Response In Visceral Leishmaniasis Research Articles

Immunogenicity and protective efficacy of tuzin protein as a vaccine candidate in Leishmania donovani-infected BALB/c mice.

Visceral leishmaniasis (VL) is referred to as the most severe and fatal type of leishmaniasis basically caused by Leishmania donovani and L. infantum. The most effective method for preventing the spread of the disease is vaccination. Till today, there is no promising licensed vaccination for human VL. Hence, investigation for vaccines is necessary to enrich the therapeutic repertoire against leishmaniasis. Tuzin is a rare trans-membrane protein that has been reported in Trypanosoma cruzi with unknown function. However, tuzin is not characterized in Leishmania parasites. In this study, we for the first time demonstrated that tuzin protein was expressed in both stages (promastigote and amastigote) of L. donovani parasites. In-silico studies revealed that tuzin has potent antigenic properties. Therefore, we analyzed the immunogenicity of tuzin protein and immune response in BALB/c mice challenged with the L. donovani parasite. We observed that tuzin-vaccinated mice have significantly reduced parasite burden in the spleen and liver compared with the control. The number of granulomas in the liver was also significantly decreased compared with the control groups. We further measured the IgG2a antibody level, a marker of Th1 immune response in VL, which was significantly higher in the serum of immunized mice when compared with the control. Splenocytes stimulated with soluble Leishmania antigen (SLA) displayed a significant increase in NO and ROS levels compared with the control groups. Tuzin-immunized and parasite-challenged mice exhibit a notable rise in the IFN-γ/IL-10 ratio by significantly suppressing IL-10 expression level, an immunosuppressive cytokine that inhibits leishmanicidal immune function and encourages disease progression. In conclusion, tuzin immunizations substantially increase the protective immune response in L. donovani-challenged mice groups compared with control.

NLRP12-expressing dendritic cells mediate both dissemination of infection and adaptive immune responses in visceral leishmaniasis.

The NLR protein NLRP12 contributes to innate immunity, but the mechanism remains elusive. Infection of Nlrp12 -/- or wild-type mice with Leishmania infantum led to aberrant parasite tropism. Parasites replicated to higher levels in livers of Nlrp12 -/- mice than in the livers of WT mice and failed to disseminate to spleens. Most retained liver parasites resided in dendritic cells (DCs), with correspondingly fewer infected DCs in spleens. Furthermore, Nlrp12 -/- DCs expressed lower CCR7 than WT DCs, failed to migrate toward CCL19 or CCL21 in chemotaxis assays, and migrated poorly to draining lymph nodes after sterile inflammation. Leishmania-infected Nlpr12 -/- DCs were significantly less effective at transporting parasites to lymph nodes than WT DCs. Consistently, adaptive immune responses were also impaired in infected Nlrp12 -/- mice. We hypothesize that Nlrp12-expressing DCs are required for efficient dissemination and immune clearance of L.infantum from the site of initial infection. This is at least partly due to the defective expression of CCR7.

Use of N-acetylcysteine as treatment adjuvant regulates immune response in visceral leishmaniasis: Pilot clinical trial and in vitro experiments.

This investigation aimed to assess the effect of N-acetylcysteine (NAC) as an adjuvant treatment to alleviate visceral leishmaniasis (VL). The present work includes both blinded randomized clinical intervention and experimental in vitro studies. The clinical trial included 60 patients with VL randomly allocated into two groups: a test group (n = 30) treated with meglumine antimoniate plus NAC (SbV + NAC) and a control group (n = 30) treated with meglumine antimoniate only (SbV). The primary outcome was clinical cure (absence of fever, spleen and liver sizes reduction, and hematological improvement) in 180 days. The cure rate did not differ between the groups; both groups had similar results in all readout indices. The immunological parameters of the patients treated with SbV + NAC showed higher sCD40L in sera during treatment, and the levels of sCD40L were negatively correlated with Interleukin-10 (IL-10) serum levels. In addition, data estimation showed a negative correlation between the sCD40L levels and the spleen size in patients with VL. For the in vitro experiments, peripheral blood mononuclear cells (PBMCs) or PBMC-derived macrophages from healthy donors were exposed to soluble Leishmania antigen (SLA) or infected with stationary promastigotes of Leishmania infantum in the presence or absence of NAC. Results revealed that NAC treatment of SLA-stimulated PBMCs reduces the frequency of monocytes producing IL-10 and lowers the frequency of CD4+ and CD8+ T cells expressing (pro-)inflammatory cytokines. Together, these results suggest that NAC treatment may modulate the immune response in patients with VL, thus warranting additional investigations to support its case use as an adjuvant to antimony therapy for VL.

