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Shyam Sundar - One of the best experts on this subject based on the ideXlab platform.

  • SEE PROFILE
    2016
    Co-Authors: Mitali Chatterjee, Shyam Sundar, Chitra Mandal, Available From Mitali Chatterjee
    Abstract:

    A Leishmania donovani species-specific monoclonal antibody (monoclonal antibody D2) was evaluated for its diagnostic and prognostic potential by a competitive enzyme-linked immunosorbent assay (C-ELISA) in sera from Indian patients with visceral leishmaniasis (VL) and seven patients with post-kala-azar dermal leish-maniasis (PKDL). These results were compared with those obtained by microscopy with Giemsa-stained tissue smears and a direct enzyme-linked immunosorbent assay (direct ELISA) with crude parasite antigen. Of 121 patients with clinically diagnosed VL examined, 103 (85.1%) were positive and 11 (9.1%) were negative by all three methods. An additional 7 (5.8%) who were negative by microscopy were positive by both C-ELISA and direct ELISA. Seven PKDL patients were also examined and were found to be positive by all three methods. Analysis of the chemotherapeutic response to sodium Antimony Gluconate of these 110 serologically positive VL patients showed that 57 (51.8%) were drug responsive and 53 (48.2%) were drug resistant. The C-ELISA wit

  • The overexpression of genes of thiol metabolism contribute to drug resistance in clinical isolates of visceral leishmaniasis (kala azar) in India
    Parasites & Vectors, 2014
    Co-Authors: Neeloo Singh, Mitali Chatterjee, Shyam Sundar
    Abstract:

    Background Visceral leishmaniasis (VL), also called Kala Azar (KA) or black fever in India, claims around 20,000 lives every year. Chemotherapy remains one of the most important tools in the control of VL. Current chemotherapy for Kala Azar in India relies on a rather limited arsenal of drugs including sodium Antimony Gluconate and amphotericin B in addition to the very expensive drug miltefosine. Pentavalent antimonials have been used for more than half a century in the therapy of leishmaniasis as it is relatively safe and inexpensive, however, the spread of resistance to this drug is forcing clinicians in India to abandon this treatment. Consequently, improvement of antimonial chemotherapy has become a major challenging area of study by leishmaniacs worldwide. The alarming emergence of resistance to the commonly used antleishmanial drug, sodium Antimony Gluconate, in India, has led us to elucidate the resistance mechanism(s) in clinical isolates. Studies on laboratory mutants have shown that resistance to antimonials is highly dependent on thiol levels. The parasite evades cytotoxic effects of antimonial therapy by enhanced efflux of drug upon conjugation with thiols, through overexpressed membrane proteins belonging to the superfamily of ABC transporters. Methods We have carried out functional studies to determine the activity of the efflux pumps in antimonial resistant clinical isolates collected from disease endemic areas in India and also carried out molecular characterization of thiol levels in these parasites. Results Overexpression of the gene coding for γ glutamylcysteine synthetase was observed in these resistant clinical isolates thereby establishing that thiols represent the key determinants of antimonial resistance. The SbIII/thiol conjugates can be sequestered by ABC transporter multidrug resistance protein A (MRPA) into intracellular organelles or can be directly pumped out by an uncharacterized transporter. Conclusions Our studies investigating antimonial resistance in different L. donovani clinical isolates suggest that over functioning of MRP plays a role in generation of Antimony resistance phenotype in some L. donovani clinical isolates.

  • elucidation of cellular mechanisms involved in experimental paromomycin resistance in leishmania donovani
    Antimicrobial Agents and Chemotherapy, 2014
    Co-Authors: Vasundhra Bhandari, Shyam Sundar, Jeanclaude Dujardin, Poonam Salotra
    Abstract:

    Leishmania donovani is the causative agent of the potentially fatal disease visceral leishmaniasis (VL). Chemotherapeutic options available to treat VL are limited and often face parasite resistance, inconsistent efficacy, and toxic side effects. Paromomycin (PMM) was recently introduced to treat VL as a monotherapy and in combination therapy. It is vital to understand the mechanisms of PMM resistance to safeguard the drug. In the present study, we utilized experimentally generated PMM-resistant L. donovani to elucidate the mechanisms of resistance and parasite biology. We found increased membrane fluidity accompanied by decreased intracellular drug accumulation in the PMM-resistant parasites. There were marked increases in gene expression of ATP-binding cassette (ABC) transporters (MDR1 and MRPA) and protein phosphatase 2A that evince increased drug efflux. Further, evaluation of parasite tolerance toward host leishmanicidal mechanisms revealed PMM-resistant parasites as being more tolerant to nitrosative stress at the promastigote and amastigote stages. The PMM-resistant parasites also predicted a better survival capacity, as indicated by resistance to complement-mediated lysis and increased stimulation of host interleukin-10 (IL-10) expression. The susceptibilities of PMM-resistant isolates to other antileishmanial agents (sodium Antimony Gluconate and miltefosine) remained unchanged. The data implicated the roles of altered membrane fluidity, decreased drug accumulation, increased expression of ABC transporters, and greater tolerance of parasites to host defense mechanisms in conferring PMM resistance in Leishmania.

