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Biomedical subjects

Daniel K Podolsky

Publications and source records attributed to Daniel K Podolsky.

At least 37 records · Page 2Linked to original sources
PubMed2004

Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C.

BACKGROUND & AIMS: Protein kinase C (PKC) has been implicated in regulation of intestinal epithelial integrity in response to lumenal bacteria. Intestinal epithelial cells (IECs) constitutively express Toll-like receptor (TLR)2, which contains multiple potential PKC binding sites. The aim of this study was to determine whether TLR2 may activate PKC in response to specific ligands, thus potentially modulating barrier function in IECs. METHODS: TLR2 agonist (synthetic bacterial lipopeptide Pam(3)CysSK4, peptidoglycan)-induced activation of PKC-related signaling cascades were assessed by immunoprecipitation, Western blotting, immunofluorescence, and kinase assays-combined with functional transfection studies in the human model IEC lines HT-29 and Caco-2. Transepithelial electrical resistance characterized intestinal epithelial barrier function. RESULTS: Stimulation with TLR2 ligands led to activation (phosphorylation, enzymatic activity, translocation) of specific PKC isoforms (PKCalpha and PKCdelta). Phosphorylation of PKC by TLR2 ligands was blocked specifically by transfection with a TLR2 deletion mutant. Ligand-induced activation of TLR2 greatly enhanced transepithelial resistance in IECs, which was prevented by pretreatment with PKC-selective antagonists. This effect correlated with apical tightening and sealing of tight junction (TJ)-associated ZO-1, which was mediated via PKC in response to TLR2 ligands, whereas morphologic changes of occludin, claudin-1, or actin cytoskeleton were not evident. Downstream the endogenous PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS), but not transcriptional factor activator protein-1 (AP-1), was activated significantly on stimulation. CONCLUSIONS: The present study provides evidence that PKC is an essential component of the TLR2 signaling pathway with the physiologic consequence of directly enhancing intestinal epithelial integrity through translocation of ZO-1 on activation.

Caco-2 Cells↗
PubMed2004

An analysis of the potential impact of computed tomographic colonography (virtual colonoscopy) on colonoscopy demand.

BACKGROUND AND AIMS: There has been much speculation about the potential impact on the use of conventional colonoscopy if "virtual" computed tomographic colonography (CTC) became a widely accepted modality for colorectal cancer (CRC) screening. However, no formal analysis of the impact of CTC on colonoscopy demand has been reported. METHODS: A mathematical model to predict colonoscopy demand based on several relevant input parameters was constructed. Current national colonoscopy practice, estimated using various published reports, was used as the foundation to project colonoscopy demand if CTC were implemented as the primary CRC screening modality. RESULTS: In the base-case analysis, if CTC were used as the primary modality for CRC screening, 1.78 million colonoscopies could be eliminated from the total 6.47 million in 2003. Depending on the polyp size threshold used to define a CTC study as positive (6 or 10 mm), this loss would be partially offset by 1.21 million (6 mm) or .34 million (10 mm) follow-up colonoscopies for CTC examinations with positive findings, resulting in a net loss of .57 million (8.8% decrease) (6 mm) or 1.44 million (22.3% decrease) (10 mm). Extensive sensitivity analyses showed that the findings of this model were robust and insensitive to most parameters tested but were sensitive to a few parameters, including the percentage of CTC examinations with positive findings. CONCLUSIONS: Wide-scale implementation of CTC for CRC screening would likely lead to a decrease in use of conventional colonoscopy. The percentage of CTC studies with positive findings seemed to be a pivotal variable, which would be determined in large part by the polyp size ultimately established to define a positive finding.

Colonography, Computed Tomographic↗
PubMed2004

Functional modulation of enterocytes by gram-positive and gram-negative microorganisms.

