Browsing by Author "Smriti Sharma"

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A subset of neutrophils expressing markers of antigen-presenting cells in human visceral leishmaniasis
(Oxford University Press, 2016) Smriti Sharma; Richard E. Davis; Shweta Srivastva; Susanne Nylén; Shyam Sundar; Mary E. Wilson
Background. Visceral leishmaniasis (VL) is a potentially fatal parasitic disease associated with fever, cachexia and impaired protective T-cell responses against the parasite. Methods. Peripheral blood leukocytes from 105 subjects with VL and healthy control subjects from the endemic region of Muzaffarpur, Bihar, India, were compared using flow cytometry and reverse-transcriptase quantitative polymerase chain reaction. Findings were correlated with clinical data. Results. An expanded population of low-density neutrophils that expressed HLA-DR, CD80 and CD86 was observed in subjects with VL. This neutrophil population contracted after successful treatment of disease. Plasma from patients with acute VL was able to induce similar high-level HLA-DR expression in neutrophils from healthy subjects. HLA-DR+ neutrophils from subjects with VL did not stimulate T-cell proliferation, but they did express higher programmed cell death ligand-1 (PDL1) than other neutrophils, and lymphocytes of the same subjects expressed high programmed cell death 1 (PD1). Conclusions. Patients with acute VL have expanded circulating low-density neutrophils expressing markers of antigen presentation, which diminish after treatment. Development of HLA-DR+ neutrophils is stimulated, at least in part, by components of plasma from patients with acute disease. Although we found no evidence that they act as antigen-presenting cells, these neutrophils expressed markers implicating a role in T-cell exhaustion. © 2016 Published by Oxford University Press for the Infectious Diseases Society of America.
Evaluation of a diospyrin derivative as antileishmanial agent and potential modulator of ornithine decarboxylase of Leishmania donovani
(2013) Sudipta Hazra; Subhalakshmi Ghosh; Madhushree Das Sarma; Smriti Sharma; Mousumi Das; Prakash Saudagar; Vijay Kumar Prajapati; Vikash Kumar Dubey; Shyam Sundar; Banasri Hazra
World health organization has called for academic research and development of new chemotherapeutic strategies to overcome the emerging resistance and side effects exhibited by the drugs currently used against leishmaniasis. Diospyrin, a bis-naphthoquinone isolated from Diospyros montana Roxb., and its semi-synthetic derivatives, were reported for inhibitory activity against protozoan parasites including Leishmania. Presently, we have investigated the antileishmanial effect of a di-epoxide derivative of diospyrin (D17), both in vitro and in vivo. Further, the safety profile of D17 was established by testing its toxicity against normal macrophage cells (IC50~20.7μM), and also against normal BALB/c mice in vivo. The compound showed enhanced activity (IC50~7.2μM) as compared to diospyrin (IC50~12.6μM) against Leishmania donovani promastigotes. Again, D17 was tested on L. donovani BHU1216 isolated from a sodium stibogluconate-unresponsive patient, and exhibited selective inhibition of the intracellular amastigotes (IC50~0.18μM). Also, treatment of infected BALB/c mice with D17 at 2mg/kg/day reduced the hepatic parasite load by about 38%. Subsequently, computational docking studies were undertaken on selected enzymes of trypanothione metabolism, viz. trypanothione reductase (TryR) and ornithine decarboxylase (ODC), followed by the enzyme kinetics, where D17 demonstrated non-competitive inhibition of the L. donovani ODC, but could not inhibit TryR. © 2013 Elsevier Inc.
Evaluation of two novel rapid rKE16 antigen-based tests for diagnosis of visceral leishmaniasis in India
(2012) Manisha Vaish; Smriti Sharma; Jaya Chakravarty; Shyam Sundar
We compared the two formats of rKE16 antigen-based rapid tests, a flowthrough test (KEFT) and a lateral flow test (KELF), with the rK39 rapid test for the diagnosis of visceral leishmaniasis. Sensitivities with KEFT (99%, 198/200) and rK39 (99.5%, 199/200) were comparable and higher than that with KELF (95.5%, 191/200). In the control groups comprising subjects with diseases from areas of nonendemicity or endemicity and subjects with different diseases, the specificities were comparable for all three rapid tests, except that specificity was higher with KELF in the controls from areas of endemicity. Copyright © 2012, American Society for Microbiology. All Rights Reserved.
