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Shanthi V Sitaraman

Publications and source records attributed to Shanthi V Sitaraman.

34 records · Page 2Linked to original sources
PubMed2004

Elevated flagellin-specific immunoglobulins in Crohn's disease.

Crohn's disease (CD) is driven by seemingly aberrant immune responses directed toward commensal enteric microflora. However, the specific antigens targeted by this immune response remain largely undefined. Herein, we demonstrate that common enteric flagellins are one such target of the CD-associated immune response. Thus flagellin may not only drive acute inflammation via activation of Toll-like receptor 5-mediated gene expression but may also serve as a target of the adaptive immune response that maintains the chronic inflammation characteristic of CD.

Antibody Specificity↗
PubMed2004

ADAM-15 inhibits wound healing in human intestinal epithelial cell monolayers.

The disintegrin metalloproteases (or ADAMs) are membrane-anchored glycoproteins that have been implicated in cell-cell or cell-matrix interactions and in proteolysis of molecules on the cell surface. The expression and/or the pathophysiological implications of ADAMs are not known in intestinal epithelial cells. Therefore, our aim was to investigate the expression and the role of ADAMs in intestinal epithelial cells. Expression of ADAMs was assessed by RT-PCR, Western blot analysis, and immunufluorescence experiments. Wound-healing experiments were performed by using the electric cell substrate impedence sensing technology. Our results showed that ADAMs-10, -12, and -15 mRNA are expressed in the colonic human cell lines Caco2-BBE and HT29-Cl.19A. An ADAM-15 complementary DNA cloned from Caco2-BBE poly(A)+ RNA, and encompassing the entire coding region, was found to be shorter and to present a different region encoding the cytoplasmic tail compared with ADAM-15 sequence deposited in the database. In Caco2-BBE cells and colonic epithelial cells, ADAM-15 protein was found in the apical, basolateral, and intracellular compartments. We also showed that the overexpression of ADAM-15 reduced cell migration in a wound-healing assay in Caco2-BBE monolayers. Our data show that 1) ADAM-15 is expressed in human intestinal epithelia, 2) a new variant of ADAM-15 is expressed in a human intestinal epithelial cell line, and 3) ADAM-15 is involved in intestinal epithelial cells wound-healing processes. Together, these results suggest that ADAM-15 may have important pathophysiological roles in intestinal cells.

ADAM Proteins↗
PubMed2004

Polarized fibronectin secretion induced by adenosine regulates bacterial-epithelial interaction in human intestinal epithelial cells.

Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface. However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated. In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN. Polarized monolayers of T84 cells were used as an intestinal epithelial model. Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine. Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine. The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide. Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated. Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells. In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine. FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role in mucosal immune responses during inflammation.

Adenosine↗
PubMed2004

Leptin as a novel profibrogenic cytokine in hepatic stellate cells: mitogenesis and inhibition of apoptosis mediated by extracellular regulated kinase (Erk) and Akt phosphorylation.

A key feature in the molecular pathogenesis of liver fibrosis requires maintenance of the activated hepatic stellate cell (HSC) phenotype by both proliferation and inhibition of apoptosis. We provide evidence that leptin is a potent HSC mitogen and dramatically inhibits stellate cell apoptosis. Leptin proved to be as potent an HSC mitogen as platelet-derived growth factor (PDGF) as assessed by bromodeoxyuridine (BrdU) incorporation in isolated primary HSCs; data using fluorescent propidium iodide (PI) uptake revealed that leptin, like PDGF, increased HSC populations in the S- and G2/M-phases of the cell cycle. Leptin resulted in a robust increase in cyclin D1 expression. Using the chemical inhibitor of Janus kinase 2 (Jak2) activity, AG 490, and overexpression of the suppressor of cytokine signaling 3 (SOCS-3), we show that blockade of leptin receptor (Ob-Rb) phosphorylation blocks leptin-induced HSC proliferation. Leptin-associated phosphorylation of both extracellular regulated kinase (p44/p42, Erk) and Akt is also prohibited. Further, the PI-3 kinase inhibitor LY294002 and MAPK inhibitor PD98059 were found to significantly reduce leptin-induced HSC proliferation, thereby indicating that leptin induced HSC proliferation is Akt- and Erk-dependent. Akt was also protective against HSC apoptosis. Leptin abolished both cycloheximide-induced and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, demonstrated by reduced caspase-3 activity, HSC-TUNEL staining, and DNA fragmentation. We conclude that leptin acts as a direct hepatic stellate cell survival agonist. Importantly, we have demonstrated that leptin-induced HSC proliferation and survival by Ob-Rb phosphorylation are both Erk- and Akt-dependent.