Clinical studies have suggested that so-called probiotic bacteria may be effective as therapy in inflammatory bowel disease. However, the molecular mechanisms of their interaction with the intestinal surface remain undefined. The influence of whole probiotic bacteria [Escherichia coli Nissle 1917 (EcN); probiotic mixture VSL#3 (PM)], bacterial cell lysates, and conditioned media on transepithelial resistance (TER), IL-8 secretion, mucin gene expression, and tight junction proteins were determined in T84 and HT-29 intestinal epithelial cells (IEC). In addition, effects on pathogen (Salmonella dublin)-induced alterations were analyzed. EcN as well as debris and cell extracts induced IL-8 secretion from IEC, whereas no such effect was observed following incubation with the PM. The PM and soluble protein(s) released from the PM increased TER, prevented pathogen-induced decrease in TER, and were shown to stabilize tight junctions. The PM induced expression of mucins in IEC, and these organisms as well as EcN diminished S. dublin-induced cell death. Inhibition of MAPKs with PD-98059 or SB-203580 significantly decreased alterations in IL-8 synthesis and mucin expression and affected the regulation of TER. Probiotics and protein(s) released by these organisms may functionally modulate the intestinal epithelium of the host by different mechanisms, including the competition of whole organisms for contact with the epithelial surface as well as stabilization of the cytoskeleton and barrier function and the induction of mucin expression. Gram-negative and gram-positive organisms differ in the mechanisms activated, and a combination of organisms might be more effective than the application of a single strain.

Blotting, Western↗
PubMed2003

Interferon-gamma augments CARD4/NOD1 gene and protein expression through interferon regulatory factor-1 in intestinal epithelial cells.

Although intestinal epithelial cells appear to be functionally hyporesponsive to normal intestinal flora, human intestinal epithelial cells can respond to enteroinvasive bacteria and induce an inflammatory response. This initial inflammatory response leads to the recruitment of polymorphonuclear leukocytes to the affected site in vitro and in vivo. CARD4/NOD1 is a potential cytosolic receptor for peptidoglycan in mammalian cells that resembles pathogen-resistant proteins of plants. In this context, CARD4/NOD1 is a candidate for a recognition protein of intracellular bacteria or peptidoglycan in intestinal epithelial cells. In this study, we demonstrate that CARD4/NOD1 is constitutively expressed in intestinal epithelial cell lines and isolated primary intestinal epithelial cells. Interferon-gamma (IFN gamma), which is a potent pro-inflammatory cytokine in intestinal mucosal inflammation, activates CARD4/NOD1 mRNA transcription in a time- and dose-dependent manner and results in augmentation of CARD4/NOD1 protein in SW480 cells. Promoter analysis of CARD4/NOD1 indicates that interferon regulatory factor-1 (IRF-1) binding motif (-791 to -782) is essential for the effect of IFN gamma. Nuclear extracts from SW480 cells treated with IFN gamma show specific binding of oligonucleotides corresponding to this IRF-1-binding motif, which was supershifted by anti-IRF-1 antibody in electrophoretic mobility shift assay. Overexpression of IRF-1 protein activates the CARD4/NOD1 promoter but not the deletion mutant of the IRF-1-binding site in a co-transfection assay of IRF-1 expression plasmid with CARD4/NOD1 promoter. These studies suggest that the Th1 cytokine, IFN gamma, activates CARD4/NOD1 transcription and regulate innate immune mechanisms in the condition of intestinal mucosal inflammation.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Transforming growth factor-beta mediates intestinal healing and susceptibility to injury in vitro and in vivo through epithelial cells.

