmania. In this paper, we also discuss some of the difficulties associated with isolating and stocking parasites., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Parasites 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., Methods: The 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., Results: The 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., Conclusions: Notwithstanding 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.

Background: Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease., Methodology/principal Findings: Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages., Conclusions/significance: Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.

This study investigated the presence of the black-pigmented bacteria Prevotella nigrescens and Prevotella intermedia, the non-black-pigmented bacteria Actinomyces spp and particularly the cariogenic pathogen Streptococcus mutans in the dental biofilms of patients with or without black extrinsic tooth stains, using the multiplex polymerase chain reaction (PCR) technique. Analysis of the dental biofilms of patients with (n=26) or without (n=26) black tooth stains was performed using duplex PCR for the 16S ribosomal RNA gene (P. nigrescens, P. intermedia, Actinomyces spp) and glucosyltransferase-I gene for S. mutans. P. nigrescens and S. mutans were the most frequent bacteria detected in both groups. The least frequently detected were P. intermedia and Actinomyces spp. The similar bacterial composition of dental biofilms of black tooth stains and healthy tooth surfaces indicates that black tooth stains are not free of cariogenic bacteria.

Leishmania spp are protozoans capable of carbohydrates degradation and as energy source they can use glucose, aminoacids or lipids from the environment. The products of the metabolic pathways such as organic acids may be used as an index of their energetic metabolic profile. Therefore, in this study a metabolic profile comparison was made between promastigotes from one reference strain (MHOM/BR/1975/M2903) and two different isolates of Leishmania (Viannia) braziliensis (MHOM/BR/2003/IMG3 and MHOM/BR/2005/RPL5). The parasites culture was performed in complete Grace's culture media seeded in 24-well plates at 26°C. During the growth curve performance samples were collected from the logarithmic and stationary phases of culture and therefore analyzed by high performance liquid chromatography (HPLC) and spectrophotometry assays to determine the concentrations of glucose, lactate, citrate, α-ketoglutarate, succinate, fumarate, malate, oxaloacetate and β-hydroxybutirate which are indicative of the energetic pathways. It was possible to detect an increase in the glucose from the stationary phase from the M2903 strain when compared to the logarithmic phase while in the IMG3 and RPL5 isolates there was a decrease (p<0.05). The spectrophotometric and chromatographic results indicated that the logarithmic phase which presents higher energy consumption due to the intense replication rate have the energetic pathways intensified. It was also possible to note some metabolic differences between the analyzed parasites which may indicate possible adaptations of the parasite when facing different environmental and physiological conditions during its life cycle and that these differences may help in the understanding of the diversity of the host-parasite relationship from Leishmania parasites., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mycoplasmal lipid-associated membrane proteins (LAMPs) and Mycoplasma arthritidis mitogen (MAM superantigen) are potent stimulators of the immune system. The objective of this work was to detect antibodies to MAM and LAMPs of Mycoplasma hominis and M. fermentans in the sera of patients affected by rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) to identify mycoplasmal products that can be involved in the etiopathogenesis of these autoimmune diseases. Serum samples from female RA and SLE patients and controls, recombinant MAM, and LAMPs of M. hominis PG21 and M. fermentans PG18 were used in Western blot assays. A similar frequency of sera from patients and controls reactive to MAM was detected. A larger number of M. hominis and M. fermentans LAMPs were recognized by sera from RA patients than controls, but no differences were detected between sera from SLE patients and controls. Among the LAMPs recognized by IgG antibodies from RA patients, proteins of molecular masses in a range of <49 and ≥20 KDa (M. hominis) and <102 and ≥58 KDa (M. fermentans) were the most reactive. These preliminary results demonstrate the strong reactivity of antibodies of RA patients with some M. hominis and M. fermentans LAMPs. These LAMPs could be investigated as mycoplasmal antigens that can take part in the induction or amplification of human autoimmune responses.

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNgamma KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNgamma KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNgamma KO mice to improve the possibility to isolate New World Leishmania species.

