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The forces that are reshaping the delivery of health care through much of the developed world are especially acute within academic centers that carry the responsibility for delivering that care while advancing medical knowledge and ensuring well-trained physicians. Gastroenterology will not be spared any of those forces, and in some ways represents the leading edge of their impact. Though the dynamics vary within the context of the health-care delivery and scientific enterprise of individual countries, common elements are demands for greater accountability and transparency in how academic medical centers demonstrate their value while assuring broad access to their expertise. In the United States, underlying many forms of change in the payment scheme are the common elements that will increasingly place the risk for the cost of care on providers rather than on the payers, be it government or private, as has historically been true. At the same time, academic medical centers, with gastroenterology responsible for addressing the burden of digestive diseases, must remain the stem cells for health care integrating all their missions and providing the foundation of medical advances which will ultimately improve human welfare. What will academic gastroenterology units look like if they are able to effectively respond to these forces? Gastrointestinal (GI) divisions and faculty will own new roles including responsibility for system success in caring for patients. They will evolve their training programs to provide the next generation with skills needed to succeed, including the discipline of system improvement, team leadership and others. And there will be new models that will drive the organization of research that are not as conventionally self-contained within the gastroenterology units, but fostering research teams that have hubs and spokes. The vitality of GI divisions will depend on the willingness to seize ownership of the new value proposition of disease management ensuring that each patient achieves the best outcome with the most effective use of resources and endeavor within their systems to capture some of that value to invest in their training and research missions. In the course of that evolution, gastroenterology will be well served by rebalancing the dependence on existing modalities. If procedural gastroenterology becomes the sole value proposition, it will lead to an increasingly narrow view of the field.

Background The integrity of the gut barrier in patients with inflammatory bowel disease is known to be impaired but the exact mechanisms remain mostly unknown. SHANK3 mutations are associated with autism, and patients with autism are known to have higher proportions of inflammatory bowel disease. Here, we explore the role of SHANK3 in inflammatory bowel disease, both in vivo and in vitro. Methods Dextran sulfate sodium colitis was induced in SHANK3 knockout mice. Transepithelial electrical resistance, paracellular permeability, and Salmonella invasion assays were used to evaluate epithelial barrier function, in vitro and in vivo. Expression of tight junction proteins, protein kinases, and MAP kinase phosphorylation changes were analyzed by immunoblotting after overexpression or knockdown of SHANK3 expression. SHANK3 expression in intestinal tissue from patients with Crohn's disease was analyzed by quantitative polymerase chain reaction and immunohistochemistry. Results SHANK3 knockout mice were more susceptible to dextran sulfate sodium. SHANK3 knockout resulted in a leaky epithelial barrier phenotype, as demonstrated by decreased transepithelial electrical resistance, increased paracellular permeability, and increased Salmonella invasion. Overexpression of SHANK3 enhanced ZO-1 expression, and knockdown of SHANK3 resulted in decreased expression of ZO-1. Regulation of ZO-1 expression by SHANK3 seems to be mediated through a PKCe-dependent pathway. SHANK3 expression correlated with ZO-1 and PKCe in colonic tissue of patients with Crohn's disease. Conclusions The expression level of SHANK3 affects ZO-1 expression and the barrier function in intestinal epithelial cells. This may provide novel insights in Crohn's disease pathogenesis and treatment.

Fourteen editors-in-chiefs have steered G astroenterology to success since its inception in 1943. Five (Alvarez, Ivy, Aaron, Grossman, and Donaldson) are no longer with us. Their personalities and editorships, along with those of Marvin Sleisenger, are presented by their admirers. Fordtran, Ockner, Goyal, LaRusso, Podolsky, Brenner, Rustgi, and Omary describe their own backgrounds, experiences, and personal reflections on serving as editor-in-chief of G astroenterology.

Background & Aims IgG4-related cholangitis is a chronic inflammatory biliary disease that involves different parts of the pancreatobiliary system, but little is known about its mechanisms of pathogenesis. A T-helper (Th) 2 cell cytokine profile predominates in liver tissues from these patients. We investigated whether Th2 cytokines disrupt the barrier function of biliary epithelial cells (BECs) in patients with IgG4-related cholangitis. Methods We assessed the Th2 cytokine profile in bile samples and brush cytology samples from 16 patients with IgG4-related cholangitis and respective controls, and evaluated transcription of tight junction (TJ)–associated proteins in primary BECs from these patients. The effect of Th2 cytokines on TJ-mediated BEC barrier function and wound closure was examined by immunoblot, transepithelial resistance, charge-selective Na + /Cl − permeability, and 4-kDa dextran flux analyses. Results Bile samples from patients with IgG4-related cholangitis had significant increases in levels of Th2 cytokines, interleukin (IL)-4, and IL-5. IL-13 was not detected in bile samples, but polymerase chain reaction analysis of whole-brush cytology samples from patients with IgG4-related cholangitis revealed increased levels of IL-13 mRNA, compared with controls. BECs isolated from the brush cytology samples revealed decreased levels of claudin-1 and increased levels of claudin-2 mRNAs. In vitro, IL-4 and IL-13 significantly reduced TJ-associated BEC barrier function by activating claudin-2–mediated paracellular pore pathways. Th2 cytokines also impaired wound closure in BEC monolayers. Conclusions Th2 cytokines predominate in bile samples from patients with IgG4-related cholangitis and disrupt the TJ-mediated BEC barrier in vitro. Subsequent increases in biliary leaks might contribute to the pathogenesis of chronic biliary inflammation in these patients.