In vitro studies suggest that transforming growth factor (TGF)-beta has potent effects on gastrointestinal mucosal integrity, wound repair, and neoplasia. However, the multiplicity of actions of this peptide on many different cell types confounds efforts to define the role of TGF-beta within the intestinal epithelium in vivo. To delineate these effects selective blockade of intestinal epithelial TGF-beta activity was undertaken through targeted expression of a dominant-negative (DN) TGF-beta RII to intestinal epithelial cells in vitro and in vivo. Stable intestinal epithelial cell (IEC)-6 lines overexpressing TGF-beta RII-DN (nucleotides -7 to 573) were established. Transgenic mice overexpressing TGF-beta RII-DN under the regulation of a modified liver fatty acid-binding promoter (LFABP-PTS4) were constructed. In vitro healing was assessed by wounding of confluent monolayers. Colitis was induced by the addition of dextran sodium sulfate (2.5 to 7.5% w/v) to their drinking water. Overexpression of TGF-beta RII-DN in intestinal epithelial cell-6 cells resulted in a marked reduction in cell migration and TGF-beta-stimulated wound healing in vitro. TGF-beta RII-DN transgenic mice did not exhibit baseline intestinal inflammation or changes in survival, body weight, epithelial cell proliferation, aberrant crypt foci, or tumor formation. TGF-beta RII-DN mice were markedly more susceptible to dextran sodium sulfate-induced colitis and exhibited impaired recovery after colonic injury. TGF-beta is required for intestinal mucosal healing and TGF-beta modulation of the intestinal epithelium plays a central role in determining susceptibility to injury.

Animals↗
PubMed2003

Intestinal myofibroblasts in innate immune responses of the intestine.

BACKGROUND & AIMS: Intestinal myofibroblasts are known to respond to inflammatory signals and may play a role in Crohn's disease-associated fibrosis. However, putative involvement by myofibroblasts in innate immune responses as part of intestinal host defense has not been characterized. We therefore analyzed expression and regulation of toll-like receptors (TLRs) in colonic human myofibroblasts (CCD-18) and primary human colonic myofibroblasts in comparison with human lung myofibroblasts (CCD-37). METHODS: Expression of TLRs (1-10) and NOD 1 and 2 was assessed before and after stimulation with either lipopolysaccharide (LPS) or lipoteichoic acid (LTA) by using a custom microarray, reverse-transcription polymerase chain reaction, Northern blot and Western blot analysis, and immunohistochemistry. Activation of signaling pathways, translocation of p65, and secretion of interleukin (IL)-8 were determined. RESULTS: Messenger RNAs encoding for TLR1-9, as well as NOD1 and NOD2, were amplified from cultured and primary human intestinal myofibroblasts. After stimulation with LPS or LTA, a 1.5-4.2-fold up-regulation of TLRs (2, 3, 4, 6, 7) and elements of the signaling cascade (MyD88, TIR domain-containing adapter protein [TIRAP]) was observed. CCD-18 and CCD-37 cells expressed TLR 2 and 4 protein, which were located primarily on the cell membrane. Stimulation with LTA or LPS resulted in activation of the mitogen-activated protein kinases pathway, nuclear translocation of p65, and significantly increased IL-8 secretion. CONCLUSIONS: Bacterial components directly activate intestinal myofibroblasts expressing TLRs. These cells may therefore participate in innate immune responses by sensing and responding to bacterial products that have penetrated into the subepithelial compartment.

Cells, Cultured↗
PubMed2003

Colonic epithelial functional phenotype varies with type and phase of experimental colitis.

BACKGROUND & AIMS: Colonic crypt elongation occurs during both chronic colitis and in the recovery phase of acute colitis. The impact of these alterations on epithelial cell functions is not fully defined. METHODS: DNA microarray analysis of freshly isolated colonic epithelial cells (CECs) from acute and chronic colitis was performed, and the results were confirmed by reverse transcription polymerase chain reaction. Localization of the selected molecules was examined by immunohistochemistry using newly generated antibodies. The function of selected molecules detected in this study was examined by administering the specific inhibitors in dextran sodium sulfate (DSS) colitis. RESULTS: Several detoxification-associated molecules, which contribute to prevent inflammation by regulating physiological balance under normal conditions, were markedly down-regulated, and anti-inflammatory molecules, which are not normally expressed, were up-regulated in the CEC under the chronic colitis. Among the detoxification-associated molecules, carbonic anhydrase IV was specifically down-regulated in CEC of Th2- but not Th1-mediated colitis. Functionally, inhibition of carbonic anhydrase activity led to the enhancement of recovery from DSS-induced acute colitis by directly stimulating CEC proliferation. Increased expression of regeneration-associated molecules such as regenerating gene-III gamma was detectable in the CEC of acute and chronic colitis but not in the recovery phase of colitis. The expression of this molecule was restricted in surface epithelium and upper crypts but not lower crypts. CONCLUSIONS: These studies suggest that functional alterations, which result in either the exacerbation or the suppression of colitis, coexist in the CEC during chronic colitis. CEC functions are likely to be differentially regulated in the context of the stage and mechanism of colitis.

