Browsing by Author "Mitali Chatterjee"

Now showing 1 - 14 of 14

Antibodies directed against O-acetylated sialoglycoconjugates accelerate complement activation in Leishmania donovani promastigotes
(2004) Sumi Bandyopadhyay; Mitali Chatterjee; Tanusree Das; Suman Bandyopadhyay; Shyam Sundar; Chitra Mandal
Background. An enhanced presence of 9-O-acetylated Sialoglycoconjugates (9-O-AcSGs) triggers the alternate pathway (AP) in Indian visceral leishmaniasis (VL). Antibodies directed against these epitopes are present in high titers. The biological relevance of these antibodies, with regard to activation of the classical pathway (CP), was investigated. Methods. Complement activators were affinity purified, complement activation via the CP, AP, and lectin-mediated complement pathway was measured by use of an anti-C3 radio-binding assay, and the number of C3 molecules was quantitated by Scatchard analysis. Cell death induced via the complement pathways was measured by use of MTT (tetrazolium salt 3- [4, 5-dimethylthiazol-2-yl] -2, 5-diphenyltetrazolium bromide) assay, and uptake of propidium iodide (PI) was measured by flow cytometry. Results. Anti-O-AcSGs from both healthy donors and patients with VL elicited C3 deposition as early as 3 min, which triggered parasite lysis, as demonstrated by use of MTT assay and corroborated by the high rate of uptake of PI. Analysis of complement activation by mannan-binding lectin and C-reactive protein demonstrated their negligible contribution during the 3-min time frame. Conclusions. Anti-O-AcSGs were identified as an important source of CP activation under normal physiological conditions, suggesting that they play a role in conferring host protection against parasite infection.
Development of an assay for quantification of linkage-specific O-acetylated sialoglycans on erythrocytes; its application in Indian visceral leishmaniasis
(2002) Anil Kumar Chava; Mitali Chatterjee; Shyam Sundar; Chitra Mandal
We have developed a noninvasive approach for the quantification of linkage-specific 9-O-acetylated sialoglycans on mammalian erythrocytes using a lectin, Achatinin-H, whose lectinogenic epitope has previously been defined as 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) α2→6 linked to subterminal GalNAc. Titration and checkerboard analysis were performed to optimize the assay using rabbit, rat and human erythrocytes that contain differing amounts of this glycotope. Assay specificity was established by decreased binding of erythrocytes to immobilised Achatinin-H when pre-incubated with excess lectin. The intra-assay coefficient of variation (CV) for rat and human erythrocytes was 8.6-9.2% and 11.1-13.0%, respectively. The inter-assay CV for rat and human erythrocytes was 9.9-10.1% and 15.2-16.6%, respectively. In previous studies, we have identified an enhanced presence of cell surface 9-O-AcSGs on the erythrocytes of patients with visceral leishmaniasis (VL) [Am. J. Trop. Med. Hyg. 58 (1998) 551]. Our assay when evaluated on erythrocytes from VL patients (n=30) showed a fourfold increase in lectin binding as compared to endemic controls. The mean±S.E.M. of the A405 nm value was 1.14±0.04 vs. 0.23±0.03, respectively (p<0.0001). Following effective chemotherapy, a significant reduction of this glycotope on the erythrocytes of VL patients indicates that this assay has both a diagnostic and prognostic potential. Taken together, we conclude that this antigen-based assay is a specific and reproducible method for monitoring the disease status of VL patients and could be used in retrospective and prospective trials. © 2002 Elsevier Science B.V. All rights reserved.
Diagnostic and prognostic potential of a competitive enzyme-linked immunosorbent assay for leishmaniasis in India
(American Society for Microbiology, 1999) Mitali Chatterjee; Charles L. Jaffe; Shyam Sundar; Debasis Basu; Sandeep Sen; Chitra Mandal
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.
Elevated levels of tryparedoxin peroxidase in antimony unresponsive Leishmania donovani field isolates
(2010) Susan Wyllie; Goutam Mandal; Neeloo Singh; Shyam Sundar; Alan H. Fairlamb; Mitali Chatterjee
Enhancement of the anti-oxidant metabolism of Leishmania parasites, dependent upon the unique dithiol trypanothione, has been implicated in laboratory-generated antimony resistance. Here, the role of the trypanothione-dependent anti-oxidant pathway is studied in antimony-resistant clinical isolates. Elevated levels of tryparedoxin and tryparedoxin peroxidase, key enzymes in hydroperoxide detoxification, were observed in antimonial resistant parasites resulting in an increased metabolism of peroxides. These data suggest that enhanced anti-oxidant defences may play a significant role in clinical resistance to antimonials. © 2010 Elsevier B.V.