  • Summary of results from IgG1 rapid diagnostic tests (RDT) prototypes.
    2014
    Co-Authors: Tapan Bhattacharyya, Shyam Sundar, Armon Ayandeh, Andrew K. Falconar, Sayda El-safi, Marissa A. Gripenberg, Duncan E. Bowes, Caroline Thunissen, Om Prakash Singh, Rajiv Kumar
    Abstract:

    aTherapy: sodium Antimony Gluconate n = 8; miltefosine n = 10; amphotericin B n = 3; combination therapy n = 2.b22 of these samples were also used with prototype 1.cthese samples were also negative with prototype 1.dthese 5 samples were also used with prototype 1.emalaria n = 3; hepatitis n = 1; TB n = 2; dengue n = 1.Summary of results from IgG1 rapid diagnostic tests (RDT) prototypes.

  • Antimony Resistant Leishmania donovani but Not Sensitive Ones Drives Greater Frequency of Potent T-Regulatory Cells upon Interaction with Human PBMCs: Role of IL-10 and TGF-β in Early Immune Response
    2014
    Co-Authors: Rajan Guha, Shyam Sundar, Jeanclaude Dujardin, Shantanabha Das, June Ghosh, Syamal Roy
    Abstract:

    In India the sand fly, Phlebotomus argentipes, transmitted parasitic disease termed kala-azar is caused by Leishmania donovani (LD) in humans. These immune-evading parasites have increasingly developed resistance to the drug sodium Antimony Gluconate in endemic regions.Lack of early diagnosis methods for the disease limits the information available regarding the early interactions of this parasite with either human tissues or cell lineages. We reasoned that peripheral blood mononuclear cells (PBMCs) from healthy human beings could help compare some of their immune signatures once they were exposed for up to 8 days, to either pentavalent Antimony sensitive (SbS-LD) or resistant (SbR-LD) Leishmania donovani isolates.At day 2, PBMC cultures exposed to SbS-LD and SbR-LD stationary phase promastigotes had four and seven fold higher frequency of IL-10 secreting monocyte-macrophage respectively, compared to cultures unexposed to parasites. Contrasting with the CD4+CD25−CD127− type-1 T-regulatory (Tr1) cell population that displayed similar features whatever the culture conditions, there was a pronounced increase in the IL-10 producing CD4+CD25+CD127low/− inducible T-regulatory cells (iTregs) in the PBMC cultures sampled at day 8 post addition of SbR-LD.Sorted iTregs from different cultures on day 8 were added to anti-CD3/CD28 induced naïve PBMCs to assess their suppressive ability. We observed that iTregs from SbR-LD exposed PBMCs had more pronounced suppressive ability compared to SbS-LD counterpart on a per cell basis and is dependent on both IL-10 and TGF-β, whereas IL-10 being the major factor contributing to the suppressive ability of iTregs sorted from PBMC cultures exposed to SbS–LD. Of note, iTreg population frequency value remained at the basal level after addition of genetically modified SbR-LD lacking unique terminal sugar in surface glycan.Even with limitations of this artificial in vitro model of L. donovani-human PBMC interactions, the present findings suggest that SbR-LD have higher immunomodulatory capacity which may favour aggressive pathology.

Poonam Salotra - One of the best experts on this subject based on the ideXlab platform.

  • elucidation of cellular mechanisms involved in experimental paromomycin resistance in leishmania donovani
    Antimicrobial Agents and Chemotherapy, 2014
    Co-Authors: Vasundhra Bhandari, Shyam Sundar, Jeanclaude Dujardin, Poonam Salotra
    Abstract:

    Leishmania donovani is the causative agent of the potentially fatal disease visceral leishmaniasis (VL). Chemotherapeutic options available to treat VL are limited and often face parasite resistance, inconsistent efficacy, and toxic side effects. Paromomycin (PMM) was recently introduced to treat VL as a monotherapy and in combination therapy. It is vital to understand the mechanisms of PMM resistance to safeguard the drug. In the present study, we utilized experimentally generated PMM-resistant L. donovani to elucidate the mechanisms of resistance and parasite biology. We found increased membrane fluidity accompanied by decreased intracellular drug accumulation in the PMM-resistant parasites. There were marked increases in gene expression of ATP-binding cassette (ABC) transporters (MDR1 and MRPA) and protein phosphatase 2A that evince increased drug efflux. Further, evaluation of parasite tolerance toward host leishmanicidal mechanisms revealed PMM-resistant parasites as being more tolerant to nitrosative stress at the promastigote and amastigote stages. The PMM-resistant parasites also predicted a better survival capacity, as indicated by resistance to complement-mediated lysis and increased stimulation of host interleukin-10 (IL-10) expression. The susceptibilities of PMM-resistant isolates to other antileishmanial agents (sodium Antimony Gluconate and miltefosine) remained unchanged. The data implicated the roles of altered membrane fluidity, decreased drug accumulation, increased expression of ABC transporters, and greater tolerance of parasites to host defense mechanisms in conferring PMM resistance in Leishmania.

