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ABSTRACT: Background. Over expression of Programmed death-1 (PD-1) receptor is thought to inhibit effector T cell response in human tuberculosis. However, the precise mechanism of such inhibition remains unclear. The present study aimed at addressing the role of PD-1 in dampening host T cell function among tuberculosis patients.Methods. Expression of PD-1 and its ligands (PD-L1/L2) on T cells, B cells and monocytes was evaluated by flow cytometry (FACS). In vitro stimulation of PBMCs in presence of M. tuberculosis antigens was performed with and without blocking PD-1 and intracellular cytokine production was measured by FACS.Results. We showed higher frequencies of PD-1 and its ligand(s) expressing T cells, monocytes and B cells among PTB patients. Live infections with M. tuberculosis up regulate PD-L1 expression on monocytes. In vitro PD-1 blocking rescued M. tuberculosis specific IFN-γ producing T cells from undergoing apoptosis. Number of PD-1(+) T cells decreased significantly during therapy and inversely correlated with IFN-γ dominant T cell response against M.tuberculosis.Conclusions. Manipulating PD-1 signalling may restore host T cells response, thus may have therapeutic potential. PD-1 also may serve as biomarker to monitor host immunity among tuberculosis patients during therapeutic and vaccination protocols.
The Journal of Infectious Diseases 05/2013; · 6.41 Impact Factor
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ABSTRACT: Selective recruitment of IFN-γ biased Th1 effector cells at the pathologic site(s) determines the local immunity of tuberculosis (TB). We observed the enrichment of CXCR3, CCR5 and CD11a(high) T cells in the peripheral blood, pleural fluid and bronchoalveolar lavage of TB pleural effusion (TB-PE) and miliary tuberculosis (MTB) patients respectively. CXCR3(+)CCR5(+) T cells were significantly high at the local disease site(s) in both the forms of TB and their frequency was highest among activated lymphocytes in TB-PE. Interestingly, all CCR5(+) cells were invariably positive for CXCR3 but all CXCR3(+) cells did not co-express CCR5 in pleural fluid whereas the situation was reverse in bronchoalveolar lavage. These CXCR3(+)CCR5(+) cells dominantly produced IFN-γ in response to Mycobacterium tuberculosis antigen. In vitro chemotaxis assay indicates dominant role of RANTES and IP-10 in the selective recruitment of CXCR3(+)CCR5(+)cells at the tubercular pathologic sites.
Cytokine 05/2013; · 3.02 Impact Factor
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Pradeep Sharma,
Shavait Yamini,
Divya Dube,
Amar Singh,
Gorakh Mal,
Nisha Pandey,
Mau Sinha,
Abhay Kumar Singh,
Sharmistha Dey,
Punit Kaur, Dipendra K Mitra,
Sujata Sharma,
Tej P Singh
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ABSTRACT: Short peptidoglycan recognition protein (PGRP-S) is a member of the mammalian innate immune system. PGRP-S from Camelus dromedarius (CPGRP-S) has been shown to bind to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Its structure consists of four molecules A, B, C and D with ligand binding clefts situated at A-B and C-D contacts. It has been shown that LPS, LTA and PGN bind to CPGRP-S at C-D contact. The cleft at the A-B contact indicated features that suggested a possible binding of fatty acids including mycolic acid of Mycobacterium tuberculosis. Therefore, binding studies of CPGRP-S were carried out with fatty acids, butyric acid, lauric acid, myristic acid, stearic acid and mycolic acid which showed affinities in the range of 10(-5)M to 10(-8)M. Structure determinations of the complexes of CPGRP-S with above fatty acids showed that they bound to CPGRP-S in the cleft at the A-B contact. The flow cytometric studies showed that mycolic acid induced the production of pro-inflammatory cytokines, TNF-α and IFN- by CD3+ T cells. The concentrations of cytokines increased considerably with increasing concentrations of mycolic acid. However, their levels decreased substantially on adding CPGRP-S.
