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S Keshav

Publications and source records attributed to S Keshav.

At least 19 recordsLinked to original sources
PubMed2006

Review article: maintenance therapy in patients with ulcerative colitis.

British Society of Gastroenterology guidelines recommend that all patients with ulcerative colitis should receive long-term therapy with a 5-aminosalicylic acid compound to maintain remission. Recent studies have shown that time spent in remission is longer when the maintenance dose is increased from 1.2 to 2.4 g/day, with patients with extensive disease benefiting most from an increase with dosage. A retrospective analysis also found that the frequency of relapse was lower in patients taking more than the median dose of 5-aminosalicylic acid (1.6 g/day) compared with those taking less than the median dose. Similarly, when 5-aminosalicylic acids are used to induce remission, continuing the induction dosage for an extra 4 weeks prolongs remission and reduces the frequency of relapse. However, patients rarely comply fully with the prescribed dose regimen, which can lead to effective under-dosing. The recent discovery that 5-aminosalicylic acids may act in ulcerative colitis by activating peroxisome proliferator-activated receptor-gamma, a nuclear receptor that plays a role in the control of cell proliferation and apoptosis, has given new impetus to the idea that long-term therapy with 5-aminosalicylic acid may reduce the risk of colorectal cancer. Epidemiological studies are beginning to provide evidence to support this view. Accumulating evidence suggests that the next revision of the clinical guidelines should suggest life-long doses of 5-aminosalicylic acid of > or =2 g/day for maintenance of remission in patients with ulcerative colitis.

Administration, Oral↗
PubMed2003

Expression of NOD2 in Paneth cells: a possible link to Crohn's ileitis.

BACKGROUND AND AIMS: Genetic variation in NOD2 has been associated with susceptibility to Crohn's disease (CD) and specifically with ileal involvement. The reason for the unique association of NOD2 mutations with ileal disease is unclear. To identify a possible link, we tested expression of NOD2 in intestinal tissue of CD patients and controls. PATIENTS AND METHODS: Fifty five specimens of ileum or colon from 21 CD patients, seven ulcerative colitis (UC) patients, and five controls with pathology other than CD or UC were stained for NOD2 using an immunoperoxidase method. RESULTS: Using a monoclonal antibody against NOD2 developed in our laboratory, we detected uniform expression of NOD2 in terminal ileum Paneth cells from controls and patients as well as in metaplastic Paneth cells in the colon. Mechanical purification showed enriched expression of NOD2 mRNA in ileal crypts. In Paneth cells, NOD2 was located in the cytosol in close proximity to the granules that contain antimicrobial peptides. We detected minimal NOD2 in the villous epithelium of the ileum or in the colonic epithelium from both CD patients and controls. CONCLUSIONS: These results suggest a role for NOD2 in the regulation of Paneth cell mediated responses against intestinal bacteria and a plausible mechanism to explain the selective association of NOD2 mutations with ileal disease. The impaired capacity of CD associated mutations to sense luminal bacteria may result in increased susceptibility to certain gut microbes.

Adolescent↗
PubMed1999

Correction of both prothrombin time and primary haemostasis by recombinant factor VII during therapeutic alcohol injection of hepatocellular cancer in liver cirrhosis.

We evaluated the efficacy of recombinant factor VII to correct impaired haemostasis in a patient with liver cirrhosis requiring an invasive procedure. A test intravenous bolus of 80 microg/kg of recombinant factor VII was given to a Jehovah's Witness, with a solitary 4.4-cm hepatocellular carcinoma and underlying hepatitis C virus cirrhosis, in an attempt to correct his haemostatic disorders and safely inject the tumour with alcohol. An extensive portal block had precluded consideration of liver transplantation. Haemostasis was evaluated by clotting assays, bleeding time and thromboelastography 10 min before and 10 min and 1, 2, 4, 8 and 24 h after factor VII infusion. Parameters of both coagulation (prothrombin time) and platelet function (bleeding time and the alpha and ma parameters of thrombelastography) were improved 10 min after factor VII infusion; improvements lasted 4 to 8 h or more. Platelet count did not change and there was no evidence of disseminated intravascular coagulation. The improvements in haemostatic parameters correlated significantly with the increases in factor VII plasma concentrations (p<0.04). Factor VII clearance was 25.1 U/h/kg and its half-life was 5.8 h. The same dose of recombinant factor VII was given to the patient 1 week later, just before the alcohol injections. The patient had no subsequent bleeding or other complication, with no change in haemoglobin levels over 24 h. Thus, recombinant factor VII represents a therapeutic advance, as it can correct fully both coagulation and platelet function defects in cirrhosis and allow invasive procedures to be performed safely.

