Epithelial Gap Density Research Articles

Evidence of Duodenal Epithelial Barrier Impairment and Increased Pyroptosis in Patients With Functional Dyspepsia on Confocal Laser Endomicroscopy and "Ex Vivo" Mucosa Analysis.

Duodenal epithelial barrier impairment and immune activation may play a role in the pathogenesis of functional dyspepsia (FD). This study was aimed to evaluate the duodenal epithelium of patients with FD and healthy individuals for detectable microscopic structural abnormalities. This is a prospective study using esophagogastroduodenoscopy enhanced with duodenal confocal laser endomicroscopy (CLE) and mucosal biopsies in patients with FD (n = 16) and healthy controls (n = 18). Blinded CLE images analysis evaluated the density of epithelial gaps (cell extrusion zones), a validated endoscopic measure of the intestinal barrier status. Analyses of the biopsied duodenal mucosa included standard histology, quantification of mucosal immune cells/cytokines, and immunohistochemistry for inflammatory epithelial cell death called pyroptosis. Transepithelial electrical resistance (TEER) was measured using Ussing chambers. Epithelial cell-to-cell adhesion proteins expression was assessed by real-time polymerase chain reaction. Patients with FD had significantly higher epithelial gap density on CLE in the distal duodenum than that of controls (P = 0.002). These mucosal abnormalities corresponded to significant changes in the duodenal biopsy samples of patients with FD, compared with controls, including impaired mucosal integrity by TEER (P = 0.009) and increased number of epithelial cells undergoing pyroptosis (P = 0.04). Reduced TEER inversely correlated with the severity of certain dyspeptic symptoms. Furthermore, patients with FD demonstrated altered duodenal expression of claudin-1 and interleukin-6. No differences in standard histology were found between the groups. This is the first report of duodenal CLE abnormalities in patients with FD, corroborated by biopsy findings of epithelial barrier impairment and increased cell death, implicating that duodenal barrier disruption is a pathogenesis factor in FD and introducing CLE a potential diagnostic biomarker in FD.

Increased Epithelial Gap Density in the Noninflamed Duodenum of Children With Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD) present commonly in childhood, with unknown etiology, but an important role for the epithelial lining is suggested. Epithelial cell extrusion, measured by counting gaps between epithelial cells, is higher in adult patients with Crohn disease (CD) than in controls. Our objectives were to compare epithelial gaps in the duodenum of IBD and non-IBD pediatric patients, to study the correlation between epithelial gaps, inflammation, and disease activity, and identify potential mechanisms. Epithelial gap density of the duodenum was evaluated using probe-based confocal laser endomicroscopy in 26 pediatric patients with IBD (16 CD, 10 ulcerative colitis [UC]) and 17 non-IBD controls during endoscopy. Epithelial gaps were correlated with serum inflammatory markers, disease activity indices, and intraepithelial lymphocytes. A panel of 10 inflammatory cytokines and expression of TNFAIP3 (A20; inhibits NF-κβ-induced inflammation) were analyzed in duodenal and ileal biopsies. Confocal imaging showed significantly higher epithelial gap density in patients with IBD, including UC. Interleukin (IL)-2 and IL-8 were higher in duodenal but not ileal biopsies of patients with UC. No significant correlation was present between C-reactive protein, erythrocyte sedimentation rate, disease activity indices, and epithelial gaps in patients with UC. In patients with CD, C-reactive protein positively correlated with epithelial gaps. A20 expression in the duodenum was unchanged among non-IBD and IBD cases. Duodenal epithelial gaps are increased in pediatric patients with IBD (including UC) but are unrelated to inflammation. This suggests that altered epithelial barrier is an important systemic feature of pediatric IBD and is not only secondary to inflammation.

A Pilot Study of Confocal Laser Endomicroscopy to Predict Barrier Dysfunction and Relapse in Pediatric Inflammatory Bowel Disease.

