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Objectives: Intestinal epithelial cells (IEC) express toll-like receptors (TLR) that facilitate microbial recognition. Stimulation of TLR ligands induces a transient increase in epithelial cell shedding, a mechanism that serves the antibacterial and antiviral host defence of the epithelium and promotes elimination of intracellular pathogens. Although activation of the extrinsic apoptosis pathway has been described during inflammatory shedding, its functional involvement is currently unclear., Design: We investigated the functional involvement of caspase-8 signalling in microbial-induced intestinal cell shedding by injecting Lipopolysaccharide (LPS) to mimic bacterial pathogens and poly(I:C) as a probe for RNA viruses in vivo., Results: TLR stimulation of IEC was associated with a rapid activation of caspase-8 and increased epithelial cell shedding. In mice with an epithelial cell-specific deletion of caspase-8 TLR stimulation caused Rip3-dependent epithelial necroptosis instead of apoptosis. Mortality and tissue damage were more severe in mice in which IECs died by necroptosis than apoptosis. Inhibition of receptor-interacting protein (Rip) kinases rescued the epithelium from TLR-induced gut damage. TLR3-induced necroptosis was directly mediated via TRIF-dependent pathways, independent of Tnf-α and type III interferons, whereas TLR4-induced tissue damage was critically dependent on Tnf-α., Conclusions: Together, our data demonstrate an essential role for caspase-8 in maintaining the gut barrier in response to mucosal pathogens by permitting inflammatory shedding and preventing necroptosis of infected cells. These data suggest that therapeutic strategies targeting the cell death machinery represent a promising new option for the treatment of inflammatory and infective enteropathies., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objectives: Loss of intestinal barrier function plays an important role in the pathogenesis of inflammatory bowel disease (IBD). Shedding of intestinal epithelial cells is a potential cause of barrier loss during inflammation. The objectives of the study were (1) to determine whether cell shedding and barrier loss in humans can be detected by confocal endomicroscopy and (2) whether these parameters predict relapse of IBD., Methods: Confocal endomicroscopy was performed in IBD and control patients using intravenous fluorescein to determine the relationship between cell shedding and local barrier dysfunction. A grading system based on appearances at confocal endomicroscopy in humans was devised and used to predict relapse in a prospective pilot study of 47 patients with ulcerative colitis and 11 patients with Crohn's disease., Results: Confocal endomicroscopy in humans detected shedding epithelial cells and local barrier defects as plumes of fluorescein effluxing through the epithelium. Mouse experiments demonstrated inward flow through some leakage-associated shedding events, which was increased when luminal osmolarity was decreased. In IBD patients in clinical remission, increased cell shedding with fluorescein leakage was associated with subsequent relapse within 12 months after endomicroscopic examination (p<0.001). The sensitivity, specificity and accuracy for the grading system to predict a flare were 62.5% (95% CI 40.8% to 80.4%), 91.2% (95% CI 75.2 to 97.7) and 79% (95% CI 57.7 to 95.5), respectively., Conclusions: Cell shedding and barrier loss detected by confocal endomicroscopy predicts relapse of IBD and has potential as a diagnostic tool for the management of the disease.

Objective: To assess the safety and efficacy of the B lymphocyte (anti-CD20) antibody, rituximab, in the treatment of steroid-resistant moderately active ulcerative colitis (UC)., Methods: A double-blinded, randomised controlled trial with a 2:1 ratio of treatment:placebo (phase II) was carried out in the setting of a University teaching hospital. The subjects comprised 24 patients with moderately active UC who have either failed to respond to conventional corticosteroid therapy or who have relapsed during corticosteroid withdrawal. Five of 8 placebo-treated patients and 12 of 16 rituximab-treated patients were receiving azathioprine, 6-mercaptopurine or methotrexate. Two infusions of rituximab 1 g in 500 ml of 0.9% saline intravenously over 4 h (n=16) or saline placebo (n=8) were given at 0 and 2 weeks. Patients still receiving corticosteroids on entry (placebo group 7/8; rituximab group 14/16) continued a standard steroid tapering regimen. The primary end point was remission (Mayo score ≤ 2) at 4 weeks. Secondary end points included response (Mayo score reduced ≥ 3) at 4 and 12 weeks., Results: Mayo score at entry was higher in rituximab-treated patients (mean 9.19; 95% CI 8.31 to 10.06) than for placebo patients (7.63; 6.63 to 8.62, p=0.03). At week 4 only 1/8 placebo-treated patients and 3/16 rituximab-treated patients were in remission (p=1.0), but 8/16 rituximab-treated patients had responded compared with 2/8 placebo-treated patients, with a median reduction in Mayo score of 2.5 (rituximab) compared with 0 (placebo; p=0.07). This response was only maintained to week 12 in 4/16. Mucosal healing was seen at week 4 in 5/16 rituximab-treated patients and 2/8 placebo-treated patients (non-significant). Rituximab was well tolerated, with one chest infection, three mild infusion reactions plus one case of (probably unrelated) non-fatal pulmonary embolism. CONCLUSIONS Rituximab has no significant effect on inducing remission in moderately active UC not responding to oral steroids. There was a possible short-term response that was not sustained. Rituximab is well tolerated in UC., Clinical Trial Number: NCT00261118.