Carcinoma, Hepatocellular↗
PubMed1997

The macrophage scavenger receptor type A is expressed by activated macrophages and protects the host against lethal endotoxic shock.

During gram-negative bacterial infections, lipopolysaccharide (LPS) stimulates primed macrophages (Mphi) to release inflammatory mediators such as tumor necrosis factor (TNF)-alpha, which can cause hypotension, organ failure, and often death. Several different receptors on Mphi have been shown to bind LPS, including the type A scavenger receptor (SR-A). This receptor is able to bind a broad range of polyanionic ligands such as modified lipoproteins and lipoteichoic acid of gram-positive bacteria, which suggests that SR-A plays a role in host defense. In this study, we used mice lacking the SR-A (SRKO) to investigate the role of SR-A in acquired immunity using a viable bacillus Calmette Guérin (BCG) infection model. We show that activated Mphi express SR-A and that this molecule is functional in assays of adhesion and endocytic uptake. After BCG infection, SRKO mice are able to recruit Mphi to sites of granuloma formation where they become activated and restrict BCG replication. However, infected mice lacking the SR-A are more susceptible to endotoxic shock and produce more TNF-alpha and interleukin-6 in response to LPS. In addition, we show that an antibody which blocks TNF-alpha activity reduces LPS-induced mortality in these mice. Thus SR-A, expressed by activated Mphi, plays a protective role in host defense by scavenging LPS as well as by reducing the release by activated Mphi of proinflammatory cytokines. Modulation of SR-A may provide a novel therapeutic approach to control endotoxic shock.

Animals↗
PubMed1994

BCG-induced granuloma formation in murine tissues.

BCG infection of mice provides a convenient model to study natural and cellular immunity to mycobacteria and the mechanisms of granuloma formation and repair. We have used a range of macrophage (M phi) membrane molecules and secretory products to investigate resident M phi-pathogen interactions and T lymphocyte-dependent recruitment and activation of M phi in different tissues of immature, normal adult and gamma interferon deficient animals. In situ hybridization (ISH), RT-PCR and immunocytochemical analysis of M phi gene and product expression have been correlated with in vitro study of endocytic and secretory activity in which biogel polyacrylamide bead-elicited peritoneal M phi are exposed to Th1 and Th2 cytokines, LPS, BCG and other stimuli. The role of resident and newly recruited M phi responding to BCG in liver, spleen, lung and brain has been defined by means of antigen markers expressed by M phi (F4/80, 7/4, CR3, macrosialin, sialoadhesin and scavenger receptor) and/or T and B lymphoid cells (MHC Class II, CD4, CD8, B220). Heterogeneity in M phi secretory activity was revealed by ISH analysis of lysozyme, TNF-alpha, IL-1 IL-6 and MCP-1, by in vitro assay of NO and superoxide anion production, and by RT-PCR studies of Th1 (interferon gamma) and Th2 (IL-4, IL-13, IL-10) lymphokine mRNA in tissues. Our studies confirm the importance of interferon gamma as a critical mediator of host resistance to mycobacterial infection and raise intriguing questions in regard to T cell and M phi functional heterogeneity in distinct tissue microenvironments.

Animals↗
PubMed1994

HIV-1 induces tumour necrosis factor and IL-1 gene expression in primary human macrophages independent of productive infection.