Flow cytometric determination of intracellular non-protein thiols in Leishmania promastigotes using 5-chloromethyl fluorescein diacetate
(2009) Avijit Sarkar; Goutam Mandal; Neeloo Singh; Shyam Sundar; Mitali Chatterjee
Leishmania parasites lack catalase and therefore, their anti-oxidant system hinges primarily upon non-protein thiols; accordingly, depletion of thiols could potentially serve as an effective drug target. We have developed a flow cytometry based assay using 5-chloromethyl fluorescein diacetate based upon its selective staining of non-protein thiols. Its specificity was confirmed using buthionine sulphoximine (a γ-glutamyl cysteine synthetase inhibitor), diamide (an oxidizing agent of intracellular thiols) and N-ethylmaleimide (a covalent modifier of cysteine residues) as evidenced by reduction in fluorescence; furthermore, restoration of fluorescence by N-acetyl cysteine corroborated specificity of 5-chloromethyl fluorescein diacetate to measure non-protein thiols. Differences in basal level of thiols in antimony sensitive and antimony resistant Leishmania field isolates were detected. The depletion of non-protein thiols by conventional anti-leishmanial drugs e.g. antimony and miltefosine was demonstrated. Furthermore, fluorescence was unaffected by depletion of ATP in majority of the strains studied, indicating that 5-chloromethyl fluorescein diacetate is not a substrate for the pump operative in most Leishmania donovani strains. Taken together, measurement of 5-chloromethyl fluorescein diacetate fluorescence is an effective method for monitoring non-protein thiols in Leishmania promastigotes. © 2009 Elsevier Inc. All rights reserved.
Functionality of drug efflux pumps in antimonial resistant Leishmania donovani field isolates
(2009) Goutam Mandal; Avijit Sarkar; Piu Saha; Neeloo Singh; Shyam Sundar; Mitali Chatterjee
The recent upsurge of antimony (Sb) resistance is a major impediment to successful chemotherapy of visceral leishmaniasis (VL). Mechanisms involved in antimony resistance have demonstrated an upregulation of drug efflux pumps; however, the biological role drug efflux pumps in clinical isolates remains to be substantiated. Thus, in this study, the functionality of drug efflux pumps was measured in promastigotes and axenic amastigotes isolated from VL patients, who were either Sb-sensitive (AG83, 2001 and MC9) or resistant (NS2, 41 and GE1) using rhodamine 123 as a substrate for multidrug resistant (MDR) pumps and calcein as a substrate for multidrug resistance-associated proteins (MRP) respectively; their specificity was confirmed using established blockers. Sb-resistant (Sb-R) isolates accumulated higher amounts of R123, as compared to Sb-sensitive (Sb-S) isolates. Verapamil, a MDR inhibitor failed to alter R123 accumulation, suggesting absence of classical MDR activity. In Sb-R isolates, both promastigotes and axenic amastigotes accumulated significantly lower amounts of calcein than Sb-S isolates and probenecid, an established pan MRP blocker, marginally increased calcein accumulation. Depletion of ATP dramatically increased calcein accumulation primarily in Sb-R isolates, indicating existence of a MRP-like pump, which was more active in Sb-R isolates. In conclusion, our data suggested that overfunctioning of a MRP-like pump contributed towards generation of Sb-R phenotype in L. donovani field isolates.
Identification of 9-O-acetylated sialoglycans on peripheral blood mononuclear cells in Indian Visceral Leishmaniasis
(2003) Sumi Bandyopadhyay; Mitali Chatterjee; Shyam Sundar; Chitra Mandal
Although the existence of O-acetylated sialic acids is well known, it is only in recent years that steady refinement of analytical techniques have enabled detailed mapping of their structural diversity [1]. Fluorimetric analysis of peripheral blood mononuclear cells (PBMC) of patients with Visceral Leishmaniasis (VL) showed six fold increase in the percentage of surface 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) as compared to normal human donors. Using Achatinin-H, a 9-O-acetyl sialic acid- binding lectin, an enhanced presence of 9-O-AcSGs in an α2 → 6 linkage was demonstrated by flow cytometry; abolition of its binding by pretreatment with a recombinant 9-O-acetylesterase corroborated the presence of this glycotope. Western blotting of PBMC from VL patients indicated the presence of five O-acetylated sialoglycans corresponding to 144, 65, 56, 36 and 19 kDa as compared to 144 and 36 kDa in normal individuals. Taken together our data indicates that during active disease, there is an overexpression of 9-O-AcSGs on the surface of PBMC of VL patients, thus opening up new research avenues wherein the expression of this biomarker could be exploited to monitor the clinical status of VL patients. © 2004 Kluwer Academic Publishers.
