Biomedical subjects

Shailesh Singh

Publications and source records attributed to Shailesh Singh.

13 recordsLinked to original sources

PubMed2006

Inhibition of cyclooxygenase-2 decreases breast cancer cell motility, invasion and matrix metalloproteinase expression.

BACKGROUND: Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the formation of prostaglandins. The inducible isoform of COX (COX-2) is highly expressed in aggressive metastatic breast cancers and may play a critical role in cancer progression (i.e. growth and metastasis). However, the exact mechanism(s) for COX-2-enhanced metastasis has yet to be clearly defined. It is well established that one of the direct results of COX-2 action is increased prostaglandin production, especially prostaglandin E2 (PGE2). Here, we correlate the inhibition of COX-2 activity with decreased breast cancer cell proliferation, migration, invasion and matrix metalloproteinase (MMP) expression. METHODS: Breast cancer cells (Hs578T, MDA-MB-231 and MCF-7) were treated with selective COX-2 inhibitors (NS-398 and Niflumic acid, NA). Cell proliferation was measured by staining with erythrosin B and counting the viable cells using a hemacytometer. Cell migration and invasion were measured using migration and invasion chamber systems. MMP expression was determined by enzyme immunoassay (secreted protein) and real-time quantitative polymerase chain reaction (mRNA). RESULTS: Our results show that there is a decline in proliferation, migration and invasion by the Hs578T and MDA-MB-231 breast cancer cell lines in the presence of either low concentrations (1 microM or lower) NA or NS-398. We also report that MMP mRNA and protein expression by Hs578T cells is inhibited by NS-398; there was a 50% decrease by 100 muM NS-398. PGE2 completely reversed the inhibitory effect of NS-398 on MMP mRNA expression. CONCLUSION: Our data suggests that COX-2-dependent activity is a necessary component for cellular and molecular mechanisms of breast cancer cell motility and invasion. COX-2 activity also modulates the expression of MMPs, which may be a part of the molecular mechanism by which COX-2 promotes cell invasion and migration. The studies suggest that COX-2 assists in determining and defining the metastatic signaling pathways that promote the breast cancer progression to metastasis.

Breast Neoplasms↗

PubMed2006

CCL5 modulates pneumococcal immunity and carriage.

Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Recently, it has been shown that genetic polymorphisms can result in diminished expression of CCL5, which results in increased susceptibility to and progression of infectious diseases. We show that CCL5, together with Th cytokine mRNA expression, is temporally up-regulated during pneumococcal carriage. To determine the contribution of CCL5 to pneumococcal surface antigen A-specific humoral and cellular pneumococcal immunity, mice were treated with anti-CCL5 or control Abs before and during Streptococcus pneumoniae strain EF3030-challenge for the initiation of carriage. CCL5 blockade resulted in a decrease of CD4(+) and CD8(+) T cells as well as CD11b(+) cells in the spleen, cervical lymph node, lung, and nasopharyngeal associated lymphoid tissue during the recognition phase of the pneumococcal adaptive immune response. CCL5 blockade significantly reduced the Ag-specific IgG2a and IgG1 Abs in serum and IgA Ab levels in nasal washes. These decreases also corresponded to reductions in Ag-specific T cell (mucosal and systemic) responses. CCL5 inhibition resulted in decreasing the quantity of IL-4- and IFN-gamma-secreting CD4(+) T cells and increasing the number of Ag-specific IL-10-producing CD4(+) T cells; these changes combined also corresponded with the transition from pneumococcal carriage to lethal pneumonia. These data suggest that CCL5 is an essential factor for the induction and maintenance of protective pneumococcal immunity.

Animals↗

PubMed2005

Plasmodium yoelii 17XL infection up-regulates RANTES, CCR1, CCR3 and CCR5 expression, and induces ultrastructural changes in the cerebellum.

