Radheshyam Maurya

Splenic accumulation of IL-10 mRNA in T cells distinct from CD4+CD25+ (Foxp3) regulatory T cells in human visceral leishmaniasis.

Visceral leishmaniasis (VL) is a life-threatening disease characterized by uncontrolled parasitization of the spleen, liver, and bone marrow. Interleukin (IL)-10 has been implicated in the suppression of host immunity in human VL based on the elevated levels of IL-10 observed in plasma and lesional tissue, and its role in preventing clearance of Leishmania donovani in murine models of VL. The aim of this study was to identify the cellular source of IL-10 in human VL and determine if CD4+CD25+ (Foxp3high) regulatory T (T reg) cells are associated with active disease. We analyzed surface marker and gene expression in peripheral blood mononuclear cells and splenic aspirates from Indian VL patients before and 3–4 wk after treatment with Amphotericin B. The results did not point to an important role for natural CD4+CD25+ (Foxp3high) T reg cells in human VL. They did not accumulate in and were not a major source of IL-10 in the spleen, and their removal did not rescue antigen-specific interferon γ responses. In contrast, splenic T cells depleted of CD25+ cells expressed the highest levels of IL-10 mRNA and were the predominant lymphocyte population in the VL spleen. The elevated levels of IL-10 in VL plasma significantly enhanced the growth of L. donovani amastigotes in human macrophages. The data implicate IL-10–producing CD25−Foxp3− T cells in the pathogenesis of human VL.

IL-10 neutralization promotes parasite clearance in splenic aspirate cells from patients with visceral leishmaniasis.

The mechanisms underlying the failure to contain the growth of Leishmania parasites in human visceral leishmaniasis (VL) are not understood. L donovani amastigotes were quantified in cultured splenic aspirate cells to assess the function of IL-10 in lesional tissue ex vivo. In 67 patients with active VL, IL-10 neutralization promoted parasite killing in 73% and complete clearance in 30%, while 18% had more parasites and 9% did not change. The splenic cells secreted increased levels of both tumor necrosis factor α (TNFα) and interferon γ (IFNγ) under IL-10-neutralizing conditions. These findings provide direct support for targeting IL-10 as an approach to therapy in human VL.

Rapid, Noninvasive Diagnosis of Visceral Leishmaniasis in India: Comparison of Two Immunochromatographic Strip Tests for Detection of Anti-K39 Antibody

ABSTRACT Used with blood or serum, a new anti-K39 antibody immunochromatographic strip test (IT-Leish; DiaMed AG) proved sensitive (range, 99 to 100%) and specific (range, 95 to 100%) for the noninvasive serodiagnosis of visceral leishmaniasis in India. Used with serum, the IT-Leish test and the existing Kalazar Detect test (InBios International, Inc.) yielded comparable results for symptomatic infection and identified apparent subclinical infection in 15 to 32% of healthy residents in a region where visceral leishmaniasis is highly endemic.

Evaluation of PCR for Diagnosis of Indian Kala-Azar and Assessment of Cure

ABSTRACT This study was done to evaluate PCR with Ld1 primers for the diagnosis of Indian visceral leishmaniasis (VL) and to assess its role in prediction of the disease outcome. The PCR assay was performed with DNA isolated from the peripheral blood of parasitologically confirmed cases of VL before the initiation of treatment, just after the end of treatment, and at 3 and 6 months of follow-up. The pretreatment PCR result was positive for 100 of 101 patients (sensitivity, 99%; confidence interval [CI], 94 to 100%). None of the 150 negative controls tested were PCR positive (specificity, 100%; CI, 96.8 to 100%). Of 60 patients who were treated at our center, 51 (85%; CI, 73 to 93%) became negative immediately after treatment and continued to be negative at 3 and 6 months of follow-up. At the 3-month follow-up, two of the remaining nine patients were PCR positive, making 58 (96.7%; CI, 87 to 100%) patients PCR negative. At the 6-month follow-up, all patients became PCR negative. One patient who was PCR negative immediately after the end of treatment relapsed 11 months later. This limited prospective study with VL patients suggests that the PCR assay is a highly sensitive and specific (99% and 100%, respectively) tool for the diagnosis of VL. In the majority of patients, it can identify a successful disease outcome; however, its translation into the field setting remains a major challenge.