  • increased parasite surface antigen 2 expression in clinical isolates of leishmania donovani augments Antimony resistance
    Biochemical and Biophysical Research Communications, 2013
    Co-Authors: Vasundhra Bhandari, Ruchi Singh, Dhiraj Kumar, Sandeep Verma, Gurumurthy Srividya, N S Negi, Poonam Salotra
    Abstract:

    Abstract Resistance to sodium Antimony Gluconate (SAG) is a major cause of therapeutic failure in a large proportion of visceral leishmaniasis (VL) cases. Determinants of SAG resistance have been widely studied; however, the mechanism operating in clinical isolates is poorly understood. In the present study, expression of parasite surface antigen-2 (PSA-2) gene was studied in clinical isolates of Leishmania donovani comprising of Antimony resistant (n = 10) and sensitive (n = 4) parasites. The expression of PSA-2 gene was found to be consistently high in SAG resistant clinical isolates (⩾1.5-fold) at both transcript and protein level. Further, over-expression of PSA-2 in L. donovani isolates (LdPSA-2++) resulted in conversion of SAG sensitive phenotype to resistant. The LdPSA-2++ parasites showed significantly decreased susceptibility towards SAG (>12-fold), amphotericin B (>4-fold) and miltefosine (>2.5-fold). Marked decrease in Antimony accumulation and enhanced tolerance towards complement mediated lysis was evident in LdPSA-2++ parasites. The study established the role of PSA-2 gene in SAG resistance and its potential as a biomarker to distinguish resistant and sensitive clinical isolates of L. donovani.

  • genetic typing reveals monomorphism between Antimony sensitive and resistant leishmania donovani isolates from visceral leishmaniasis or post kala azar dermal leishmaniasis cases in india
    Parasitology Research, 2012
    Co-Authors: B Subba V Raju, Vasundhra Bhandari, Srividya Gurumurthy, Katrin Kuhls, Gabriele Schnonian, Poonam Salotra
    Abstract:

    Resistance to pentavalent antimonials has emerged as a major hurdle to the treatment and control of visceral leishmaniasis (VL), also known as kala-azar (KA), caused by Leishmania donovani. In India, over 60 % of KA patients are unresponsive to the first-line drug sodium Antimony Gluconate (SAG). Resistance determinants in laboratory strains are partly known; however, the mechanism operating in field isolates is not well understood. In this study, we attempted to analyze the genetic polymorphism between SAG sensitive and resistant parasites using a total of 52 L. donovani isolates obtained either from bone marrow of VL patients or from skin lesions of post kala-azar dermal leishmaniasis (PKDL) patients that constitute an important reservoir of parasite. The clinical isolates were analyzed in comparison with L. donovani parasites from reference strains belonging to distinct geographical locations, at internal transcribed spacer 1 region; coding region of gp63 and nine microsatellite repeat regions. Our results demonstrated that both SAG resistant (n = 26) and sensitive (n = 19) Indian isolates, whether causing VL or PKDL, were monomorphic at all the genetic loci tested, unlike the L. donovani in East African or Leishmania infantum in Mediterranean countries where intraspecies variations exist at these loci. Further, the Indian isolates were found closest to the Kenyan isolates of L. donovani on the basis of fragment analysis of microsatellite markers.

  • Antimony resistant clinical isolates of Leishmania donovani are susceptible to paromomycin and sitamaquine
    Antimicrobial agents and chemotherapy, 2011
    Co-Authors: Arpita Kulshrestha, Ruchi Singh, Dhiraj Kumar, Narender Singh Negi, Poonam Salotra
    Abstract:

    Widespread antimonial resistance in anthroponotic visceral leishmaniasis (VL) makes it critical to monitor the susceptibility of prevailing field isolates to upcoming antileishmanials in order to frame the right treatment policies to protect these drugs against development of resistance. We aimed to generate the baseline data on natural in vitro susceptibility to paromomycin and sitamaquine in Leishmania donovani field isolates from VL patients (n = 20) coming from zones of varying sodium Antimony Gluconate (SAG) resistance. We further monitored nitric oxide (NO) release in infected macrophages treated with these drugs. Field isolates exhibited variable sensitivity to paromomycin and sitamaquine with respective mean 50% effective dose (ED50) values ± standard error of the mean (SEM) of 3.9 ± 0.3 μM and 2.1 ± 0.2 μM at the intracellular amastigote stage and 29.8 ± 2.5 μM and 17.7 ± 1.0 μM at the promastigote stage. Susceptibilities at the two parasite stages did not correlate for either drug. Isolates from high SAG resistance zones exhibited significantly lower susceptibility to sitamaquine than those from low SAG resistance zones, while isolates from different zones showed similar susceptibilities to paromomycin. NO release was promoted in L. donovani-infected macrophages upon treatment with paromomycin/sitamaquine. NO inhibitors significantly compromised amastigote killing by sitamaquine, but not by paromomycin. In conclusion, SAG-resistant/sensitive VL isolates were susceptible to both paromomycin and sitamaquine. Paromomycin, exhibiting higher efficacy against SAG-resistant parasites and having a distinct mechanism of action, appears to be a promising drug for combination therapy.