BACKGROUND: Malaria afflicts 300-500 million people causing over 1 million deaths globally per year. The immunopathogenesis of malaria is mediated partly by co mplex cellular and immunomodulator interactions involving co-regulators such as cytokines and adhesion molecules. However, the role of chemokines and their receptors in malaria immunopathology remains unclear. RANTES (Regulated on Activation Normal T-Cell Expressed and Secreted) is a chemokine involved in the generation of inflammatory infiltrates. Recent studies indicate that the degradation of cell-cell junctions, blood-brain barrier dysfunction, recruitment of leukocytes and Plasmodium-infected erythrocytes into and occlusion of microvessels relevant to malaria pathogenesis are associated with RANTES expression. Additionally, activated lymphocytes, platelets and endothelial cells release large quantities of RANTES, thus suggesting a unique role for RANTES in the generation and maintenance of the malaria-induced inflammatory response. The hypothesis of this study is that RANTES and its corresponding receptors (CCR1, CCR3 and CCR5) modulate malaria immunopathogenesis. A murine malaria model was utilized to evaluate the role of this chemokine and its receptors in malaria. METHODS: The alterations in immunomodulator gene expression in brains of Plasmodium yoelii 17XL-infected mice was analysed using cDNA microarray screening, followed by a temporal comparison of mRNA and protein expression of RANTES and its corresponding receptors by qRT-PCR and Western blot analysis, respectively. Plasma RANTES levels was determined by ELISA and ultrastructural studies of brain sections from infected and uninfected mice was conducted. RESULTS: RANTES (p < 0.002), CCR1 (p < 0.036), CCR3 (p < 0.033), and CCR5 (p < 0.026) mRNA were significantly upregulated at peak parasitaemia and remained high thereafter in the experimental mouse model. RANTES protein in the brain of infected mice was upregulated (p < 0.034) compared with controls. RANTES plasma levels were significantly upregulated; two to three fold in infected mice compared with controls (p < 0.026). Some distal microvascular endothelium in infected cerebellum appeared degraded, but remained intact in controls. CONCLUSION: The upregulation of RANTES, CCR1, CCR3, and CCR5 mRNA, and RANTES protein mediate inflammation and cellular degradation in the cerebellum during P. yoelii 17XL malaria.

Animals↗

PubMed2005

Molecular basis for the potency of IL-10-deficient dendritic cells as a highly efficient APC system for activating Th1 response.

Identification and targeting of novel immunobiological factors that regulate the induction of Th1 cells are crucial for designing effective vaccines against certain intracellular pathogens, including Chlamydia. IL-10-deficient dendritic cells (DC) are potent APCs and effective cellular vaccines that activate a high frequency of specific Th1 cells. To elucidate the molecular basis for the potency of the IL-10-deficient APC system, we tested the hypothesis that Chlamydia Ag-primed IL-10 knockout (IL-10KO) DC are quantitatively and qualitatively distinct in their metabolic characteristics relating to T cell activation. Using a combination of RT-PCR, two-dimensional gel electrophoresis, and MALDI-TOF-based proteomics analyses, the transcriptional and translational activities of Chlamydia-pulsed DC from wild-type and IL-10KO mice were assessed. IL-10 deficiency caused early maturation and activation of pulsed DC (i.e., high CD11c, CD40, CD80, CD83, CD86, IL-1, IL-12, and the T cell-attracting chemokine CCL27/CTACK) and consequently an enhanced ability to process and present Ags for a rapid and robust T cell activation. Supporting comparative proteomics revealed further that IL-10 deficient DC possess specific immunobiological properties, e.g., the T cell-attracting chemokine CCL27/CTACK, calcium-dependent protein kinase, and the IL-1/IL-12 inducer, NKR-P1A (CD161), which differentiated them immunologically from wild-type DC that express molecules relating to anti-inflammatory, differentiative, and metabolic processes, e.g., the anti-IL-12 molecule peroxisome proliferator-activated receptor-alpha and thymidine kinase. Collectively, these results provide a molecular basis for the high Th1-activating capacity of IL-10KO APC and may provide unique immunomodulation targets when designing vaccines against pathogens controlled by T cell immunity.

Animals↗

PubMed2005

Differential PsaA-, PspA-, PspC-, and PdB-specific immune responses in a mouse model of pneumococcal carriage.

Larger numbers of pneumococci were detected in the nasal tract compared to the lung, cervical lymph nodes, and spleen 1, 2, 4, 7, 14, and 21 days after nasal challenge with Streptococcus pneumoniae strain EF3030. In this mouse model of pneumococcal carriage, peripheral S. pneumoniae pneumococcal surface adhesin A (PsaA)-specific humoral responses (immunoglobulin G2a [IgG2a] >> IgG1 = IgG2b > IgG3) were significantly higher than pneumococcal surface protein A (PspA)-specific, genetic toxoid derivative of pneumolysin (PdB)-specific, or pneumococcal surface protein C (PspC)-specific serum antibody levels. However, PspA-specific mucosal IgA antibody levels were significantly higher than those against PsaA, PdB, and PspC. In general, both PsaA- and PspA-specific lung-, cervical lymph node-, nasal tract-, and spleen-derived CD4(+) T-cell cytokine (interleukin-4, interleukin-6, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha) and proliferative responses were higher than those for either PspC or PdB. Taken together, these findings suggest that PsaA- and PspA-specific mucosal responses as well as systemic humoral and T helper cell cytokine responses are predominantly yet differentially induced during pneumococcal carriage.