Evaluation of Blood Agar Microtiter Plates for Culturing Leishmania Parasites To Titrate Parasite Burden in Spleen and Peripheral Blood of Patients with Visceral Leishmaniasis

ABSTRACT Serial dilution of blood and spleen biopsy specimens, plated on Novy-MacNeal-Nicolle (NNN) blood agar using microtiter culture plates, is a sensitive and reproducible method for detection and growth of Leishmania parasites. Plates could be easily monitored, and growth could be rapidly detected. Moreover, parasite number may be estimated using this technique.

An in vitro study of apoptotic like death in Leishmania donovani promastigotes by withanolides

The aim of this study was to isolate and evaluate the withanolides in inducing apoptotic like death in Leishmania donovani in vitro. Withanolides were fractionated and isolated from the leaves of Withania somnifera and LC-MS/MS analysis of two fractions namely, F5 and F6 of ethanolic extracts, obtained through column chromatography with silica gel, was performed. The antileishmanial effect of withanolides on L. donovani promastigotes was assessed in vitro using PI dye exclusion test. The effect of withanolides on promastigote morphology was determined by scanning electron microscopy. To understand their mode of action against L. donovani, DNA fragmentation, quantification of parasites at sub G0/G1 phase, determination of phosphatidylserine externalization, measurement of reactive oxygen species (ROS) and mitochondrial membrane potential (Ψm) were done. Results showed that LC-MS/MS analysis confirmed the presence of withanolides in isolated fractions. Treatment with withanolides resulted in morphological alterations from spindle to round shape and loss of flagella/cell integrity in promastigotes. Moreover, it induced DNA nicks, cell cycle arrest at sub G0/G1 phase and externalization of phosphatidylserine in dose and time dependent manner via increase in ROS and decrease in Ψm. Results of this study indicate that withanolides induce apoptotic like death through the production of ROS from mitochondria and disruption of Ψm in promastigotes of L donovani.

Human visceral leishmaniasis is not associated with expansion or accumulation of Foxp3 + CD4 cells in blood or spleen

Natural regulatory T cells (CD4+ CD25+ Foxp3+), natural regulatory T cells (nTreg), play an important role in the regulation of inflammatory immune responses. However, the immunosuppressive properties of nTreg may unfavourably affect the host’s ability to clear certain infections. In human visceral leishmaniasis (VL), reports on the frequency and function of nTreg are not conclusive. A limitation of our own previous studies that did not indicate a major role for Foxp3+ nTreg in VL pathogenesis was that Foxp3 was measured by mRNA expression alone, as other tools were not available at the time. We have in this study assessed CD4+CD25+Foxp3+ cells in splenic aspirates and peripheral blood mononuclear cells (PBMC) from an extensive series of patients with VL and endemic controls (EC) by flow cytometry (FACS). The results do not show increased frequencies of Foxp3+ cells in patient with VL pre‐ and post‐treatment, neither were they elevated when compared to PBMC of EC. We conclude that active VL is not associated with increased frequencies of peripheral Foxp3 Treg or accumulation at the site of infection.