First person – Pragya Chandrakar

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Pragya Chandrakar is first author on ‘Jagged–Notch-mediated divergence of immune cell crosstalk maintains the anti-inflammatory response in visceral leishmaniasis’, published in JCS. Pragya conducted the research described in this article while a Senior Research Fellow in Dr Susanta Kar's lab at the CSIR Central Drug Research Institute, Lucknow, India. She is now a Postdoctoral Research Fellow in the lab of Dr John Chan at Albert Einstein College of Medicine, New York, USA, investigating the different possible mechanisms by which foreign pathogens breach the immune system.

Jagged-Notch-mediated divergence of immune cell crosstalk maintains the anti-inflammatory response in visceral leishmaniasis.

Notch signaling governs crucial aspects of intercellular communication spanning antigen-presenting cells and T-cells. In this study, we investigate how Leishmaniadonovani takes advantage of this pathway to quell host immune responses. We report induction of the Notch ligand Jagged1 in L. donovani-infected bone marrow macrophages (BMMϕs) and subsequent activation of RBPJκ (also known as RBPJ) in T cells, which in turn upregulates the transcription factor GATA3. Activated RBPJκ also associates with the histone acetyltransferase p300 (also known as EP300), which binds with the Bcl2l12 promoter and enhances its expression. Interaction of Bcl2L12 with GATA3 in CD4+ T cells facilitates its binding to the interleukin (IL)-10 and IL-4 promoters, thereby increasing the secretion of these cytokines. Silencing Jagged1 hindered these events in a BMMϕ-T cell co-culture system. Upon further scrutiny, we found that parasite lipophosphoglycan (LPG) induces the host phosphoinositide 3-kinase (PI3K)/Akt pathway, which activates β-catenin and Egr1, the two transcription factors responsible for driving Jagged1 expression. In vivo morpholino-silencing of Jagged1 suppresses anti-inflammatory cytokine responses and reduces organ parasite burden in L. donovani-infected Balb/c mice, suggesting that L. donovani-induced host Jagged1-Notch signaling skews macrophage-T cell crosstalk into disease-promoting Th2 mode in experimental visceral leishmaniasis.This article has an associated First Person interview with the first author of the paper.

Exploring new immunological insight on SP15 (∼14 kDa) family protein in saliva of Indian sand-fly (Phlebotomus argentipes) in experimental visceral leishmaniasis

Visceral leishmaniasis (VL) is a disease caused by protozoan species of the genus Leishmania and is transmitted through bites from the Phlebotomus sand fly; it is associated with considerable morbidity and mortality in many parts of world, including India. Reports on the protective role played by saliva proteins of Lutozomyia longipalpis, Phlebotomus papatasi and Phlebotomus duboscqi. are available. However, no studies have explored the salivary proteins of P. argentipes, which is the known proven vector for the transmission of VL in the Indian sub-continent. Herein we revealed the presence of two proteins of 14.2 and one protein of 13.6 kDa in Indian strain P. argentipes which is absolute identical to previously reported protein of SP15 family (PagSP01, PagSP02 and PagSP07) of P. argentipes of NIH colony, USA. In an experimental study on P. argentipes from Bihar, India, we demonstrated that a strong humoral and cellular immune response was triggered to reduce the concomitant Leishmania load in groups of immunized mice. The immunized group produced a considerable amount of IgG antibodies, and their splenocytes generated TH1 cytokines (IL-12, IFN-γ) with the support of delayed-type hypersensitivity (DTH) reactivity in such mice at the challenged site. We summarize from our data that some identical proteins to previous from SP15 family protein of 14.2 and 13.6 kDa molecular size, derived from Indian P. argentipes and reported its first time, can also be significant in resolution of VL infection after modulation of host protective T cell response in VL.