Cytokines such as tumour necrosis factor-alpha (TNF-alpha) and IL-1 beta may play a role in immunopathogenesis of AIDS. We studied early effects (0.5-48 h) of monocytotropic (ADA) or lymphotropic (IIIB) strains of HIV-1 on TNF-alpha and IL-1 beta mRNA expression in primary human macrophages by a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Three-day-old monocyte-derived macrophages were exposed either to tissue culture supernatants containing virus (at multiplicity of infection (m.o.i.) of 0.05) or to control supernatants free of virions and gp120. ADA strain, but not IIIB, replicated in primary tissue culture-differentiated macrophages (TCDM). Soluble CD4 (sCD4) was used to inhibit binding of both strains to macrophages. We found that TNF-alpha and IL-1 beta gene expression was induced by both strains 0.5-3 h after addition of virus, and that enhanced expression of both cytokines was inhibited by sCD4. We conclude that CD4-dependent binding to the cell surface is sufficient to enhance TNF-alpha and IL-1 beta mRNA, whereas productive viral replication in primary human macrophages is not required. Therefore, similar pathways regulate gene expression of TNF-alpha and IL-1 beta by macrophages during initial infection by HIV-1 in vitro.

Base Sequence↗
PubMed1993

Multiple defects of immune cell function in mice with disrupted interferon-gamma genes.

Interferon-gamma (IFN-gamma) is a pleiotrophic cytokine with immunomodulatory effects on a variety of immune cells. Mice with a targeted disruption of the IFN-gamma gene were generated. These mice developed normally and were healthy in the absence of pathogens. However, mice deficient in IFN-gamma had impaired production of macrophage antimicrobial products and reduced expression of macrophage major histocompatibility complex class II antigens. IFN-gamma-deficient mice were killed by a sublethal dose of the intracellular pathogen Mycobacterium bovis. Splenocytes exhibited uncontrolled proliferation in response to mitogen and alloantigen. After a mixed lymphocyte reaction, T cell cytolytic activity was enhanced against allogeneic target cells. Resting splenic natural killer cell activity was reduced in IFN-gamma-deficient mice. Thus, IFN-gamma is essential for the function of several cell types of the murine immune system.

Animals↗
PubMed1992

Precursor structure, expression, and tissue distribution of human guanylin.

Heat-stable enterotoxins (STa) are small, cysteine-rich peptides secreted by Escherichia coli that are able to induce diarrhea through the stimulation of an intestine-specific receptor-guanylyl cyclase known as STaR. A 15-amino acid peptide, guanylin, was recently purified from rat jejunum and proposed to be a potential endogenous activator of this receptor. We describe here the cloning and characterization of human and mouse cDNAs encoding precursor proteins of 115 and 116 amino acids, respectively, having guanylin present at their C termini. Expression of the human cDNA in mammalian cells leads to the secretion of proguanylin, an inactive 94-amino acid protein. Guanylin generated by either trypsin or acid treatment of proguanylin was purified and found to bind to, and activate, STaR. Northern blot and in situ hybridization show high-level expression of guanylin mRNA restricted to the intestine, with localization to Paneth cells at the base of the small intestinal crypts. These results demonstrate that guanylin is an endogenous activator of STaR.

Amino Acid Sequence↗
PubMed1992

Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation.

Expression of the macrophage mannose receptor is inhibited by interferon gamma (IFN-gamma), a T helper type 1 (Th-1)-derived lymphokine. Interleukin 4 (IL-4), a Th-2 lymphocyte product, upregulates major histocompatibility class II antigen expression but inhibits inflammatory cytokine production by macrophages. We have studied the effect of IL-4 on expression of the macrophage mannose receptor (MMR) by elicited peritoneal macrophages. We found that recombinant murine IL-4 enhances MMR surface expression (10-fold) and activity (15-fold), as measured by the respective binding and degradation of 125I-mannose-bovine serum albumin. Polymerase chain reaction analysis of cDNAs from purified primary macrophage populations revealed that MMR, but not lysozyme or tumor necrosis factor alpha, mRNA levels were markedly increased by IL-4. The above effects were associated with morphologic changes. These data establish IL-4 as a potent and selective enhancer of murine MMR activity in vitro. IL-4 induces inflammatory macrophages to adopt an alternative activation phenotype, distinct from that induced by IFN-gamma, characterized by a high capacity for endocytic clearance of mannosylated ligands, enhanced (albeit restricted) MHC class II antigen expression, and reduced proinflammatory cytokine secretion.