Adhesins, Bacterial↗

PubMed2004

Expression and functional role of CCR9 in prostate cancer cell migration and invasion.

PURPOSE: Metastasis is responsible for most cancer-related deaths; hence, therapies designed to minimize metastasis are greatly needed. The precise cellular and molecular mechanisms used by cancer cells for metastasis are not fully understood; however, the metastatic spread of neoplastic cells is probably related to the ability of these cells to migrate, invade, home, and survive locally. The migration of tumor cells shares many similarities with leukocyte trafficking, which is regulated by chemokine receptor-ligand interactions. The current study evaluates the molecular mechanisms of CCL25 and CCR9 in prostate cancer cell migration and invasion. EXPERIMENTAL DESIGN: In the current study, real-time quantitative polymerase chain reaction, flow cytometry analysis, and in vitro migration as well as invasion chamber analysis (with and without antibody-mediated inhibition) were used to ascertain the biological and functional significance of CCR9 expression by normal prostatic epithelial cells (PrEC) or prostate cancer cell lines (LNCaP-10995 and PC3). RESULTS: We report that functional CCR9 is highly expressed by LNCaP cells and modestly, yet significantly, expressed by PC3 cells when compared with PrEC cells. Neutralization of CCL25-CCR9 interactions impaired the migration and invasion potential of the LNCaP and PC3 cell lines. CCL25 differentially modulated the expression of collagenase-1 or matrix metalloproteinase (MMP)-1, collagenase-3 (MMP-13), stromalysin-2 (MMP-10), stromalysin-3 (MMP-11), and gelatinase-A (MMP-2), but not MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, or MMP-14 in prostate cancer cells. CONCLUSIONS: These studies suggest that the expression and activation of CCR9 affect cancer cell migration, invasion, and MMP expression, which together may affect prostate cancer metastasis.

Adenocarcinoma↗

PubMed2004

Viral macrophage-inflammatory protein-II: a viral chemokine that differentially affects adaptive mucosal immunity compared with its mammalian counterparts.

Chemokines play a profound role in leukocyte trafficking and the development of adaptive immune responses. Perhaps due to their importance in host defense, viruses have adopted many of the hallmarks displayed by chemokines. In particular, viral MIP-II (vMIP-II) is a human chemokine homologue that is encoded by human herpes virus 8. vMIP-II is angiogenic, selectively chemotactic for Th2 lymphocytes, and a homologue of human I-309 and mouse TCA-3, which also differentially attracts Th2 cells. To better understand the effect of viral chemokines on mucosal immunity, we compared the affects of vMIP-II, I-309, and TCA-3 on cellular and humoral immune responses after nasal immunization with OVA. These CCR8 ligands significantly enhanced Ag-specific serum and mucosal Abs through increasing Th2 cytokine secretion by CD4+ T cells. These alterations in adaptive humoral and cellular responses were preceded (12 h after immunization) by an increase in CD4+ T and B cells in nasal tracts with decreases of these leukocyte populations in the lung. Interestingly, vMIP-II increased neutrophil infiltration in the lung and Ag-specific IL-10-secreting CD4+ T cells after immunization. Although I-309 increased the number of CD28-, CD40L-, and CD30-positive, Ag-stimulated naive CD4+ T cells, vMIP-II and TCA-3 decreased the number of CD28-, CD40L-, and CD30-positive, resting naive CD4+ T cells. Taken together, these studies suggest that CCR8 ligands direct host Th2 responses, and vMIP-II up-regulates IL-10 responses and limits costimulatory molecule expression to mitigate host immunity.

Adjuvants, Immunologic↗

PubMed2004

Granulocyte chemotactic protein-2 mediates adaptive immunity in part through IL-8Rbeta interactions.