Cytosolic tryparedoxin of Leishmania donovani modulates host immune response in visceral leishmaniasis

Leishmaniasis is a neglected tropical disease caused by the unicellular protozoan parasite of genus Leishmania. Tryparedoxin (TXN) is a low molecular mass dithiol protein belonging to oxidoreductases super-family; which function in concert with tryparedoxin peroxidase (TXNPx) as a system in protozoan parasites including Leishmania. Leishmanial hydroperoxides detoxification cascade uses trypanothione as electron donor to reduce hydroperoxide inside the macrophages during infection. However, the mechanism by which tryparedoxin can contribute in progression of visceral leishmaniasis (VL) and its impact on host’s cellular immune response during infection in Indian VL patient is unknown. In this study, we purified a ∼17 kDa recombinant cytosolic tryparedoxin (cTXN) protein of Leishmania donovani (rLdcTXN) and investigated its immunological responses in peripheral blood monocytes (PBMC) isolated from VL patients. The protein significantly enhanced the promastigotes count after 96 h of culture showing a direct correlation with parasite growth. Furthermore, stimulation of PBMC isolated from VL patients with rLdcTXN resulted in up-regulation of IL-4 and IL-10 production whereas IL-12 and IFN-γ was significantly down-regulated suggesting a pivotal role of cTXN in provoking the immune suppression during VL. Our study demonstrates the importance of cTXN protein which can potentially modulate the outcome of disease through suppressing host protective Th1 response in VL patients.

Leptin regulates Granzyme-A, PD-1 and CTLA-4 expression in T cell to control visceral leishmaniasis in BALB/c Mice

Visceral leishmaniasis (VL) is responsible for several deaths in malnourished children accompanied by diminished circulating leptin and impaired cell-mediated immunity. Typically, leptin deficiency is associated with the Th2 polarization that markedly coincides with the pathogenesis of VL. The aim of the present study was to unravel the prophylactic role of leptin in malnutrition-coupled VL mice. Interestingly, we observed that L. donovani infection itself reduces the serum leptin levels in malnutrition. Exogenous leptin restored severe body weight loss and parasite load in the spleen and liver of malnourished infected mice compared to controls. Leptin increases functional CD8+ T-cell population, Granzyme-A expression down-regulates anergic T-cell markers such as PD-1 and CTLA-4. It was also noticed that, leptin suppresses GM-CSF mRNA expression in parasite favored monocytes and reduced arginase activity in bone marrow derived macrophage indicate macrophages dependent T-cell activation and proliferation. Leptin-induced IFN-γ, IL-2, and TNF-α cytokines in the culture supernatant of splenocytes upon soluble leishmanial antigen (SLA) stimulation and significantly up-regulates serum IgG2a titers, which help to generate Th1 immune response in VL. Furthermore, leptin induced a granulomatous response and restored L. donovani induced tissue degeneration in the liver. Altogether, our findings suggest the exogenous leptin can restore T cell mediated immunity in malnourished VL mice.

A New Vaccine Strategy of Dendritic Cell Presented Kinetoplastid Membrane (KMP-11) as Immunogen for Control against Experimental Visceral Leishmaniasis

Available reports suggest that, Leishmania donovani antigen KMP-11 may be significant in the modulation of immune responses in visceral leishmaniasis (VL). This study evaluated vaccine prospect of presentation of KMP-11 antigen through murine dendritic cells against VL in infected BALB/c mice. We report here that immunization with KMP-11 delivered through bone marrow derived dendritic cells can lead to killing of L. donovani in infected BALB/c mice. Furthermore, the strategy to use KMP-11 as vaccine delivered through DCs can stimulate the production of IFN-g, IL-12, IL-2R and TNF-α with concomitant down-regulation of IL-10 and IL-4. Furthermore, anti-leishmanial defence function (ROS) of splenocytes was observed increased in the presence of DC-delivered KMP-11 vaccination accompanied with an increased p38-MAPK signalling in vaccinated splenocytes. We summarized from our data that KMP-11 delivered through DCs has potential for eliciting protective immunity through pro-inflammatory cytokines (IFN-γ, IL-12, IL-2, TNF-α) following an up-regulation in signalling event of p38-MAPK. Therefore the study suggests a new control strategy against VL in future.