  • in vitro susceptibility of field isolates of leishmania donovani to miltefosine and amphotericin b correlation with sodium Antimony Gluconate susceptibility and implications for treatment in areas of endemicity
    Antimicrobial Agents and Chemotherapy, 2009
    Co-Authors: Dhiraj Kumar, Ruchi Singh, Arpita Kulshrestha, Poonam Salotra
    Abstract:

    Indian Leishmania donovani isolates (n = 19) from regional zones representing various levels of Antimony resistance displayed significantly (P < 0.01) correlated results with respect to in vitro susceptibility to the antileishmanial drugs sodium Antimony Gluconate, amphotericin B, and Miltefosine, raising the possibility of cross-resistance mechanisms operating in the field isolates. The results of gene expression analysis of LdMT and LdRos3 were suggestive of alternate mechanisms of Miltefosine susceptibility in the isolates.

Anuradha Dube - One of the best experts on this subject based on the ideXlab platform.

  • Over-expression of 60s ribosomal L23a is associated with cellular proliferation in SAG resistant clinical isolates of Leishmania donovani
    2013
    Co-Authors: Sanchita Das, Shyam Sundar, Priyanka Shah, Rajendra K. Baharia, Rati T, Prashant Khare, Amogh A. Sahasrabuddhe, M. I. Siddiqi, Anuradha Dube
    Abstract:

    Background: Sodium Antimony Gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism. Methodology and principal findings: The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode. Conclusion/Significance: This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosoma

  • proteome mapping of overexpressed membrane enriched and cytosolic proteins in sodium Antimony Gluconate sag resistant clinical isolate of leishmania donovani
    British Journal of Clinical Pharmacology, 2010
    Co-Authors: Awanish Kumar, Shyam Sundar, Ajit Kumar Shasany, Brijesh S Sisodia, Pragya Misra, Anuradha Dube
    Abstract:

    WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Over 60% of patients with visceral leishmaniasis (VL) in India and Sudan have become unresponsive to treatment with pentavalent antimonials, the first line of drugs for over 60 years. The drug resistance mechanism, studied so far in in vitro selected laboratory strains, has been attributed to various biochemical parameters. The resistance to Sb (V) in Leishmania field isolates is still unexplored. WHAT THIS STUDY ADDS In order to elucidate for the first time the mechanism of drug resistance in field isolates, this study was done in those clinically relevant field isolates which were either responsive or non responsive to SAG. A comparison of proteome profiles of membrane-enriched as well as cytosolic protein fractions of these isolates has pinpointed the multiple overexpressed proteins in resistant isolates. This study has indicated their possible essential role in Antimony resistance of the parasite and provides a vast field to be exploited to find much needed novel treatment strategies against VL. AIMS This study aimed to identify differentially overexpressed membrane-enriched as well as cytosolic proteins in SAG sensitive and resistant clinical strains of L. donovani isolated from VL patients which are involved in the drug resistance mechanism. METHODS The proteins in the membrane-enriched as well as cytosolic fractions of drug-sensitive as well as drug-resistant clinical isolates were separated using two-dimensional gel electrophoresis and overexpressed identified protein spots of interest were excised and analysed using MALDI-TOF/TOF. RESULTS Six out of 12 overexpressed proteins were identified in the membrane-enriched fraction of the SAG resistant strain of L. donovani whereas 14 out of 18 spots were identified in the cytosolic fraction as compared with the SAG sensitive strain. The major proteins in the membrane-enriched fraction were ABC transporter, HSP-83, GPI protein transamidase, cysteine–leucine rich protein and 60S ribosomal protein L23a whereas in the cytosolic fraction proliferative cell nuclear antigen (PCNA), proteasome alpha 5 subunit, carboxypeptidase, HSP-70, enolase, fructose-1,6-bisphosphate aldolase, tubulin-beta chain have been identified. Most of these proteins have been reported as potential drug targets, except 60S ribosomal protein L23a and PCNA which have not been reported to date for their possible involvement in drug resistance against VL. CONCLUSION This study for the first time provided a cumulative proteomic analysis of proteins overexpressed in drug resistant clinical isolates of L. donovani indicating their possible role in Antimony resistance of the parasite. Identified proteins provide a vast field to be exploited for novel treatment strategies against VL such as cloning and overexpression of these targets to produce recombinant therapeutic/prophylactic proteins.