Resistance of Leishmania parasites to specific chemotherapy has become a well-documented problem in the Indian subcontinent in recent years but only a few studies have focused on the susceptibility of American Leishmania isolates. Our susceptibility assays to meglumine antimoniate were performed against intracellular amastigotes after standardizing an in vitro model of macrophage infection appropriate for Leishmania (Viannia) braziliensis isolates. For the determination of promastigote susceptibility to amphotericin B, we developed a simplified MTT-test. The sensitivity in vitro to meglumine antimoniate and amphotericin B of 13 isolates obtained from Brazilian patients was determined. L. (V.) braziliensis isolates were more susceptible to meglumine antimoniate than Leishmania (Leishmania) amazonensis. EC(50), EC(90) and activity indexes (calculated over the sensitivity of reference strains), suggested that all isolates tested were susceptible in vitro to meglumine antimoniate, and did not show association with the clinical outcomes. Isolates were also uniformly susceptible in vitro to amphotericin B.

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.

Mycoplasma arthritidis causes autoimmune arthritis in rodents. It produces a superantigen (MAM) that simultaneously activates antigen presenting cells and T cells inducing nitric oxide and cytokine release. Nitric oxide is a key inducer and regulator of the immune system activation. Here, we investigated nitric oxide and cytokine production and interactions of these molecules in MAM-stimulated co-cultures of macrophages (J774A.1 cell line) with spleen lymphocytes. We found that: a) MAM-induced nitric oxide, interferon-gamma, membrane-associated tumor necrosis factor and interleukin-2 production in co-cultures of macrophages with lymphocytes from BALB/c and C3H/HePas but not from C57Bl/6 mice; b) production of nitric oxide was dependent on interferon-gamma whereas that of interferon-gamma was dependent on interleukin-2 and membrane-associated tumor necrosis factor; c) these cytokines up regulated MAM-induced nitric oxide production. Unraveling the mechanisms of cell activation induced by MAM might be helpful to design strategies to prevent immune system activation by superantigens and therefore in seeking amelioration of associated immunopathologies.

The production of interleukin-12 and interferon-gamma is a key event for controlling leishmaniasis. Here, we tested the hypothesis that after murine infection with Leishmania major, cell migration into draining lymph nodes is crucial for early production of those cytokines. We showed that inflammatory cells carrying the marker of recently migrated cells, the Gr-1 antigen, including polymorphonuclear and mononuclear cells, migrate rapidly into the site of promastigote infection and, subsequently, into draining lymph nodes. Treatment with RB6-8C5 monoclonal antibody reduced local inflammation and migration of Gr-1+ cells into the draining lymph nodes. This reduction was associated with a decrease of interleukin-12 production by draining lymph node cells from BALB/c mice but not C57BL/6 mice. Additionally, interferon-gamma was also reduced in both mouse strains after depletion of Gr-1+ cells, suggesting that these cells are important for early interleukin-12 and interferon-gamma production. Our findings suggest that recently migrated myeloid cells, more than resident cells, are the major source of the early IL-12 production after L. major infection.

TLRs may contribute to the progression of rheumatoid arthritis through recognition of microbial or host-derived ligands found in arthritic joints. Here, we show that TLR2 and TLR4, but not TLR9, are involved in the pathogenesis of autoimmune arthritis and play distinct roles in the regulation of T cells and cytokines. We investigated the involvement of TLR2, TLR4, and TLR9 in the progression of arthritis using IL-1 receptor antagonist-knockout (IL1rn-/-) mice, which spontaneously develop an autoimmune T cell-mediated arthritis. Spontaneous onset of arthritis was dependent on TLR activation by microbial flora, as germ-free mice did not develop arthritis. Clinical and histopathological evaluation of IL1rn-/-Tlr2-/- mice revealed more severe arthritis, characterized by reduced suppressive function of Tregs and substantially increased IFN-gamma production by T cells. IL1rn-/-Tlr4-/- mice were, in contrast, protected against severe arthritis and had markedly lower numbers of Th17 cells and a reduced capacity to produce IL-17. A lack of Tlr9 did not affect the progression of arthritis. While any therapeutic intervention targeting TLR2 still seems complicated, the strict position of TLR4 upstream of a number of pathogenic cytokines including IL-17 provides an interesting potential therapeutic target for rheumatoid arthritis.