Probe-based confocal laser endomicroscopy (pCLE) is a novel imaging modality that enables virtual optical biopsy in vivo. Loss of barrier function of the small bowel observed via pCLE as increased density of epithelial gaps (extrusion zones left in the intestinal lining after cells are shed) is predictive of relapse in adult patients with inflammatory bowel disease (IBD). This study aims to determine whether such observations on pCLE are similarly predictive of disease relapse in pediatric patients with IBD. Pediatric patients with biopsy-proven IBD underwent pCLE during colonoscopy and subsequent clinical follow-up every 6 months. Relapse was defined as moderate to severe flare with endoscopic evidence of inflammation during the follow-up period. The relations between epithelial gap density, disease relapse, and imaging parameters were determined using Cox models. Twenty-four patients with IBD (13 with Crohn disease, 11 with ulcerative colitis) with a median age of 14 years (range 10-21) were studied for a median of 13 (4-33) months. The median duration of disease was 2.9 years (range 0-9). Increased epithelial gap density in the terminal ileum on pCLE of normal endoscopic appearing terminal ileum mucosa (N = 19) was predictive of disease relapse when 3 or more areas were imaged (N = 6, log-rank P = 0.02, C-statistic = 0.94). In pediatric patients with IBD, barrier dysfunction observed on pCLE imaging of the small bowel was predictive of disease relapse.

Epithelial Cell Extrusion Zones Observed on Confocal Laser Endomicroscopy Correlates with Immunohistochemical Staining of Mucosal Biopsy Samples.

The density of epithelial cell extrusion zones in the intestinal lining, also known as gap density (number of gaps/1000 epithelial cells counted), can be quantitated using probe-based confocal laser endomicroscopy (pCLE). Gap density has been reported to be higher than normal in both inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) patients. Epithelial cells destined for extrusion from the intestinal surface would stain positive for either activated caspase-1 or caspase-3 on mucosal biopsy samples. The aim of this study was to determine whether epithelial gap density on pCLE correlates with quantitative analysis of activated caspase staining of mucosal biopsy samples from patients. We obtained pCLE images and biopsy samples of the terminal ileum during colonoscopies of healthy controls and patients with either IBD or IBS. The pCLE images and biopsy samples were blindly analyzed for gap density and for cells staining positive for activated caspases, respectively. The degree of correlation was determined using nonparametric statistical tests. The median results were 10gaps/1000 cells counted for controls versus 33 gaps/1000 cells counted for chronic intestinal disorder patients (p=0.02). Activated caspase staining showed 13positive cells/1000 epithelial cells counted versus 26positive cells/1000 epithelial cells counted, respectively (p=0.02), thus showing a strong correlation with a Spearman's coefficient ρ of 0.61 (strong correlation for ρ=0.4-0.75, p=0.01). Intestinal epithelial gap density via pCLE correlated strongly with quantitative analysis of immunohistochemical staining of mucosal biopsy samples.

Mo2013 Live, Dynamic Bioluminescent Imaging of Pancreatic NF-KB Activation in Mice Using Novel Gene Delivery via Intraductal Infusion of an Adeno-Associated Viral Reporter

Introduction: IBS is a symptom-based diagnosis characterized by chronic abdominal pain, bloating, and alterations of the bowel habits without any organic cause. CLE allows in vivo visualization of microscopic features of the intestinal epithelium in real time during endoscopy. Aims: Primary study objective was to assess the potential of CLE to demask subtle mucosal changes of the small and large bowel mucosa in patients with established diagnosis of IBS. Material & Methods: Patients with established diagnosis of IBS according to RomeIII criteria and control patients underwent ileocolonoscopy. Fluoresceine guided CLE was performed in the terminal ileum and random optical biopsies were additionally performed in the colon and rectum. Attention was paid to presence or absence of epithelial gaps, intramucosal bacteria, crypt and vessel morphology, goblet cells and cellular infiltrate within the lamina propria. Physical biopsies were additionally taken for histopathological analysis. Results: In a subgroup (44%) of patients with IBS according to Rome-III epithelial gap density and the microvascular pattern were increased compared to control patients suggesting an altered intestinal permeability. No differences were observed regarding the presence of intramucosal bacteria, colonic crypt morphology, presence of goblet cells or the cellular infiltrate within the lamina propria (P >0.05). Conclusion: Confocal laser endomicroscopy revealed subtle mucosal changes in patients with IBS. These findings were not visible in all IBS patients suggesting that only a subgroup of IBS patients may have an organic cause of the disease.

