Mukesh Samant

Publications of Mukesh Samant

• Leishmania donovani: immunostimulatory cellular responses of membrane and soluble protein fractions of splenic amastigotes in cured patient and hamsters.

  Authors: Shraddha Kumari, Pragya Misra, Rati Tandon, Mukesh Samant, Shyam Sundar, Anuradha Dube

  PloS one. 01/2012; 7(1):e30746.

  Visceral leishmaniasis (VL), caused by the intracellular parasite Leishmania donovani, L. chagasi and L. infantum is characterized by defective cell-mediated immunity (CMI) and is usually fatal ifVisceral leishmaniasis (VL), caused by the intracellular parasite Leishmania donovani, L. chagasi and L. infantum is characterized by defective cell-mediated immunity (CMI) and is usually fatal if not treated properly. An estimated 350 million people worldwide are at risk of acquiring infection with Leishmania parasites with approximately 500,000 cases of VL being reported each year. In the absence of an efficient and cost-effective antileishmanial drug, development of an appropriate long-lasting vaccine against VL is the need of the day. In VL, the development of a CMI, capable of mounting Th1-type of immune responses, play an important role as it correlate with recovery from and resistance to disease. Resolution of infection results in lifelong immunity against the disease which indicates towards the feasibility of a vaccine against the disease. Most of the vaccination studies in Leishmaniasis have been focused on promastigote--an infective stage of parasite with less exploration of pathogenic amastigote form, due to the cumbersome process of its purified isolation. In the present study, we have isolated and purified splenic amastigotes of L. donovani, following the traditional protocol with slight modification. These were fractionated into five membranous and soluble subfractions each i.e MAF1-5 and SAF1-5 and were subjected for evaluation of their ability to induce cellular responses. Out of five sub-fractions from each of membrane and soluble, only four viz. MAF2, MAF3, SAF2 and SAF3 were observed to stimulate remarkable lymphoproliferative, IFN-γ, IL-12 responses and Nitric Oxide production, in Leishmania-infected cured/exposed patients and hamsters. Results suggest the presence of Th-1 type immunostimulatory molecules in these sub-fractions which may further be exploited for developing a successful subunit vaccine from the less explored pathogenic stage against VL.

• Treatment of Leishmania donovani-infected hamsters with miltefosine: analysis of cytokine mRNA expression by real-time PCR, lymphoproliferation, nitrite production and antibody responses.

  Authors: Reema Gupta, Pramod K Kushawaha, Mukesh Samant, Anil K Jaiswal, Rajendra K Baharia, Anuradha Dube

  The Journal of antimicrobial chemotherapy. 11/2011; 67(2):440-3.

  Miltefosine, an orally effective antileishmanial drug, works directly on the parasite by impairing membrane synthesis and subsequent apoptosis of the parasite and has also been reported to haveMiltefosine, an orally effective antileishmanial drug, works directly on the parasite by impairing membrane synthesis and subsequent apoptosis of the parasite and has also been reported to have macrophage-activating functions that aid parasite killing. We investigated the type of immunological responses generated in miltefosine-treated Leishmania donovani-infected hamsters, which simulate the clinical situation of human kala-azar. Twenty-five-day-old infected hamsters, treated with miltefosine at 40 mg/kg for 5 consecutive days, were euthanized on days 30 and 45 post treatment (p.t.) and checked for parasite clearance and for real-time analysis of mRNAs of the Th1/Th2 cytokines interferon-γ (IFN-γ), interleukin-12 (IL-12), tumour necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), IL-4, IL-10 and transforming growth factor-β (TGF-β), nitric oxide (NO) production, the lymphocyte transformation test (LTT) and antibody responses. Responses were compared with the normal and Leishmania-infected groups at the same time points. By day 45 p.t. there was a significant increase in the mRNA expression of iNOS, IFN-γ, IL-12 and TNF-α, whereas there were significant decreases in IL-4, IL-10 and TGF-β in cured hamsters as compared with their infected counterparts. In vitro stimulation of lymphocytes with concanavalin A and soluble Leishmania donovani antigen showed a maximum LTT response and there was a gradual increase in the NO level (∼7-fold compared with infected counterparts). Anti-Leishmania IgG and IgG1 levels, found to be elevated in the infected group, decreased significantly after treatment but there was a significant increase in IgG2 isotype. Treatment of Leishmania-infected hamsters with miltefosine reverses the Th2-type response into a strong Th1-type immune response.