The immunology of post-kala-azar dermal leishmaniasis (PKDL).

Post-kala-azar dermal leishmaniasis (PKDL) is a common complication of visceral leishmaniasis (VL) caused by Leishmania donovani. Because of its possible role in transmission it is considered a public health problem in VL endemic areas. The clinical features include a skin rash consisting of macules, papules or nodules in an otherwise healthy individual; this presentation is determined by the immune response towards parasites in the skin that probably persisted from the previous VL episode. The immune response in VL, cured VL and PKDL is the result of changes in the cytokine profile that only in part can be captured under the Th1 and Th2 dichotomy. Regulatory T cells and Th 17 cells also play a role. VL is characterized by an absent immune response to Leishmania with a predominantly Th2 type of response with high levels of IL-10; after successful treatment the patient will be immune with in vitro features of a Th1 type of response and in vivo a positive leishmanin skin test. PKDL takes an intermediate position with a dissociation of the immune response between the skin and the viscera, with a Th2 and Th1 type of response, respectively. It is likely that immune responses determine the different epidemiological and clinical characteristics of PKDL in Asia and Africa; various risk factors for PKDL may influence this, such as incomplete and inadequate treatment of VL, parasite resistance and genetic factors. It should be noted that PKDL is a heterogeneous and dynamic condition and patients differ with regard to time of onset after visceral leishmaniasis (VL), chronicity, extent and appearance of the rash including related immune responses, all of which may vary over time. Better understanding of these immune responses may offer opportunities for manipulation including combined chemotherapy and immunotherapy for VL to prevent PKDL from occurring and similarly in the treatment of chronic or treatment resistant PKDL cases.

DNA vaccine against visceral leishmaniasis: a promising approach for prevention and control.

The visceral leishmaniasis (VL) caused by Leishmania donovani parasite severely affects large populations in tropical and subtropical regions of the world. The arsenal of drugs available is limited, and resistance is common in clinical field isolates. Therefore, vaccines could be an important alternative for prevention against VL. Recently, some investigators advocated the protective efficacy of DNA vaccines, which induces the T cell-based immunity against VL. The vaccine antigens are selected as conserved in various Leishmania species and provide a viable strategy for DNA vaccine development. Our understanding for DNA vaccine development against VL is not enough and much technological advancement is required. Improved formulations and methods of delivery are required, which increase the uptake of DNA vaccine by cells; optimization of vaccine vectors/encoded antigens to augment and direct the host immune response in VL. Despite the many genes identified as vaccine candidates, the disappointing potency of the DNA vaccines in VL underscores the challenges encountered in the efforts to translate efficacy in preclinical models into clinical realities. This review will provide a brief background of DNA vaccines including the insights gained about the design, strategy, safety issues, varied candidates, progress and challenges that play a role in their ability against VL.

Expression of leukosialin (CD43) defines a major intrahepatic T cell subset associated with protective responses in visceral leishmaniasis.

BackgroundLeishmaniasis is a neglected vector-borne tropical disease caused by Leishmania protozoa that are transmitted to mammalian hosts by infected sand flies. Infection is associated with distinct clinical manifestations that include cutaneous, mucocutaneous and visceral lesions. Visceral leishmaniasis (VL) is the most severe form of the disease and is considered second in terms of mortality and fourth in terms of morbidity among tropical diseases. IFN-γ-producing T cells are involved in protection against the disease.MethodsCD43+/+ and CD43-/- mice on a C57BL/6 background were intravenously injected with 5 × 10 7 amastigotes of Leishmania (L.) infantum chagasi, and 30 days after infection the clinical signs of disease were examined; the splenocytes were isolated and assayed for cytokine production; and the livers were removed for phenotypic analysis of T cell subsets by flow cytometry.ResultsWe report that mice lacking CD43 display increased susceptibility to infection by Leishmania (L.) infantum chagasi, with higher parasite burdens than wild-type mice. The increased susceptibility of CD43−/− mice were associated with a weakened delayed hypersensitivity response and reduced levels of IgG2a antibodies to leishmania antigens. We further showed that expression of CD43 defines a major intrahepatic CD4+ and CD8+ T cell subsets with pro-inflammatory phenotypes and leads to increased levels of IFN-γ secretion by activated splenocytes.ConclusionsOur findings point to a role of CD43 in the development of host resistance to visceral leishmaniasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0721-9) contains supplementary material, which is available to authorized users.