Identification of antibodies directed against O-acetylated sialic acids in visceral leishmaniasis: Its diagnostic and prognostic role
(1998) Mitali Chatterjee; Vineeta Sharma; Chitra Mandal; Shyam Sundar; Sandeep Sen
A significantly increased O-acetylated sialic acid (O-AcSA) binding fraction was purified from serum of visceral leishmaniasis (VL) patients by affinity chromatography on immobilized bovine submaxillary mucin (BSM) and found to be immunoglobulin in origin. The serodiagnostic and prognostic potential of BSM as a capture antigen was established by ELISA with no cross reactivity with coendemic diseases like malaria, tuberculosis, leprosy, chagas disease and cutaneous leishmaniasis; however, a strong cross reactivity was present with trypanosomiasis patients. In 56 clinically diagnosed VL patients, the BSM-ELISA was compared with diagnosis by microscopy using Giemsa stained tissue smears and direct ELISA using crude parasite antigen (parasite-ELISA); 49/56(87.5%) and 5/56(9.0%) were positive and negative respectively by all 3 methods. The BSM-ELISA failed to diagnose 2/56(3.5%) patients which were biopsy and parasite-ELISA positive. The prognostic potential of the BSM-ELISA in 18 longitudinally monitored VL patients before and after conventional antimonial treatment showed a significant decrease in anti O-AcSA titres in drug responsive patients whereas anti O-AcSA levels persisted in drug unresponsive patients. The IgG subclass distribution of antibodies directed against O-AcSA showed increased lgG2 levels in VL patients as compared to healthy controls. The BSM-based ELISA holds great promise as a serodiagnostic and prognostic assay for VL.
Purification, characterization of O-acetylated sialoglycoconjugates- specific IgM, and development of an enzyme-linked immunosorbent assay for diagnosis and follow-up of indian visceral leishmaniasis patients
(2004) Sumi Bandyopadhyay; Mitali Chatterjee; Santanu Pal; Ross F. Waller; Shyam Sundar; Malcolm J. McConville; Chitra Mandal
The surface expression of 9-O-acetylated sialic acid (9-OAcSA) is elevated on hematopoietic cells and erythrocytes of visceral leishmaniasis (VL) patients. In this study, we show that VL patients contain elevated levels of IgM antibodies directed against 9-O-acetylated sialoglycoconjugates (9-OAcSG). These antibodies were affinity purified with bovine submaxillary protein as the affinity matrix containing the terminal epitope, 9-OAcSAα2-6GalNAc. They also bound to 9-OAcSGs on hematopoietic cells of patients with VL and to epitopes in the cytosol of Leishmania donovani promastigotes. A novel enzyme-linked immunosorbent assay was employed that showed 4-fold higher anti-OAcSG titers in VL patients (n = 38), mean ± S.E.M. being 0.83 ± 0.09 vs. 0.21 ± 0.04 detected in normal donors (n = 20) and patients with cross-reactive diseases such as malaria (n = 4) or tuberculosis (n = 4). Assay specificity and sensitivity was 100% and 92%, respectively, whereas positive and negative predictive values were 100% and 90%, respectively. Significantly, anti-OAcSG titers declined 30 days after completion of anti-leishmanial treatment, indicating that monitoring of anti-9-OAcSGs may be a valuable alternative toward increasing the efficiency of diagnosis and follow-up of VL. © 2004 Elsevier Inc. All rights reserved.