• Immunization with the DNA-encoding N-terminal domain of proteophosphoglycan of Leishmania donovani generates Th1-Type immunoprotective response against experimental visceral leishmaniasis.

  Authors: Mukesh Samant, Reema Gupta, Shraddha Kumari, Pragya Misra, Prashant Khare, Pramod Kumar Kushawaha, Amogh Anant Sahasrabuddhe, Anuradha Dube

  Journal of immunology (Baltimore, Md. : 1950). 08/2009; 183(1):470-9.

  Leishmania produce several types of mucin-like glycoproteins called proteophosphoglycans (PPGs) which exist as secretory as well as surface-bound forms in both promastigotes and amastigotes. TheLeishmania produce several types of mucin-like glycoproteins called proteophosphoglycans (PPGs) which exist as secretory as well as surface-bound forms in both promastigotes and amastigotes. The structure and function of PPGs have been reported to be species and stage specific as in the case of Leishmania major and Leishmania mexicana; there has been no such information available for Leishmania donovani. We have recently demonstrated that PPG is differentially expressed in sodium stibogluconate-sensitive and -resistant clinical isolates of L. donovani. To further elucidate the structure and function of the ppg gene of L. donovani, a partial sequence of its N-terminal domain of 1.6 kb containing the majority of antigenic determinants, was successfully cloned and expressed in prokaryotic as well as mammalian cells. We further evaluated the DNA-encoding N-terminal domain of the ppg gene as a vaccine in golden hamsters (Mesocricetus auratus) against the L. donovani challenge. The prophylactic efficacy to the tune of approximately 80% was observed in vaccinated hamsters and all of them could survive beyond 6 mo after challenge. The efficacy was supported by a surge in inducible NO synthase, IFN-gamma, TNF-alpha, and IL-12 mRNA levels along with extreme down-regulation of TGF-beta, IL-4, and IL-10. A rise in the level of Leishmania-specific IgG2 was also observed which was indicative of enhanced cellular immune response. The results suggest the N-terminal domain of L. donovani ppg as a potential DNA vaccine against visceral leishmaniasis.

• Photodynamic vaccination of hamsters with inducible suicidal mutants of Leishmania amazonensis elicits immunity against visceral leishmaniasis.

  Authors: Shraddha Kumari, Mukesh Samant, Prashant Khare, Pragya Misra, Sujoy Dutta, Bala Krishna Kolli, Sharad Sharma, Kwang-Poo Chang, Anuradha Dube

  European journal of immunology. 01/2009;

  Leishmania, naturally residing in the phagolysosomes of macrophages, is a suitable carrier for vaccine delivery. Genetic complementation of these trypanosomatid protozoa to partially rectify theirLeishmania, naturally residing in the phagolysosomes of macrophages, is a suitable carrier for vaccine delivery. Genetic complementation of these trypanosomatid protozoa to partially rectify their defective heme-biosynthesis renders them inducible with delta-aminolevulinate to develop porphyria for selective photolysis, leaving infected host cells unscathed. Delivery of released "vaccines" to antigen-presenting cells is thus expected to enhance immune response, while their self-destruction presents added advantages of safety. Such suicidal L. amazonensis was found to confer immunoprophylaxis and immunotherapy on hamsters against L. donovani. Neither heat-killed nor live parasites without suicidal induction were effective. Photodynamic vaccination of hamsters with the suicidal mutants reduced the parasite loads by 99% and suppressed the development of disease. These suppressions were accompanied by an increase in Leishmania-specific delayed-type hypersensitivity and lymphoproliferation as well as in the levels of splenic iNOS, IFN-gamma, and IL-12 expressions and of Leishmania-specific IgG2 in the serum. Moreover, a single intravenous administration of T cells from vaccinated hamsters was shown to confer on naïve animals an effective cellular immunity against L. donovani challenges. The absence of lesion development at vaccination sites and parasites in the draining lymphnodes, spleen and liver further indicates that the suicidal mutants provide a safe platform for vaccine delivery against experimental visceral leishmaniasis.

• Discovery of novel vaccine candidates and drug targets against visceral leishmaniasis using proteomics and transcriptomics.

  Authors: Shraddha Kumari, Awanish Kumar, Mukesh Samant, Neeloo Singh, Anuradha Dube

  Current drug targets. 12/2008; 9(11):938-47.