Animals↗
PubMed1992

Lysozyme gene expression in inflammatory bowel disease.

Riboprobe in situ hybridization (rISH) demonstrates active lysozyme synthesis in ulcerative colitis and Crohn's disease. Maximal labeling was seen in Paneth cells, macrophages, and granulomas. Diffuse infiltration of the mucosa by lysozyme-rich polymorphs characterizes ulcerative colitis but obscures reactivity in other cell lineages in immunohistochemical studies; lysozyme mRNA is not detected in polymorphs, rISH giving a clearer picture than immunohistochemical studies of the active synthesis of lysozyme within the gut in inflammatory bowel disease. In ulcerative colitis, strong signals localized to Paneth cell metaplasia were found in 11 of 20 cases and to a lesser degree in non-Paneth cell lineages in regenerative mucosa in 13 of 20 cases. In Crohn's disease, abundant labeling was seen in tuberculoid granulomas (5 of 20) and over macrophage aggregates in the lamina propria in another 7, characteristic patterns not encountered in ulcerative colitis. Low levels of lysozyme messenger RNA were found in the ulceration-associated cell lineage ("pseudopyloric metaplasia"). These results support the view that neutrophils are largely responsible for elevated fecal lysozyme levels in ulcerative colitis and macrophages for elevated serum lysozyme levels in Crohn's disease.

Colitis, Ulcerative↗
PubMed1992

Detection of mRNAs for macrophage products in inflammatory bowel disease by in situ hybridisation.

In situ hybridisation has been used to detect mRNAs to the macrophage secretory products, lysozyme, interleukin 1 beta and tumour necrosis factor-alpha. Sections of paraformaldehyde fixed, frozen colonoscopic biopsies from patients with ulcerative colitis, Crohn's disease or normal controls were hybridised with specific radiolabelled probes and the signal detected by autoradiography. Lysozyme mRNA expression was more common in ulcerative colitis (22/27) and Crohn's disease (eight of eight) compared with controls (17/27). Positive cells were found mainly in the subepithelial region in normal colon, while in inflammatory bowel disease they also appeared in the deeper lamina propria. Immunocytochemistry in parallel sections showed that lysozyme mRNA was expressed only in macrophages or in metaplastic Paneth cells in longstanding inflammatory bowel disease. Tissue neutrophils did not express the lysozyme mRNA, though they have large stores of the protein. Tumour necrosis factor mRNA was detected in four of nine controls compared with 11/15 inflammatory bowel disease patients. For interleukin 1 beta, three of eight controls were positive compared with 10/13 with ulcerative colitis. The tumour necrosis factor signal was located mainly in the deeper lamina propria whereas the interleukin 1 beta was seen in subepithelial macrophages. These results confirm increased macrophage activation in inflammatory bowel disease and suggest functional heterogeneity within the intestinal macrophage population.

Adult↗
PubMed1992

The product of the cellular crk gene consists primarily of SH2 and SH3 regions.

We have cloned and sequenced a complementary DNA encoding the cellular homologue of the transforming oncogene v-crk of avian sarcoma virus CT10. This complementary DNA contains an open reading frame of 915 base pairs that encodes a polypeptide of 305 amino acids. The first 205 amino acids of this c-Crk protein were identical to those of the CT10 encoded v-Crk protein, with the exception of 5 amino acids. Like v-Crk, this portion of c-Crk contained one each of the Src homology domains SH2, SH2', and SH3. The 100 carboxy-terminal amino acids of c-Crk protein, which are not coded for in the CT10 viral genome, contain another SH3 region. We found limited sequence homology between c-crk and the avian retrovirus genome, which explains recombination events in the transduction of this protooncogene. Using a polyclonal antiserum made against bacterially expressed v-crk, we identified a 35-kilodalton protein in normal chicken embryo fibroblasts and in all embryonic chicken tissues examined. This 35-kilodalton protein was indistinguishable from a polypeptide made by in vitro translation of c-crk complementary DNA.