Evaluation of interleukin-8 -251 t/a polymorphisms in visceral leishmaniasis.

Interleukin (IL)-8 plays important roles in the recruitment and activation of immune cells during visceral leishmaniasis (VL). Genetic variations in IL-8 modulate the expression of IL-8 protein and may be associated with VL. This study aimed to evaluate polymorphisms at the IL-8 -251 position in VL patients. This cross-sectional study was performed on three groups: Leishmania-seropositive patients with clinical symptoms of VL (n = 91), seropositive patients without clinical symptoms (n = 104), and healthy controls (n = 110). Polymorphisms at the IL-8-251 position were analyzed using allele-specific polymerase chain reaction (PCR). Anti-Leishmania antibody titers were assessed by immunofluorescence. IL-8-251 polymorphism was significantly associated with VL (P<0.002). The IL-8-251 T/T genotype was significantly higher in group 1 than in groups 2 and 3 (P<0.002). The validity of the data was analyzed using Hardy-Weinberg equilibrium and one-way analysis of variance (ANOVA), as well as χ2 tests. IL-8-251 polymorphism was significantly associated with impaired immune responses in VL and might be considered a risk factor for disease development.

Leishmania donovani: impairment of the cellular immune response against recombinant ornithine decarboxylase protein as a possible evasion strategy of Leishmania in visceral leishmaniasis

Ornithine decarboxylase, the rate limiting enzyme of the polyamine biosynthesis pathway, is significant in the synthesis of trypanothione, T(SH)2, the major reduced thiol which is responsible for the modulation of the immune response and pathogenesis in visceral leishmaniasis. Data on the relationship between ornithine decarboxylase and the cellular immune response in VL patients are limited. Therefore, we purified a recombinant ornithine decarboxylase from Leishmania donovani (r-LdODC) of approximately 77kDa and examined its effects on the immunological responses in peripheral blood mononuclear cells of human visceral leishmaniasis cases. For these studies, α-difluoromethylornithine was tested as an inhibitor and was used in parallel in all experiments. The r-LdODC was identified as having a direct correlation with parasite growth and significantly increased the number of promastigotes as well as axenic amastigotes after 96h of culture. The stimulation of peripheral blood mononuclear cells with r-LdODC up-regulated IL-10 production but not IFN-γ production from CD4+ T cells in active as well as cured visceral leishmaniasis cases, indicating a pivotal role for r-LdODC in causing strong immune suppression in a susceptible host. In addition, severe hindrance of the immune response and anti-leishmanial macrophage function due to r-LdODC, as revealed by decreased IL-12 and nitric oxide production, and down-regulation in mean fluorescence intensities of reactive oxygen species, occurred in visceral leishmaniasis patients. There was little impact of r-LdODC in the killing of L. donovani amastigotes in macrophages of visceral leishmaniasis patients. In contrast, when cultures of promastigotes and amastigotes, and patients’ blood samples, were directed against α-difluoromethylornithine, parasite numbers significantly reduced in culture, whereas the levels of IFN-γ and IL-12, and the production of reactive oxygen species and nitric oxide, were significantly elevated. Therefore, we demonstrated cross-talk with the use of α-difluoromethylornithine which can reduce the activity of ornithine decarboxylase of L. donovani, eliminating the parasite-induced immune suppression and inducing collateral host protective responses in visceral leishmaniasis.