Archives of Biochemistry and Biophysics 11/2012; · 2.93 Impact Factor
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ABSTRACT: An inefficient Th1 response, coupled with skewed Th2 cytokine production, has been implicated to increase susceptibility to visceral leishmaniasis (VL) infection. The expression of the dipeptidyl peptidase Cd26 by polarized Th1 activates a chemokine cascade that recruits Th1 recruitment to the pathologic site. Here, we studied 42 patients with confirmed VL (mean age 24.80 ± 16.26 years; range 3-70 years; 25 males and 17 females), 30 endemic controls, and 10 nonendemic controls. We observed a decrease in constitutive and antigen-induced expression of CD26 on the T cells of VL patients. Soluble CD26 (sCD26) levels in serum and BM were also found to be significantly lower in VL patients. Following successful therapy, increased sCD26 expression was observed. Tuberculosis pleural effusion derived CCR5(+) CXCR3(+) effector T cells showed enhanced chemokine-driven migration in the presence of posttreatment BM aspirate containing high levels of sCD26. Moreover, T-cell migration could be inhibited by blocking RANTES, IP-10, and CD26 signaling from the posttreatment aspirate with Ab. Our results indicate that, in VL patients, impaired expression and secretion of CD26 compromises chemokine activation and thus T-cell recruitment, eventually resulting in a deficient state of local immunity at pathologic sites.
European Journal of Immunology 07/2012; 42(10):2782-91. · 5.10 Impact Factor
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ABSTRACT: Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc, and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3 × 10(-9), 2.6 × 10(-7), and 1.8 × 10(-7) M, respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules, A, B, C, and D, in the asymmetric unit that exists as A-B and C-D units of two neighboring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms, indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α and IL-6 from human peripheral blood mononuclear cells. The introduction of CPGRP-S to the PGN-challenged cultured peripheral blood mononuclear cells reduced the expressions of proinflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and down-regulated macrophage-mediated inflammation, indicating its potential applications as an antibacterial agent.
Journal of Biological Chemistry 05/2012; 287(26):22153-64. · 4.77 Impact Factor
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ABSTRACT: Suppression of T cell response is thought to be involved in the pathogenesis of visceral leishmaniasis (VL). Regulatory T cell (Treg) mediated immune-suppression is reported in animal models of Leishmania infection. However, their precise role among human patients still requires pathologic validation. The present study is aimed at understanding the frequency dynamics and function of Treg cells in the blood and bone marrow (BM) of VL patients. The study included 42 parasitologically confirmed patients, 17 healthy contact and 9 normal bone marrow specimens (NBM). We show i) the selective accumulation of Treg cells at one of the disease inflicted site(s), the BM, ii) their in vitro expansion in response to LD antigen and iii) persistence after successful chemotherapy. Results indicate that the Treg cells isolated from BM produces IL-10 and may inhibit T cell activation in IL-10 dependent manner. Moreover, we observed significantly higher levels of IL-10 among drug unresponsive patients, suggesting their critical role in suppression of immunity among VL patients. Our results suggest that IL-10 plays an important role in suppression of host immunity in human VL and possibly determines the efficacy of chemotherapy.
PLoS ONE 01/2012; 7(2):e31551. · 4.09 Impact Factor
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ABSTRACT: Sperm DNA integrity is a prerequisite for normal spermatozoal function. The aim of the study was to evaluate the role of sperm chromatin damage, its cut-off level and its effect on sperm parameters in men with idiopathic infertility by analyzing 100 idiopathic infertile men and 50 fertile controls. Semen samples were analyzed as per WHO 1999 guidelines and sperm chromatin structure assay (SCSA) was applied to measure DNA fragmentation index (DFI) in sperm. The mean DFI of infertile men (35.75) was significantly (P < .0001) higher as compared to controls (26.22). The threshold level of 30.28% was obtained as cut-off value to discriminate infertile men from fertile controls. Sperm count, forward motility, and normal morphology found to be negatively associated with DFI in overall study subjects. Infertile men with severe oligozoospermia had higher mean DFI (40.01 ± 11.31) than infertile men with oligozoospermia (35.11 ± 10.05) and normal sperm count (33.99 ± 9.96). Moreover 64% of infertile men have DFI > 30 against 6% of fertile controls (P < .0001). Higher sperm DNA fragmentation may be the underlying cause for poor semen quality in idiopathic infertile men and the threshold value of 30.28% is a clear discriminator to distinguish infertile men from fertile men of Indian population. Thus, DFI is a good prognostic marker as cases with higher sperm DFI may have poor success rate even after assisted conception and may experience recurrent pregnancy loss (RPL) and should be counseled accordingly.