Evolutionary genomics of epidemic visceral leishmaniasis in the Indian subcontinent
(eLife Sciences Publications Ltd, 2016) Hideo Imamura; Tim Downing; Frederik van den Broeck; Mandy J. Sanders; Suman Rijal; Shyam Sundar; An Mannaert; Manu Vanaerschot; Maya Berg; Géraldine de Muylder; Franck Dumetz; Bart Cuypers; Ilse Maes; Malgorzata Domagalska; Saskia Decuypere; Keshav Rai; Surendra Uranw; Narayan Raj Bhattarai; Basudha Khanal; Vijay Kumar Prajapati; Smriti Sharma; Olivia Stark; Gabriele Schönian; Harry P. de Koning; Luca Settimo; Benoit Vanhollebeke; Syamal Roy; Bart Ostyn; Marleen Boelaert; Louis Maes; Matthew Berriman; Jean-Claude Dujardin; James A. Cotton
Leishmania donovani causes visceral leishmaniasis (VL), the second most deadly vector- borne parasitic disease. A recent epidemic in the Indian subcontinent (ISC) caused up to 80% of global VL and over 30,000 deaths per year. Resistance against antimonial drugs has probably been a contributing factor in the persistence of this epidemic. Here we use whole genome sequences from 204 clinical isolates to track the evolution and epidemiology of L. donovani from the ISC. We identify independent radiations that have emerged since a bottleneck coincident with 1960s DDT spraying campaigns. A genetically distinct population frequently resistant to antimonials has a two base-pair insertion in the aquaglyceroporin gene LdAQP1 that prevents the transport of trivalent antimonials. We find evidence of genetic exchange between ISC populations, and show that the mutation in LdAQP1 has spread by recombination. Our results reveal the complexity of L. donovani evolution in the ISC in response to drug treatment. © Imamura et al.
IL-10 and TGF-β Induced Arginase Expression Contributes to Deficient Nitric Oxide Response in Human Visceral Leishmaniasis
(Frontiers Media S.A., 2021) Manu Kupani; Smriti Sharma; Rajeev Kumar Pandey; Rajiv Kumar; Shyam Sundar; Sanjana Mehrotra
Nitric oxide (NO) is an anti-microbial effector of the innate immune system which plays major role in non-specific killing of various pathogens including protozoan parasites. However, due to subversion of the host’s immune processes by pathogens, suboptimal production of NO is frequently found in many infection models. Previous studies have shown suppressed NO production during Leishmania donovani infection, the causative agent of visceral leishmaniasis (VL). Availability of L-Arginine, a semi-essential amino acid is required for inducible nitric oxide synthase (iNOS) mediated NO production. However, arginase is another enzyme, which if expressed concomitantly, may strongly compete for L-Arginine, and suppress NO production by iNOS. In the present study, plasma nitrite and arginase levels were measured in VL patients before and after successful drug treatment, endemic and non-endemic healthy donors. We observed significantly lower NO levels in the plasma of VL patients as compared to endemic controls, which improved significantly post-treatment. Significantly elevated arginase activity was also observed in the plasma of VL patients, which may be associated with NO deficiency. VL patients also showed significantly higher levels of IL-10 and TGF-β, which are known to regulate expression of arginase in various immune cells. In vitro studies with human peripheral blood mononuclear cells (PBMCs) further corroborated the role of IL-10 and TGF-β in arginase mediated suppression of NO production. © Copyright © 2021 Kupani, Sharma, Pandey, Kumar, Sundar and Mehrotra.
In vitro susceptibility of leishmania donovani to miltefosine in Indian visceral leishmaniasis
(2013) Vijay Kumar Prajapati; Smriti Sharma; Madhukar Rai; Bart Ostyn; Poonam Salotra; Manu Vanaerschot; Jean-Claude Dujardin; Shyam Sundar
Promastigote miltefosine (MIL) susceptibility was performed on Leishmania donovani isolates from Indian patients with visceral leishmaniasis treated with MIL. Isolates that were obtained before the onset of MIL treatment, after completion of treatment (29th day), or at the time of treatment failure, were screened using in vitro promastigote assay. The MIL susceptibility of the pre-treatment isolates (N = 24, mean IC50 ± SEM= 3.74 ± 0.38 mM) was significantly higher than that of the post-treatment group (N = 26, mean IC50 ± SEM = 6.15 ± 0.52 mM; P = 0.0006) but was similar in the cured patients (N = 22, mean IC50 ± SEM= 5.58 ± 0.56 mM) and those who failed treatment (N = 28, mean IC50 ± SEM= 4.53 ± 0.47 mM). The pre/post-treatment results thus showed a 2-fold difference, whereas isolated from cured versus failed patients showed a similar susceptibility, suggesting that this higher tolerance is not responsible forMIL-treatment failure. Our work highlights the need for careful monitoring of MIL susceptibility for implementation in national VL elimination programs. Copyright © 2013 by The American Society of Tropical Medicine and Hygiene.
