Breaks in the wall: increased gaps in the intestinal epithelium of irritable bowel syndrome patients identified by confocal laser endomicroscopy (with videos).

BACKGROUND: Altered intestinal permeability and mucosal inflammation have been reported in irritable bowel syndrome (IBS) patients. Increased cell extrusion in the epithelium as measured by epithelial gaps may be associated with barrier dysfunction and may lead to mucosal inflammation. Confocal laser endomicroscopy can be used to identify and quantitate epithelial gaps in the small intestine. OBJECTIVE: To determine the epithelial gap density in IBS and healthy control patients. DESIGN: Prospective, controlled cohort study. SETTING: A tertiary referral center. PATIENTS: In IBS and control patients undergoing routine colonoscopy, probe-based confocal laser endomicroscopy was used to image the terminal ileum. MAIN OUTCOME MEASUREMENTS: The primary outcome was the density of epithelial gaps (gaps/cells counted) in adequately imaged villi using pCLE. Images were reviewed by 2 blinded reviewers. RESULTS: We recruited 18 healthy controls and 16 IBS patients. The median epithelial gap densities for control and IBS patients were 6 and 32 gaps per 1000 cells, respectively (P < .001). There was a trend toward higher gap density in female (P = .07) and younger (ρ = -0.43, P = .07) patients. Using 3% (90% of the control population) as the cutoff for abnormal gap density, we found the diagnostic accuracy for IBS to be as follows: 62% sensitivity, 89% specificity, 83% positive predictive value, and 73% negative predictive value. LIMITATIONS: A single-center study, small number of patients. CONCLUSIONS: IBS patients have significantly more epithelial gaps in their small intestine compared with healthy controls, which suggests that increased epithelial cell extrusion may be a cause of altered intestinal permeability observed in IBS.

Increased epithelial gaps in the small intestine are predictive of hospitalization and surgery in patients with inflammatory bowel disease.

OBJECTIVES: Epithelial gaps resulting from intestinal cell extrusions can be visualized with confocal laser endomicroscopy (CLE) during colonoscopy and increased in normal-appearing terminal ileum of inflammatory bowel disease (IBD) patients. Cell-shedding events on CLE were found to be predictive of disease relapse. The aim of this study was to assess the prognostic value of epithelial gap densities for major clinical events (hospitalization or surgery) in follow-up. METHODS: We prospectively followed IBD patients undergoing colonoscopy with probe-based CLE (pCLE) for clinical events including symptom flares, medication changes, hospitalization, or surgery. Survival analysis methods were used to compare event times for the composite outcome of hospitalization or surgery using log-rank tests and Cox proportional hazards models. We also examined the relationship of gap density with IBD flares, need for anti-tumor necrosis factor therapy, disease duration, gender and endoscopic disease severity, and location. RESULTS: A total of 21 Crohn's disease and 20 ulcerative colitis patients with a median follow-up of 14 (11-31) months were studied. Patients with elevated gap density were at significantly higher risk for hospitalization or surgery (log-rank test P=0.02). Gap density was a significant predictor for risk of major events, with a hazard ratio of 1.10 (95% confidence interval=1.01, 1.20) associated with each increase of 1% in gap density. Gap density was also correlated with IBD disease duration (Spearman's correlation coefficient rho=0.44, P=0.004), and was higher in male patients (9.0 vs. 3.6 gaps per 100 cells, P=0.038). CONCLUSIONS: Increased epithelial gaps in the small intestine as determined by pCLE are a predictor for future hospitalization or surgery in IBD patients.

Increased epithelial gaps in the small intestines of patients with inflammatory bowel disease: density matters.

BACKGROUND: Epithelial gaps created by shedding of epithelial cells in the small intestine can be visualized by using confocal laser endomicroscopy (CLE). The density of epithelial gaps in the small bowels of patients with inflammatory bowel disease (IBD) and controls without IBD is unknown. OBJECTIVE: To determine whether the epithelial gap density in patients with IBD is different from that in controls. DESIGN: Prospective, controlled, cohort study. SETTING: A tertiary-care referral center. PATIENTS: This study involved patients with IBD and control patients without IBD undergoing colonoscopy. INTERVENTION: Probe-based CLE (pCLE) was used to image the terminal ileum. MAIN OUTCOME MEASUREMENTS: The primary outcome of the study was gap density, defined as the total number of gaps per 1000 cells counted in adequately imaged villi by using pCLE. The pCLE images were blindly reviewed, and the number of epithelial gaps and cells were manually counted. The secondary outcomes were correlation of gap density with disease activity, location, and severity of clinical disease. RESULTS: There were 30 controls and 28 patients with IBD. Of the patients with IBD, 16 had Crohn's disease, and 12 had ulcerative colitis. The median epithelial gap densities for controls and patients with IBD were 18 and 61 gaps/1000 cells, respectively (P < .001). Gap density did not correlate with disease activity. Patients with ulcerative pan-colitis tended toward gap densities lower than those of patients with limited colitis (32 versus 97 gaps/1000 cells, P = .06). Patients with IBD with severe clinical disease also had lower median gap densities (37 vs 90 gaps/1000 cells, P = .04). LIMITATIONS: A single-center study. CONCLUSION: The epithelial gap density was significantly increased in patients with IBD compared with controls. ( CLINICAL TRIAL REGISTRATION NUMBER: NCT00988273.).