The topological insulator is a fundamentally new phase of matter, with the striking property that the conduction of electrons occurs only on its surface, not within the bulk, and that conduction is topologically protected. Topological protection, the total lack of scattering of electron waves by disorder, is perhaps the most fascinating and technologically important aspect of this material: it provides robustness that is otherwise known only for superconductors. However, unlike superconductivity and the quantum Hall effect, which necessitate low temperatures or magnetic fields, the immunity to disorder of topological insulators occurs at room temperature and without any external magnetic field. For this reason, topological protection is predicted to have wide-ranging applications in fault-tolerant quantum computing and spintronics. Recently, a large theoretical effort has been directed towards bringing the concept into the domain of photonics: achieving topological protection of light at optical frequencies. Besides the interesting new physics involved, photonic topological insulators hold the promise for applications in optical isolation and robust photon transport. Here, we theoretically propose and experimentally demonstrate the first photonic topological insulator: a photonic lattice exhibiting topologically protected transport on the lattice edges, without the need for any external field. The system is composed of an array of helical waveguides, evanescently coupled to one another, and arranged in a graphene-like honeycomb lattice. The chirality of the waveguides results in scatter-free, one-way edge states that are topologically protected from scattering., Comment: 21 pages, 5 figures

Background & Aims The Toll-like receptor (TLR) 4 mediates homeostasis of the intestinal epithelial cell (IEC) barrier. We investigated the effects of TLR4-D299G on IEC functions. Methods We engineered IECs (Caco-2) to stably overexpress hemagglutinin-tagged wild-type TLR4, TLR4-D299G, or TLR4-T399I. We performed gene expression profiling using DNA microarray analysis. Findings were confirmed by real-time, quantitative, reverse-transcriptase polymerase chain reaction, immunoblot, enzyme-linked immunosorbent assay, confocal immunofluorescence, and functional analyses. Tumorigenicity was tested using the CD1 nu/nu mice xenograft model. Human colon cancer specimens (N = 214) were genotyped and assessed for disease stage. Results Caco-2 cells that expressed TLR4-D299G underwent the epithelial-mesenchymal transition and morphologic changes associated with tumor progression, whereas cells that expressed wild-type TLR4 or TLR4-T399I did not. Caco-2 cells that expressed TLR4-D299G had significant increases in expression levels of genes and proteins associated with inflammation and/or tumorigenesis compared with cells that expressed other forms of TLR4. The invasive activity of TLR4-D299G Caco-2 cells required Wnt-dependent activation of STAT3. In mice, intestinal xenograft tumors grew from Caco-2 cells that expressed TLR4-D299G, but not cells that expressed other forms of TLR4; tumor growth was blocked by a specific inhibitor of STAT3. Human colon adenocarcinomas from patients with TLR4-D299G were more frequently of an advanced stage (International Union Against Cancer [UICC] ≥III, 70% vs 46%; P = .0142) with metastasis (UICC IV, 42% vs 19%; P = .0065) than those with wild-type TLR4. Expression of STAT3 messenger RNA was higher among colonic adenocarcinomas with TLR4-D299G than those with wild-type TLR4. Conclusions TLR4-D299G induces features of neoplastic progression in intestinal epithelial Caco-2 cells and associates with aggressive colon cancer in humans, implying a novel link between aberrant innate immunity and colonic cancerogenesis.