ROS and RNS induced apoptosis through p53 and iNOS mediated pathway by a dibasic hydroxamic acid molecule in leukemia cells
(Elsevier B.V., 2014) Kaushik Banerjee; Avishek Ganguly; Paramita Chakraborty; Avijit Sarkar; Suryabhan Singh; Mitali Chatterjee; Subrato Bhattacharya; Soumitra Kumar Choudhuri
Anticancer drugs induce apoptosis to cancer cells and also exhibit undesired toxicity to normal cells. Therefore development of novel agents triggering apoptosis and have low toxicity towards normal cells is most important. Hydroxamic acids suppress tumour cell growth through apoptosis but the underlying mechanism is poorly understood. Herein, we describe the apoptotic potential of a dibasic hydroxamic acid derivative, viz., oxayl bis (N-phenyl) hydroxamic acid (OBPHA), which induces apoptosis through generation of both ROS and NO in doxorubicin resistant T-lymphoblastic leukemia, CEM/ADR5000 cells. Present study discloses that OBPHA selectively kills cancerous cells irrespective of their drug resistant phenotype. We also determined the crystal structure of OBPHA to understand the structural requirements for apoptosis; the study reveals that the presence of substituted hydroxamic acid groups (-CO-NH-OH) favours the generation of NO possibly through auto degeneration. Along with the induction of caspase 3 mediated intrinsic apoptosis; OBPHA also activates p53 dependent signalling cascade and downregulates HDAC3 expression in a time dependent manner possibly due to increased ROS and NO production and simultaneous decrease in cellular GSH level. Thus ROS and NO mediated downstream signalling are essential for the anticancer effect of OBPHA. Therefore OBPHA, having a structurally relevant pharmacophore provides important insight into the development of new ROS and RNS generating chemicals inducing p53 dependent apoptosis. © 2013 Elsevier B.V. All rights reserved.
The development of a global research agenda and individual participant data platform for visceral leishmaniasis: challenges and future opportunities
(BioMed Central Ltd, 2025) Sauman Singh-Phulgenda; Prabin Dahal; Brittany J. Maguire; Jorge Alvar; Fabiana Piovesan Alves; Mitali Chatterjee; Carlos Henrique Nery Costa; Simon L. Croft; Philippe Jean Jean Guérin; Dinesh K. Mondal; Ahmed Mudawi Musa; Krishna Pandey; Koert Ritmeijer; G. A.s. Romero; Santhanam Sundar
Background: Visceral leishmaniasis (VL) is one of the neglected tropical diseases (NTDs) listed by the World Health Organization (WHO). The disease is currently in the elimination phase in the Indian subcontinent (ISC) and being targeted for elimination by 2030 in East Africa (EA). Maintaining the necessary financial and political commitments to achieve and sustain the current elimination efforts remains challenging. As with other NTDs, VL research is constrained by limited funding, and drug development has relied largely on partnerships between not-for-profit organisations and the pharmaceutical industry. Conducting robust clinical studies remains difficult, and therapeutic innovations have been limited. However, re-use of existing data offers an untapped opportunity to generate new evidence. Methods: We describe the process of developing a global VL research agenda and the establishment of an individual participant data (IPD) platform at the Infectious Diseases Data Observatory (IDDO). Key steps included a systematic scoping review of VL clinical trials, consultations with the Scientific Advisory Committee, expert and public reviews, and implementation of an equitable governance framework to harmonise and share IPD. Results: The VL research agenda, finalised in 2019, identified priority methodological and clinical questions suited to IPD analyses. The IDDO VL platform currently hosts harmonised data from nearly 15,000 patients across more than 50 studies (VL and post-kala-azar dermal leishmaniasis, PKDL). The platform is an inclusive resource guided by an equitable governance framework and provides a critical asset to support new evidence generation and can serve as a historical data to support accelerated drug development. Conclusions: The development of a global VL research agenda has provided an inventory of priority research questions of public health importance. A shared IPD platform aligned with this agenda was developed to complement ongoing global efforts. In addition, such a platform can accelerate secondary evidence generation, support methodological innovation and inform future trial designs and policy. Sustained collaboration and investment are needed to maximise the scientific and public health value of data re-use in VL and PKDL. © The Author(s) 2025.