Background and Objectives: Interaction of bacteria with the immune system within the intestinal mucosa plays a key role in the pathogenesis of inflammatory bowel disease (IBD). The aim of the current study was to develop a fluorescein-aided confocal laser endomicroscopy (CLE) method to visualise intramucosal enteric bacteria in vivo and to determine the involved mucosal area in the colon and ileum in patients with ulcerative colitis (UC) and Crohn's disease (CD)., Methods: Initially, E coli strains expressing enhanced green fluorescent protein (pEGFP) were endomicroscopically imaged in mice. In addition, ex vivo and in vivo imaging of fluorescent human enteric bacteria was performed to specify the distinct endomicroscopic appearance of enteral bacteria. Targeted mucosal biopsies towards endomicroscopic identifiable intramucosal bacteria and negative mucosal areas were prospectively obtained during colonoscopy and correlated with bench-top fluorescence microscopy (FISH) to prove the endomicroscopic visualisation of intramucosal bacteria. Finally, a retrospective analysis as well as a prospective study was performed in patients with UC and CD to confirm the presence and distribution of intramucosal bacteria within the gut., Results: Confocal endomicroscopy was able to identify intramucosal pEGFP E coli in mice and strains of enteric microflora in the mucosa of humans. Using FISH as the gold standard, evaluation of 21 patients showed that CLE had a sensitivity of 89% and specificity of 100% to identify intramucosal bacteria. In a retrospective study, 113 patients with CD and UC had intramucosal bacteria significantly more often than 50 control patients (66% vs 60% vs 14%, p<0.001). This result was confirmed in a prospective study in which 10 patients with CD and 10 with UC had a significantly wider distribution of involvement with intramucosal bacteria in the colon and terminal ileum compared with 10 controls (85.2% vs 75.9% vs 16.8%, p<0.0001)., Conclusions: CLE is a new tool that can image intramucosal bacteria in vivo in patients with IBD. Intramucosal bacteria are found more frequently and with a wider distribution in patients with IBD than in patients with a normal intestine.

Reactive oxygen metabolites have been implicated in causing epithelial cell injury in colonic inflammation. A model of oxidant injury in intestinal epithelial cells has been developed in which HT-29-18-C1 cells are injured with graded concentrations of hydrogen peroxide and characterised by the MTT test. The MTT test was validated as a cytotoxicity assay and has a similar sensitivity to hydrogen peroxide induced injury as the assay of intracellular adenosine triphosphate. Exposure to a range of hydrogen peroxide concentrations (0.05-20 mM) for varying duration (5-120 min) showed that injury was dependent on time and concentration. The median lethal dose (LD50) for one hour exposure to hydrogen peroxide was approximately 0.1 mM. Injury from hydrogen peroxide was only partially reversible as determined by the MTT test and assay of cellular proliferation by crystal violet staining. There was an exponential loss of hydrogen peroxide when incubated with HT-29-18-C1 cells (t1/2 35 min). Experiments with 0.5 mg/ml aminotriazole and 0.5-2 mM buthionine sulphoximine suggested hydrogen peroxide breakdown was predominantly caused by catalase rather than glutathione peroxidase. Injury resulting from 1 mM hydrogen peroxide could be reduced by either coincubation of cells with 1,10-phenanthroline, an Fe2+ chelator, or preincubation with deferoxamine, and Fe3+ chelator, suggesting the participation of Fe2+ and Fe3+ in hydrogen peroxide induced injury. In conclusion, hydrogen peroxide induces injury in HT-29-18-C1 cells both directly and by generation of the hydroxyl radical.