Objective Trefoil factor (TFF) peptides are expressed in gastric tissues, where they are part of the epithelial defences. To complement previous in vitro work, the goal of the present study was to examine directly if TFF2 was essential for gastric restitution in vivo during the recovery from microscopic damage. Design TFF2 mutant (KO) mice were examined to study the epithelial repair process in vivo after laser-induced photodamage (LPD). Using two-photon laser energy absorption (710 nm), LPD was imposed on an ∼3–5 cell region of surface epithelium in anaesthetised mouse stomach. Responses to damage were evaluated during confocal time-lapse microscopy; including area of damage and the extracellular pH adjacent to the damaged surface (Cl-NERF pH sensor). Results In control (TFF2+/+ and TFF2+/–) mice, damaged cells were exfoliated and the damaged epithelium was repaired by indomethacin. The resting surface pH was similar between control and TFF2-KO animals, but the post-LPD alkalisation of surface pH observed in control mice (∆pH 0.3±0.05, n=21) was attenuated in the TFF2-KO stomach (∆pH −0.08±0.09, n=18). Recobinant rat TFF3 partially rescued the attenuated surface pH change in TFF2-KO stomach, in the presence or absence of indomethacin. Conclusions In the gastric epithelium in vivo, TFFs promote epithelial restitution via a mechanism that does not require cyclooxygenase activation. A novel role for TFFs to affect gastric surface pH is observed.

Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1alpha-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.

Background & Aims Goblet cells (GC) facilitate mucosal protection and epithelial barrier repair, yet the innate immune mechanisms that selectively drive GC functions have not been defined. The aim of this study was to determine whether Toll-like receptor (TLR) 2 and modulation of GC-derived trefoil factor (TFF) 3 are functionally linked in the intestine. Methods GC modulation was assessed using quantitative real-time polymerase chain reaction analysis (qRT-PCR), Western blotting, and confocal microscopy. Dextran sulfate sodium (DSS) colitis was induced in wild-type, TFF3 −/− , and TLR2 −/− mice. Recombinant TLR2 ligand or TFF3 peptide were orally administered after DSS termination. Caco-2 cells overexpressing full-length TLR2 or mutant TLR2-R753Q were tested for TFF3 synthesis and functional-related effects in a wounding assay. Results Data from in vitro (Ls174T) and ex vivo models of murine and human GC reveal that TLR2 activation selectively induces synthesis of TFF3. In vivo studies using TFF3 −/− or TLR2 −/− mice demonstrate the ability for oral treatment with a TLR2 agonist to confer antiapoptotic protection of the intestinal mucosa against inflammatory stress-induced damage through TFF3. Recombinant TFF3 rescues TLR2-deficient mice from increased morbidity and mortality during acute colonic injury. Severe ulcerative colitis (UC) has recently been found to be associated with the R753Q polymorphism of the TLR2 gene. The relevance of the observed functional effect of TLR2 in regulating GC is confirmed by the finding that the UC-associated TLR2-R753Q variant is functionally deficient in the ability to induce TFF3 synthesis, thus leading to impaired wound healing. Conclusions These data demonstrate a novel function of TLR2 in intestinal GC that links products of commensal bacteria to innate immune protection of the host via TFF3.

Background & Aims: Ligation of tumor necrosis factor (TNF) receptors (TNFRs) with TNF plays a critical role in the pathogenesis of human inflammatory bowel disease (IBD). However, it remains unclear which cell types activated through TNFR-associated signaling cascades are involved in the pathogenesis of colitis. Methods: Recombination activating gene-1 (RAG) knockout (KO) (no T or B cells)-based TNFR double and triple KO mice were generated. Bone marrow (BM) chimera mice in which BM-derived myeloid cells, but not colonic epithelial cells (CECs), express TNFRs were also generated. Colitis was induced by administration of dextran sodium sulfate (DSS) in distilled water. Murine lines and chimeras were assessed for disease severity, histopathology, apoptotic cell rate, epithelial proliferation, and bacterial invasion rate. Results: Following DSS administration, mice lacking both RAG and TNFR1 exhibited a high mortality (>80%) rate with an impaired CEC regeneration compared with RAG KO and RAG × TNFR2 double KO (DKO) mice. Transplantation of RAG KO-derived BM cells restored CEC regeneration and rescued the majority of recipient RAG × TNFR1 DKO mice from DSS-induced mortality. After BM transplantation, RAG × TNFR1 DKO mice exhibited an increased rate of apoptosis in the colonic lamina propria macrophages in association with the activation of caspases. In addition, BM reconstitution directly or indirectly enhanced the proliferation of CECs by activating mitogen-activated protein kinase and phosphoinositide-3 kinase/Akt pathways. Conclusions: TNFR1-signaling cascade in colonic myeloid lineage cells contributes to the suppression of acute damage-associated mortality presumably by controlling CEC homeostasis.

Recently, substantial advances in the understanding of the molecular pathogenesis of inflammatory bowel disease (IBD) have been made owing to three related lines of investigation. First, IBD has been found to be the most tractable of complex disorders for discovering susceptibility genes, and these have shown the importance of epithelial barrier function, and innate and adaptive immunity in disease pathogenesis. Second, efforts directed towards the identification of environmental factors implicate commensal bacteria (or their products), rather than conventional pathogens, as drivers of dysregulated immunity and IBD. Third, murine models, which exhibit many of the features of ulcerative colitis and seem to be bacteria-driven, have helped unravel the pathogenesis/mucosal immunopathology of IBD.

Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize conserved molecular motifs on microorganisms. Ligand binding to TLRs initiates signaling cascades that activate NF-κB, MAPK, and interferon response factors. These culminate in cellular responses including activation of antimicrobial killing mechanisms, production of cytokines and chemokines, maturation of antigen presenting cells, and the recruitment of the adaptive immune response. Intestinal epithelial cells represent a unique population of cells that exist in direct contact with a biomass of bacteria. Initiation of TLR signaling is tightly regulated because prolonged and excessive activation of TLRs can lead to uncontrolled inflammation detrimental to the host. Varied mechanisms appear to contribute to control of TLR activation in the intestinal epithelium. These include the collective effects of several negative regulators that include IRAK-M, TOLLIP, SIGIRR, A20, Nod2, and PPARγ. However, it remains to be determined whether they comprise the entire spectrum of negative control mechanisms and how they are bypassed to trigger activation during challenge by pathogens.

The intestinal flora has long been thought to play a role either in initiating or in exacerbating the inflammatory bowel diseases (IBD). Host defenses, such as those mediated by the Toll-like receptors (TLR), are critical to the host/pathogen interaction and have been implicated in IBD pathophysiology. To explore the association of genetic variation in TLR pathways with susceptibility to IBD, we performed a replication study and pooled analyses of the putative IBD risk alleles in NFKB1 and TLR4, and we performed a haplotype-based screen for association to IBD in the TLR genes and a selection of their adaptor and signaling molecules. Our genotyping of 1539 cases of IBD and pooled analysis of 4805 cases of IBD validates the published association of a TLR4 allele with risk of IBD (odds ratio (OR): 1.30, 95% confidence interval (CI): 1.15 - 1.48; P = 0.00017) and Crohn's disease (OR: 1.33, 95% CI: 1.16 - 1.54; P = 0.000035) but not ulcerative colitis. We also describe novel suggestive evidence that TIRAP (OR: 1.16, 95% CI: 1.04 - 1.30; P = 0.007) has a modest effect on risk of IBD. Our analysis, therefore, offers additional evidence that the TLR4 pathway - in this case, TLR4 and its signaling molecule TIRAP - plays a role in susceptibility to IBD. ispartof: Genes and immunity vol:8 issue:5 pages:387-397 ispartof: location:England status: published

Background & Aims: Toll-like receptors (TLRs) represent a class of transmembrane pattern recognition receptors essential for microbial recognition and control of innate immune responses. Commensal bacteria play an important role in maintaining tolerance and active stability of the intestinal epithelial barrier by suppressing intestinal inflammation, yet the mechanisms of action are unknown. The aim of this study was to determine the functional relevance of TLR2 to control tight junction (TJ)-associated intestinal epithelial barrier integrity to balance mucosal homeostasis against inflammatory stress-induced damage. Methods: TLR2 ligand (synthetic Pam3Cys-SK4 [PCSK])-induced activation of signaling cascades and TJ-associated distribution was assessed by using Western blotting and confocal microscopy combined with functional transfection and inhibitor studies in model intestinal epithelial cell (IEC) lines (IEC-6, Caco-2) or primary IEC cultured short-term ex vivo. DSS colitis was induced by standard protocol in wild-type, TLR2−/−, and MyD88−/− mice. Spontaneous apoptosis was assessed by terminal deoxinucleotidyl-transferase-mediated dUTP-biotin nick end-labeling. Results: Data from in vitro and ex vivo models of intestinal epithelial cells revealed that TLR2 stimulation effectively preserves TJ-associated barrier assembly against stress-induced damage through promotion of PI3K/Akt-mediated cell survival via MyD88. Furthermore, in vivo studies underscored that TLR2-mediated TJ regulation critically determines susceptibility to intestinal injury and inflammation. Inflammatory stress in mice deficient of TLR2 or MyD88 induced early TJ-associated disruption interrelated with anti-apoptotic failure of the intestinal epithelial barrier. Oral treatment of colitis with the TLR2 ligand PCSK significantly suppressed mucosal inflammation and apoptosis by efficiently restoring TJ-associated integrity of the intestinal epithelium in vivo. Conclusion: TLR2 may provide a target to pharmacologically modulate mucosal injury and intestinal inflammation.

This review discusses the current progress in the understanding of how commensal-mediated activation of toll-like receptors (TLRs) may be involved in the regulation of physiological and pathophysiological processes of the intestinal mucosa including tissue regeneration and inflammation. While regulation of TLRs and their downstream signaling mediators might be used to prevent and treat inflammatory bowel diseases, paradoxically, at this time, it remains uncertain whether this would be more effectively accomplished by enhancing or inhibiting these pathways.