Mo2012 Confocal Laser Endomicroscopy in Patients With Irritable Bowel Syndrome (IBS)

Introduction: IBS is a symptom-based diagnosis characterized by chronic abdominal pain, bloating, and alterations of the bowel habits without any organic cause. CLE allows in vivo visualization of microscopic features of the intestinal epithelium in real time during endoscopy. Aims: Primary study objective was to assess the potential of CLE to demask subtle mucosal changes of the small and large bowel mucosa in patients with established diagnosis of IBS. Material & Methods: Patients with established diagnosis of IBS according to RomeIII criteria and control patients underwent ileocolonoscopy. Fluoresceine guided CLE was performed in the terminal ileum and random optical biopsies were additionally performed in the colon and rectum. Attention was paid to presence or absence of epithelial gaps, intramucosal bacteria, crypt and vessel morphology, goblet cells and cellular infiltrate within the lamina propria. Physical biopsies were additionally taken for histopathological analysis. Results: In a subgroup (44%) of patients with IBS according to Rome-III epithelial gap density and the microvascular pattern were increased compared to control patients suggesting an altered intestinal permeability. No differences were observed regarding the presence of intramucosal bacteria, colonic crypt morphology, presence of goblet cells or the cellular infiltrate within the lamina propria (P >0.05). Conclusion: Confocal laser endomicroscopy revealed subtle mucosal changes in patients with IBS. These findings were not visible in all IBS patients suggesting that only a subgroup of IBS patients may have an organic cause of the disease.

Tu1952 Title: Increased Epithelial Gap Density in the Small Bowel Is Mediated by Caspase-1 Activation of Intestinal Epithelial Cells: A Quantitative Analysis of Confocal Laser Endomicroscopy Images and Mucosal Biopsy Samples

BACKGROUND AND AIMS: There are only few case reports describing the frequency and the clinical features of pancreatic involvement (PI) in inflammatory bowel disease (IBD). We aimed to estimate the prevalence and to characterize IBD pediatric patients with PI. METHODS: We retrospectively reviewed data collected in the IBD web-registry of the Italian Society for Pediatric Gastroenterology, Hepatology and Nutrition. All children presenting with hyperamylasemia and/or hyperlipasemia were identified. Demographic and clinical data, IBD type, disease exstension and activity, laboratory data, IBD therapy, imaging findings and therapeutic interventions were evaluated. Acute pancreatitis (AP) was defined by the occurrence of at least two of the following findings: acute epigastric abdominal pain, elevated serum amylase and/or lipase ≥ 3 times the upper level of normal, and characteristic radiological changes. RESULTS: We found 27 children out of 649 (4.1%) with an increased value of amylase and/or lipase [Median Age: 148, range 65-191, mths; F/M: 14/13; Ulcerative colitis (UC): 12; Crohn's disease (CD): 13; Unclassified Colitis (IBDU): 2)]. Mean serum amylase level was 205.3 ± 91.5 (range 61-413 IU/L) and mean serum lipase level was 526.8 ± 598.8 (range 37-2565 IU/L). Eleven patients (1.6%) fulfilled diagnostic criteria for AP (6 CD, 4 UC, 1 IBDU). Ten (37%) patients underwent imaging ultrasound scan, 8 (29.6%) patients had magnetic resonance cholangiopancreatography and 2 (7.4%) patients had a computed tomography scan. Pancreatic pathological findings were found in all subjects with AP. The mean lag time period between the diagnosis of IBD and the PI was 12.1 mths (range 0-65). Five patients out of 27 (18.5%) showed PI at diagnosis of IBD. Twenty-four out of 27 (88.8%) had colonic disease [10 CD, 12 UC (8 pancolitis) and 2 IBDU]. Ten patients were receiving mesalamine (ASA) and 16 patients were under immunosuppressive therapy [3 streoids, 6 azathioprine (AZT), 5 AZT + ASA, 1 methotrexate, 1 infliximab (IFX), and 1 IFX+ASA+AZT], while 1 patient was not treated. Comparing with the patients with an exclusive hyperamylasemia and/or hyperlipasemia, AP was significantly associated with female gender (p=0.018), whereas type of IBD, ongoing treatments, activity and extension of disease did not result significant risk factors. Seven out of 11 (63.6%) patients with AP needed therapeutic measures including AZT and/or ASA withdrawal, compared to only 4 (25%) patients out of 16 with serum hyperamylasemia and/or hyperlipasemia (p=0.06). CONCLUSIONS: Our study suggests that prevalence of AP in children is similar to that reported in adults. PI was more common in colonic disease. Female gender seems to be a risk factor for developing AP in children. In contrast to previous study, PI was not associated with any type of IBD.