Leishmania donovani phosphoproteins pp41 and pp29 re-establishes host protective immune response in visceral leishmaniasis

As phospho proteins are reported to be involved in virulence and survival, the ability of Leishmania to inhibit macrophage effector functions may result from a direct interference of leishmanial molecules with macrophage signal transduction pathways. Several such proteins such as pp63, pp41 and pp29 have also been identified as a Th1 stimulatory protein in the Leishmania donovani. In the present study, the immunogenicity of a cocktail of pp63+pp41+pp29 was assessed by estimation of serum antibody titre, nitric oxide(NO) production, estimation of Th1 cytokine(IFN-γ) as well as Th2 cytokines(IL-4), and determination of parasite load in L. donovani infected mice. In the group immunized with antigenic cocktail there was a sharp rise in antibody titer up to Day 20 which reduced considerably by Day 50. Groups of mice vaccinated with pp63, pp41, pp29 and the antigenic cocktail expressed 10-fold, 16-fold, 22-fold and 25-fold increase respectively in NO production by splenocytes. The animal groups immunized with pp63, pp41, pp29 and the antigenic cocktail showed reduced parasite load in the liver and spleen, as well as increased IFN-gamma production in the spleen. Furthermore immunized animals remained with a normal hematological profile, whereas L. donovani in unimmunized mice lead to significant anemia.

Class II MHC/peptide interaction in Leishmania donovani infection: implications in vaccine design.

We show that Leishmania donovani-infected macrophages (MΦs) are capable of stimulating MHC class II (MHC-II)-restricted T cells at 6 h of infection. At 48 h, infected MΦs (I-MΦs) failed to stimulate MHC-II-restricted T cells but not MHC class I-restricted ones, in contrast to normal MΦs. Such I-MΦs could stimulate T cells at a higher Ag concentration, indicating that general Ag processing and trafficking of peptide-MHC-II complexes are not defective. Analysis of the kinetic parameters, like "kon" and "koff," showed that peptide-MHC-II complex formation is compromised in I-MΦs compared with normal MΦs. This indicates interference in loading of the cognate peptide to MHC-II, which may be due to the presence of a noncognate molecule. This notion received support from the finding that exposure of I-MΦs to low pH or treatment with 2-(1-adamantyl)-ethanol, a molecule that favors peptide exchange, led to T cell activation. When treated with 2-(1-adamantyl)-ethanol, splenocytes from 8 wk-infected BALB/c mice showed significantly higher antileishmanial T cell expansion in vitro compared with untreated controls. Hence, it is tempting to speculate that high, but not low, concentrations of cognate peptide may favor peptide exchange in I-MΦs, leading to expansion of the antileishmanial T cell repertoire. The results suggest that a high Ag dose may overcome compromised T cell responses in visceral leishmaniasis, and this has an important implication in therapeutic vaccine design.

Evaluation of Leishmania donovani disulfide isomerase as a potential target of cellular immunity against visceral leishmaniasis

In Leishmania species, protein disulfide isomerase (PDI) – a redox chaperone is primarily associated with virulence and survival. The precise mechanism, especially in relation to redox changes and its effects on immunological responses in visceral leishmaniasis (VL) is not completely understood as yet. Therefore, we purified a recombinant PDI from Leishmania donovani (r-LdPDI) which was of ∼15kDa molecular size and examined its effects on immunological responses in peripheral blood (PBMC) of human VL cases. For these studies, alanine was tested as an inhibitor and was used in parallel to all experiments. This protein was identified to have a direct correlation with parasite growth which significantly increased number of promastigotes as well as axenic amastigotes after 96h of culture. Our experiments examining the immunological response against r-LdPDI also indicate the activation of pro-L. donovani dictated immunological responses in VL. The stimulation of PBMC with r-LdPDI induced lactate dehydrogenase (LDH) activities and up regulated interleukin-10 (IL-10) production but not the HLA-DR expression, Nitric oxide (NO) release and IFN-γ production indicating a pivoted role for r-LdPDI in causing a strong immunosuppression in a susceptible host. Further, we observed that an addition of alanine in L. donovani culture offers a significant inhibition in growth of parasite and helps in reconstitution of protective immune response in VL cases. Therefore, we demonstrate a future cross talk on use of alanine which can reduce the activities of PDI of L. donovani, eliminating the parasite induced immunosuppression and inducing collateral host protective response in VL.