Apoptosis↗
PubMed2004

Agonist-induced polarized trafficking and surface expression of the adenosine 2b receptor in intestinal epithelial cells: role of SNARE proteins.

Adenosine, acting through the A2b receptor, induces vectorial chloride and IL-6 secretion in intestinal epithelia and may play an important role in intestinal inflammation. We have previously shown that apical or basolateral adenosine receptor stimulation results in the recruitment of the A2b receptor to the plasma membrane. In this study, we examined domain specificity of recruitment and the role of soluble N-ethylmaleimide (NEM) attachment receptor (SNARE) proteins in the agonist-mediated recruitment of the A2b receptor to the membrane. The colonic epithelial cell line T84 was used because it only expresses the A2b-subtype adenosine receptor. Cell fractionation, biotinylation, and electron microscopic studies showed that the A2b receptor is intracellular at rest and that apical or basolateral adenosine stimulation resulted in the recruitment of the receptor to the apical membrane. Upon agonist stimulation, the A2b receptor is enriched in the vesicle fraction containing vesicle-associated membrane protein (VAMP)-2. Furthermore, in cells stimulated with apical or basolateral adenosine, we demonstrate a complex consisting of VAMP-2, soluble NEM-sensitive factor attachment protein (SNAP)-23, and A2b receptor that is coimmunoprecipitated in cells stimulated with adenosine within 5 min and is no longer detected within 15 min. Inhibition of trafficking with NEM or nocodazole inhibits cAMP synthesis induced by apical or basolateral adenosine by 98 and 90%, respectively. cAMP synthesis induced by foskolin was not affected, suggesting that generalized signaling is not affected under these conditions. Collectively, our data suggest that 1) the A2b receptor is intracellular at rest; 2) apical or basolateral agonist stimulation induces recruitment of the A2b receptor to the apical membrane; 3) the SNARE proteins, VAMP-2 and SNAP-23, participate in the recruitment of the A2b receptor; and 4) the SNARE-mediated recruitment of the A2b receptor may be required for its signaling.

Adenosine↗
PubMed2004

STAT signaling underlies difference between flagellin-induced and tumor necrosis factor-alpha-induced epithelial gene expression.

Both bacterial flagellin and the cytokine tumor necrosis factor-alpha (TNFalpha) are potent activators of intestinal epithelial cell pro-inflammatory gene expression in general; nonetheless, there seem to be distinct differences in the specific patterns of gene expression induced by these agonists. The goal of this study was to define one such difference and elucidate the signaling mechanism responsible for such differential gene induction by these agonists. We observed that expression of inducible nitric-oxide synthase is substantially induced by flagellin but only minimally expressed in response to TNFalpha. This difference seemed to be underlain by differential induction of signal transducers and activators of transcription (STAT) activation in that, whereas flagellin and TNFalpha seemed to be equipotent activators of p38 mitogen-activated protein kinase and nuclear factor-kappaB, flagellin induced substantially higher levels of STAT-1 and -3 tyrosine phosphorylation. Such flagellin-induced STAT activation exhibited delayed kinetics and was ablated by treatment with cycloheximide. Flagellin-induced activation of STAT-3 was abolished via neutralizing antibodies to interleukin (IL)-6, but not interferon (IFN)beta nor IFNgamma; none of these neutralizing antibodies had any effect on flagellin-induced STAT-1 tyrosine phosphorylation. Flagellin induced substantially more IL-6 expression than did TNFalpha, but neither agonist elicited detectable levels of IFN expression. Flagellin-induced expression of inducible nitric-oxide synthase but not IL-6, was abolished by blocking STAT activation with AG490, and was reduced by blocking STAT-3 activation with anti-IL-6. Together, these results indicate that epithelial cell induction of flagellin-specific gene expression is mediated, in part, by STAT activation that results from autocrine activation via IL-6.

Autocrine Communication↗
PubMed2003

TGF-beta down-regulates IL-6 signaling in intestinal epithelial cells: critical role of SMAD-2.