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

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. To determine the epithelial gap density in IBS and healthy control patients. Prospective, controlled cohort study. A tertiary referral center. In IBS and control patients undergoing routine colonoscopy, probe-based confocal laser endomicroscopy was used to image the terminal ileum. 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. 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. A single-center study, small number of patients. 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.

23 Increased Epithelial Gap Density as Measured by Probe-Based Confocal Laser Endomicroscopy Predicts the Need for Future Hospitalization in Patients With Inflammatory Bowel Disease (IBD)

23 Increased Epithelial Gap Density as Measured by Probe-Based Confocal Laser Endomicroscopy Predicts the Need for Future Hospitalization in Patients With Inflammatory Bowel Disease (IBD)

Salivary VEGF Plays an Essential Role in Oral Mucosal Wound Healing

Introduction: Palatal mucosal wound healing occurs more rapidly than in cutaneous wounds. Vascular endothelial growth factor (VEGF) induced angiogenesis is critical for cutaneous wound healing. We have previously demonstrated 1) salivary glands produce high levels of VEGF and 2) an essential role for salivary VEGF (salVEGF) in the intestinal response to injury. This has led us to hypothesize that salVEGF is critical in oral mucosal wound healing and neovascularization. to test this hypothesis, we performed a loss-of-function experiment by selectively inhibiting salVEGF using a novel adenoviral construct (AdVegfTrap) and a gain-of-function experiment by supplementing oral VEGF following submandibular gland (SMG) resection. Methods: In the loss-of-function experiment, C57/BL6 mice underwent SMG duct cannulation and were treated with 1x108 PFU AdVegfTrap, AdLacZ or PBS (n=5/group). 1.5 mm palatal mucosal wounds were created. in the gain-of-function experiment, C57/BL6 mice underwent SMG sialoadenectomy (n=4) or sham operation (n=5). 1.5 mm palatal wounds were created and oral VEGF protein was supplemented. AdVegfTrap was not used due to anticipated consumption of the orally replaced VEGF. in both experiments, salVEGF protein levels were quantified by ELISA. Wounds were harvested at 3 days and analyzed for epithelial gap and vessel density. Data were expressed as mean+SEM. Results: Retrograde SMG duct injection of AdLacZ results in 100% transduction efficiency confirming effective transgene delivery. SMG duct cannulation, SMG administration of AdLacZ and sham operation controls do not result in significantly decreased of salVEGF (PBS513pg/ml±141 vs AdLacZ353pg/ml±56 vs sham472pg/ml±40 vs control514pg/ml±89;p=NS). Administration of AdVegfTrap results in depletion of salVEGF comparable to SMG sialoadenectomy (AdVegfTrap122pg/ml±42 vs SMG resection136pg/ml±15.9;p=NS). Selective inhibition of salVEGF by AdVegfTrap impairs wound closure (AdVegfTrap947μm±22 vs AdLacZ411μm±21 vs PBS355μm±31;p<0.001) and vessel density (AdVegfTrap15.95caps/hpf±0.83 vs AdLacZ30.87caps/hpf±1.1 vs PBS30.5caps/hpf±0.95;p<0.001). Oral replacement of VEGF in SMG resected mice rescues the impaired reepithelialization and neovascularization phenotype (SMG with oral VEGF194μm±43 vs sham350μm±46,p=NS; SMG with oral VEGF24.0caps/hpf±1.4 vs sham18.6caps/hpf±2.1,p=NS). Conclusions: Selective inhibition of salVEGF significantly impairs palatal reepithelialization and neovascularization. Oral supplementation of VEGF rescues the impaired wound healing phenotype in SMG sialoadenectomized mice. Taken together, these findings suggest that salVEGF is an essential growth factor in oral mucosal wound healing which may be a surrogate for intestinal wound healing. the palatal wound model may be a valuable tool in understanding the critical role of salVEGF in the alimentary tract response to injury.