A 71-year-old woman with Crohn's disease, who had been receiving treatment with mercaptopurine and infliximab, had fatigue, blurred vision, headache and mood changes, and an inguinal mass. She had a sudden onset of left-sided weakness; brain imaging disclosed a mass, and she was transferred to this hospital. Shortly after admission, she had a seizure and became unresponsive.

With recent advances in the understanding of its pathophysiology, inflammatory bowel disease has become a very active area for the development of novel therapeutic agents. New targets for biologics include cytokines involved in T-cell activation, with antibodies directed against IL-12 and interferon-gamma. Selective adhesion molecule blockade has produced promising, though mixed, results. Recombinant human granulocyte-macrophage colony-stimulating factor might be effective in active Crohn's disease, presumably through stimulation of intestinal innate immune responses. With increasing evidence for a crucial role for luminal flora in maintaining the health of the bowel, strategies to manipulate intestinal bacteria using probiotics and prebiotics are being actively investigated as well.

Although our understanding of the pathogenesis of the chief forms of inflammatory bowel disease, Crohn's disease, and ulcerative colitis remains incomplete, progress is being made in identifying es...

Nucleotide oligomerization domain 2 (NOD2) functions as a mammalian cytosolic pathogen recognition molecule, and variants have been associated with risk for Crohn disease. We recently demonstrated that NOD2 functions as an anti-bacterial factor limiting survival of intracellular invasive bacteria. To gain further insight into the mechanism of NOD2 activation and signal transduction, we performed yeast two-hybrid screening. We demonstrate that GRIM-19, a protein with homology to the NADPH dehydrogenase complex, interacts with endogenous NOD2 in HT29 cells. GRIM-19 is required for NF-kappaB activation following NOD2-mediated recognition of bacterial muramyl dipeptide. GRIM-19 also controls pathogen invasion of intestinal epithelial cells. GRIM-19 expression is decreased in inflamed mucosa of patients with inflammatory bowel diseases. GRIM-19 may be a key component in NOD2-mediated innate mucosal responses and serve to regulate intestinal epithelial cell responses to microbes.

Nucleotide oligomerization domain (NOD) 2 functions as a mammalian cytosolic pathogen recognition molecule, and mutant forms have been genetically linked to Crohn's disease (CD). NOD2 associates with the caspase activation and recruitment domain of RIP-like interacting caspase-like apoptosis regulatory protein kinase (RICK)/RIP2 and activates nuclear factor (NF)–κB in epithelial cells and macrophages, whereas NOD2 mutant 3020insC, which is associated with CD, shows an impaired ability to activate NF-κB. To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants. NOD2, but not NOD2 3020insC mutant, associated with cell surface membranes of intestinal epithelial cells. Membrane targeting and subsequent NF-κB activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2. The membrane targeting of NOD2 is required for NF-κB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

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.

The “Immune Regulatory Networks” meeting in Boston gathered scientists with a passion for regulatory T cells to discuss the latest information on an increasingly important subject.

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.

We report the experience of 11 patients (of 60 planned patients) enrolled in a double-blind, placebo-controlled clinical trial of infliximab in patients with severe, active steroid-refractory ulcerative colitis. The study was terminated prematurely because of slow enrollment. Patients having active disease for at least 2 weeks and receiving at least 5 days of intravenous corticosteroids were eligible to receive a single intravenous infusion of infliximab at 5, 10, or 20 mg/kg body weight. The primary endpoint used in this study was treatment failure at 2 weeks after infusion. Treatment failure was defined as 1) unachieved clinical response as defined by a modified Truelove and Witts severity score, 2) increase in corticosteroid dosage, 3) addition of immunosuppressants, 4) colectomy, or 5) death. Safety evaluations included physical examination, clinical chemistry and hematology laboratory tests, and occurrence of adverse experiences. Four of 8 patients (50%) who received infliximab were considered treatment successes at 2 weeks, compared with none of 3 patients who received placebo. Improvement in erythrocyte sedimentation rates and serum concentrations of C-reactive protein and interleukin-6 correlated with the clinical response observed in patients receiving infliximab. Infusion with infliximab produced no significant adverse events. Infliximab was well tolerated and may provide clinical benefit for some patients with steroid-refractory ulcerative colitis.

Summary Intestinal epithelial cells are capable of producing a variety of cytokines and other regulatory factors that can affect functional regulation of the epithelium itself, through autocrine and paracrine mechanisms, as well as functional integration with lamina propria populations. The bi-directional nature of this cytokine network is now apparent, with the demonstration that both rat and human intestinal epithelium-derived cell lines possess a much greater array of cytokine receptors than previously anticipated.

