Goutam Mandal

Publications of Goutam Mandal

• Monitoring of intracellular nitric oxide in leishmaniasis: its applicability in patients with visceral leishmaniasis.

  Authors: Avijit Sarkar, Piu Saha, Goutam Mandal, Debanjan Mukhopadhyay, Susmita Roy, Sunny Kumar Singh, Sukhen Das, R P Goswami, Bibhuti Saha, Deepak Kumar, Padma Das, Mitali Chatterjee

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 01/2011; 79(1):35-45.

  Nitric oxide (NO) has been demonstrated to be a principal effector molecule responsible for mediating intracellular killing of Leishmania parasites, the causative organism of leishmaniasis. AsNitric oxide (NO) has been demonstrated to be a principal effector molecule responsible for mediating intracellular killing of Leishmania parasites, the causative organism of leishmaniasis. As measurement of intracellular NO remains a challenge to biologists, we have developed a flow cytometric approach to perform real time biological detection of NO within Leishmania parasites and parasitized macrophages using a membrane permeable derivative of diaminofluorescein [4,5-diaminofluorescein diacetate (DAF-2DA)]. Initially, assay optimization was performed in Leishmania donovani promastigotes, assay specificity being confirmed using both a NO donor [S-nitroso-N-acetyl-penicillamine (SNAP)] and a NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, C-PTIO]. Using 40 μM DAF-2DA, basal levels of intracellular NO were measured which varied in different Leishmania species; addition of conventional anti-leishmanial drugs, antimony and miltefosine translated into a dramatic increase in DAF-2T fluorescence. Furthermore, the assay also measured levels of NO in macrophages, but needed a 20 fold lower concentration of DAF-2DA, being 2 μM. Following parasitization, levels of NO decreased which was normalized following treatment with anti-leishmanial drugs. Similarly monocytes of patients with visceral leishmaniasis at disease presentation showed decreased levels of NO which too reverted on completion of treatment. Taken together, this study opens new perspectives of research regarding monocyte function and provides a real time approach for monitoring the effect of anti-leishmanial compounds.

• Elevated levels of tryparedoxin peroxidase in antimony unresponsive Leishmania donovani field isolates.

  Authors: Susan Wyllie, Goutam Mandal, Neeloo Singh, Shyam Sundar, Alan H Fairlamb, Mitali Chatterjee

  Molecular and biochemical parasitology. 10/2010; 173(2):162-4.

  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 ofEnhancement 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.

• Flow cytometric determination of intracellular non-protein thiols in Leishmania promastigotes using 5-chloromethyl fluorescein diacetate.

  Authors: Avijit Sarkar, Goutam Mandal, Neeloo Singh, Shyam Sundar, Mitali Chatterjee

  Experimental parasitology. 05/2009;

  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 drugLeishmania 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 gamma 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 antileishmanial 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.

• Functionality of drug efflux pumps in antimonial resistant Leishmania donovani field isolates.

  Authors: Goutam Mandal, Avijit Sarkar, Piu Saha, Neeloo Singh, Shyam Sundar, Mitali Chatterjee

  Indian journal of biochemistry & biophysics. 03/2009; 46(1):86-92.

  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 anThe 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 rhodamine123 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.

• Cytotoxicity of Senecio in macrophages is mediated via its induction of oxidative stress.

  Authors: Samiran Bandyopadhyay, Sudipto Ganguly, Goutam Mandal, Rupashree Sen, Piu Saha, Monoj Kumar Ghosh, Mihir Sarkar, Mitali Chatterjee

  Research in veterinary science. 02/2009;

  In Arunachal Pradesh and other sub-Himalayan areas of India, accidental consumption of Senecio plants by yaks is often fatal as the plant contains toxic alkaloids like Seneciophylline. The presentIn Arunachal Pradesh and other sub-Himalayan areas of India, accidental consumption of Senecio plants by yaks is often fatal as the plant contains toxic alkaloids like Seneciophylline. The present investigation was undertaken to demonstrate the pro-oxidant effects of an ethanolic extract of Seneciochrysanthemoides (S-EtOH). S-EtOH impaired viability in macrophages, the IC(50) being 13.8+/-1.11mug/mL. The effect of S-EtOH (1mug/mL) on generation of reactive oxygen species (ROS) in macrophages was measured by flow cytometry using 2',7'-dichlorofluorescein diacetate (H(2)DCFDA) where it caused a significant increase in the mean fluorescence channel (MFC) from 8.55+/-0.03 to 47.32+/-2.25 (p<0.001). S-EtOH also effected a 3.8-fold increase in extracellular nitric oxide (NO) generation from 4.90+/-0.72muM to 18.79+/-0.32muM (p<0.001), a 2.2-fold increase in intracellular NO production, the MFC increasing from 14.95+/-0.48 to 33.34+/-1.66 (p<0.001), and concomitantly depleted non protein thiols as analyzed by flow cytometry using mercury orange, with a reduction in MFC from 632.5+/-49.44 to 407.4+/-12.61 (p<0.01). Additionally, S-EtOH (14mug/mL, 24h) caused apoptosis as evident by increased Annexin V binding and terminal deoxynucleotidyl transferase mediated dUTP DNA nick end labeling. Taken together, the cytotoxicity of S-EtOH can be partly attributed to its capacity to inflict oxidative damage via generation of both reactive oxygen and nitrogen species culminating in apoptosis.