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.

Increased Epithelial Gaps in the Small Intestine 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.) (Gastrointest Endosc 2011;73:1174-80.)

Increased Small Intestinal Permeability and Liver Damage After Exhaustive Physical Exercise in Healthy Individuals

Purpose: TNF-α is a pro-inflammatory cytokine believed to be an important mediator of inflammatory bowel disease (IBD). TNF is known to induce cell shedding and increase permeability of the small intestine. We hypothesize that TNF induced intestinal cell shedding contributes to barrier dysfunction and increased antigen presentation. In this study, we used confocal laser endomicroscopy (CLE) and light microscopy (LM), to quantitate TNF induced epithelial cell shedding in the small intestine, and correlated cell shedding with dextran absorption and translocation of commensal bacteria. Methods: 129 Sv/Ev mice were divided into two groups: the treatment group received intraperitoneal injection of TNF-α (0.175μg/ g), while the control group received an equivalent volume of normal saline injection. CLE were performed using Optiscan confocal endomicroscope on exteriorized intestine stained with acriflavine. Epithelial cell shedding was quantitated using density of epithelial gaps created by shedding of intestinal cells. Epithelial density was defined as the total number of epithelial gaps per 1000 cells counted in 5-10 villi per mouse. For LM, frozen sections of the intestine were stained with alcian blue and nuclear fast red. 3D reconstructed CLE images and 2D LM sections were manually counted for epithelial gaps and cells. For FITCdextran absorption study, mice were gavaged with 0.6 mg/g dextran after an overnight fast, blood samples were collected after 4 hr for serum FITC-dextran determination. For translocation studies, mice were gavaged after an overnight fast with 10^10 GFP labelled E. coli JM 109 suspended in 0.17mL LB broth. After 24 hours, liver, spleen and blood samples were obtained for culture. Results: For saline and TNF treated mice (n=6), the CLEdetermined epithelial gap density (mean+SD) was 10.6 ± 2.4 and 47.1 ± 7.4 gaps/1000 cells (P = 0.001), respectively. The LM-determined epithelial gap density was 34.8 ± 6.0 and 64.2 ± 7.4 gaps/1000 cells (P <0.0001), respectively. The discrepancy in the gap density observed between CLE and LM were due to the 3D and 2D nature of image analysis. Serum dextran concentration in the saline and TNF treated mice were 0.59 ± 0.05, and 3.3 ± 0.5 μg/mL (P <0.001), respectively. The liver, spleen and blood cultures from the saline treated mice were all negative, while the TNF treated mice grew 1.4 ± 0.94 E 3 and 1.1 ± 0.5 E 3 cfu/mL of GFP labelled E. colifrom liver and spleen cultures, confirming translocation of commensal bacteria. Conclusion: TNF induced epithelial cell shedding in the small intestine as observed by significant increases in the epithelial gap density of 129 Sv/Ev mice using CLE and LM. This increase in gap density was associated with increased permeability to macromolecules as measured by dextran absorption and translocation of commensal bacteria.