Animals↗
PubMed2003

Trefoil factors: initiators of mucosal healing.

Maintaining the integrity of the gastrointestinal tract, despite the continual presence of microbial flora and injurious agents, is essential. Epithelial continuity depends on a family of small, yet abundant, secreted proteins--the trefoil factors (TFFs). TFFs protect mucous epithelia from a range of insults and contribute to mucosal repair, although the signalling events that mediate these responses are only partially understood.

Animals↗
PubMed2003

CARD15/NOD2 functions as an antibacterial factor in human intestinal epithelial cells.

BACKGROUND & AIMS: Mutations in the CARD15/NOD2 gene, a putative intracellular pattern recognition receptor, have been linked to the risk for Crohn's disease. Because intestinal epithelial cells play a role as the barrier to luminal microorganisms, we investigated the expression and function of CARD15/NOD2 in intestinal epithelial cells. METHODS: Expression of CARD15/NOD2 messenger RNA (mRNA) in intestinal epithelial cell lines and primary intestinal epithelial cells was assessed by reverse-transcription polymerase chain reaction (RT-PCR). Regulation of expression of CARD15/NOD2 by cytokines was determined by Northern blot using the SW480 cell line. Active CARD15/NOD2 protein in SW480 cells was assessed by the combination of immunoprecipitation and immunoblotting using anti-CARD15/NOD2 antisera. To identify the functional role of CARD15/NOD2 in intestinal epithelial cells, gentamicin protection assays of Salmonella typhimurium were performed using Caco2 cells stably transfected with either wild-type CARD15/NOD2 or the 3020insC mutant associated with Crohn's disease. RESULTS: CARD15/NOD2 mRNA was expressed in both intestinal epithelial cell lines and primary intestinal epithelial cells. CARD15/NOD2 mRNA and protein were up-regulated by tumor necrosis factor alpha (TNFalpha) in SW480 cells. The number of viable internalized S. typhimurium in Caco2 cells stably transfected with CARD15/NOD2 expression plasmid was lower than untransfected Caco2 cells or MOCK transfectant. In contrast, expression of a variant associated with Crohn's disease was unable to constrain bacterial survival. CONCLUSIONS: CARD15/NOD2 is expressed in intestinal epithelial cells and may serve as a key component of innate mucosal responses to luminal bacteria as an antibacterial factor. Failure in this activity may contribute to the development of Crohn's disease.

Caco-2 Cells↗
PubMed2003

Gamma interferon augments the intracellular pathway for lipopolysaccharide (LPS) recognition in human intestinal epithelial cells through coordinated up-regulation of LPS uptake and expression of the intracellular Toll-like receptor 4-MD-2 complex.