Amino Acid Sequence↗
PubMed1991

Lysozyme is an inducible marker of macrophage activation in murine tissues as demonstrated by in situ hybridization.

This study demonstrates the induction of lysozyme mRNA expression in situ in tissue macrophages (M phi) of mice following in vivo stimulation. The resting resident tissue M phi of most tissues do not contain enough lysozyme mRNA to be detected by in situ hybridization using 35S-labeled RNA probes. Following Bacille Calmette Guerin or Plasmodium yoelli infection, however, M phi recruited to liver and spleen hybridize strongly to the lysozyme probe. Within 24 h of infection, cells found in the marginal zone of the spleen begin to produce lysozyme mRNA. This response is also evoked by a noninfectious agent (intravenously injected sheep erythrocytes), and is possibly the result of an early phagocytic interaction. Later in the infection, other cells in the red and white pulp of the spleen, and cells in granulomas in the liver, become lysozyme-positive. Kupffer cells are rarely lysozyme-positive. Lysozyme mRNA levels in liver granulomas remain relatively constant during the infection, and lysozyme is produced by most granuloma cells. This contrasts with tumor necrosis factor alpha (TNF alpha) mRNA, which is produced by fewer cells in the granuloma, and which can be massively induced by lipopolysaccharide administration. The production of lysozyme, previously considered a constitutive function of M phi, is therefore an indicator of M phi activation in vivo, where immunologically specific and nonspecific stimuli both stimulate lysozyme production at high levels in subpopulations of cells occupying discrete anatomical locations.

Animals↗
PubMed1990

Tumor necrosis factor mRNA localized to Paneth cells of normal murine intestinal epithelium by in situ hybridization.

Paneth cells in normal murine small intestine contain TNF mRNA that is readily detectable by in situ hybridization, unlike resident macrophages in lamina propria, which are negative. Northern blot analysis of whole tissue shows the presence of mRNA that has the same electrophoretic mobility as TNF mRNA from activated macrophages. A low level of TNF bioactivity, but no immunoreactivity, was detected in normal small intestine, and TNF production in resting Paneth cells appears to be post-transcriptionally controlled. Typical leukocyte surface membrane markers were not found on Paneth cells, but were expressed by the surrounding lamina propria macrophages. Paneth cells are thus epithelial cells with leukocyte-like secretory potential that may be important in intestinal physiology and pathology.

Animals↗
PubMed1988

Cloning the human lysozyme cDNA: inverted Alu repeat in the mRNA and in situ hybridization for macrophages and Paneth cells.

Lysozyme is a major secretory product of human and rodent macrophages and a useful marker for myelomonocytic cells. Based on the known human lysozyme amino acid sequence, oligonucleotides were synthesized and used as probes to screen a phorbol 12-myristate 13-acetate-treated U937 cDNA library. A full-length human lysozyme cDNA clone, pHL-2, was obtained and characterized. Sequence analysis shows that human lysozyme, like chicken lysozyme, has an 18-amino-acid-long signal peptide, but unlike the chicken lysozyme cDNA, the human lysozyme cDNA has a greater than 1-kilobase-long 3' nontranslated sequence. Interestingly, within this 3' region, an inverted repeat of the Alu family of repetitive sequences was discovered. In RNA blot analyses, DNA probes prepared from pHL-2 can be used to detect lysozyme mRNA not only from human but also from mouse and rat. Moreover, by in situ hybridization, complementary RNA transcripts have been used as probes to detect lysozyme mRNA in mouse macrophages and Paneth cells. This human lysozyme cDNA clone is therefore likely to be a useful molecular probe for studying macrophage distribution and gene expression.

Amino Acid Sequence↗