TNF Alpha Induced Epithelial Cell Shedding in the Small Intestine Correlates With Increased Permeability to Macromolecules and Translocation of Commensal Bacteria

Purpose: TNF-α is a pro-inflammatory cytokine believed to be an important mediator of inflammatory bowel disease (IBD). TNF is known to induce cell shedding and increase permeability of the small intestine. We hypothesize that TNF induced intestinal cell shedding contributes to barrier dysfunction and increased antigen presentation. In this study, we used confocal laser endomicroscopy (CLE) and light microscopy (LM), to quantitate TNF induced epithelial cell shedding in the small intestine, and correlated cell shedding with dextran absorption and translocation of commensal bacteria. Methods: 129 Sv/Ev mice were divided into two groups: the treatment group received intraperitoneal injection of TNF-α (0.175μg/ g), while the control group received an equivalent volume of normal saline injection. CLE were performed using Optiscan confocal endomicroscope on exteriorized intestine stained with acriflavine. Epithelial cell shedding was quantitated using density of epithelial gaps created by shedding of intestinal cells. Epithelial density was defined as the total number of epithelial gaps per 1000 cells counted in 5-10 villi per mouse. For LM, frozen sections of the intestine were stained with alcian blue and nuclear fast red. 3D reconstructed CLE images and 2D LM sections were manually counted for epithelial gaps and cells. For FITCdextran absorption study, mice were gavaged with 0.6 mg/g dextran after an overnight fast, blood samples were collected after 4 hr for serum FITC-dextran determination. For translocation studies, mice were gavaged after an overnight fast with 10^10 GFP labelled E. coli JM 109 suspended in 0.17mL LB broth. After 24 hours, liver, spleen and blood samples were obtained for culture. Results: For saline and TNF treated mice (n=6), the CLEdetermined epithelial gap density (mean+SD) was 10.6 ± 2.4 and 47.1 ± 7.4 gaps/1000 cells (P = 0.001), respectively. The LM-determined epithelial gap density was 34.8 ± 6.0 and 64.2 ± 7.4 gaps/1000 cells (P <0.0001), respectively. The discrepancy in the gap density observed between CLE and LM were due to the 3D and 2D nature of image analysis. Serum dextran concentration in the saline and TNF treated mice were 0.59 ± 0.05, and 3.3 ± 0.5 μg/mL (P <0.001), respectively. The liver, spleen and blood cultures from the saline treated mice were all negative, while the TNF treated mice grew 1.4 ± 0.94 E 3 and 1.1 ± 0.5 E 3 cfu/mL of GFP labelled E. colifrom liver and spleen cultures, confirming translocation of commensal bacteria. Conclusion: TNF induced epithelial cell shedding in the small intestine as observed by significant increases in the epithelial gap density of 129 Sv/Ev mice using CLE and LM. This increase in gap density was associated with increased permeability to macromolecules as measured by dextran absorption and translocation of commensal bacteria.

Overexpression of PepT1 Increases the Susceptibility of Mice to Colitis in a Process Involving NOD2 Activity