The overexpression of genes of thiol metabolism contribute to drug resistance in clinical isolates of visceral leishmaniasis (kala azar) in India
(BioMed Central Ltd., 2014) Neeloo Singh; Mitali Chatterjee; Shyam Sundar
Background: Visceral leishmaniasis (VL), also called Kala Azar (KA) or black fever in India, claims around 20,000 live every year. Chemotherapy remains one of the most important tools in the control of VL. Current chemotherapy fo Kala Azar in India relies on a rather limited arsenal of drugs including sodium antimony gluconate and amphoterici B in addition to the very expensive drug miltefosine. Pentavalent antimonials have been used for more than half century in the therapy of leishmaniasis as it is relatively safe and inexpensive, however, the spread of resistance to thi drug is forcing clinicians in India to abandon this treatment. Consequently, improvement of antimonial chemotherap has become a major challenging area of study by leishmaniacs worldwide. The alarming emergence of resistance t the commonly used antleishmanial drug, sodium antimony gluconate, in India, has led us to elucidate the resistanc mechanism(s) in clinical isolates. Studies on laboratory mutants have shown that resistance to antimonials is highl dependent on thiol levels. The parasite evades cytotoxic effects of antimonial therapy by enhanced efflux of drug upo 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 resistan clinical isolates collected from disease endemic areas in India and also carried out molecular characterization of thio levels in these parasites Results: Overexpression of the gene coding for ? glutamylcysteine synthetase was observed in these resistant clinica isolates thereby establishing that thiols represent the key determinants of antimonial resistance. The SbIII/thio conjugates can be sequestered by ABC transporter multidrug resistance protein A (MRPA) into intracellular organelle or can be directly pumped out by an uncharacterized transporter Conclusions: Our studies investigating antimonial resistance in different L. donovani clinical isolates suggest that ove functioning of MRP plays a role in generation of antimony resistance phenotype in some L. donovani clinical isolates. © 2014 Singh et al.; licensee BioMed Central.
Variable degree of alternative complement pathway-mediated hemolysis in Indian visceral leishmaniasis induced by differential expression of 9-O-acetylated sialoglycans
(2004) Anil Kumar Chava; Mitali Chatterjee; Vineeta Sharma; Shyam Sundar; Chitra Mandal
Background. Increased expression of linkage-specific 9-O-acetylated sialoglycans (9-O-AcSGs) has been demonstrated on erythrocytes from patients with visceral leishmaniasis (VL) by use of Achatinin-H. We assessed the capacity of this glycotope to influence hemolysis via activation of the alternative complement pathway in patients with VL, compared with that in healthy control subjects. Methods. The differential expression of 9-O-AcSGs on surfaces of erythrocytes was measured, 9-O-AcSGs were affinity purified, and the molecular determinants were identified by Western blotting. The degree of alternative complement pathway-mediated hemolysis was compared with expression of 9-O-AcSGs on erythrocytes. Results. Enhanced expression of linkage-specific 9-O-AcSGs was demonstrated on erythrocytes from patients with active VL. Six distinct molecular determinants present only on diseased erythrocytes were affinity purified and were absent after elimination of parasite burden. A correlation (r 2 = 0.9) was observed between the presence of 9-O-AcSGs and the degree of alternative complement pathway-mediated hemolysis. Conclusion. The 9-O-AcSGs expressed on erythrocytes from patients with VL are potent complement activators, causing enhanced hemolysis via activation of the alternative complement pathway, and may account for the anemia that is a common manifestation of VL.
Visceral leishmaniasis - Current therapeutic modalities
(2006) Shyam Sundar; Mitali Chatterjee
Major therapeutic obstacles in the treatment of visceral leishmaniasis (VL) include the alarming increase in antimonial unresponsiveness especially in Bihar, India and relapses in HIV-Leishmania co-infected patients. The therapeutic armamentarium for VL is currently plagued with several limitations as the available drugs are toxic, majority are effective only parenterally and need to be administered for extended periods. The first orally effective drug, miltefosine has been approved for treating VL. In antimony refractory zones, pentavalent antimony has been largely replaced by amphotericin B deoxycholate, but prolonged hospitalization, toxic effects, and requirement for monitoring greatly hamper its widespread application in endemic regions. Lipid formulations of amphotericin B, a remarkable advance in amphotericin B therapy, have greatly reduced toxicity enabling large doses to be delivered over a short period. Even a single dose treatment with liposomal amphotericin B cures >90 per cent patients; however, the stumbling block is its prohibitive cost that precludes its widespread accessibility in endemic countries. Studies using paromomycin in VL are encouraging, and judging by the preliminary results of a recently concluded phase III trial, it could be an extremely useful and affordable antileishmanial drug. Other orally effective drugs include the azoles and allopurinol but these have met with limited success owing to either poor efficacy or unacceptable toxicity. Sitamaquine has undergone limited evaluation, and the data suggest effective antileishmanial activity; its role has to be delineated for which additional developmental studies are proposed. This review highlights the progress made in the treatment of VL, including the multiple mechanisms of action of antileishmanial drugs with a view to enable the researcher to undertake the challenge of providing affordable and effective chemotherapy.