Reproductive sciences (Thousand Oaks, Calif.) 10/2011; 18(10):1005-13. · 2.31 Impact Factor
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ABSTRACT: Absence of an effective Th-1 response has been demonstrated as a major cause for the disease pathology among patients with visceral leishmaniasis (VL). Defining strategies to prevent the development of Th-2 response and/or initiate/activate effective Th-1 response may be of help to reduce the growing incidence of drug unresponsiveness. Adenosine, which is considered as an endogenous anti-inflammatory agent is generated in injured/inflamed tissues by the enzymatic catabolism of adenosine triphosphate (ATP), and it suppresses inflammatory responses of essentially all immune cells. The extracellular adenosine-producing pathway comprises two major enzymes CD39 (ATP → ADP → AMP) and CD73 (AMP → Adenosine). In contrast, the adenosine-degrading pathway contains only one major enzyme adenosine deaminase (ADA). Our study shows high concentration of adenosine in diseased condition, varying expression of enzyme involved in adenosine-producing (CD73↓) and adenosine-degrading (ADA↑) pathways. These are less studied in infections like VL but are very important in terms of endogenous regulation of immune response among patients.
Parasite Immunology 07/2011; 33(11):632-6. · 2.60 Impact Factor
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Pradeep Sharma,
Divya Dube,
Amar Singh,
Biswajit Mishra,
Nagendra Singh,
Mau Sinha,
Sharmistha Dey,
Punit Kaur, Dipendra K. Mitra,
Sujata Sharma,
Tej P. Singh
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ABSTRACT: Peptidoglycan recognition proteins (PGRPs) are involved in the recognition of pathogen-associated molecular patterns. The
well known pathogen-associated molecular patterns include LPS from Gram-negative bacteria and lipoteichoic acid (LTA) from
Gram-positive bacteria. In this work, the crystal structures of two complexes of the short form of camel PGRP (CPGRP-S) with
LPS and LTA determined at 1.7- and 2.1-Å resolutions, respectively, are reported. Both compounds were held firmly inside the
complex formed with four CPGRP-S molecules designated A, B, C, and D. The binding cleft is located at the interface of molecules
C and D, which is extendable to the interface of molecules A and C. The interface of molecules A and B is tightly packed,
whereas that of molecules B and D forms a wide channel. The hydrophilic moieties of these compounds occupy a common region,
whereas hydrophobic chains interact with distinct regions in the binding site. The binding studies showed that CPGRP-S binds
to LPS and LTA with affinities of 1.6 × 10−9 and 2.4 × 10−8 m, respectively. The flow cytometric studies showed that both LPS- and LTA-induced expression of the proinflammatory cytokines
TNF-α and IL-6 was inhibited by CPGRP-S. The results of animal studies using mouse models indicated that both LPS- and LTA-induced
mortality rates decreased drastically when CPGRP-S was administered. The recognition of both LPS and LTA, their high binding
affinities for CPGRP-S, the significant decrease in the production of LPS- and LTA-induced TNF-α and IL-6, and the drastic
reduction in the mortality rates in mice by CPGRP-S indicate its useful properties as an antibiotic agent.
Journal of Biological Chemistry 05/2011; 286(18):16208-16217. · 4.77 Impact Factor
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Pradeep Sharma,
Divya Dube,
Amar Singh,
Biswajit Mishra,
Nagendra Singh,
Mau Sinha,
Sharmistha Dey,
Punit Kaur, Dipendra K Mitra,
Sujata Sharma,
Tej P Singh
[show abstract]
[hide abstract]
ABSTRACT: Peptidoglycan recognition proteins (PGRPs) are involved in the recognition of pathogen-associated molecular patterns. The well known pathogen-associated molecular patterns include LPS from Gram-negative bacteria and lipoteichoic acid (LTA) from Gram-positive bacteria. In this work, the crystal structures of two complexes of the short form of camel PGRP (CPGRP-S) with LPS and LTA determined at 1.7- and 2.1-Å resolutions, respectively, are reported. Both compounds were held firmly inside the complex formed with four CPGRP-S molecules designated A, B, C, and D. The binding cleft is located at the interface of molecules C and D, which is extendable to the interface of molecules A and C. The interface of molecules A and B is tightly packed, whereas that of molecules B and D forms a wide channel. The hydrophilic moieties of these compounds occupy a common region, whereas hydrophobic chains interact with distinct regions in the binding site. The binding studies showed that CPGRP-S binds to LPS and LTA with affinities of 1.6 × 10(-9) and 2.4 × 10(-8) M, respectively. The flow cytometric studies showed that both LPS- and LTA-induced expression of the proinflammatory cytokines TNF-α and IL-6 was inhibited by CPGRP-S. The results of animal studies using mouse models indicated that both LPS- and LTA-induced mortality rates decreased drastically when CPGRP-S was administered. The recognition of both LPS and LTA, their high binding affinities for CPGRP-S, the significant decrease in the production of LPS- and LTA-induced TNF-α and IL-6, and the drastic reduction in the mortality rates in mice by CPGRP-S indicate its useful properties as an antibiotic agent.