Leishmania donovani: Dynamics of L. donovani evasion of innate immune cell attack due to malnutrition in visceral leishmaniasis

ObjectiveMalnutrition may be significant in the modulation of immune responses in visceral leishmaniasis (VL). Data on the relationship between malnutrition and innate immune response in VL are limited. The aim of this study was to examine the effect of malnutrition on the profile of innate immune functions of polymorphonuclear neutrophil granulocytes (PMNs) and monocytes through comparison of well-nourished and malnourished Indian patients with VL. MethodsForty individuals were enrolled comprising 20 active and untreated cases of VL and 20 non-VL individuals from the endemic region of Bihar, India. The patients with VL were segregated into two groups of 10 well-nourished and 10 malnourished participants. Patients' blood samples were directed against a crude Leishmania donovani extract (soluble leishmanial antigen) and phorbol 12-myristate-13-acetate plus ionomycin. The transendothelial cell adherence migration abilities of the PMNs and monocytes directed against these antigens were determined in whole-blood assays by flow cytometry. The chemokine (interleukin [IL]-8, macrophage inflammatory protein [MIP]-1 α) and cytokine support (tumor necrosis factor -α, interferon [IFN]-γ, IL-10), which could be significant in transendothelial cell migration, and efficacies of antileishmanial phagocytic function and reactive oxygen species (ROS) generation were also determined. ResultsSevere hindrance in the adherence of innate immune cells to the endothelial wall due to Leishmania parasites, as revealed by decreased shedding of l-selectin (CD62 L) and down-regulation of CD11 b expression on the surface of the PMNs and monocytes, occurred in malnourished VL patients. The production of MIP-1 α and IL-8 in response to L. donovani antigen was reduced in malnourished patients. In contrast, malnutrition in VL patients significantly reduced the IFN-γ and TNF-α produced by these immune cells, whereas the levels of IL-10 were significantly elevated. Malnourished VL patients were observed with severely dysfunctional PMNs and monocytes in terms of ROS activity that could not be recovered by stimulation with L. donovani antigen. ConclusionsMalnutrition linked to VL can be a decisive factor in the dynamics of L. donovani evasion of innate immune cell function in VL patients.

Effectiveness of miltefosine treatment in targeting anti-leishmanial HO-1/Nrf-2-mediated oxidative responses in visceral leishmaniasis patients

Miltefosine, an alkylphosphocholine anti-cancer drug, exhibits direct activity against Leishmania donovani and also promotes anti-leishmanial host immunomodulatory functions. Nuclear factor-erythroid 2-related factor 2 (Nrf-2), a redox-sensitive transcription factor, regulates the host stress responses leading to effective microbial clearance by a positive effect on haem oxygenase-1 (HO-1) enzyme expression/activity. We aimed to investigate the role of miltefosine in regulating HO-1/Nrf-2-mediated oxidative responses in visceral leishmaniasis (VL) patients in vivo and in vitro. Splenic aspirate and bone marrow aspirate cells of VL patients (n = 23) were used in the study. RT-PCR of HO-1, Nrf-2 translocation analysis and HO-1 ELISA were used to investigate the HO-1/Nrf-2-mediated modulation of oxidative responses by miltefosine in vivo. Fluorometric measurement of reactive oxygen species (ROS) was performed, determination of glutathione peroxidase (GPx) activity was performed, and bilirubin and superoxide dismutase (SOD) levels were determined. The in vitro HO-1/Nrf-2-dependent anti-leishmanial effect of miltefosine was assessed by the use of specific inhibitors/inducers and subsequent microscopic measurement of parasite killing and Th1/Th2 cytokine regulation by ELISA. Increased levels of transcript and serum HO-1, Nrf-2 nuclear translocation, serum bilirubin, GPx and SOD activity in untreated VL patients were reversed after miltefosine chemotherapy. The effectiveness of miltefosine for positive induction of ROS via NADPH correlated with a decrease in HO-1/ERK/Nrf-2-dependent parasite load. Furthermore, HO-1 blockade by miltefosine led to suppression of interleukin-10 and transforming growth factor-β, but enhanced interleukin-12 and tumour necrosis factor-α production, in VL patients. The antioxidant promoting property of L. donovani is crucial for protection against the mounting redox threat in the host. Therefore, these findings provide direct evidence for targeting HO-1/Nrf-2 as an anti-leishmanial approach for chemotherapy in human VL.