Well-coordinated mechanisms have evolved that provide both innate protection against gastrointestinal mucosal injury and facilitation of rapid mucosal repair following mucosal damage. Generic protection from injury is provided by intrinsic structural features of the epithelium that form a highly competent barrier and a complex formed at the apical surface by trefoil peptides that comprise the interface between mucosa and lumen. When the epithelial barrier has been broken, regardless of the nature of the injury, epithelial surface continuity is rapidly reestablished through restitution as cells migrate and elongate. This process is promoted by trefoil peptides at the apical surface and a large array of cytokines and growth factors acting at the basolateral pole. Many of these regulatory peptides are products of the immune and other lamina propria cell populations, which are activated following disruption of the mucosal barrier. Thus efforts to repair the epithelium follow inherently from inflammatory effects after initial damage; the repair process in turn may allow abrogation of further inflammation. Ultimate repair of injury requires both proliferative replacement of damaged epithelial cells and remodeling of extracellular matrix and deeper cell populations to restore normal architecture and a fully functional mucosa.

Background Enterocutaneous fistulas are a serious complication of Crohn's disease and are difficult to treat. Infliximab, a chimeric monoclonal antibody to tumor necrosis factor alpha, has recently been developed as a treatment for Crohn's disease. We conducted a randomized, multicenter, double-blind, placebo-controlled trial of infliximab for the treatment of fistulas in patients with Crohn's disease. Methods The study included 94 adult patients who had draining abdominal or perianal fistulas of at least three months' duration as a complication of Crohn's disease. Patients were randomly assigned to receive one of three treatments: placebo (31 patients), 5 mg of infliximab per kilogram of body weight (31 patients), or 10 mg of infliximab per kilogram (32 patients); all three were to be administered intravenously at weeks 0, 2, and 6. The primary end point was a reduction of 50 percent or more from base line in the number of draining fistulas observed at two or more consecutive study visits. A secondary end point was the closure of all fistulas. Results Sixty-eight percent of the patients who received 5 mg of infliximab per kilogram and 56 percent of those who received 10 mg per kilogram achieved the primary end point, as compared with 26 percent of the patients in the placebo group (P=0.002 and P=0.02, respectively). In addition, 55 percent of the patients assigned to receive 5 mg of infliximab per kilogram and 38 percent of those assigned to 10 mg per kilogram had closure of all fistulas, as compared with 13 percent of the patients assigned to placebo (P=0.001 and P=0.04, respectively). The median length of time during which the fistulas remained closed was three months. More than 60 percent of patients in all the groups had adverse events. For patients treated with infliximab, the most common were headache, abscess, upper respiratory tract infection, and fatigue. Conclusions Infliximab is an efficacious treatment for fistulas in patients with Crohn's disease.

It seems that until relatively recently, mentorship was hardly a topic of comment in academic discourse. Without doubt, the historical neglect of the role of mentors as appropriate for explicit discussion was a significant omission. However, Although my tutelage in having Kurt as a mentor may have been longer in duration than any others (as I believe) I am but one of legions of students, GI and research fellows, and faculty whose careers have been touched, and guided by Kurt as a mentor. Without a doubt, Kurt’s vicarious impact is truly remarkable. His nearly 150 fellows and postdoctoral trainees include (by latest count) 2 university presidents, 1 medical center president, 9 center directors or department chairs, 26 division chiefs, 24 research laboratory directors, and 92 academic full-time faculty including 48 professors. In addition to those who have pursued their careers within academe, there are also many who learned to become expert and empathic clinicians and have gone on to serve as clinical

The gastrointestinal epithelium produces a wide variety of peptides which may contribute to protection from injury as well as repair after injury occurs. Restitution, the initial phase of mucosal repair, is accomplished by rapid migration of the epithelium to re-establish surface epithelial continuity. A wide variety of growth factors and cytokines, which are produced both by the epithelium itself and by lamina propria cell populations, promote restitution in models of epithelial injury. These include members of the epidermal growth factor (EGF)/transforming growth factor (TGF)alpha, and the fibroblast growth factor (FGF) families, as well as a variety of cytokines (interleukin [IL]-1, IL-2, IL-4, IL-15, and interferon gamma) which interact with their cognate receptors on the intestinal epithelial basolateral surface. These growth factors and cytokines appear to promote restitution through a TGF beta-dependent pathway and act to both enhance expression of TGF beta and to entrance its bioactivation. In contrast, trefoil peptides, members of a recently recognized family of small proteins produced by goblet cells, both protect the epithelium and promote restitution following secretion onto the apical surface through mechanisms distinct from those peptides acting through TGF beta. Thus, rapid repair after epithelial injury is achieved through complementary mechanisms acting at the basolateral and apical surfaces of the epithelium.