• Anti-inflammatory effect of allylpyrocatechol in LPS-induced macrophages is mediated by suppression of iNOS and COX-2 via the NF-kappaB pathway.

  Authors: Debjani Sarkar, Piu Saha, Sunita Gamre, Surajit Bhattacharjee, Chellaram Hariharan, Sudipto Ganguly, Rupashree Sen, Goutam Mandal, Subrata Chattopadhyay, Subrata Majumdar, Mitali Chatterjee

  International immunopharmacology. 10/2008; 8(9):1264-71.

  The crude ethanol extract of Piper betle leaf is reported to possess anti-inflammatory activity which has been suggested to be mediated by allylpyrocatechol (APC). In the present study, we haveThe crude ethanol extract of Piper betle leaf is reported to possess anti-inflammatory activity which has been suggested to be mediated by allylpyrocatechol (APC). In the present study, we have demonstrated the anti-inflammatory effects of APC (10 mg/kg, p.o.) in an animal model of inflammation. To investigate the mechanism(s) of this anti-inflammatory activity, we examined its effects on the lipopolysaccaride (LPS)-induced production of NO and PGE(2) in a murine macrophage cell line, RAW 264.7. APC inhibited production of NO and PGE(2) in a dose dependent manner as also decreased mRNA expression of iNOS, COX-2, IL-12p40 and TNF-alpha. Since nuclear factor-kappaB (NF-kappaB) appears to play a central role in transcriptional regulation of these proteins, we investigated the effects of APC on this transcription factor. APC inhibited LPS induced nuclear factor-kappaB (NF-kappaB) activation, by preventing degradation of the inhibitor kappaB (IkappaB). Taken together, our data indicates that APC targets the inflammatory response of macrophages via inhibition of iNOS, COX-2 and IL-12 p40 through down regulation of the NF-kappaB pathway, indicating that APC may have therapeutic potential in inflammation associated disorders.

• An ethanolic extract of leaves of Piper betle (Paan) Linn mediates its antileishmanial activity via apoptosis.

  Authors: Avijit Sarkar, Rupashree Sen, Piu Saha, Sudipto Ganguly, Goutam Mandal, Mitali Chatterjee

  Parasitology research. 05/2008; 102(6):1249-55.

  An unprecedented increase in the incidence of unresponsiveness to antimonial compounds has highlighted the urgent need to develop new antileishmanial agents. The leaves of Piper betle (locally knownAn unprecedented increase in the incidence of unresponsiveness to antimonial compounds has highlighted the urgent need to develop new antileishmanial agents. The leaves of Piper betle (locally known as Paan) have long been in use in the Indian indigenous system of medicine for its antimicrobial properties but its antileishmanial potential has not been studied. Accordingly, an ethanolic extract of leaves of Piper betle (PB) was tested for its antileishmanial activity that was evidenced in both promastigotes and amastigotes, with IC50 values of 9.8 and 5.45 microg/ml, respectively; importantly, it was accompanied by a safety index of >12-fold. This leishmanicidal activity of PB was mediated via apoptosis as evidenced by morphological changes, loss of mitochondrial membrane potential, in situ labeling of DNA fragments by terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling, and cell-cycle arrest at the sub-G0/G1 phase. Taken together, the data indicate that PB has promising antileishmanial activity that is mediated via programmed cell death and, accordingly, merits consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.

• Artemisinin triggers induction of cell-cycle arrest and apoptosis in Leishmania donovani promastigotes.

  Authors: Rupashree Sen, Samiran Bandyopadhyay, Avijit Dutta, Goutam Mandal, Sudipto Ganguly, Piu Saha, Mitali Chatterjee

  Journal of medical microbiology. 10/2007; 56(Pt 9):1213-8.