Patients with visceral leishmaniasis present an increased expression of TLR2 and TLR4 in lymphocytes and lipid and protein changes

Event Abstract Back to Event Patients with visceral leishmaniasis present an increased expression of TLR2 and TLR4 in lymphocytes and lipid and protein changes Mariana Gatto1, Mariana M. Abreu1, Marjorie D. Golim2, José C. Simão3, Karen I. Tasca1*, Loraine Gollino1, Carlos M. Fortaleza1, Paulo C. Pereira1 and Sueli A. Calvi1 1 Univ Estadual Paulista - UNESP, Tropical Disease, Brazil 2 Univ Estadual Paulista - UNESP, Hemocenter Laboratory of Flow Citometry, Brazil 3 Manoel de Abreu Hospital, Brazil In order to have the development of adaptive immune response in visceral leishmaniasis is necessary that cellular receptors such as TLR2 and TLR4 recognize and play the parasite effector mechanisms. Moreover, the nutritional profile also has impact on immune response. Our objective was to evaluate the expression of TLR2 and TLR4 in CD14 and CD3 cells and correlate with nutritional status in patients with visceral leishmaniasis, pre and post-treatment. Were evaluated 8 patients pre-treatment, 3 post-treatment and 8 healthy subjects. Analysis of the expression of TLR2 and TLR4 receptors on CD3 and CD14 cells was performed by flow cytometry and the nutritional profile was performed by bioelectrical impedance and biochemical tests. In CD3 cells, expression of TLR2 was higher in patients pre-treatment than those post-treatment (p <0.05) and controls (p <0.05); TLR4 expression was higher in pre-treatment compared to controls (p <0.05). In CD14 cells, TLR2 expression was lower in pre-treatment compared to controls (p <0.05) and TLR4 expression did not differ between groups. Levels of HDL and albumin were lower in pre-treatment compared to post-treatment (p <0.05) and controls (p <0.05), phase angle was lower in pre-treatment than those post-treatment (p <0.05) and triglycerides were higher in pre-treatment compared to controls (p <0.05). Our results suggest that patients with LV activates expressed primarily TLR2 and TLR4 in lymphocytes, suggesting a possible involvement of these receptors in the development of the immune response. Patients also showed nutritional changes that could be interfering with the immune response against the parasite. Keywords: Visceral leishmaniasis, Innate response, TLRs (Toll-like receptors), Nutritional Status, pre and pós-treatment Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Gatto M, Abreu MM, Golim MD, Simão JC, Tasca KI, Gollino L, Fortaleza CM, Pereira PC and Calvi SA (2013). Patients with visceral leishmaniasis present an increased expression of TLR2 and TLR4 in lymphocytes and lipid and protein changes. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00517 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 02 May 2013; Published Online: 22 Aug 2013. * Correspondence: Miss. Karen I Tasca, Univ Estadual Paulista - UNESP, Tropical Disease, Botucatu, SP, Brazil, karenitasca@hotmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Mariana Gatto Mariana M Abreu Marjorie D Golim José C Simão Karen I Tasca Loraine Gollino Carlos M Fortaleza Paulo C Pereira Sueli A Calvi Google Mariana Gatto Mariana M Abreu Marjorie D Golim José C Simão Karen I Tasca Loraine Gollino Carlos M Fortaleza Paulo C Pereira Sueli A Calvi Google Scholar Mariana Gatto Mariana M Abreu Marjorie D Golim José C Simão Karen I Tasca Loraine Gollino Carlos M Fortaleza Paulo C Pereira Sueli A Calvi PubMed Mariana Gatto Mariana M Abreu Marjorie D Golim José C Simão Karen I Tasca Loraine Gollino Carlos M Fortaleza Paulo C Pereira Sueli A Calvi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.