In situ perfusion of whole rat small intestine was used to compare the efficacy of five oral rehydration solutions in promoting water and sodium absorption in normal intestine and secreting intestine after exposure to cholera toxin. Solutions varied in their sodium (35-90 mmol/l) and glucose (111-200 mmol/l) concentrations, molar ratio of glucose:sodium (1.2-5.8), and osmolality (281-331 mOsmol/kg), and contained either bicarbonate (18-30 mmol/l) or citrate (10 mmol/l). In normal intestine all solutions promoted net water absorption. Cholera toxin induced reproducible water secretion but all solutions reversed this to absorption. Water absorption was greatest with solutions containing sodium 60 mmol/l and glucose 111 or 140 mmol/l, and with a glucose:sodium ratio approximately 2, in both normal and secreting intestine. All solutions promoted net glucose absorption in both normal and secreting intestine. Net sodium absorption occurred with solutions containing greater than or equal to 60 mmol/l sodium in normal intestine but sodium secretion occurred from all solutions in secreting intestine. Sodium movement was directly related to the sodium concentration of the solution and sodium secretion occurred despite net water and glucose absorption. We consider that these studies may guide future development of oral rehydration solutions.

The mechanism of acetate absorption in the small intestine is not yet established. One possible mechanism is by carrier mediated Na(+)-acetate cotransport since acetate, like glucose, stimulates intestinal Na+ and water absorption in vivo. Uptake of radioactive carbon acetate by small intestinal brush border membrane vesicles was not saturable or Na+ dependent and did not respond to osmotic shrinkage of the vesicles. This suggests that acetate binds to the membranes but is not transported into the intravesicular space and argues against carrier mediated Na+ acetate cotransport. These results are consistent with acetate absorption by a non-mediated diffusion and suggest that the stimulation of water and Na+ absorption by acetate in vivo is largely due to osmotic forces and solvent drag.

Acetate absorption was studied in rat jejunum using steady state perfusion in vivo. Absorption conformed to apparent saturation kinetics and was similar in magnitude to glucose absorption. When compared with normal saline, acetate perfusion was associated with luminal alkalinisation. There was no difference in total CO2 secretion when similar rates of acetate and glucose absorption were compared, suggesting that total CO2 secretion was the result of mucosal metabolism. Absorption of acetate and propionate were mutually inhibitory. Acetate absorption was also inhibited by Tris-Hepes pH 7.0. When the gut was pretreated with cholera toxin to induce a secretory state, acetate absorption was reduced by 41.9%. This effect could be reproduced if similar water secretion was osmotically induced by the addition of mannitol. These data suggest that acetate is absorbed, at least, partially by non-ionic diffusion in the rat jejunum and that its absorption is reduced in the secreting intestine by solvent drag.

In situ perfusion of rat intestine was used to evaluate the effect of bicarbonate on the efficacy of a low sodium (35 mmol/l) glucose-electrolyte oral rehydration solution in normal and cholera toxin-treated rat small intestine. In normal intestine, absorption of water was greater (108 (8.1) microliters/min/g; p less than 0.01) and sodium secretion less (-4.3 (0.3) mumol/min/g; p less than 0.01) from the oral rehydration solution containing bicarbonate than from the solution in which bicarbonate was replaced by chloride ions (59.5 (7.2) microliters/min/g and -7.8 (0.8) mumol/min/g, respectively). Glucose absorption in normal intestine was similar with both solutions. In the secreting intestine, both oral rehydration solutions reversed net water secretion to absorption, but inclusion of bicarbonate resulted in significantly less net absorption of both water (2.18 (6.9) microliters/min/g; p less than 0.05) and glucose (18.7 (2.1) mumol/min/g; p less than 0.001) compared with bicarbonate free oral rehydration solution (19.4 (3.9) microliters/min/g and 35.8 (3.7) mumol/min/g, respectively). Net sodium secretion occurred in normal and secreting intestine but was significantly less with the bicarbonate containing oral rehydration solution. These findings suggest that the demonstrable advantage of bicarbonate in promoting water absorption from this oral rehydration solution in normal rat intestine does not apply to cholera toxin treated secreting intestine.

Evidence is presented of a higher than normal incidence both of clinical coeliac disease and of small-intestinal mucosal abnormalities in relatives of children with coeliac disease. In such relatives the incidence of mucosal abnormality may differ from the incidence of clinical coeliac disease. The data show an absence of any simple Mendelian pattern of inheritance: in place of the hypothesis that inheritance is through a dominant gene of reduced penetrance, it is argued that the pathogenesis of coeliac disease is multifactorial, the genetic basis of susceptibility being polygenic and interacting with environmental factors. On this hypothesis the relative contributions of inheritance and environment to liability to the clinical condition are estimated, the genetic component being 45% +/- 9. Environmental factors appear more important in the development of mucosal abnormality.