Journal of Biological Chemistry 03/2011; 286(18):16208-17. · 4.77 Impact Factor
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ABSTRACT: Complications of amphotericin B limit its wide application in the treatment of patients with kala-azar. This study was undertaken with an aim to minimize anti-renal complications and severe rigor in course of treatment with this drug. Parasitologically confirmed kala-azar cases (n = 230) were randomized equally into two groups: a control group received amphotericin B only at a dose of 1 mg/kg of body weight/day for 20 days and a patient (test) group received 500 mL of physiologic saline and 30 mL (60 meq/L) of KC1 with amphotericin B. We observed a significantly lower increase in serum creatinine levels (P = 0.0001) and a lower incidence of severe rigor and fever (P = 0.0165) in the test group than in the control group. However, the ultimate cure rate was not significantly different (P = 0.5637) between two groups after 12 months of follow-up. Relapses occurred after even after six months in both groups. Persons with relapses were treated with 25 infusions of amphotericin B and cured. Supplementation of amphotericin B with 500 mL of physiologic saline and 30 mL (60 meq/L) of KCl during treatment could help prevent an increase in serum creatinine levels and severe rigor and would make the treatment of kala-azar with amphotericin B easier.
The American journal of tropical medicine and hygiene 11/2010; 83(5):1040-3. · 2.59 Impact Factor
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ABSTRACT: We tested the hypothesis that dengue haemorrhagic fever (DHF) is associated with a T(H)1-skewed immune response as opposed to dengue fever (DF).
We estimated intracellular (in T-cells) and serum levels of designate T(H)1/T(H)2 cytokines [interferon-γ (IFN-γ), interleukin-4 (IL-4), and tumor necrosis factor-α] and macrophage inflammatory protein-1α (MIP-1α) at admission, 48 h, and day 5 in 20 adults with dengue (DF=10, DHF=10) and 10 dengue-naïve healthy controls.
At admission, intracellular IFN-γ/IL-4 ratio in CD4+ T-cells and proportion of MIP-1α-positive CD8+ T-cells were significantly higher in patients with DHF [7.21 (5.36~10.81) vs. 3.04 (1.75~4.02); p=0.011 and 6.2% (3.2~8.2%) vs. 2.4% (2.0~3.6%); p=0.023]. The latter showed a significant positive correlation with IFN-γ/IL-4 ratio in CD4+ T-cells (Spearman's rho=0.64; p=0.003), percentage-change in haematocrit (rho=0.47; p=0.048), and serum alanine aminotransferase level (rho=0.61; p=0.009).
We conclude that DHF is associated with a T(H)1-skewed immune response. Further, MIP-1α in CD8+ T-cells is an important immunologic correlate of disease severity in dengue.
Immune Network 10/2010; 10(5):164-72.
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ABSTRACT: Delayed Type Hypersensitivity (DTH) and protective immunity are thought to be tightly linked. Remarkable similarity exists between their cellular and immune mechanisms. However, their dissociation is also well known. Here we investigate the immunological mechanisms relevant for their dissociation in a group of non-relapsing cured lepromatous leprosy (CLL) patients. In these patients, using lepromin reaction as a model system of DTH we report critical role of tissue chemokine response in synchronous manifestation of these linked phenomena. Results indicate elevation of the threshold of tissue chemokine induction thus dissociating DTH from protective immunity in lepromin -ive CLL patients. We also show that the DTH anergy in these subjects is not an absolute one but depends on the strength of the stimulus. Our data provide insights into the intricate relationship between DTH and immunity and highlight the persistent presence of effector immune mechanisms involving these two pathways in apparently unresponsive lepromatous leprosy patients.