  • identification of genetic markers in sodium Antimony Gluconate sag sensitive and resistant indian clinical isolates of leishmania donovani through amplified fragment length polymorphism aflp
    Acta Tropica, 2009
    Co-Authors: Awanish Kumar, Shyam Sundar, Vijay Raju Boggula, Ajit Kumar Shasany, Anuradha Dube
    Abstract:

    Sodium Antimony Gluconate (SAG) is currently used worldwide as the first-line drugs for the treatment of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) since 1940s. Unfortunately, the resistance of Leishmania parasite to this drug is increasing in several parts of the world. The mechanism of drug resistance in clinical isolates is still not very clear. Earlier, we have established a differentiation between six clinical isolates as sensitive and resistant on the basis of their sensitivity to SAG in vitro and in vivo as well as expression of proteophosphoglycan contents. In this preliminary study, we have further analyzed these isolates on the basis of their genetic diversity, molecular variance and phylogenetic structure using for the first time, a fingerprinting approach – amplified fragment length polymorphism (AFLP). Altogether 2338 informative AFLP bands were generated using 10 selective primer combinations. Percentage of polymorphism was 55.35%. A number of unique AFLP markers (217) were also identified in these strains. It was deduced that a higher rate of variations occurred among Leishmania clinical isolates which indicate the shifting of drug sensitive nature of parasite towards resistant condition.

  • Over-expression of 60s ribosomal L23a is associated with cellular proliferation in SAG resistant clinical isolates of Leishmania donovani.
    Public Library of Science (PLoS), 1
    Co-Authors: Sanchita Das, Shyam Sundar, Priyanka Shah, Rajendra K. Baharia, Prashant Khare, Amogh A. Sahasrabuddhe, M. I. Siddiqi, Rati Tandon, Anuradha Dube
    Abstract:

    Sodium Antimony Gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism.The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode.This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism

Syamal Roy - One of the best experts on this subject based on the ideXlab platform.

  • co administration of glycyrrhizic acid with the antileishmanial drug sodium Antimony Gluconate sag cures sag resistant visceral leishmaniasis
    International Journal of Antimicrobial Agents, 2015
    Co-Authors: Amrita Bhattacharjee, Saikat Majumder, Suchandra Bhattacharyya Majumdar, Soumitra Kumar Choudhuri, Syamal Roy, Subrata Majumdar
    Abstract:

    Since there are very few affordable antileishmanial drugs available, antimonial resistance has crippled antileishmanial therapy, thereby emphasising the need for development of novel therapeutic strategies. This study aimed to evaluate the antileishmanial role of combined therapy with sodium Antimony Gluconate (SAG) and the triterpenoid glycyrrhizic acid (GA) against infection with SAG-resistant Leishmania (GE1F8R). Combination therapy with GA and SAG successfully limited infection with SAG-resistant Leishmania in a synergistic manner (fractional inhibitory concentration index <1.0). At the same time, mice infected with SAG-resistant Leishmania and co-treated with GA and SAG exhibited a significant reduction in hepatic and splenic parasite burden. In probing the mechanism, it was observed that GA treatment suppressed the expression and efflux activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1), two host ABC transporters responsible for Antimony efflux from host cells infected with SAG-resistant parasites. This suppression correlated with greater intracellular Antimony retention during SAG therapy both in vitro and in vivo, which was reflected in the reduced parasite load. Furthermore, co-administration of GA and SAG induced a shift in the cytokine balance towards a Th1 phenotype by augmenting pro-inflammatory cytokines (such as IL-12, IFNγ and TNFα) and inducing nitric oxide generation in GE1F8R-infected macrophages as well as GE1F8R-infected mice. This study aims to provide an affordable leishmanicidal alternative to expensive antileishmanial drugs such as miltefosine and amphotericin B. Furthermore, this report explores the role of GA as a resistance modulator in MRP1- and P-gp-overexpressing conditions.

  • Antimony Resistant Leishmania donovani but Not Sensitive Ones Drives Greater Frequency of Potent T-Regulatory Cells upon Interaction with Human PBMCs: Role of IL-10 and TGF-β in Early Immune Response
    2014
    Co-Authors: Rajan Guha, Shyam Sundar, Jeanclaude Dujardin, Shantanabha Das, June Ghosh, Syamal Roy
    Abstract:

    In India the sand fly, Phlebotomus argentipes, transmitted parasitic disease termed kala-azar is caused by Leishmania donovani (LD) in humans. These immune-evading parasites have increasingly developed resistance to the drug sodium Antimony Gluconate in endemic regions.Lack of early diagnosis methods for the disease limits the information available regarding the early interactions of this parasite with either human tissues or cell lineages. We reasoned that peripheral blood mononuclear cells (PBMCs) from healthy human beings could help compare some of their immune signatures once they were exposed for up to 8 days, to either pentavalent Antimony sensitive (SbS-LD) or resistant (SbR-LD) Leishmania donovani isolates.At day 2, PBMC cultures exposed to SbS-LD and SbR-LD stationary phase promastigotes had four and seven fold higher frequency of IL-10 secreting monocyte-macrophage respectively, compared to cultures unexposed to parasites. Contrasting with the CD4+CD25−CD127− type-1 T-regulatory (Tr1) cell population that displayed similar features whatever the culture conditions, there was a pronounced increase in the IL-10 producing CD4+CD25+CD127low/− inducible T-regulatory cells (iTregs) in the PBMC cultures sampled at day 8 post addition of SbR-LD.Sorted iTregs from different cultures on day 8 were added to anti-CD3/CD28 induced naïve PBMCs to assess their suppressive ability. We observed that iTregs from SbR-LD exposed PBMCs had more pronounced suppressive ability compared to SbS-LD counterpart on a per cell basis and is dependent on both IL-10 and TGF-β, whereas IL-10 being the major factor contributing to the suppressive ability of iTregs sorted from PBMC cultures exposed to SbS–LD. Of note, iTreg population frequency value remained at the basal level after addition of genetically modified SbR-LD lacking unique terminal sugar in surface glycan.Even with limitations of this artificial in vitro model of L. donovani-human PBMC interactions, the present findings suggest that SbR-LD have higher immunomodulatory capacity which may favour aggressive pathology.

  • Leishmania donovani Isolates with Antimony-Resistant but Not-Sensitive Phenotype Inhibit Sodium Antimony
    2013
    Co-Authors: Gluconate-induced Dendritic, Pradip Sen, Rajatava Basu, Cell Activation, Syamal Roy
    Abstract:

    The inability of sodium Antimony Gluconate (SAG)-unresponsive kala-azar patients to clear Leishmania donovani (LD) infection despite SAG therapy is partly due to an ill-defined immune-dysfunction. Since dendritic cells (DCs) typically initiate anti-leishmanial immunity, a role for DCs in aberrant LD clearance was investigated. Accordingly, regulation of SAG-induced activation of murine DCs following infection with LD isolates exhibiting two distinct phenotypes such as Antimony-resistant (Sb R LD) and Antimony-sensitive (Sb S LD) was compared in vitro. Unlike Sb S LD, infection of DCs with Sb R LD induced more IL-10 production and inhibited SAG-induced secretion of proinflammatory cytokines, up-regulation of co-stimulatory molecules and leishmanicidal effects. Sb R LD inhibited these effects of SAG by blocking activation of PI3K/AKT and NF-kB pathways. In contrast, Sb S LD failed to block activation of SAG (20 mg/ml)-induced PI3K/AKT pathway; which continued to stimulate NF-kB signaling, induce leishmanicidal effects and promote DC activation. Notably, prolonged incubation of DCs with Sb S LD also inhibited SAG (20 mg/ml)-induced activation of PI3K/AKT and NF-kB pathways and leishmanicidal effects, which was restored by increasing the dose of SAG to 40 mg/ml. In contrast, Sb R LD inhibited these SAG-induced events regardless of duration of DC exposure to Sb R LD or dose of SAG. Interestingly, the inhibitory effects of isogenic Sb S LD expressing ATP-binding cassette (ABC) transporter MRPA on SAG-induced leishmanicidal effects mimicked that of Sb R LD to some extent, although Antimony resistance in clinical LD isolates is known to be multifactorial. Furthermore, NF-kB wa

  • leishmania donovani isolates with Antimony resistant but not sensitive phenotype inhibit sodium Antimony Gluconate induced dendritic cell activation
    PLOS Pathogens, 2010
    Co-Authors: Arun Kumar Haldar, Vinod Yadav, Eshu Singhal, Kamlesh Bisht, Alpana Singh, Suniti Bhaumik, Rajatava Basu, Pradip Sen, Syamal Roy
    Abstract:

    The inability of sodium Antimony Gluconate (SAG)-unresponsive kala-azar patients to clear Leishmania donovani (LD) infection despite SAG therapy is partly due to an ill-defined immune-dysfunction. Since dendritic cells (DCs) typically initiate anti-leishmanial immunity, a role for DCs in aberrant LD clearance was investigated. Accordingly, regulation of SAG-induced activation of murine DCs following infection with LD isolates exhibiting two distinct phenotypes such as Antimony-resistant (SbRLD) and Antimony-sensitive (SbSLD) was compared in vitro. Unlike SbSLD, infection of DCs with SbRLD induced more IL-10 production and inhibited SAG-induced secretion of proinflammatory cytokines, up-regulation of co-stimulatory molecules and leishmanicidal effects. SbRLD inhibited these effects of SAG by blocking activation of PI3K/AKT and NF-κB pathways. In contrast, SbSLD failed to block activation of SAG (20 µg/ml)-induced PI3K/AKT pathway; which continued to stimulate NF-κB signaling, induce leishmanicidal effects and promote DC activation. Notably, prolonged incubation of DCs with SbSLD also inhibited SAG (20 µg/ml)-induced activation of PI3K/AKT and NF-κB pathways and leishmanicidal effects, which was restored by increasing the dose of SAG to 40 µg/ml. In contrast, SbRLD inhibited these SAG-induced events regardless of duration of DC exposure to SbRLD or dose of SAG. Interestingly, the inhibitory effects of isogenic SbSLD expressing ATP-binding cassette (ABC) transporter MRPA on SAG-induced leishmanicidal effects mimicked that of SbRLD to some extent, although Antimony resistance in clinical LD isolates is known to be multifactorial. Furthermore, NF-κB was found to transcriptionally regulate expression of murine γglutamylcysteine synthetase heavy-chain (mγGCShc) gene, presumably an important regulator of Antimony resistance. Importantly, SbRLD but not SbSLD blocked SAG-induced mγGCS expression in DCs by preventing NF-κB binding to the mγGCShc promoter. Our findings demonstrate that SbRLD but not SbSLD prevents SAG-induced DC activation by suppressing a PI3K-dependent NF-κB pathway and provide the evidence for differential host-pathogen interaction mediated by SbRLD and SbSLD.