Epithelial gaps in a rodent model of inflammatory bowel disease: a quantitative validation study.

OBJECTIVES: Confocal laser endomicroscopy (CLE) is a non-invasive imaging modality of the gastrointestinal tract. Epithelial gaps in the small intestine of patients and rodents have been demonstrated using CLE. The goal of this study was to quantitatively validate the findings of epithelial gap density observed with CLE against confocal microscopy (CM) and light microscopy. METHODS: Two strains of mice (control 129 Sv/Ev and interleukin 10 knockout (IL-10(-/-))) underwent CLE of the terminal ileum. Adjacent ileal tissues were examined using CM and light microscopy. The total number of gaps and cells in the villi were manually counted from the three-dimensional reconstruction of cross-sectional CLE and CM images. The histology specimens were reviewed for epithelial gap and cell counts by a pathologist blinded to the study groups. The inter- and intra-observer variability for cell and gap counts were determined. RESULTS: For CLE, the gap densities (mean±s.d.) in the ileum for control and IL-10(-/-) mice were: 9.5±1.3 gaps per 1,000 cells and 20.6±2.1 gaps per 1,000 cells counted (P<0.001), respectively. For CM, the ileal gap densities were 7.3±1.3 gaps per 1,000 cells and 22.8±6.2 gaps per 1,000 cells (P=0.03), respectively. For light microscopy, the ileal gap densities were 29.2±5.9 gaps per 1,000 cells and 51.5±6.4 gaps per 1,000 cells for the two strains. CONCLUSION: CLE can be used to quantitatively assess epithelial cells and gaps with accuracy comparable to CM and light microscopy. In a mouse model of inflammatory bowel disease, the epithelial gap density in the terminal ileum is significantly increased when examined using all three modalities.

Glutamate receptor trafficking: endoplasmic reticulum quality control involves ligand binding and receptor function.

The glutamate receptor (GluR) agonist-binding site consists of amino acid residues in the extracellular S1 and S2 domains in the N-terminal and M3-M4 loop regions, respectively. In the present study, we sought to confirm that the conserved ligand-binding residues identified in the AMPA receptor S1S2 domains also participate in ligand binding of GluR6 kainate receptors. Amino acid substitutions were made in the GluR6 parent at R523, T690, and E738 to alter their potential interactions with ligand. Mutant receptors were expressed in human embryonic kidney 293 cells, confirmed by Western blot analysis, and tested by [3H]kainate binding and patch-clamp recording. Each of the binding site mutations was sufficient to reduce [3H]kainate binding to undetectable levels and eliminate functional responses to glutamate or kainate. As with our studies of other nonfunctional mutants (Fleck et al., 2003), immunocytochemical staining and cell-surface biotinylation studies showed that the mutant receptors were retained intracellularly and did not traffic to the cell surface. Endoglycosidase-H digests and colocalization with endoplasmic reticulum (ER) markers demonstrated that the mutant receptors are immaturely glycosylated and retained in the ER. Immunoprecipitation, native PAGE, and functional studies confirmed that the GluR6-binding site mutants are capable of multimeric assembly, indicating their retention in the ER does not result from a gross protein folding error. Together, these results confirm the role of R523, T690, and E738 directly in ligand binding to GluR6 and further support our previous report that nonfunctional GluRs are retained intracellularly by a functional checkpoint in ER quality control.

Amino-acid residues involved in glutamate receptor 6 kainate receptor gating and desensitization.

The glutamate receptor (GluR) agonist-binding site consists of amino acid residues in the extracellular S1 and S2 segments in the N-terminal and M3-M4 loop regions, respectively. Molecular and atomic level structural analyses have identified specific S1 and S2 residues that interact directly with ligands, interact with one another in a dimeric configuration, and influence channel gating and desensitization properties of AMPA receptors. Other studies suggest that KA receptor gating and desensitization may differ mechanistically. In particular, a leucine (L) to tyrosine (Y) mutation in the S1 segment of AMPA receptors is sufficient to block desensitization, whereas KA receptors naturally contain a tyrosine residue at the equivalent position (Y751 in GluR6) but retain the fast-desensitizing phenotype. We hypothesized that KA receptor desensitization is preserved by a compensatory substitution in the S2 segment. We generated a series of GluR6 mutants that converted individual S2 domain residues to their AMPA receptor equivalents. Various S2 mutations had effects on the kinetics of desensitization and recovery from desensitization, but no single amino acid substitution was found to block desensitization, as in the L/Y mutant AMPA receptors, or to prevent desensitization to KA. Other mutations designed to neutralize residues thought to interact across the dimer interface had dramatic effects on channel gating and desensitization. These results are consistent with a close but imperfect structural homology between AMPA and KA receptors and support the role of conserved S1S2 domain interactions at the dimer interface in GluR channel function.