Microbes and Infection 09/2009; 11(14-15):1122-30. · 3.10 Impact Factor
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ABSTRACT: The inadequacy of effector T-cell response in containment of tubercle bacilli is believed to result in the development of disseminated forms of tuberculosis (TB), such as miliary tuberculosis (MTB). Regulatory T cells (Treg) plausibly play a critical role in the immunopathogenesis of disseminated TB by suppression of effector immune response against Mycobacterium tuberculosis at the pathologic site(s). To understand the role of Treg cells in disseminated tuberculosis, we studied the frequency and function of Treg cells derived from the local disease site specimens (LDSS) of patients with TB pleural effusion and MTB as clinical models of contained and disseminated forms of disease, respectively.
To (1) enumerate the frequency of Treg cells in bronchoalveolar lavage (BAL) fluid of patients with MTB and compare with that of peripheral blood, (2) study the role of Treg cells in suppression of local T-cell response, and (3) study the selective recruitment of Treg cells at the local disease site(s).
Flow cytometry, reverse transcriptase polymerase chain reaction, and 3-(4,5-dimethylthythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based cell proliferation assay.
Frequency of Treg cells (CD4(+)CD25(+)FoxP3(+)) was significantly higher in LDSS in MTB along with higher levels of FoxP3 mRNA. Importantly, FoxP3(+) Treg cells obtained from the BAL of patients with MTB predominantly produced IL-10 and could suppress the autologous T-cell proliferation in response to M. tuberculosis antigen.
Our results highlight the importance of Treg cells in suppression of effector immune response and their influence on bacillary dissemination, disease manifestation, and severity.
American Journal of Respiratory and Critical Care Medicine 03/2009; 179(11):1061-70. · 11.08 Impact Factor
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ABSTRACT: Containment of Mycobacterium tuberculosis critically depends on orchestrated generation of Th1 cells and their selective recruitment at the pathologic sites. Understanding the mechanism involved in this process is important for defining better intervention strategies. We investigated the surface phenotype of Th1 cells and the role of chemotactic factors in their selective recruitment in tuberculosis pleural effusion and tuberculin site. Memory T cells obtained from the pleural fluid expressed a battery of homing receptors such as CD11a, CCR5 and CXCR3. Similar expression profile was noted on T cells infiltrating the tuberculin site. Expression of their respective ligands such as ICAM-1, RANTES, MIP1-alpha, Mig and IP-10 were detected at pathologic sites. In vitro assay of T cell adherence to activated human umbilical vein endothelial cells (HUVEC) expressing chemotactic ligands suggests an important role of these homing molecules in their selective trafficking. Here, we demonstrate a hierarchy of CXCR3 in effector cell adhesion to HUVEC in vitro, although CD11a and CCR5 were also observed to mediate cell adhesion in an additive fashion. Findings of the present study provide mechanistic insights into the critical events of T cell trafficking in tuberculosis and may help designing better therapeutic modalities.
European Journal of Immunology 09/2005; 35(8):2367-75. · 5.10 Impact Factor
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ABSTRACT: Delayed Type Hypersensitivity (DTH) and protective immunity are thought to be tightly linked. Remarkable similarity exists between their cellular and immune mechanisms. However, their dissociation is also well known. Here we investigate the immunological mechanisms relevant for their dissociation in a group of non-relapsing cured lepromatous leprosy (CLL) patients. In these patients, using lepromin reaction as a model system of DTH we report critical role of tissue chemokine response in synchronous manifestation of these linked phenomena. Results indicate elevation of the threshold of tissue chemokine induction thus dissociating DTH from protective immunity in lepromin -ive CLL patients. We also show that the DTH anergy in these subjects is not an absolute one but depends on the strength of the stimulus. Our data provide insights into the intricate relationship between DTH and immunity and highlight the persistent presence of effector immune mechanisms involving these two pathways in apparently unresponsive lepromatous leprosy patients.
Microbes and Infection.