  Among the three clinical forms (cutaneous, mucosal and visceral) of leishmaniasis visceral (VL) one is the most devastating type caused by the invasion of the reticuloendothelial system of human byAmong the three clinical forms (cutaneous, mucosal and visceral) of leishmaniasis visceral (VL) one is the most devastating type caused by the invasion of the reticuloendothelial system of human by Leishmania donovani, L. infantum and L. chagasi. India and Sudan account for about half the world's burden of VL. Current control strategy is based on chemotherapy, which is difficult to administer, expensive and becoming ineffective due to the emergence of drug resistance. An understanding of resistance mechanism(s) operating in clinical isolates might provide additional leads for the development of new drugs. Further, due to the lack of fully effective treatment the search for novel immune targets is also needed. So far, no vaccine exists for VL despite indications of naturally developing immunity. Therefore, an urgent need for new and effective leishmanicidal agents and for this identification of novel drug and vaccine targets is imperative. The availability of the complete genome sequence of Leishmania has revolutionised many areas of leishmanial research and facilitated functional genomic studies as well as provided a wide range of novel targets for drug designing. Most notably, proteomics and transcriptomics have become important tools in gaining increased understanding of the biology of Leishmania to be explored on a global scale, thus accelerating the pace of discovery of vaccine/drug targets. In addition, these approaches provide the information regarding genes and proteins that are expressed and under which conditions. This review provides a comprehensive view about those proteins/genes identified using proteomics and transcriptomic tools for the development of vaccine/drug against VL.

• Induction of Th1-type cellular responses in cured/exposed Leishmania-infected patients and hamsters against polyproteins of soluble Leishmania donovani promastigotes ranging from 89.9 to 97.1 kDa.

  Authors: Shraddha Kumari, Mukesh Samant, Prashant Khare, Shyam Sundar, Sudhir Sinha, Anuradha Dube

  Vaccine. 09/2008; 26(37):4813-8.

  Earlier, we have identified soluble antigenic fraction ranging from 68 to 97.4 kDa (F2-fraction) of Leishmania donovani promastigote, which induced Th1-immunostimulatory cellular responses in bothEarlier, we have identified soluble antigenic fraction ranging from 68 to 97.4 kDa (F2-fraction) of Leishmania donovani promastigote, which induced Th1-immunostimulatory cellular responses in both cured Leishmania patients/hamsters and exhibited significant prophylactic potential against experimental visceral leishmaniasis (VL). In the present study, we have further fractionated F2-fraction by continuous elution SDS-PAGE and subjected them to re-evaluation for their ability to induce cellular responses. Out of seven sub-fractions: F2.1-F2.7, only four F2.4-F2.7 (89.9-97.1 kDa), individually or in pooled sub-fractions (P4-7), stimulated Th1-type remarkable lymphoproliferative, NO, IFN-gamma and IL-12 responses in Leishmania-infected cured/exposed patients and hamsters. Interestingly, optimum Th1-responses were obtained with P4-7 suggesting the presence of immunostimulatory molecules in it and may be exploited for developing a successful subunit vaccine against VL.

• Th1-stimulatory polyproteins of soluble Leishmania donovani promastigotes ranging from 89.9 to 97.1kDa offers long-lasting protection against experimental visceral leishmaniasis.

  Authors: Shraddha Kumari, Mukesh Samant, Pragya Misra, Prashant Khare, Brijesh Sisodia, Ajit K Shasany, Anuradha Dube

  Vaccine. 09/2008;

  Our earlier studies identified a fraction (F2) of Leishmania donovani soluble promastigote antigen belonging to 97.4-68kDa for its ability to stimulate Th1-type cellular responses in cured visceralOur earlier studies identified a fraction (F2) of Leishmania donovani soluble promastigote antigen belonging to 97.4-68kDa for its ability to stimulate Th1-type cellular responses in cured visceral leishmaniasis (VL) patients as well as in cured hamsters. A further fractionation of F2-fraction into seven subfractions (F2.1-F2.7) and re-assessment for their immunostimulatory responses revealed that out of these, only four (F2.4-F2.7) belonging to 89.9-97.1kDa, stimulated remarkable Th1-type cellular responses either individually or in a pooled form (P4-7). In this study these potential subfractions were further assessed for their prophylactic potential in combination with BCG against L. donovani challenge in hamsters. Optimum parasite inhibition ( approximately 99%) was obtained in hamsters vaccinated with pooled subfractions and they survived for 1 year. The protection was further supported by remarkable lymphoproliferative, IFN-gamma and IL-12 responses along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody as observed on days 45, 90 and 120 post-challenge suggesting that a successful subunit vaccine against VL may require multiple Th1-immunostimulatory proteins. MALDI-TOF-MS/MS analysis of these subfractions further revealed that of the 19 identified immunostimulatory proteins, Elongation factor-2, p45, Heat shock protein-70/83, Aldolase, Enolase, Triosephosphate isomerase, Disulfideisomerase and Calreticulin were the major ones in these subfractions.