Interleukin-6 (IL-6) is a pro-inflammatory cytokine that plays an important role in the pathogenesis of inflammatory bowel disease. TGF-beta, a multifunctional cytokine, is a potent negative regulator of mucosal inflammation in the intestine. The aim of the present study is to determine possible cross-talk between IL-6 and TGF-beta signaling pathways. Model intestinal epithelial cell lines, Caco2-BBE were used. We show that TGF-beta receptor Type II is predominantly present in the basolateral membrane of intestinal epithelial cells. TGF-beta1 induces a time-dependent phosphorylation of Smad2 and co-immunoprecipitation of SMAD-2 with Smad-4 and its subsequently translocation to the nucleus. We show that pretreatment of cells with TGF-beta1 is associated with a down-regulation of IL-6 induced tyrosine phosphorylation of STAT1 and STAT3 and suppression of ICAM-1 expression. Furthermore, TGF-beta1 pretreatment resulted in a significant inhibition of IL-6 induced ICAM-1 promoter activity. TGF-beta mediated inhibition of IL-6 induced ICAM-1 expression was reversed by transfection with dominant negative Smad2 constructs. In conclusion, we show that: 1) TGF-beta receptor Type II is predominantly located on basolateral membrane and receptor stimulation activates Smad pathway; 2) TGF-beta1 down-regulates IL-6-induced tyrosine phoshorylation of STAT1 and STAT3 and ICAM-1 expression; and 3) Smad2 is required for the down-regulation of IL-6 signaling by TGF-beta. Collectively, our data demonstrate a cross-talk between TGF-beta and IL-6, and TGF-beta may play a role in the negative regulation of IL-6 signaling in intestinal epithelial cells.

Active Transport, Cell Nucleus↗
PubMed2003

IL-6 induces NF-kappa B activation in the intestinal epithelia.

IL-6 is a potent proinflammatory cytokine that has been shown to play an important role in the pathogenesis of inflammatory bowel disease (IBD). It is classically known to activate gene expression via the STAT-3 pathway. Given the crucial role of IL-6 in the pathogenesis of chronic intestinal inflammation, it is not known whether IL-6 activates NF-kappaB, a central mediator of intestinal inflammation. The model intestinal epithelial cell line, Caco2-BBE, was used to study IL-6 signaling and to analyze whether suppressor of cytokine signaling 3 (SOCS-3) proteins play a role in the negative regulation of IL-6 signaling. We show that IL-6 receptors are present in intestinal epithelia in a polarized fashion. Basolateral IL-6 and, to a lesser extent, apical IL-6 induces the activation of the NF-kappaB pathway. Basolateral IL-6 stimulation results in a maximal induction of NF-kappaB activation and NF-kappaB nuclear translocation at 2 h. IL-6 induces polarized expression of ICAM-1, an adhesion molecule shown to be important in the neutrophil-epithelial interactions in IBD. Using various deletion constructs of ICAM-1 promoter, we show that ICAM-1 induction by IL-6 requires the activation of NF-kappaB. We also demonstrate that overexpression of SOCS-3, a protein known to inhibit STAT activation in response to IL-6, down-regulates IL-6-induced NF-kappaB activation and ICAM-1 expression. In summary, we demonstrate the activation of NF-kappaB by IL-6 in intestinal epithelia and the down-regulation of NF-kappaB induction by SOCS-3. These data may have mechanistic and therapeutic implications in diseases such as IBD and rheumatoid arthritis in which IL-6 plays an important role in the pathogenesis.

Caco-2 Cells↗
PubMed2003

Semapimod. Cytokine.

Cytokine PharmaSciences is developing semapimod (CNI-1493), a cytokine inhibitor and synthetic guanylhydrazone mitogen-activated protein kinase blocker, as a potential treatment for Crohn's disease and other inflammatory conditions. As of December 2001, a phase I study demonstrating the safety of the compound had been completed and phase II trials for psoriasis and Crohn's disease were ongoing. In April 2003, preclinical and early clinical studies were underway for a variety of indications, including congestive heart failure and pancreatitis.

Animals↗
PubMed2002

The adenosine 2b receptor is recruited to the plasma membrane and associates with E3KARP and Ezrin upon agonist stimulation.