The mechanisms that maintain the epithelial integrity of the gastrointestinal tract remain largely undefined. The gene encoding intestinal trefoil factor (ITF), a protein secreted throughout the small intestine and colon, was rendered nonfunctional in mice by targeted disruption. Mice lacking ITF had impaired mucosal healing and died from extensive colitis after oral administration of dextran sulfate sodium, an agent that causes mild epithelial injury in wild-type mice. ITF-deficient mice manifested poor epithelial regeneration after injury. These findings reveal a central role for ITF in the maintenance and repair of the intestinal mucosa.

The unique three-loop structure of the trefoil motif, formed by intrachain disulfide bonds in a 1-5, 2-4, 3-6 configuration between six conserved cysteine residues, is the defining feature of a recently recognized family of peptides. Expression of trefoil peptides is closely related to that of mucin glycoproteins in diverse biological sources. Three distinct members of the family (pS2, intestinal trefoil factor, and spasmolytic polypeptide) are produced in the mammalian gastrointestinal tract by mucus-secreting cells and targeted primarily for luminal secretion. The compact structure of the trefoil motif may be responsible for marked resistance of trefoil peptides to proteolytic digestion, enabling them to function in the harsh environment of the gastrointestinal lumen. Trefoil peptides are ectopically expressed adjacent to areas of inflammation within the gastrointestinal tract and may play an important role in both maintaining the barrier function of mucosal surfaces and facilitating healing after injury.

Recent studies have suggested that nitric oxide (NO.), the product of nitric oxide synthase in inflammatory cells, may play a part in tissue injury and inflammation through its oxidative metabolism. In this study the colonic generation of oxides of nitrogen (NOx) and nitric oxide synthase activity was determined in ulcerative colitis and Crohn's disease. Colonic biopsy specimens were obtained from inflammatory bowel disease patients and from normal controls. Mucosal explants were cultured in vitro for 24 hours and NOx generation was determined. Nitric oxide synthase activity was monitored by the conversion of [3H]-L-arginine to citrulline. Median NOx generation by inflamed colonic mucosa of patients with active ulcerative colitis and Crohn's colitis was 4.2- and 8.1-fold respectively higher than that by normal human colonic mucosa. In ulcerative colitis and Crohn's colitis nitric oxide synthase activity was 10.0- and 3.8-fold respectively higher than in normal subjects. Colonic NOx generation is significantly decreased by methylprednisolone and ketotifen. The decrease in NOx generation by cultured colonic mucosa induced by methylprednisolone suggests that NO synthase activity is induced during the culture and the steroid effect may contribute to its therapeutic effect. Enhanced colonic NOx generation by stimulated nitric oxide synthase activity in ulcerative colitis and Crohn's disease may contribute to tissue injury.

The epithelium of the gastrointestinal tract mucosa is a highly dynamic and diverse mixture of cell populations requiring exquisite integration of the processes of cellular proliferation, differentiation, and senescence. It is likely that the proliferative compartment of the intestinal epithelium encompasses a hierarchy of totipotent and pluripotent stem cells in a manner similar to that which generates diversity in hematopoietic cell populations. Identification and characterization of the stem cell and progenitor populations in the intestine has been limited by the absence of markers or culture systems to identify these cells. Regulation of the proliferative compartment may be accomplished through the combined integration of key peptide growth factors and constituents of the extracellular matrix. The relative contribution of the epithelial populations themselves and the contributions made by associated cell populations such as pericryptal fibroblasts remain unclear. Recent studies have suggested that the transforming growth factors-alpha and -beta, two structurally unrelated peptide growth factors, might serve to regulate the balanced proliferation and turnover of intestinal epithelial cells. The proproliferative effects of TGF-alpha may be counterbalanced by the proliferation-inhibiting TGF-beta. Recent studies have demonstrated close interregulation of these peptides, which act through autocrine and paracrine mechanisms in model intestinal epithelial cell lines.

Chronic inflammatory bowel diseases appear to result from inappropriate immune responses driven by apparently normal intestinal microflora in genetically susceptible hosts. This review focuses on recently described mechanisms balancing toll-like receptor and nucleotide-binding-oligomerization domain activation in the face of ubiquitous enteric flora.Toll-like receptor and nucleotide-binding-oligomerization domain signaling plays an integral role in the close collaboration between the intestinal epithelial cell monolayer and adjacent mucosal immune cells. Pathways activated by functional cytosolic nucleotide-binding-oligomerization domain proteins appear to interact with those mediated by membrane-associated toll-like receptors in the innate and adaptive immune defense against intra-and extracellular pathogens. Nucleotide-binding-oligomerization domain-mediated signaling may also control toll-like receptor-induced proinflammatory pathways.Intersections between toll-like receptor and nucleotide-binding-oligomerization domain pathways may exist to refine the host immune response to pathogens and prevent undesired immune stimulation driven by the intestinal microbiota. Deficient toll-like receptor and nucleotide-binding-oligomerization domain function due to genetic variability is associated with an increased susceptibility to the development of inflammatory bowel disease.