• Antileishmanial activity mediated by apoptosis and structure-based target study of peganine hydrochloride dihydrate: an approach for rational drug design.

  Authors: Pragya Misra, Tanvir Khaliq, Anshuman Dixit, Souvik Sengupta, Mukesh Samant, Shraddha Kumari, Awanish Kumar, Pramod K Kushawaha, H K Majumder, Anil K Saxena, T Narender, Anuradha Dube

  The Journal of antimicrobial chemotherapy. 09/2008;

  Objectives The aim of this study was to resolve the putative pathway responsible for death induced by peganine hydrochloride dihydrate isolated from Peganum harmala seeds at cellular, structural andObjectives The aim of this study was to resolve the putative pathway responsible for death induced by peganine hydrochloride dihydrate isolated from Peganum harmala seeds at cellular, structural and molecular level in Leishmania donovani, a causative agent of fatal visceral leishmaniasis. Methods The mode of action was assessed using various biochemical approaches including phosphatidylserine exposure, estimation of mitochondrial transmembrane potential and in situ dUTP nick end labelling staining of nicked DNA in the parasite. Molecular modelling and molecular dynamics studies were conducted with DNA topoisomerase I to identify the target of peganine hydrochloride dihydrate mediating apoptosis. Further, DNA topoisomerase I inhibition by peganine hydrochloride dihydrate was also assessed using an L. donovani topoisomerase I relaxation assay. Results Peganine hydrochloride dihydrate, besides being safe, was found to induce apoptosis in both the stages of L. donovani via loss of mitochondrial transmembrane potential. Molecular docking studies suggest that a binding interaction with DNA topoisomerase I of L. donovani (binding energy of -79 kcal/mol) forms a stable complex, indicating a possible role in apoptosis. The compound also inhibits L. donovani topoisomerase I. Conclusions The compound induces apoptosis in L. donovani and inhibits DNA topoisomerase I.

• Proteomic approaches for discovery of new targets for vaccine and therapeutics against visceral leishmaniasis.

  Authors: Shraddha Kumari, Awanish Kumar, Mukesh Samant, Shyam Sundar, Neeloo Singh, Anuradha Dube

  Proteomics. Clinical applications. 03/2008; 2(3):372-86.

  Visceral leishmaniasis (VL) is the most devastating type caused by Leishmania donovani, Leishmania infantum, and Leishmania chagasi. The therapeutic mainstay is still based on the antiquatedVisceral leishmaniasis (VL) is the most devastating type caused by Leishmania donovani, Leishmania infantum, and Leishmania chagasi. The therapeutic mainstay is still based on the antiquated pentavalent antimonial against which resistance is now increasing. Unfortunately, due to the digenetic life cycle of parasite, there is significant antigenic diversity. There is an urgent need to develop novel drug/vaccine targets against VL for which the primary goal should be to identify and characterize the structural and functional proteins. Proteomics, being widely employed in the study of Leishmania seems to be a suitable strategy as the availability of annotated sequenced genome of Leishmania major has opened the door for dissection of both protein expression/regulation and function. Advances in clinical proteomic technologies have enable to enhance our mechanistic understanding of virulence/pathogenicity/host-pathogen interactions, drug resistance thereby defining novel therapeutic/vaccine targets. Expression proteomics exploits the differential expression of leishmanial proteins as biomarkers for application towards early diagnosis. Further using immunoproteomics efforts were also focused on evaluating responses to define parasite T-cell epitopes as vaccine/diagnostic targets. This review has highlighted some of the relevant developments in the rapidly emerging field of leishmanial proteomics and focus on its future applications in drug and vaccine discovery against VL.

• Evaluation of antileishmanial potential of Tinospora sinensis against experimental visceral leishmaniasis.

  Authors: Nasib Singh, Awanish Kumar, Prasoon Gupta, Kailash Chand, Mukesh Samant, Rakesh Maurya, Anuradha Dube

  Parasitology research. 03/2008; 102(3):561-5.

