Epithelial-specific Toll-like Receptor (TLR)5 Activation Mediates Barrier Dysfunction in Experimental Ileitis.

BACKGROUND: A large body of evidence supports a central role of TLR5 and its natural ligand, flagellin, in Crohn's disease (CD), with the precise mechanism(s) still unresolved. METHODS: We investigated the role of flagellin/TLR5 in SAMP1/YitFc (SAMP) mice, a spontaneous model of Crohn's disease-like ileitis. RESULTS: Ileal Tlr5 and serum antiflagellin IgG antibodies were increased in SAMP before the onset of inflammation and during established disease; these trends were abrogated in the absence of colonizing commensal bacteria. Irradiated SAMP receiving either wild-type (AKR) or SAMP bone marrow (BM) developed severe ileitis and displayed increased ileal Tlr5 compared with AKR recipients of either SAMP or AKR bone marrow, neither of which conferred ileitis, suggesting that elevated TLR5 in native SAMP is derived primarily from a nonhematopoietic (e.g., epithelial) source. Indeed, ileal epithelial TLR5 in preinflamed SAMP was increased compared with age-matched AKR and germ-free SAMP. TLR5-specific ex vivo activation of SAMP ileal tissues decreased epithelial barrier resistance, indicative of increased permeability, and was accompanied by altered expression of the tight junction proteins, claudin-3, occludin, and zonula occludens-1. CONCLUSIONS: Our results provide evidence that aberrant, elevated TLR5 expression is present in the ileal epithelium of SAMP mice, is augmented in the presence of the gut microbiome, and that TLR5 activation in response to bacterial flagellin results in a deficiency to maintain appropriate epithelial barrier integrity. Together, these findings represent a potential mechanistic pathway leading to the exacerbation and perpetuation of chronic gut inflammation in experimental ileitis and possibly, in patients with Crohn's disease.

Abnormal intestinal permeability in Crohn's disease pathogenesis.

Increased small intestinal permeability is a longstanding observation in both Crohn's disease patients and in their healthy, asymptomatic first-degree relatives. However, the significance of this compromised gut barrier function and its place in the pathogenesis of the disease remains poorly understood. The association between abnormal small intestinal permeability and a specific mutation in the NOD2 gene, which functions to modulate both innate and adaptive immune responses to intestinal bacteria, suggests a common, genetically determined pathway by which an abnormal gut barrier could result in chronic intestinal inflammation. Furthermore, rodent colitis models show that gut barrier defects precede the development of inflammatory changes. However, it remains possible that abnormal permeability is simply a consequence of mucosal inflammation. Further insight into whether abnormal barrier function is the cause or consequence of chronic intestinal inflammation will be crucial to understanding the role of intestinal permeability in the pathogenesis of Crohn's disease.

Targeted epithelial tight junction dysfunction causes immune activation and contributes to development of experimental colitis.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is a multifactorial disease thought to be caused by alterations in epithelial function, innate and adaptive immunity, and luminal microbiota. The specific role of epithelial barrier function remains undefined, although increased activity of intestinal epithelial myosin light chain kinase (MLCK), which is the primary mechanism of tumor necrosis factor-induced barrier dysfunction, occurs in human IBD. Our aim was to determine whether, in an intact epithelium, primary dysregulation of the intestinal epithelial barrier by pathophysiologically relevant mechanisms can contribute to development of colitis. METHODS: We developed transgenic (Tg) mice that express constitutively active MLCK (CA-MLCK) specifically within intestinal epithelia. Their physiology, immune status, and susceptibility to disease were assessed and compared with non-Tg littermate controls. RESULTS: CA-MLCK Tg mice demonstrated significant barrier loss but grew and gained weight normally and did not develop spontaneous disease. CA-MLCK Tg mice did, however, develop mucosal immune activation demonstrated by increased numbers of lamina propria CD4(+)lymphocytes, redistribution of CD11c+cells, increased production of interferon-gamma and tumor necrosis factor, as well as increased expression of epithelial major histocompatibility complex class I. When challenged with CD4+CD45+Rb(hi) lymphocytes, Tg mice developed an accelerated and more severe form of colitis and had shorter survival times than non-Tg littermates. CONCLUSIONS: Primary pathophysiologically relevant intestinal epithelial barrier dysfunction is insufficient to cause experimental intestinal disease but can broadly activate mucosal immune responses and accelerate the onset and severity of immune-mediated colitis.