Purpose: TNF-α is a pro-inflammatory cytokine believed to be an important mediator of inflammatory bowel disease (IBD). TNF is known to induce cell shedding and increase permeability of the small intestine. We hypothesize that TNF induced intestinal cell shedding contributes to barrier dysfunction and increased antigen presentation. In this study, we used confocal laser endomicroscopy (CLE) and light microscopy (LM), to quantitate TNF induced epithelial cell shedding in the small intestine, and correlated cell shedding with dextran absorption and translocation of commensal bacteria. Methods: 129 Sv/Ev mice were divided into two groups: the treatment group received intraperitoneal injection of TNF-α (0.175μg/ g), while the control group received an equivalent volume of normal saline injection. CLE were performed using Optiscan confocal endomicroscope on exteriorized intestine stained with acriflavine. Epithelial cell shedding was quantitated using density of epithelial gaps created by shedding of intestinal cells. Epithelial density was defined as the total number of epithelial gaps per 1000 cells counted in 5-10 villi per mouse. For LM, frozen sections of the intestine were stained with alcian blue and nuclear fast red. 3D reconstructed CLE images and 2D LM sections were manually counted for epithelial gaps and cells. For FITCdextran absorption study, mice were gavaged with 0.6 mg/g dextran after an overnight fast, blood samples were collected after 4 hr for serum FITC-dextran determination. For translocation studies, mice were gavaged after an overnight fast with 10^10 GFP labelled E. coli JM 109 suspended in 0.17mL LB broth. After 24 hours, liver, spleen and blood samples were obtained for culture. Results: For saline and TNF treated mice (n=6), the CLEdetermined epithelial gap density (mean+SD) was 10.6 ± 2.4 and 47.1 ± 7.4 gaps/1000 cells (P = 0.001), respectively. The LM-determined epithelial gap density was 34.8 ± 6.0 and 64.2 ± 7.4 gaps/1000 cells (P <0.0001), respectively. The discrepancy in the gap density observed between CLE and LM were due to the 3D and 2D nature of image analysis. Serum dextran concentration in the saline and TNF treated mice were 0.59 ± 0.05, and 3.3 ± 0.5 μg/mL (P <0.001), respectively. The liver, spleen and blood cultures from the saline treated mice were all negative, while the TNF treated mice grew 1.4 ± 0.94 E 3 and 1.1 ± 0.5 E 3 cfu/mL of GFP labelled E. colifrom liver and spleen cultures, confirming translocation of commensal bacteria. Conclusion: TNF induced epithelial cell shedding in the small intestine as observed by significant increases in the epithelial gap density of 129 Sv/Ev mice using CLE and LM. This increase in gap density was associated with increased permeability to macromolecules as measured by dextran absorption and translocation of commensal bacteria.

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

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. To determine whether the epithelial gap density in patients with IBD is different from that in controls. Prospective, controlled, cohort study. A tertiary-care referral center. This study involved patients with IBD and control patients without IBD undergoing colonoscopy. Probe-based CLE (pCLE) was used to image the terminal ileum. 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. 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). A single-center study. The epithelial gap density was significantly increased in patients with IBD compared with controls. ( NCT00988273.).

Mind The Gaps

Confocal endomicroscopy can be used to image intestinal mucosa. Epithelial gaps resulting from shedding of epithelial cells have been reported in patients. We hypothesize that the rate of epithelial cell shedding increases in patients with Crohn's disease, leading to more epithelial gaps and barrier dysfunction. In this study, we used probe-based confocal laser endomicroscopy to quantify epithelial cells and gaps in patients with Crohn's disease compared with controls. We also determined the density of epithelial gaps in a mouse model of inflammatory bowel disease-interleukin-10-deficient (IL-10) mice, versus the background strain using rigid probe confocal endomicroscopy. Probe-based confocal laser endomicroscopy of the terminal ileum of both patients with Crohn's disease and controls was performed by a single endoscopist during colonoscopy. In mice, sections of the small intestine were imaged using a rigid confocal probe. Gap density was defined as the number of epithelial gaps per 1000 cells counted. In this study, we examined 6 controls (2 male and 4 female; median age 59 y) and 8 patients with Crohn's disease (5 male and 3 female; median age 42 y). The mean gap densities (±standard error) observed for the 2 groups were 17.7±5.6 and 117±33 gaps per 1000 cells, respectively (P<0.01). For control and IL-10 mice, the gap densities were 10.5±2.2 and 17.8±1.4 gaps per 1000 cells, respectively (P<0.01). The epithelial gap density was significantly higher in patients with Crohn's disease than controls. Gap density was also elevated in the mouse model of inflammatory bowel disease.

Mind the Gaps: Increased Density of Epithelial Gaps Observed in the Terminal Ileum of Patients with Inflammatory Bowel Disease Using Confocal Endomicroscopy