Chemokines constitute a large family of structurally related proteins that play a role in leukocyte migration and differentiation. Indeed, the early expression of human CXC chemokine receptor 1 (hCXCR1) and hCXCR2 [homologous to mouse interleukin (IL)-8Rbeta] ligands by the epithelium is a hallmark of the mucosal host defense. Mice lack IL-8; however, granulocyte chemotactic protein-2 (GCP-2)/lipopolysaccharide-induced CXC chemokine, a murine homologue of human GCP-2, has 32% and 61% sequence identity to human IL-8 and GCP-2, respectively, and binds hCXCR1, hCXCR2, and mouse IL-8Rbeta. To better understand the role of GCP-2 in adaptive immunity and as a nasal adjuvant, we characterized the exogenous effects of this CXC chemokine on cellular and humoral mucosal immune responses. GCP-2 significantly enhanced serum immunoglobulin G (IgG) and mucosal IgA antibodies through increased cytokine secretion by CD4+ T cells. These alterations in humoral and cellular responses were preceded by an increase in the number of B cells in the nasal tract, a decrease in the number of CD4+ T cells in the nasal tract as well as cervical lymph nodes, and an increase in the number of neutrophils in the nasal tract 12 h after GCP-2 immunization. This chemokine also modulated CD28 expression by CD4+ T cells during CD3epsilon stimulation of wild-type mice. GCP-2 increased CD80 and CD86 expression on B cells during in vitro stimulation in a dose-dependent manner. In contrast, cytokine and costimulatory molecule enhancement by GCP-2 was not induced by lymphocytes from IL-8Rbeta-/- mice, suggesting that GCP-2 modulates cellular immunity in part through IL-8Rbeta interactions.

Adaptation, Physiological↗

PubMed2004

CXCL12-CXCR4 interactions modulate prostate cancer cell migration, metalloproteinase expression and invasion.

The mechanisms responsible for prostate cancer metastasis are incompletely understood at both the cellular and molecular levels. In this regard, chemokines are a family of small, cytokine-like proteins that induce motility of neoplastic cells, leukocytes and cancer cells. The current study evaluates the molecular mechanisms of CXCL12 and CXCR4 in prostate cancer cell migration and invasion. We report that functional CXCR4 is significantly expressed by prostate cancer cell lines, LNCaP and PC3, when compared with normal prostatic epithelial cells (PrEC). As measured using motility and invasion chamber assays, prostate cancer cells migrated and invaded through extracellular matrix components in response to CXCL12, at rates that corresponded to CXCR4 expression. Anti-CXCR4 antibodies (Abs) significantly impaired the migration and invasive potential of PC3 and LNCaP cells. CXCL12 induction also enhanced collagenase-1 (metalloproteinase-1 (MMP-1)) expression by LNCaP and PC3 cells. Collagenase-3 (MMP-13) was expressed by prostate cancer cells, but it was not expressed by PrEC cells or modulated by CXCL12. CXCL12 increased MMP-2 expression by LNCaP and PC3; however, MMP-9 expression was elevated only in PC3 cells after CXCL12-CXCR4 ligation. PC3 cells also expressed high levels of stromelysin-1 (MMP-3) after CXCL12 stimulation. CXCL12 also significantly increased stromelysin-2 (MMP-10) expression by LNCaP cells. Stromelysin-3 (MMP-11) was expressed by LNCaP cells, but not by PC3 or PrEC cells and CXCL12 induced PC3 MMP-11 expression. Membrane type-1 MMP (MMP-14) was not expressed by PrEC or LNCaP cells, but CXCL12 significantly enhanced MMP-14 expression by PC3 cells. These studies reveal important cellular and molecular mechanisms of CXCR4/CXCL12-mediated prostate cancer cell migration and invasion.

Cell Line, Tumor↗

PubMed2003

Inhibition of IFN-gamma-inducible protein-10 abrogates colitis in IL-10-/- mice.

A deficiency in understanding the steps responsible for colitis is the lack of comprehension for the role chemokines play in mucosal inflammation. IFN-gamma-inducible protein-10 (IP-10) and CXCR3 are highly expressed at sites of colitis. Our findings show that IP-10 significantly contributes to the development of Th1 and inflammatory responses. Specifically, IP-10 inhibition in IL-10(-/-) mice attenuates the associated increases in serum and/or local amyloid A, IL-2, IL-6, TNF-alpha, IFN-gamma, IL-1alpha, and IL-1beta with colitis as compared with IL-10(-/-) mice that develop colitis similar to human Crohn's disease. Correspondingly, the rate or intensity of inflammation in IL-10(-/-) mice treated with anti-IP-10 Abs showed improved scoring of inflammation, compared with control IL-10(-/-) mice. This study provides important and novel information regarding IP-10 as a target for the treatment of colitis.

Animals↗

PubMed2003

Erythrocyte membrane fatty acid profile and saturation index in gallbladder carcinogenesis: a case-control study.