A key claudin extracellular loop domain is critical for epithelial barrier integrity.

Intercellular tight junctions (TJs) regulate epithelial barrier properties. Claudins are major structural constituents of TJs and belong to a large family of tetra-spanning membrane proteins that have two predicted extracellular loops (ELs). Given that claudin-1 is widely expressed in epithelia, we further defined the role of its EL domains in determining TJ function. The effects of several claudin-1 EL mimetic peptides on epithelial barrier structure and function were examined. Incubation of model human intestinal epithelial cells with a 27-amino acid peptide corresponding to a portion of the first EL domain (Cldn-1(53-80)) reversibly interfered with epithelial barrier function by inducing the rearrangement of key TJ proteins: occludin, claudin-1, junctional adhesion molecule-A, and zonula occludens-1. Cldn-1(53-80) associated with both claudin-1 and occludin, suggesting both the direct interference with the ability of these proteins to assemble into functional TJs and their close interaction under physiological conditions. These effects were specific for Cldn-1(53-80), because peptides corresponding to other claudin-1 EL domains failed to influence TJ function. Furthermore, the oral administration of Cldn-1(53-80) to rats increased paracellular gastric permeability. Thus, the identification of a critical claudin-1 EL motif, Cldn-1(53-80), capable of regulating TJ structure and function, offers a useful adjunct to treatments that require drug delivery across an epithelial barrier.

Effects of oral adenosine 5'-triphosphate and adenosine in enteric-coated capsules on indomethacin-induced permeability changes in the human small intestine: a randomized cross-over study.

BACKGROUND: It is well-known that nonsteroidal anti-inflammatory drugs (NSAIDs) can cause damage to the small bowel associated with disruption of mucosal barrier function. In healthy human volunteers, we showed previously that topical administration of adenosine 5'-triphosphate (ATP) by naso-intestinal tube attenuated a rise in small intestinal permeability induced by short-term challenge with the NSAID indomethacin. This finding suggested that ATP may be involved in the preservation of intestinal barrier function. Our current objective was to corroborate the favourable effect of ATP on indomethacin-induced permeability changes in healthy human volunteers when ATP is administered via enteric-coated capsules, which is a more practically feasible mode of administration. Since ATP effects may have been partly mediated through its breakdown to adenosine, effects of encapsulated adenosine were tested also. METHODS: By ingesting a test drink containing 5 g lactulose and 0.5 g L-rhamnose followed by five-hour collection of total urine, small intestinal permeability was assessed in 33 healthy human volunteers by measuring the urinary lactulose/rhamnose excretion ratio. Urinary excretion of lactulose and L-rhamnose was determined by fluorescent detection high-pressure liquid chromatography (HPLC). Basal permeability of the small intestine was assessed as a control condition (no indomethacin, no ATP/adenosine). As a model of increased small intestinal permeability, two dosages of indomethacin were ingested at 10 h (75 mg) and 1 h (50 mg) before ingesting the lactulose/rhamnose test drink. At 1.5 h before indomethacin ingestion, two dosages of placebo, ATP (2 g per dosage) or adenosine (1 g per dosage) were administered via enteric-coated hydroxypropyl methylcellulose (HPMC) capsules with Eudragit L30D-55. RESULTS: Median urinary lactulose/rhamnose excretion ratio (g/g) in the control condition was 0.032 (interquartile range: 0.022-0.044). Compared to the control condition, lactulose/rhamnose ratio after ingestion of indomethacin plus placebo was significantly increased to 0.039 (0.035-0.068); P < 0.01). The indomethacin-induced increase was neither affected by administration of encapsulated ATP (0.047 (0.033-0.065)) nor adenosine (0.050 (0.030-0.067)). Differences in L/R ratios between the conditions with indomethacin plus placebo, ATP or adenosine were not significant. CONCLUSION: In this study, either ATP or adenosine administered via enteric-coated capsules had no effect on indomethacin-induced small intestinal permeability changes in healthy human volunteers. The observed lack of effect of encapsulated ATP/adenosine may have been caused by opening of the enteric-coated supplement at a site distal from the indomethacin-inflicted site. Further studies on site-specific effectiveness of ATP/adenosine on intestinal permeability changes are warranted.