We have previously shown that adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5'-AMP. Acting through the adenosine A2b receptor (A2bR), the luminally derived adenosine induces vectorial chloride secretion and a polarized secretion of interleukin-6 to the intestinal lumen. Although some G protein-coupled receptors interact with anchoring or signaling molecules, not much is known in this critical area for the A2bR. We used the model intestinal epithelial cell line, T84, and Caco2-BBE cells stably transfected with GFP-A2b receptor to study the intestinal A2bR. The A2bR is present in both the apical and basolateral membranes of intestinal epithelia. Apical or basolateral stimulation of the A2bR induces recruitment of the receptor to the plasma membrane and caveolar fractions. The A2bR co-immunoprecipitates with E3KARP and ezrin upon agonist stimulation. Ezrin interacts with E3KARP and PKA and the interaction between ezrin and E3KARP is enhanced by agonist stimulation. Our data suggest that the A2bR is recruited to the plasma membrane upon apical or basolateral agonist stimulation and interacts with E3KARP and ezrin. We speculate that such an interaction may not only anchor the A2bR to the plasma membrane but may also function to stabilize the receptor in a signaling complex in the plasma membrane.

Adenosine↗
PubMed2002

Luminal leptin enhances CD147/MCT-1-mediated uptake of butyrate in the human intestinal cell line Caco2-BBE.

In the intestine, butyrate constitutes the major energy fuel for colonocytes. However, little is known about the transport of butyrate and its regulation in the intestine. In this study we demonstrate that the monocarboxylate transporter (MCT-1) is apically polarized in model human intestinal epithelia and is involved in butyrate uptake by Caco2-BBE cell monolayers. The butyrate uptake by Caco2-BBE cell monolayers displayed conventional Michaelis-Menten kinetics and was found to be pH-dependent, Na(+)-independent, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-insensitive, and inhibited by the monocarboxylate transporter inhibitor alpha-cyano-4-hydroxycinnamate and by an excess of unlabeled butyrate. We show that MCT-1 associates with CD147 at the apical plasma membrane in Caco2-BBE cell monolayers. Using antisense CD147, we demonstrate that the association of CD147 with MCT-1 is critical for the butyrate transport activity. Interestingly, we show for the first time hormonal regulation of CD147/MCT-1 mediated butyrate uptake. Specifically, luminal leptin significantly up-regulates MCT-1-mediated butyrate uptake by increasing its maximal velocity (V(max)) without any modification in the apparent Michaelis-Menten constant (K(m)). Finally, we show that luminal leptin up-regulates butyrate uptake in Caco2-BBE monolayers by two distinct actions: (i) increase of the intracellular pool of MCT-1 protein without affecting CD147 expression and (ii) translocation of CD147/MCT-1 to the apical plasma membrane of Caco2-BBE cell monolayers.

Antigens, CD↗
PubMed2002

Intestinal epithelial pathobiology: past, present and future.

The intestinal epithelium serves as one of man's primary interfaces with the outside world. Its importance is illustrated by the fact that the proper functioning of this interface is absolutely essential for human health, and even modest perturbations in its function may lead to diarrhoea, constipation, malnutrition, dehydration, infectious disease or chronic intestinal inflammatory diseases such as Crohn's disease and ulcerative colitis, collectively referred to as inflammatory bowel disease. Both pathogen-induced intestinal inflammation and the active flares of inflammatory bowel disease are histopathologically defined, their sequellae being mediated by neutrophils that migrate across the intestinal epithelium, forming a crypt abscess. Classically, the intestinal epithelium has been thought of primarily as a barrier, and indeed this is a very important aspect of its function, but the intestinal epithelium is also a highly interactive barrier. This chapter will summarize some of the basic research conducted over the past 15 years that has revealed basic insights into how the epithelium participates in the formation of a crypt abscess and how it plays a role in causing the characteristic clinical manifestations that ensue. In addition, the chapter will discuss how this research has resurrected the 'old', yet newly emerging, concept that physiological malfunction of the intestinal epithelium can be the primary defect that leads to the innate and adaptive immune dysregulation mediating inflammatory bowel disease.

Cell Movement↗
PubMed2002

Tacrolimus Fujisawa.

Fujisawa has launched the immunosuppressant tacrolimus extensively for use in organ transplantation. The compound has also been developed for the treatment of atopic dermatitis, for which it has been launched in the US, Japan, Switzerland, Canada and Germany [425053], [426616], [435668], [447770]. Tacrolimus has also been developed for chronic rheumatoid arthritis (RA) and several other autoimmune diseases [351891], [352195], [360717]. By March 2002, tacrolimus had entered phase III trials for inflammatory bowel disease (IBD) in Japan [442503]. This evaluation will focus on the development of tacrolimus for this indication.

Animals↗
PubMedDate not supplied

Growth hormone favorably affects bone turnover and bone mineral density in patients with short bowel syndrome undergoing intestinal rehabilitation.