BACKGROUND: Gallbladder cancer is a common neoplasm of biliary tract, with an unknown etiology. PATIENTS AND METHODS: This study was carried out to evaluate the changes in the membrane fatty acid profile and saturation index in patients with gallbladder cancer. The study sample consisted of 50 newly diagnosed cases of gallbladder cancer and 50 patients undergoing cholecystectomy for cholelithiasis were recruited as controls. The fatty acid estimation was carried out by high performance liquid chromatography (HPLC). Statistical analysis was carried out by student 't' test and one-way ANOVA. Pearson's correlation coefficient was also obtained. RESULTS: A significant lowering of erythrocyte membrane stearic acid (p = 0.000), arachidonic acid (p = 0.001), and saturation index (p = 0.001) was observed in patients with gallbladder cancer compared to controls. A significant inverse relation was observed between stearic and oleic acid (r = -0.269 p = 0.007). CONCLUSION: Results of the present study suggest an increase in the delta 9 desaturation in cancer patients compared to controls however, a possible role of biliary stasis due to altered gallbladder motility or derangements of signal transduction secondary to altered membrane lipid bilayer cannot be ruled out.

Journal Article↗

PubMed2003

IFN-gamma-inducible chemokines enhance adaptive immunity and colitis.

T helper type 1 (Th1) cells secreting interferon-gamma (IFN-gamma) have been closely associated with Crohn's disease (CD). Monokine-induced by IFN-gamma (MIG), IFN-gamma-inducible T cell alpha chemoattractant (I-TAC), and IFN-gamma-inducible protein-10 (IP-10), are chemokines that bind CXCR3 and mediate the chemotaxis of leukocytes. IP-10, MIG, and CXCR3 have been shown to be expressed at sites of CD. The current study stems from our recent findings that IP-10, MIG, and I-TAC significantly contribute to the development of Th1-mediated inflammatory responses. To better understand the role of CXCR3 interactions during CD, we characterized the effects of IP-10, MIG, I-TAC, and CXCR3+ T cells on mucosal immune responses. IP-10, MIG, and I-TAC significantly enhanced antigen-specific serum and mucosal antibodies through Th1-mediated events and CD28 modulation. Additionally, the adoptive transfer of naive CXCR3+ T cells and CD4+CD45RB(HI) to T cell receptor beta (TCRbeta) x delta(-/-) mice resulted in the onset of murine colitis. Taken together, these studies suggest that IP-10, MIG, I-TAC, and CXCR3 interactions are involved in mucosal immune responses required for the induction of CD.

Adjuvants, Immunologic↗

PubMed2002

MIP-1alpha and MIP-1beta differentially mediate mucosal and systemic adaptive immunity.

Macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta are distinct but highly homologous CC chemokines produced by a variety of host cells in response to various external stimuli and share affinity for CCR5. To better elucidate the role of these CC chemokines in adaptive immunity, we have characterized the affects of MIP-1alpha and MIP-1beta on cellular and humoral immune responses. MIP-1alpha stimulated strong antigen (Ag)-specific serum immunoglobulin G (IgG) and IgM responses, while MIP-1beta promoted lower IgG and IgM but higher serum IgA and IgE antibody (Ab) responses. MIP-1alpha elevated Ag-specific IgG1 and IgG2b followed by IgG2a and IgG3 subclass responses, while MIP-1beta only stimulated IgG1 and IgG2b subclasses. Correspondingly, MIP-1beta produced higher titers of Ag-specific mucosal secretory IgA Ab levels when compared with MIP-1alpha. Splenic T cells from MIP-1alpha- or MIP-1beta-treated mice displayed higher Ag-specific Th1 (interferon-gamma [IFN-gamma]) as well as selective Th2 (interleukin-5 [IL-5] and IL-6) cytokine responses than did T cells from control groups. Interestingly, mucosally derived T cells from MIP-1beta-treated mice displayed higher levels of IL-4 and IL-6 compared with MIP-1alpha-treated mice. However, MIP-1alpha effectively enhanced Ag-specific cell-mediated immune responses. In correlation with their selective effects on humoral and cellular immune responses, these chemokines also differentially attract CD4(+) versus CD8(+) T cells and modulate CD40, CD80, and CD86 expressed by B220(+) cells as well as CD28, 4-1BB, and gp39 expression by CD4(+) and CD8(+) T cells in a dose-dependent fashion. Taken together, these studies suggest that these CC chemokines differentially enhance mucosal and serum humoral as well as cellular immune responses.

Administration, Intranasal↗