Pneumococcal surface protein A (PspA) has been considered a potential candidate for human vaccines because of its serotype-independent protective immunity. Nasopharyngeal (NP) pneumococcal colonization is highly prevalent in infants and precedes the invasive disease. Thus, prevention of NP colonization may reduce the burden of pneumococcal disease in children. Scarce information focusing on PspA from pneumococcal carriage in humans is available. We examined the genetic diversity of PspA from NP isolates obtained during an ongoing pneumococcal surveillance study with children. PspA families and clades of 183 community-acquired Streptococcus pneumoniae NP isolates from healthy children (n = 97) and children with respiratory tract infections (n = 48), pneumonia (n = 33), or meningitis (n = 5) were investigated. Overall, 79.8% (n = 146) of the pneumococcal isolates were classified as PspA family 1 (35.5%) and family 2 (44.3%), whereas 20.2% of the isolates could not be typed. The distribution of PspA families and clades did not differ significantly according to the clinical status of the children. A dendrogram comparing the genetic relationship between the amino acid sequences of the clade-defining region of PspA from NP strains together with 24 invasive reference strains (GenBank) closely reproduced the profile of the families and clades previously reported for pneumococcal invasive strains. These findings strengthen the idea that the use of PspA as a vaccine antigen may protect children against carriage as well as invasive pneumococcal disease.

Malaria causes important functional alterations of the immune system, but several of them are poorly defined. To evaluate thoroughly the natural killer cell cytotoxicity in patients with malaria, we developed a technique capable to assess both the dynamics and the kinetics of the process. For the kinetics assay, human peripheral blood mononuclear cells were previously incubated with K562 cells and kept in agarose medium, while for the dynamics assay both cells were maintained in suspension. NK activity from patients with vivax malaria presented a kinetics profile faster than those with falciparum malaria. NK cytotoxicity positively correlated with parasitemia in falciparum malaria. The dynamics of NK cytotoxicity of healthy individuals was elevated at the beginning of the process and then significantly decreased. In contrast, malaria patients presented successive peaks of NK activity. Our results confirmed the occurrence of alteration in NK cell function during malaria, and added new data about the NK cytotoxicity process.

We investigated the capacity of Mycoplasma arthritidis mitogen (MAM) to induce (a) expression of the inducible enzymes cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS), (b) production of prostaglandin E2 (PGE2) and nitric oxide (NO), and (c) involvement of platelet-activating factor (PAF) in the MAM-induced activation pathway. Resident peritoneal cells from C3H/HePas mice were incubated with MAM in the presence or absence of a PAF-antagonist (WEB2170) or COX-2 inhibitors (nimesulide or NS398). Enzyme expression was evaluated by immunoblotting, PGE2 by EIA, and NO by Griess reaction. Following MAM-stimulation of peritoneal cells, expression of COX-2 was detected at 3 h (peak levels at 12 h) and of iNOS at 6 h (peak levels at 20 h). PGE2 increased till 20 h, decreasing thereafter, whereas NO increased with time. WEB2170 (5 x 10(-5) M) treatment caused 44% inhibition of NO output and reduced iNOS expression (48% at the peak of expression). Concomitant treatment with WEB2170 and nimesulide (10(-5) M) reversed these inhibitory effects. WEB2170 reduced COX-2 expression (43% at the peak of expression) and prevented the decline in PGE2 levels after 20 h. These results suggest the involvement of PAF in the signaling pathway triggered by MAM that leads to expression of iNOS and COX-2, and show that PAF regulates the production of NO, possibly by controlling levels of PGE2.

Mycoplasma arthritidis causes arthritis in rodents that resembles human rheumatoid arthritis. It produces a superantigen (MAM) that stimulates production of cytokines by making a bridge between lymphocyte T-cell receptor with the appropriate Vbeta chain, and H-2 1-Ealpha MHC class II molecules. Here we studied MAM-induced nitric oxide (NO) production in mouse peritoneal macrophages and found that it was: (1) time and concentration dependent, (2) possibly derived from inducible NOS synthase since it was reduced significantly by amino guanidine pretreatment, (3) restricted to H-2(K) (C3H/HePas and C3H/HeJ) and H-2(d) strains (BALB/c), (4) independent of TLR4 signaling since the coisogenic strains C3H/HePas and C3H/HeJ (TLR4 deficient) produced similar levels of NO following MAM stimulation, (5) potentiated by lipopolysaccharide, and (6) dependent on the presence of nonadherent peritoneal cells. Neutralization of interferon-gamma (IFNgamma in the peritoneal cell cultures with monoclonal antibodies abolished MAM-induced NO production. Addition of rIFNgamma to the adherent cells substituted the nonadherent cells for MAM-induced NO production. A macrophage cell line, J774A.1 (H-2(d)), also produced NO upon MAM stimulation but only when BALB/c spleen lymphocytes were added. Thus, in murine macrophages, MAM induces NO production that is dependent on signaling through MHC class II molecules and IFNgamma but independent of TLR4 expression.