Leptin induces the phagocytosis and protective immune response in Leishmania donovani infected THP-1 cell line and human PBMCs

Visceral leishmaniasis (VL) is an infectious disease responsible for several deaths in malnourished children due to impaired cell-mediated immunity, which is accompanied by low circulating leptin levels. The cytokine function of leptin is implicated for several immune regulation activities such as hematopoiesis, angiogenesis, innate and adaptive immunity. Its deficiency associated with polarization of Th2 response, which coincides with VL pathogenesis. To determine the cytokine role of leptin in case of experimental VL, we tested the leptin associated Th1/Th2 type cytokine profile at mRNA level from Leishmania donovani infected human monocytic leukemia cell line (THP-1) and peripheral blood mononuclear cells (PBMCs). We also tested the effect of leptin on macrophages activation (viz. studying the phosphorylation of signaling moieties), phagocytic activity and intracellular reactive oxygen species (ROS) production during infection. We observed that leptin induced Th1 specific response by upregulation of IL-1α, IL-1β, IL-8 and TNF-α in THP-1 and IFN-γ, IL-12 and IL-2 in PBMCs. We also observed the downregulation of Th2 type cytokine i.e. IL-10 in THP-1 and unaltered expression of cytokines i.e. TGF-β, IL-10 and IL-4 in PBMCs. In addition, leptin stimulates the macrophages by inducing phosphorylation of Erk1/2 and Akt which are usually dephosphorylated in L. donovani infection. In concordance, leptin also induces the macrophage phagocytic activity by enhancing the intracellular ROS generation which helps in phagolysosome formation and oxidative killing of the parasite. In compilation, leptin is able to maintain the defensive environment against L. donovani infection through the classical macrophage activity.

In vitro and in vivo evaluation of anti-leishmanial and immunomodulatory activity of Neem leaf extract in Leishmania donovani infection

The toxicity and emergence of resistance to available chemical drugs against visceral leishmaniasis is evoking to explore herbal treatment. One such attempt with the Neem is being reported here. The current study is primarily focused to evaluate the anti-leishmanial effects of Neem leaf extracts. Among which, ethyl acetate fraction (EAF) alone was found to exhibit leishmanicidal effect validated through cytotoxicity assay and estimated its IC₅₀ to be 52.4 µg/ml on the promastigote stage. Propidium iodide (PI) staining of dead cells substantiated the aforementioned activity. Carboxy fluorescein-diaceate succinimidyl ester (CFSE) staining of promastigotes has affirmed its anti-proliferation activity. The characteristic features such as DNA fragmentation, reduced mitochondrial membrane potential, increased sub G0/G1 phase parasites and increased reactive oxygen species (ROS) production in EAF treated promastigotes indicate the apoptosis like death. In addition, the reduced parasite burden both in vitro (viz. ~45% in human monocytic leukemia cell line (THP-1) and ~50% in peripheral blood mononuclear cells) and in vivo (spleen and liver) provides the evidence for its anti-leishmanial activity on amastigote stage. The increase of ROS levels in THP-1 and nitric oxide (NO) production from J774.1 cell line (mouse macrophages) upon EAF treatment was evidenced for oxidative killing of intracellular amastigotes. Active immunomodulatory activity at m-RNA level (viz. upregulation of Th1 cytokines, and downregulation of Th2 cytokines) both in vitro and in vivo was also shown to be exhibited by EAF. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of EAF revealed the presence of 14 compounds.

Role of leptin in human visceral leishmaniasis

Human visceral leishmaniasis (VL) is frequently found in poor population who are suffering from malnutrition in endemic areas. Therefore, obviously they may have reduced levels of leptin due to reduction in number of adipocytes which are major source of leptin production. Human pathogenesis of VL and reduced levels of leptin both are associated with increase in Th2 type immune response, characterized by secretion of cytokines such as IL-4 and IL-10. Whereas, the protective immune response during visceral leishmaniasis is associated with effective Th1 type immune response characterized by secretion of IFN-γ, IL-2 and IL-12, which correlates with leptin induction of T cells polarizing to Th1 population and secretion of proinflammatory cytokines, and also inhibition of Th2 type response. Therefore, we hypothesized that leptin might be effective in treatment of visceral leishmaniasis alone or VL patients who have co-infection with other immune deficiency syndromes such as AIDS/diabetes/autoimmune disorders by regulation of Th1/Th2 homeostasis.