  • sub optimal dose of sodium Antimony Gluconate sag diperoxovanadate combination clears organ parasites from balb c mice infected with Antimony resistant leishmania donovani by expanding antileishmanial t cell repertoire and increasing ifn γ to il 10 r
    Experimental Parasitology, 2009
    Co-Authors: Arun Kumar Haldar, Subha Banerjee, Kshudiram Naskar, Diganta Kalita, Nashreen S Islam, Syamal Roy
    Abstract:

    We demonstrate that the combination of sub-optimal doses of Sodium Antimony Gluconate (SAG) and the diperoxovanadate compound K[VO(O2)2(H2O)], also designated as PV6, is highly effective in combating experimental infection of BALB/c mice with Antimony resistant (Sb(R)) Leishmania donovani (LD) as evident from the significant reduction in organ parasite burden where SAG is essentially ineffective. Interestingly, such treatment also allowed clonal expansion of antileishmanial T-cells coupled with robust surge of IFN-c and concomitant decrease in IL-10 production. The splenocytes from the treated animals generated significantly higher amounts of IFN-c inducible parasiticidal effector molecules like superoxide and nitric oxide as compared to the infected group. Our study indicates that the combination of sub-optimal doses of SAG and PV6 may be beneficial for the treatment of SAG resistant visceral leishmaniasis patients.

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  • Over-expression of cysteine leucine rich protein is related to sag resistance in clinical isolates of Leishmania donovani
    Public Library of Science, 2015
    Co-Authors: Das Sanchita, Sundar Shyam, Shah Priyanka, Tandon Rati, Sahasrabuddhe, Amogh A., Yadav, Narendra Kumar, Siddiqi, Mohammad Imran, Dube Anuradha
    Abstract:

    Background: Resistance emergence against antileishmanial drugs, particularly Sodium Antimony Gluconate (SAG) has severely hampered the therapeutic strategy against visceral leishmaniasis, the mechanism of resistance being indistinguishable. Cysteine leucine rich protein (CLrP), was recognized as one of the overexpressed proteins in resistant isolates, as observed in differential proteomics between sensitive and resistant isolates of L. donovani. The present study deals with the characterization of CLrP and for its possible connection with SAG resistance. Methodology and Principal Findings: In pursuance of deciphering the role of CLrP in SAG resistance, gene was cloned, over-expressed in E. coli system and thereafter antibody was raised. The expression profile of CLrP and was found to be over-expressed in SAG resistant clinical isolates of L. donovani as compared to SAG sensitive ones when investigated by real-time PCR and western blotting. CLrP has been characterized through bioinformatics, immunoblotting and immunolocalization analysis, which reveals its post-translational modification along with its dual existence in the nucleus as well as in the membrane of the parasite. Further investigation using a ChIP assay confirmed its DNA binding potential. Over-expression of CLrP in sensitive isolate of L. donovani significantly decreased its responsiveness to SAG (SbV and SbIII) and a shift towards the resistant mode was observed. Further, a significant increase in its infectivity in murine macrophages has been observed. Conclusion/Significance: The study reports the differential expression of CLrP in SAG sensitive and resistant isolates of L. donovani. Functional intricacy of CLrP increases with dual localization, glycosylation and DNA binding potential of the protein. Further over-expressing CLrP in sensitive isolate of L. donovani shows significantly decreased sensitivity towards SAG and increased infectivity as well, thus assisting the parasite in securing a safe niche. Results indicates the possible contribution of CLrP to antimonial resistance in L. donovani by assisting the parasite growth in the macrophages

  • Over-expression of 60s ribosomal L23a is associated with cellular proliferation in SAG resistant clinical isolates of Leishmania donovani
    'Public Library of Science (PLoS)', 2013
    Co-Authors: Das Sanchita, Sundar Shyam, Shah Priyanka, Baharia, Rajendra K., Tandon Rati, Khare Prashant, Sahasrabuddhe, Amogh A., Siddiqi M., Dube Anuradha
    Abstract:

    Background: Sodium Antimony Gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L. donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L. donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism. Methodology and principal findings: The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L. donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L. donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode. Conclusion/Significance: This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism

  • Novel mechanism of drug resistance in kala azar field isolates
    University of Chicago Press, 2003
    Co-Authors: Singh Neeloo, Ramsing Th. Singh, Sundar Shyam
    Abstract:

    Clinical resistance to pentavalent antimonial drugs in the form of sodium Antimony Gluconate (SAG) has become a major problem in the treatment of kala azar (visceral leishmaniasis) in India. The mechanism of resistance is unclear in these clinical isolates, although work has been conducted with Leishmania species mutants selected in vitro by stepwise increase of drug concentration, using Antimony-related metal arsenic and, more recently, SAG. In the present study, we investigated the molecular aspect of drug resistance in clinically confirmed SAG-resistant field isolates. Our results show that the mechanisms of resistance postulated for laboratory mutants of Leishmania species are not operating in field isolates of Leishmania donovani. Instead, we identified a novel gene amplified in these drug-resistant parasites whose locus is on chromosome 9. The significant finding was that this isolated fragment confers Antimony resistance to wild-type Leishmania species after transfection. We speculate that protein phosphorylation may play a role in signal transduction pathway in the parasite after exposure to drug-conferring resistance

  • Immunoblot Analysis of the Humoral Immune Response to Leishmania donovani Polypeptides in Cases of Human Visceral Leishmaniasis: Its Usefulness in Prognosis
    American Society for Microbiology, 2002
    Co-Authors: Kumar Promod, Pai Kalpana, Tripathi Kiran, Pandey H. P., Sundar Shyam
    Abstract:

    Sera from Indian patients with parasitologically confirmed visceral leishmaniasis were studied by immunoblot analysis in order to identify a specific pattern for Leishmania infection. A soluble extract of Leishmania donovani was used as antigen. At diagnosis the sera from patients with visceral leishmaniasis specifically recognized fractions represented by bands of 201 kDa (50% of serum samples), 193 kDa (60%), 147 kDa (50%), 120 kDa (60%), 100 kDa (50%), 80 kDa (80%), 70 kDa (70%), 65 kDa (100%), 50 kDa (50%), 36 kDa (50%), 20 kDa (70%), and 18 kDa (50%). The 65-kDa band, common to all patients infected with Leishmania parasites, was found at the time of diagnosis. However, the immunoblot pattern changed after patients were treated and cured with sodium Antimony Gluconate (SAG; n =10) or miltefosine (n =10), as was evident from blots of sera obtained pretreatment and at 1, 3, and 6 months posttreatment. At 6 months posttreatment, immunoblots of sera from patients on the SAG regimen showed the disappearance of all bands except the 70-kDa band. Similarly, sera from those on the miltefosine regimen showed the disappearance of all bands except the 65- and 70-kDa bands. This study shows that Western blot analysis is a sensitive test for detection of anti-Leishmania antibodies. Moreover, the persistence of reactivity with the 65- and 70-kDa bands in the sera of all groups shows its promise as a diagnostic and prognostic tool

  • Diagnostic and Prognostic Potential of a Competitive Enzyme-Linked Immunosorbent Assay for Leishmaniasis in India
    American Society for Microbiology, 1999
    Co-Authors: Chatterjee Mitali, Sundar Shyam, Jaffe, Charles L., Basu Debasis, Sen Sandeep, Mandal Chitra
    Abstract:

    A Leishmania donovani species-specific monoclonal antibody (monoclonal antibody D2) was evaluated for its diagnostic and prognostic potential by a competitive enzyme-linked immunosorbent assay (C-ELISA) in sera from Indian patients with visceral leishmaniasis (VL) and seven patients with post-kala-azar dermal leishmaniasis (PKDL). These results were compared with those obtained by microscopy with Giemsa-stained tissue smears and a direct enzyme-linked immunosorbent assay (direct ELISA) with crude parasite antigen. Of 121 patients with clinically diagnosed VL examined, 103 (85.1%) were positive and 11 (9.1%) were negative by all three methods. An additional 7 (5.8%) who were negative by microscopy were positive by both C-ELISA and direct ELISA. Seven PKDL patients were also examined and were found to be positive by all three methods. Analysis of the chemotherapeutic response to sodium Antimony Gluconate of these 110 serologically positive VL patients showed that 57 (51.8%) were drug responsive and 53 (48.2%) were drug resistant. The C-ELISA with sera from 20 longitudinally monitored VL patients before and after chemotherapy showed a significant decrease in percent inhibition of monoclonal antibody D2 in drug-responsive patients. However, in drug-unresponsive patients, the percent inhibition of D2 was unchanged or was slightly increased. Our results therefore indicate (i) the applicability of L. donovani species-specific monoclonal antibody D2 for sensitive and specific serodiagnosis by C-ELISA, (ii) that the C-ELISA is more sensitive than microscopy, especially for early diagnosis, (iii) that L. donovani is still the main causative agent of VL, irrespective of the chemotherapeutic response, and (iv) that the C-ELISA can be used to evaluate the success of drug treatment