  A major impediment to effective anti-leishmanial chemotherapy is the emergence of drug resistance, especially to sodium antimony gluconate, the first-line treatment for leishmaniasis. Artemisinin, aA major impediment to effective anti-leishmanial chemotherapy is the emergence of drug resistance, especially to sodium antimony gluconate, the first-line treatment for leishmaniasis. Artemisinin, a sesquiterpene lactone isolated from Artemisia annua, is an established anti-malarial compound that showed anti-leishmanial activity in both promastigotes and amastigotes, with IC(50) values of 160 and 22 microM, respectively, and, importantly, was accompanied by a high safety index (>22-fold). The leishmanicidal activity of artemisinin was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, in situ labelling of DNA fragments by terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell-cycle arrest at the sub-G(0)/G(1) phase. Taken together, these data indicate that artemisinin has promising anti-leishmanial activity that is mediated by programmed cell death and, accordingly, merits consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.

• In vitro antileishmanial activity of Aloe vera leaf exudate: a potential herbal therapy in leishmaniasis.

  Authors: Avijit Dutta, Goutam Mandal, Chitra Mandal, Mitali Chatterjee

  Glycoconjugate journal. 02/2007; 24(1):81-6.

  Aloe vera has wide spread use in health products, and despite several reports on the whole plant and inner gel, little work has been performed on the leaf exudate. Our aim was to evaluate the inAloe vera has wide spread use in health products, and despite several reports on the whole plant and inner gel, little work has been performed on the leaf exudate. Our aim was to evaluate the in vitro efficacy of Aloe vera leaf exudate (AVL) in leishmaniasis. Irrespective of the disease manifestation, promastigotes from strains responsible for cutaneous, mucocutaneous, and visceral leishmaniasis were susceptible to AVL and their IC(50) ranged from 100 to 180 microg/ml. In axenic amastigotes cultured from a L. donovani strain 2001 responsible for visceral leishmaniasis, the IC(50) was 6.0 microg/ml. AVL caused activation of host macrophages evident by an increased release of members of reactive oxygen species that was attenuated by preincubation with free radical scavengers. Collectively, our data indicates that AVL, via its direct leishmanicidal activity which can be further enhanced by activation of host macrophages, is an effective antileishmanial agent meriting further pharmacological investigations.

• Anti-inflammatory effect of allylpyrocatechol in LPS-induced macrophages is mediated by suppression of iNOS and COX-2 via the NF-κB pathway

  Authors: Debjani Sarkar, Piu Saha, Sunita Gamre, Surajit Bhattacharjee, Chellaram Hariharan, Sudipto Ganguly, Rupashree Sen, Goutam Mandal, Subrata Chattopadhyay, Subrata Majumdar, Mitali Chatterjee

  International Immunopharmacology.

  The crude ethanol extract of Piper betle leaf is reported to possess anti-inflammatory activity which has been suggested to be mediated by allylpyrocatechol (APC). In the present study, we haveThe crude ethanol extract of Piper betle leaf is reported to possess anti-inflammatory activity which has been suggested to be mediated by allylpyrocatechol (APC). In the present study, we have demonstrated the anti-inflammatory effects of APC (10 mg/kg, p.o.) in an animal model of inflammation. To investigate the mechanism(s) of this anti-inflammatory activity, we examined its effects on the lipopolysaccaride (LPS)-induced production of NO and PGE2 in a murine macrophage cell line, RAW 264.7. APC inhibited production of NO and PGE2 in a dose dependent manner as also decreased mRNA expression of iNOS, COX-2, IL-12p40 and TNF-alpha. Since nuclear factor-κB (NF-κB) appears to play a central role in transcriptional regulation of these proteins, we investigated the effects of APC on this transcription factor. APC inhibited LPS induced nuclear factor-kappaB (NF-κB) activation, by preventing degradation of the inhibitor kappaB (IκB). Taken together, our data indicates that APC targets the inflammatory response of macrophages via inhibition of iNOS, COX-2 and IL-12 p40 through down regulation of the NF-κB pathway, indicating that APC may have therapeutic potential in inflammation associated disorders.

• Flow cytometric determination of intracellular non-protein thiols in Leishmania promastigotes using 5-chloromethyl fluorescein diacetate

  Authors: Avijit Sarkar, Goutam Mandal, Neeloo Singh, Shyam Sundar, Mitali Chatterjee

  Experimental Parasitology.

  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 drugLeishmania 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.