Leishmania donovani-induced increase in macrophage Bcl-2 favors parasite survival
(Frontiers Media S.A., 2016) Rajeev Kumar Pandey; Sanjana Mehrotra; Smriti Sharma; Ramachandra Subbaraya Gudde; Shyam Sundar; Chandrima Shaha
Members of the Bcl-2 family are major regulators of apoptosis in mammalian cells, and hence infection-induced perturbations in their expression could result into elimination of the parasites or creation of a niche favoring survival. In this investigation, we uncover a novel role of host Bcl-2 in sustaining Leishmania donovani infection. A rapid twofold increase in Bcl-2 expression occurred in response to parasite challenge. Downregulation of post infection Bcl-2 increase using siRNA or functional inhibition using Bcl-2 small molecule inhibitors interfered with intracellular parasite survival confirming the necessity of elevated Bcl-2 during infection. An increased nitric oxide (NO) response and reduced parasitic burden was observed upon Bcl-2 inhibition, where restitution of the NO response accounted for parasite mortality. Mechanistic insights revealed a major role of elevated Th2 cytokine IL-13 in parasite-induced Bcl-2 expression via the transcription factor STAT-3, where blocking at the level of IL-13 receptor or downstream kinase JAK-2 dampened Bcl-2 induction. Increase in Bcl-2 was orchestrated through Toll like receptor (TLR)-2-MEK-ERK signaling, and changes in TLR-2 levels affected parasite uptake. In a mouse model of visceral leishmaniasis (VL), Bcl-2 inhibitors partially restored the antimicrobial NO response by at least a twofold increase that resulted in significantly reduced parasite burden. Interestingly, monocytes derived from the peripheral blood of six out of nine human VL subjects demonstrated Bcl-2 expression at significantly higher levels, and sera from these patients showed only marginally quantifiable nitrites. Collectively, our study for the first time reveals a pro-parasitic role of host Bcl-2 and the capacity of host-derived IL-13 to modulate NO levels during infection via Bcl-2. Here, we propose Bcl-2 inhibition as a possible therapeutic intervention for VL. © 2016 Pandey, Mehrotra, Sharma, Gudde, Sundar and Shaha.
Phenotypic and functional characteristics of HLA-DR+ neutrophils in Brazilians with cutaneous leishmaniasis
(Federation of American Societies for Experimental Biology, 2017) Richard E. Davis; Smriti Sharma; Jacilara Conceição; Pedro Carneiro; Fernanda Novais; Phillip Scott; Shyam Sundar; Olivia Bacellar; Edgar M. Carvalho; Mary E. Wilson
The protozoan Leishmania braziliensis causes cutaneous leishmaniasis (CL) in endemic regions. In murine models, neutrophils (PMNs) are recruited to the site of infection soon after parasite inoculation. However, the roles of neutrophils during chronic infection and in human disease remain undefined. We hypothesized that neutrophils help maintain a systemic inflammatory state in subjects with CL. Lesion biopsies from all patients with CL tested contained neutrophils expressing HLA-DR, a molecule thought to be restricted to professional antigen-presenting cells. Although CL is a localized disease, a subset of patients with CL also had circulating neutrophils expressing HLA-DR and the costimulatory molecules CD80, CD86, and CD40. PMNs isolated from a low-density leukocyte blood fraction (LD-PMNs) contained a higher percentage of HLA-DR+ PMNs than did normal-density PMNs. In vitro coculture experiments suggested LD-PMNs do not suppress T cell responses, differentiating them from MDSCs. Flow-sorted HLA-DR+ PMNs morphologically resembled conventional PMNs, and they exhibited functional properties of PMNs. Compared with conventional PMNs, HLA-DR+ PMNs showed increased activation, degranulation, DHR123 oxidation, and phagocytic capacity. A few HLA-DR+ PMNs were observed in healthy subjects, and that proportion could be increased by incubation in either inflammatory cytokines or in plasma from a patient with CL. This was accompanied by an increase in PMN hladrb1 mRNA, suggesting a possible connection between neutrophil “priming” and up-regulation of HLA-DR. These data suggest that PMNs that are primed for activation and that also express surface markers of antigen-presenting cells emerge in the circulation and infected tissue lesions of patients with CL. © Society for Leukocyte Biology.
The phenotype of circulating neutrophils during visceral leishmaniasis
(American Society of Tropical Medicine and Hygiene, 2017) Smriti Sharma; Shweta Srivastva; Richard E. Davis; Siddharth Sankar Singh; Rajiv Kumar; Susanne Nylén; Mary E. Wilson; Shyam Sundar
Visceral leishmaniasis (VL) is a chronic parasitic disease associated with suppressed T cell responses. Although parasites reside intracellularly in macrophages during chronic VL, neutrophils are the first host cell to in filtrate the infection site and phagocytose the parasite. Subsets of neutrophils with unusual characteristics have been documented in human VL, but whether the total neutrophil population is a berrant during disease is not known. Therefore, we examined phenotypic characteristics of unfractionated polymorphonuclear leukocyte (neutrophils) from subjects with active VL, and compared these with neutrophils from healthy controls or subjects who have been treated for VL. The data showed decreased mRNA and diminished amounts of the neutrophil chemoattractant CXCL8 (interleukin [IL]-8), increased IL-10 mRNA and protein, and elevated transcripts encoding arginase-1, which is involved in suppressing T cell responses. Neutrophils from VL subjects showed enhanced capacity to phagocytose Leishmania spp. promastigotes. The results suggest that neutrophils may contribute to immunosuppression in subjects with active VL. © Copyright 2017 by The American Society of Tropical Medicine and Hygiene.