Purpose: Epithelial cells within the small intestine undergo shedding resulting in the formation of epithelial gaps (spaces between cells). The cytokine TNF-α has been shown to accelerate this process by 20-fold. Epithelial gaps can be observed within human small and large intestine through the use of confocal endomicroscopy, and increased epithelial gap density within the duodenum has been previously reported in patients with inflammatory bowel disease (IBD). Given the role of TNF-α in the pathogenesis of IBD, we hypothesize that epithelial gap density is increased in the terminal ileum of IBD patients compared to controls. Methods: Probe-based confocal laser endomicroscopy (pCLE) is a new modality which allows in-vivo histopathologic evaluation of the intestinal mucosa. Use of the ColoFlexTM UHD miniprobe (Cellvizio, Mauna Kea Technologies, Paris, France) provides a resolution of 1.4 μm (for comparison, the diameter of a RBC is 7 μm) thus allowing visualization of epithelial cells and gaps. After intravenous injection of 5 mL of 10% fluorescein, pCLE of the terminal ileum of consecutive patients undergoing colonoscopy was performed. Controls were patients undergoing colonoscopy for non-IBD related indications; IBD patients were those with a history of biopsy-proven Crohn's disease or ulcerative colitis. We imaged endoscopically normal mucosa in the terminal ileum, as areas with visible erosions and ulcerations had significant architectural distortions making quantitative analysis of cells and gaps impossible. Over 3000 images were acquired for each patient. Gap density was defined as the number of epithelial gaps found per 1000 cells counted. Results: The experimental cohort consisted of 20 controls (9 males; mean age=51±12 years) and 15 IBD patients (9 males; mean age=39±13 years). Of the 15 IBD patients, 10 had Crohn's disease and 5 had ulcerative colitis. Endoscopically normal terminal ileal mucosa was imaged in both groups. The images were reviewed by two blinded reviewers. A minimu m of 3 confocal images with the highest epithelial gap density were analyzed for each patient; epithelial cells and gaps were manually counted. Gaps were ascertained in at least three different sequential images. The mean gap density (± SD) was 15±2 gaps per 1000 cells counted for controls versus 100 ± 8 gaps/1000 cells counted for IBD patients (p<0.0001). Conclusion: Confocal endomicroscopy revealed significantly increased epithelial gap density in endoscopically normal-appearing terminal ileum of IBD patients as compared to controls.

074 Wound Healing Enhancement by Pulsed Electromagnetic Fields

The effects of pulsed electromagnetic fields (PEMF) on soft tissue are not well characterized. This study utilized a diabetic wound model to examine the effects of PEMF effects on soft tissue healing. Methods: Wounds were created on the dorsum of db/db and wild type C57BL6 mice. Mice were exposed to PEMF (4.5 ms pulse/15 hz) for 8 hrs/day for 14 days. Gross closure was assessed with digital analysis of area changes over time. Histological examination assessed granulation and epithelial gap, cell proliferation (BrdU), and endothelial cell density (CD31). Human umbilical vein endothelial cells (HUVECs) were incubated in the presence or absence of PEMF for 8 hrs and VEGF/FGF2 was measured in culture supernatants by ELISA. Results: Mice exposed to PEMF had accelerated wound closure at day 7 (wound area as % of original, db/db: 60%(PEMF) vs. 78%(control), C57BL6: 15% vs. 42%, p &lt; 0.05) and day 14 (db/db: 21% vs. 55%, C57BL6: 8% vs. 28%, p &lt; 0.05), with increased granulation and cell proliferation (db/db day 7: 52 ± 8 vs. 31 ± 5 cells/HPF (200x)). Immunohistochemical analysis revealed significantly higher CD31 density in wounds exposed to PEMF at day 7 (vessels/HPF, db/db: 28 ± 4 vs. 17 ± 4, C57BL6: 41 ± 7 vs. 28 ± 6) and day 14 (db/db: 32 ± 6 vs. 21 ± 5, C57BL6: 48 ± 5 vs. 40 ± 5). HUVECs in PEMF exhibited 5‐fold higher levels of FGF2 compared to controls after 30 min (20.50 pg/ml ± 6.75 vs. 4.25 pg/ml ± 0.75), with no change in VEGF through 8 hrs. Conclusions: PEMF accelerates closure time and endothelial cell proliferation in wound healing. Upregulation of FGF2 in HUVECs exposed to PEMF suggests that release of angiogenic growth factors may explain increased vascular density and accelerated wound closure. Clinical uses include treatment for diabetic ulcers and other non‐healing wounds.This work is supported in part by the Alumni Fund, Alumni Association of SUNY Downstate College of Medicine (MJC).