Although some intestinal epithelial cell lines are known to respond to lipopolysaccharide (LPS), understanding of the relationship between LPS responsiveness and the expression of LPS receptors or factors regulating LPS responsiveness of intestinal epithelial cell lines is incomplete. In this study, we demonstrate that commonly studied human intestinal epithelial cell lines can be classified into at least three different types on the basis of LPS responsiveness, Toll-like receptor-4 (TLR4) expression, and the effects of gamma interferon (IFN-gamma) on LPS responsiveness. The first phenotype, which includes the HCT-116 and Caco-2 cell lines, is characterized by relative hyporesponsiveness to LPS and diminished expression of TLR4 protein. In these cells, IFN-gamma does not induce LPS responsiveness. The second phenotype, which includes cell line SW480, exhibits a highly LPS-responsive phenotype and surface expression of TLR4 protein even in unprimed conditions. These lines are functionally similar to cells of monocytic lineage. In the third phenotype, which includes the HT-29 and Colo205 cell lines, TLR4 protein is largely present in the cytoplasmic fraction and the cells are hyporesponsive to LPS in an unprimed condition. However, priming of these cells with IFN-gamma can induce LPS responsiveness through augmentation of LPS uptake and expression of MD-2 mRNA and intracellular TLR4 proteins. Finally, these findings suggest that the Th1 cytokine IFN-gamma modulates LPS responsiveness through several mechanisms in intestinal epithelial cells and that these cells may comprise different subpopulations with distinct roles in innate immune responses.

Antigens, Surface↗
PubMed2003

The future of IBD treatment.

Throughout most of the past 50 years, treatment of Crohn's disease and ulcerative colitis has been dominated by the use of agents whose efficacy was defined on an empiric basis. Many of these, including most especially 5-amino salicylic acid (5-ASA)-based agents and corticosteroids, remain mainstays of current treatment even as research is catching up with clinical experience to define the mechanistic basis of their efficacy. Other agents, also of established utility in at least a subset of patients, were developed on the basis of general inferences about disease pathogenesis. These agents are exemplified by antibiotics (metronidazole) and immunosuppressive agents (azathioprine/6-mercaptopurine, methotrexate, and cyclosporine); attention was directed on the assumption of the general likely importance of microbial species and immunoactivation, respectively. However, the rapid progress, even if still incomplete in understanding of pathophysiological mechanisms that play a role in IBD, has transformed the development of new therapeutic agents, enabling the development of several agents now available or currently in advanced clinical development. It is notable that each of the major overall thrusts in development of new therapeutic strategies parallels, and may be reasonably viewed as the partial outgrowth of, dominant areas of progress in understanding of disease mechanisms relevant to IBD.

Colitis, Ulcerative↗
PubMed2002

Trefoil peptide expression and goblet cell number in rat intestine: effects of KGF and fasting-refeeding.

The trefoil factor family peptides TFF1, TFF2, and TFF3 are important for gut mucosal protection and restitution. Keratinocyte growth factor (KGF) stimulates proliferation and differentiation of epithelial cells with potent effects on goblet cells. To investigate interactions between food intake and KGF, rats were fed ad libitum (control), fasted for 72 h, or fasted for 72 h and then refed for 72 h with or without KGF (3 mg. kg(-1). day(-1)). With fasting, goblet cell number in duodenum increased, TFF3 mRNA in duodenum and jejunum decreased, and TFF3 protein did not change or increased. KGF during fasting stimulated colonic growth, normalized TFF3 mRNA in duodenum and jejunum, and broadly upregulated gut goblet cell number and TFF3 protein expression. With fasting-refeeding, KGF increased small bowel and colonic mucosal growth, goblet cell number, and TFF3 protein but had variable effects on TFF3 mRNA. KGF induced TFF2 mRNA and protein in duodenum and jejunum with both nutritional regimens. We conclude that nutrient availability modifies rat intestinal goblet cell number, TFF3 mRNA, and the gut-trophic effects of KGF in a region-specific manner. KGF enhances TFF2 expression in proximal small bowel and increases goblet cell number and TFF3 protein content throughout the intestine independent of food intake.

Animals↗
PubMed2002

Commensal-associated molecular patterns induce selective toll-like receptor-trafficking from apical membrane to cytoplasmic compartments in polarized intestinal epithelium.