BACKGROUND: Patients with short bowel syndrome (SBS) have a high prevalence of metabolic bone disease due to nutrient malabsorption and potential effects of parenteral nutrition (PN). Human growth hormone (hGH) has been shown in some studies to have anabolic effects on bone, but hGH effects on bone in patients with SBS are unknown. METHODS: Adults with PN-dependent SBS underwent a 7-day period of baseline studies while receiving usual oral diet and PN and then began receiving modified diets designed to improve nutrient absorption and daily oral calcium/vitamin D supplements (1500 mg elemental calcium and 600 IU vitamin D, respectively). Subjects were randomized to receive in a double-blind manner either subcutaneous (sc) saline placebo as the control or hGH (0.1 mg/kg/d for 3 weeks, then 0.1 mg/kg 3 days a week for 8 subsequent weeks). Open-label hGH was given from week 13 to week 24 in subjects who required PN after completion of the 12-week double-blind phase. Markers of bone turnover (serum osteocalcin and urinary N-telopeptide [NTX]), vitamin D nutriture (serum calcium, 25-hydroxyvitamin D [25-OH D] and parathyroid hormone [PTH] concentrations), and intestinal calcium absorption were measured at baseline and at weeks 4 and 12. Dual x-ray absorptiometry (DXA) of the hip and spine was performed to determine bone mineral density (BMD) at baseline and weeks 12 and 24. RESULTS: The majority of subjects in each group exhibited evidence of vitamin D deficiency at baseline (25-OH D levels<30 ng/mL; 78% and 79% of control and hGH-treated subjects, respectively). Subjects treated with hGH demonstrated a significant increase from baseline in serum osteocalcin levels at 12 weeks (+62%; p<.05). The levels of NTX were increased over time in the hGH-treated group; however, this did not reach statistical significance. Both NTX and osteocalcin remained unchanged in control subjects. BMD of the spine and total hip was unchanged in subjects treated with placebo or hGH at 24 weeks. However, femoral neck BMD was slightly but significantly decreased in the placebo group at this time point but remained unchanged from baseline in the hGH-treated subjects. CONCLUSIONS: hGH therapy significantly increased markers of bone turnover during the initial 3 months of therapy and stabilized femoral neck bone mass over a 6-month period in patients with severe SBS undergoing intestinal rehabilitation.

Absorptiometry, Photon↗
PubMedDate not supplied

Are plasma citrulline and glutamine biomarkers of intestinal absorptive function in patients with short bowel syndrome?

Sensitive biomarkers for intestinal absorptive function would be clinically useful in short bowel syndrome (SBS). Citrulline (Cit) is a product of the metabolism of glutamine (Gln) and derived amino acids by enterocytes. Cit is produced almost exclusively by the gut, which is also a major site of Gln metabolism. The goals of this study were to examine whether plasma Cit and Gln concentrations are biomarkers of residual small intestinal length and nutrient absorptive functions in adult SBS patients followed prospectively. We studied 24 stable adults with severe SBS receiving chronic parenteral nutrition (PN) in a double-blind, randomized trial of individualized dietary modification +/- recombinant human growth hormone (GH). During a baseline week, intestinal absorption studies (% absorption of fluid, kcal, nitrogen, fat, carbohydrate, sodium, phosphorus, and magnesium) were performed and concomitant plasma Cit and Gln concentrations determined. Individualized dietary modification and treatment with subcutaneous injection of placebo (n = 9) or GH (0.1 mg/kg daily x 21 days, then 3 times/week; n = 15) were then begun. PN weaning was initiated after week 4 and continued as tolerated for 24 weeks. Repeat plasma amino acid determination and nutrient absorption studies were performed at weeks 4 and 12. Residual small bowel length at baseline was positively correlated with baseline plasma Cit (r = 0.467; p = .028). However, no significant correlations between absolute Cit or Gln concentrations and the percent absorption of nutrient substrates at any time point were observed. Similarly, no correlation between the change in Cit or GLN concentration and the change in % nutrient absorption was observed (baseline vs weeks 4 and 12, respectively). By weeks 12 and 24, 7 and 13 subjects were weaned completely from PN, respectively. However, baseline plasma Cit or Gln did not predict PN weaning at these time points. We concluded that plasma Cit (but not Gln) concentrations appeared to be an indicator of small intestinal length in adult SBS. However, neither plasma Cit nor Gln was a biomarker for intestinal absorptive function in this cohort of patients with SBS.

Biomarkers↗