  The chemotherapeutic interventions against visceral leishmaniasis (VL) are limited and facing serious concerns of toxicity, high cost, and emerging drug resistance. There is a greater interest in newThe chemotherapeutic interventions against visceral leishmaniasis (VL) are limited and facing serious concerns of toxicity, high cost, and emerging drug resistance. There is a greater interest in new drug developments from traditionally used medicinal plants which offers unprecedented diversity in structures and bioactivity. With this rationale, ethanolic extract of Tinospora sinensis Linn and its four fractions were tested in vitro against promastigotes and intracellular amastigotes and in vivo in Leishmania donovani infected hamsters. Ethanolic extract exhibited an appreciable activity against promastigotes (IC(50) 37.6+/-6.2 microg/ml) and intracellular amastigotes (IC(50) 29.8+/-3.4 microg/ml). In hamsters, it resulted in 76.2+/-9.2% inhibition at 500 mg/kg/day x 5 oral dose level. Among fractions, n-butanol imparted highest in vitro and in vivo activities. Ethanolic extract and butanol fraction also enhances reactive oxygen species (ROS) and nitric oxide (NO) release. The results indicate that T. sinensis may provide new lead molecules for the development of alternative drugs against VL.

• Age-influenced population kinetics and immunological responses of Leishmania donovani in hamsters.

  Authors: Nasib Singh, Mukesh Samant, Shraddha K Gupta, Awanish Kumar, Anuradha Dube

  Parasitology research. 10/2007; 101(4):919-24.

  Susceptibility of animals to infections depends upon various factors including sex and age of the host, which plays a pivotal role. In this communication, we have investigated the "intake" ofSusceptibility of animals to infections depends upon various factors including sex and age of the host, which plays a pivotal role. In this communication, we have investigated the "intake" of Leishmania donovani infection in young (3-4 weeks old) and adult (15-16 weeks old) golden hamsters. The splenic parasite load in young hamsters on day 15 post infection (p.i.) was 54 +/- 4 amastigotes/100 macrophage nuclei and increased to 106.3 +/- 3.5 on day 30 p.i. However, adult group showed 2.2-(P < 0.001) and 1.75-fold (P < 0.001) lesser parasite burden on these days, respectively. But as the disease progresses further, differences in parasite burden become less significant, as revealed by comparable levels of parasite loads at 2 months p.i. Spleen weight measurements correspond to the above observations. In the young group, the levels of antileishmanial antibody rise two and 4.5 times on days 15 and 30 p.i., respectively, as compared to only 1.3 and 2.3 times increase in their respective adult counterparts. However, after 2 months of infection both groups recorded analogous (12-fold) rise in antibody levels. Both mitogenic and antigenic responses in adult hamsters were less suppressed compared to young hamsters on days 15 and 30 p.i. However, both groups exhibited highly suppressed cell-mediated immune (CMI) responses after 2 months of infection. These findings implicate that age of the host may influence the susceptibility and resistance to Leishmania infection.

• Induction of Th1-type cellular responses in cured/exposed Leishmania-infected patients and hamsters against polyproteins of soluble Leishmania donovani promastigotes ranging from 89.9 to 97.1kDa

  Authors: Shraddha Kumari, Mukesh Samant, Prashant Khare, Shyam Sundar, Sudhir Sinha, Anuradha Dube

  Vaccine.

  Earlier, we have identified soluble antigenic fraction ranging from 68 to 97.4 kDa (F2-fraction) of Leishmania donovani promastigote, which induced Th1-immunostimulatory cellular responses in bothEarlier, we have identified soluble antigenic fraction ranging from 68 to 97.4 kDa (F2-fraction) of Leishmania donovani promastigote, which induced Th1-immunostimulatory cellular responses in both cured Leishmania patients/hamsters and exhibited significant prophylactic potential against experimental visceral leishmaniasis (VL). In the present study, we have further fractionated F2-fraction by continuous elution SDS-PAGE and subjected them to re-evaluation for their ability to induce cellular responses. Out of seven sub-fractions: F2.1–F2.7, only four F2.4–F2.7 (89.9–97.1 kDa), individually or in pooled sub-fractions (P4–7), stimulated Th1-type remarkable lymphoproliferative, NO, IFN-γ and IL-12 responses in Leishmania-infected cured/exposed patients and hamsters. Interestingly, optimum Th1-responses were obtained with P4–7 suggesting the presence of immunostimulatory molecules in it and may be exploited for developing a successful subunit vaccine against VL.