This brief review summarizes the current understanding of Toll-like receptor (TLRs) mediated intestinal epithelial mechanisms of commensal tolerance versus intolerance and provides an update on the downstream negative control of signaling responses through decreased surface expression, interregulation with NOD2, overexpression of Tollip, various inhibitors of NF-kappaB as well as soluble tolerizing mediators present in lumen and serum which all may maintain or--when dysregulated--impair mucosal homeostasis in health or disease, respectively.

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.

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.

Primary sclerosing cholangitis (PSC) predisposes to the development of cholangiocarcinoma, a usually fatal complication that is difficult to diagnose. Serum concentrations of CA 19-9, a tumor-associated antigen, are frequently increased in patients with only cholangiocarcinoma. The aim of this study was to assess the value of an increased serum CA 19-9 level for the diagnosis of cholangiocarcinoma in patients with preexisting PSC. We analyzed serum samples from 9 patients with PSC and superimposed cholangiocarcinoma and from 28 patients with only PSC. Serum concentrations of CA 19-9 were measured in a blinded manner with use of an immunoradiometric assay. The serum CA 19-9 concentrations were increased in 8 of 9 patients (89) with PSC and cholangiocarcinoma (mean±SE, 391 ± 86 U/ml; range, 4 to 677), whereas they were increased in only 4 of 28 patients (14) with only PSC (mean ± SE, 61 ± 16 U/ml; range, 2 to 370). The sensitivity of a CA 19-9 value greater than 100 U/ml for cholangiocarcinoma in PSC was 89, and the specificity was 86. The measurement of serum concentrations of CA 19-9 is a promising test for detecting cholangiocarcinoma in patients with PSC.

Trefoil peptides are a family of small proteins expressed by goblet cells that are secreted onto the apical gastrointestinal mucosal surface, where they are present in high concentrations. These peptides appear to both protect the epithelium and promote healing after injury. However, the factors regulating the expression and secretion of these proteins contributing to mucosal defense have not been characterized. To determine the mechanisms controlling production of trefoil peptides, the human colon cancer-derived model cell line HT-29 was exposed to a variety of potential secretagogues. Expression and secretion of human intestinal trefoil factor (hITF) as well as the intestinal apomucin MUC2 were assessed by Northern and Western blot analysis. Carbachol, an analog of acetylcholine, and the neuroendocrine peptides somatostatin and vasoactive intestinal polypeptide (VIP) stimulated increased expression of hITF mRNA within 5 min. These same factors stimulated parallel secretion of the hITF peptide, with maximal stimulation observed at concentrations ranging from 10(-6) M (carbachol and somatostatin) to 10(-7) M (VIP). Expression and secretion of hITF in response to carbachol, VIP, and somatostatin was independent of production of apomucin. hITF was not regulated by other neuroendocrine transmitters including histamine and substance P. Similarly, hITF expression and secretion was not modulated by peptide growth factors (epidermal growth factor, transforming growth factor-beta, and keratinocyte growth factor), cytokines [interleukin (IL)-1 beta, IL-2, IL-7, and IL-11], or arachidonic acid metabolites (prostaglandin E1/E2 and leukotriene B4). In conclusion, trefoil peptides appear to be integrated into mechanisms of mucosal defense and repair through the enteric neuroendocrine system and independent of the classical mucosal immune cytokine network.

Microbiologists have long been puzzled by the finding that the gut mucosa does not respond to the myriad varieties of bacteria that normally reside in the gut. As [Xavier and Podolsky][1] explain in their Perspective, this may be because bacteria that are indigenous to the gut have learned ways to switch off pathways in gut epithelial cells that lead to switching on of genes involved in inflammation ([ Neish et al ][2].). [1]: http://www.sciencemag.org/cgi/content/full/289/5484/1483 [2]: http://www.sciencemag.org/cgi/content/short/289/5484/1560

Available screening methods should make it possible to prevent most deaths from colorectal cancer. Almost all colorectal cancers arise from adenomatous polyps that develop over a period of years. During this time, polyps can be detected and then removed by colonoscopic polypectomy.1 The U.S. Preventive Services Task Force, the American Cancer Society, and a multidisciplinary expert panel assembled by the Agency for Health Care Policy and Research and the American Gastroenterological Association, as well as other groups, have all recommended screening for colorectal cancer.2–6 Standard recommendations include annual testing for fecal occult blood and periodic sigmoidoscopy after the age . . .