Commensal-associated molecular patterns, the major products of nonpathogenic bacteria, are present at high concentrations at the apical surface of the intestinal epithelium. However, the nature of the interaction of commensal-associated molecular patterns with the lumenal surface of the epithelium has not been defined. We have recently demonstrated that intestinal epithelial cells constitutively express several Toll-like receptors (TLRs) in vitro and in vivo that seem to be the key receptors responsible for immune cell activation in response to various bacterial products. In this study we characterize the subcellular distribution of two major TLRs, TLR2 and TLR4, and their ligand-specific dynamic regulation in the model human intestinal epithelial cell line T84. Immunocytochemical studies indicate that TLR2 and TLR4 are constitutively expressed at the apical pole of differentiated T84 cells. After stimulation with lipopolysaccharide or peptidoglycan, TLRs selectively traffic to cytoplasmic compartments near the basolateral membrane. Thus, we demonstrate that TLRs are positioned at the apical pole where they are poised to monitor the sensitive balance of the lumenal microbial array. The results of this dynamic epithelial surveillance can then be conveyed to the underlying cell populations of the lamina propria via these innate immune pattern recognition receptors.

Cell Membrane↗
PubMed2002

The current future understanding of inflammatory bowel disease.

Accumulating evidence indicates that the inflammatory bowel diseases result from an activation of immune and inflammatory responses initiated by a stimulation of the luminal flora or their products. Genetically determined variations in key mucosal functions, including cell activation by prototypic bacterial pattern molecules, lead to differential susceptibility to the development of these disorders, probably reflecting an interrelated activation of the innate and adaptive immune responses. The persistence and amplification of inflammation is likely to reflect the continuing presence of the driving stimulus and the complex, self-reinforcing activation of select T-helper subtypes and macrophages and other antigen-presenting cells, mediated by several cytokines. The latter include interleukins-2, 12, and 18 as well as interferon and macrophage migration inhibitory factors. The production of other broadly pro-inflammatory cytokines, most notably tumour necrosis factor and interleukins-1 and 6, enhances related inflammatory processes that eventually lead to many of the clinical manifestations of inflammatory bowel disease. The overall severity of the inflammatory process reflects a balance between leukocyte recruitment and downregulatory mucosal repair processes.

Cytokines↗
PubMed2002

Role of tumor necrosis factor receptor 2 (TNFR2) in colonic epithelial hyperplasia and chronic intestinal inflammation in mice.

BACKGROUND & AIMS: Tumor necrosis factor (TNF) induces multiple effects including cell proliferation and death by ligation with TNF receptor type II (TNFR2). We studied the role of TNFR2 in chronic inflammation-induced colonic epithelial alteration. METHODS: TNFR2 expression in colonic epithelial cells (CECs) was assessed by ribonuclease protection assay (RPA) and immunohistochemistry (IHC) in patients with inflammatory bowel disease (IBD) and murine colitis models. TNFR2 expression was also analyzed using COLO205 cells. The role of TNFR2 in colonic epithelial homeostasis was examined by generating interleukin 6-deficient TCR alpha KO (alpha IL-6DKO) or TNFR2-deficient TCR alpha (alpha TNFR2DKO) mice. RESULTS: TNFR2 expression was up-regulated in CEC in both human ulcerative colitis and Crohn's disease. In vitro studies showed that TNFR2 expression was up-regulated by a cooperative effect of key proinflammatory cytokines. By RPA, the increased expression of TNFR2 was detectable in TCR alpha KO mice with colitis compared with TCR alpha KO mice without colitis or wild-type mice. In alpha IL-6DKO mice, TNFR2 expression, proliferation, and nuclear factor kappa B activation of CECs were markedly reduced compared with TCR alpha KO mice. alpha TNFR2 mice also showed significantly less colonic epithelial proliferation compared with TCR alpha KO mice. CONCLUSIONS: Expression of TNFR2 is consistently increased on CECs in both murine colitis models as well as patients with IBD. TNFR2 may play an important role in colonic inflammation-associated alteration in the intestinal epithelium.

Animals↗