Post-endoscopy checklist reduces length of stay for non-variceal upper gastrointestinal bleeding.

OBJECTIVE: To examine the effect of improved gastroenterologist-to-admitting service communication on hospital stay for upper gastrointestinal bleeding. HYPOTHESIS: a detailed checklist addressing factors relevant to discharge planning would shorten hospital stay, when added to the procedure report. DESIGN: Pre-post intervention design, recording balance measures (potential confounders). SETTING: A Canadian university hospital. STUDY PARTICIPANTS: Intermittent 5- to 7-day batches of consecutive emergency patients presenting with non-variceal upper gastrointestinal bleeding as their primary problem. The durations of the background and intervention periods were 3 months (beginning 9 June 2003) and 4 weeks (beginning 8 September 2003), respectively. INTERVENTION: The gastrointestinal bleeding Quality Improvement and Health Information multidisciplinary team (quality improvement personnel; emergency physicians, hospitalists, gastroenterologists, in-patient and endoscopy nurses) developed a one-page checklist, outlining detailed recommendations (3-Ds-diet, drugs, discharge plan) to append to the procedure report. MAIN OUTCOME MEASURES: Difference in median length of hospital stay was the primary endpoint. As balance measures, demographics, bleeding severity, comorbidities, readmission rates, and various benchmark times were recorded prospectively. RESULTS: Thirty-nine patients met the criteria in the background period (4 months, intermittently sampled), and 22 in the intervention period (4 weeks, continuously sampled). There were no significant baseline differences. Median in-patient stay was 7.0 (95% interquartile range 2-24) versus 3.5 (95% interquartile range 1-12) days for the background and intervention periods, respectively (P = 0.003). This remained significant when outliers (stay > 10 days) were removed (P = 0.02). CONCLUSION: A checklist, with very specific recommendations to the admitting service, significantly reduced hospital stay for non-variceal gastrointestinal bleeding.

3-0 methylglucose uptake as a marker of nutrient absorption and bowel length in pediatric patients.

BACKGROUND: Inert carbohydrate probes are commonly used to assess intestinal permeability; we have previously shown that the actively transported moiety 3-0 methylglucose (3-0 MG) is a useful marker of intestinal surface area and nutrient absorption in animal models of short bowel syndrome (SBS). This study examines the correlation of 3-0 MG absorption with nutrient absorption, bowel length, and the tolerance of enteral feeds in pediatric patients. METHODS: Fifteen children (1 month to 15 years in age) were studied after intestinal surgery. All had a stoma, 2 were > 1 year of age, the remainder had surgical intervention as a neonate or within the first month of life. Eight had SBS (50% expected bowel length for age). Bowel length was measured intraoperatively. Nutrient absorption was quantified with a 48-hour bowel study, measuring fat, protein, and carbohydrate output directly. 3-0 MG absorption and intestinal permeability were quantified using a solution containing 30 mg/mL 3-0 MG, 20 mg/mL mannitol and 30 mg/mL lactulose (osmolarity 352, given at 1 mL/kg via feeding tube). Subsequent urine production was collected for 8 hours, and probe recovery measured using HPLC. RESULTS: 3-0 MG absorption was significantly correlated with nutrient absorption. The correlation with protein absorption was r2 = .59, fat r2 = .62 and carbohydrate r2 = .56. The correlation between 3-0 MG absorption and bowel length was r2 = .58. 3-0 MG absorption was significantly lower in SBS patients vs patients with normal bowel length (15.8 +/- 6.7 vs 30.5 +/- 10.2%). 3-0 MG absorption also correlated with the ability to tolerate enteral feeds (r2 = .38; p < .03 for all comparisons). CONCLUSIONS: 3-0 MG may be a useful marker of nutrient absorption and bowel length in pediatric patients with short bowel syndrome. The simplicity and reproducibility of the method make it an attractive option for following patient outcomes. Further studies are suggested to determine the utility of these markers in directing the clinical management of patients.