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828 Glucocorticoids Stimulate Tight Junction Sealing in Intestinal Epithelial Cells By Modulating Expression of Claudin-2 and Claudin-4
AGA Abstracts
the role of myosin light chain kinase (MLCK) in epithelial cell shedding and whether the tight junction potentially contributes to maintenance of barrier function at these sites. Methods: Transgenic mice were generated that expressed ZO-1 or occludin as fusion proteins with fluorescent proteins, with expression in intestinal epithelia driven by the villin promoter. In some experiments, these mice were crossed with long myosin light chain kinase (MLCK) knockout mice. After injection with TNF-α (7.5µg, i.p.) a laparotomy was performed on the anesthetised mouse and a segment of small intestine opened so that the shedding of epithelial cells could be evaluated by video confocal microscopy. Results: TNF-α increased shedding by 27-fold (p<0.007) versus non-injected controls, beginning 60 min after TNFα exposure. The fluorescent fusion proteins showed normal cellular distribution compared to immunostained wildtype mice. However during stimulated cell shedding a redistribution of both ZO-1 and occludin was observed. Over the 10 min during which shedding of a single cell occurred, both ZO-1 and occludin migrated downward along lateral membranes and condensed beneath shedding nuclei to form a funnel that moved apically with the extruding cell. Extracellular dye probes (e.g. Alexa 488 in perfusate solution) approached but did not cross the line of ZO-1 and occludin, demonstrating maintenance of the mucosal barrier at these sites. MLCK knockout mice had similar ZO-1 remodelling, suggesting this process may not require MLCK. Conclusion: We conclude that ZO-1 and occludin undergo novel MLCK-independent redistribution at sites of barrier maintenance during TNF-induced cell shedding. Supported by NIH.
826 T403/404d Mutation of Occludin Attenuates PKCη-Mediated Regulation of Epithelial Tight Junctions (TJS) Bertha Elias, Sudhir Aggarwal, Le Shen, Jerrold R. Turner, Radhakrishna (RK) Rao Occludin, an integral transmembrane protein of TJs, plays diverse roles in the regulation of epithelial cell morphology. In the intact TJs, occludin is hyper phosphorylated on Ser/ Thr residues, which undergoes dephosphorylation during the disruption by inflammatory mediators. Previous studies showed that PKCη phosphorylates occludin on T403 and T404, and its activity is required for the assembly of TJs in Caco-2 and MDCK cells. In the present study, we investigated the possible link between PKCη activity and phosphorylation of T403 and T404 in the assembly of occludin into TJs. Methods: MDCK and Caco-2 cell monolayers were transfected with GFP-OccludinWT, GFP-OccludinT403/404A or GFP-OccludinT403/ 404D. The assembly of GFP-OccludinWT and mutants into TJs was analyzed by calcium switch method. The localization of GFP-Occludin was analyzed by confocal microscopy for GFP. PKCη activity was inhibited by treatment with a PKCη-selective pseudo substrate (PKCηPS). Association of occludin with the actin cytoskeleton was determined by immunoblot analysis of detergent-insoluble fractions. Interaction between GFP-Occludin and ZO-1 was evaluated by co-immunoprecipitation. Integration of GFP-Occludin into plasma membrane was determined by biotinylation of cell surface proteins at the extracellular surface. Results: Expression of GFP-OccludinWT in both Caco-2 and MDCK cells showed its translocation into the intercellular junctions. GFP-OccludinT403/404A mutant failed to localize at the intercellular junctions with predominant localization in the intracellular compartment. GFPOccludinT403/404D mutant, on the other hand, was translocated to the intercellular junctions, which is similar to wild type occludin. PKCηPS prevented the calcium-induced assembly of GFP-OccludinWT into the intercellular junctions. On the other hand, PKCηPS failed to affect calcium-induced junctional localization of GFP-OccludinT403/404D mutant, indicating that PKCη-mediated assembly of occludin into the TJs requires its phosphorylation on T403 and T404 residues. PKCηPS induced redistribution of GFP-OccludinWT in intact cell monolayers, while it showed in effect on the junctional organization of GFP-OccludinT403/ 404D. Extracellular biotinylation studies showed that both GFP-OccludinWT and GFP-OccludinT403/404A were at least associated with the plasma membrane, thus ruling out the possibility that loss of T403/404 phosphorylation results in defective occludin trafficking to the plasma membrane. Conclusion: Therefore, phosphorylation of occludin on T403/404 is required for the assembly of occludin into TJs and PKCη activity may play a role in the phosphorylation of occludin.
824 Acetaldehyde Disrupts Tight Junctions (TJS) in CACO-2 Cell Monolayers By Pp2a Translocation and Dephosphorylation of Occludin On Threonine Residues M. Dunagan, Radhakrishna (RK) Rao, Ankur Seth Elevated intestinal permeability and endotoxemia play a crucial role in the pathogenesis of alcoholic liver disease. Acetaldehyde produced in colonic lumen via ethanol metabolism by intestinal flora increases intestinal permeability to endotoxins. In the present study we evaluated the role of PP2A in the acetaldehyde-induced disruption of TJ in Caco-2 cell monolayers. METHODS: Caco-2 cell monolayers were exposed to 400 µM acetaldehyde for varying times, and the epithelial barrier function was evaluated by measuring transepithelial electrical resistance (TER) and inulin permeability. The integrity of TJs was assessed by confocal microscopy for occludin and ZO-1. The role of PP2A was determined by evaluating the effects of fostriecin (a PP2A-selective inhibitor) and knockdown of PP2A by siRNA on acetaldehyde-induced TJ disruption. PP2A translocation to TJ was evaluated by coimmunoprecipitation and colocalization of PP2A with occludin. PP2A was detected by immunoblot analysis and its activity measured by immunocomplex PP2A-phosphatase assay. The phosphorylation state of occludin was determined by immunoprecipitation of p-Thr from the detergent-insoluble fraction followed by immunoblot analysis and densitometry. RESULTS: Acetaldehyde treatment resulted in a time-dependent increase in inulin permeability without affecting the cell viability. Increase in inulin permeability was associated with a redistribution of occludin and ZO-1 from the intercellular junction into the intracellular compartment. Preincubation of cells with 50 nM fostriecin significantly reduced acetaldehydeinduced increase in inulin permeability and redistribution of occludin and ZO-1. Selective knockdown of PP2A by siRNA also significantly attenuated acetaldehyde-induced paracellular permeability and TJ disruption. Co-immunoprecipitation studies showed that interaction of PP2A with occludin was dramatically increased following acetaldehyde treatment. PP2Aphosphatase activity associated with the occludin-immunocomplex was also increased by acetaldehyde. Acetaldehyde treatment rapidly reduced Thr-phosphorylation of occludin, but the presence of fostriecin significantly preserved Thr-phosphorylation. Acetaldehyde also reduced Thr-phosphorylation of ZO-1, but fostriecin showed no significant influence on it. Thr-phosphorylation of E-cadherin and β-catenin was unaffected by acetaldehyde. Fostriecin also attenuated acetaldehyde-induced reduction in the levels of occludin in the detergentinsoluble fractions. CONCLUSIONS: These results demonstrate that acetaldehyde-induced disruption of TJ is mediated by translocation of PP2A to the TJ and dephosphorylation of TJ on Thr residues.
827 Actomyosin-Dependent ZO-1 Stabilization At the Tight Junction Regulates Barrier Function. Dan Yu, Jerrold R. Turner The epithelial tight junction is a highly-organized structure supported by a perijunctional ring of F-actin and myosin II. Actin depolymerization disrupts tight junction structure and barrier function. The tight junction protein ZO-1, which binds directly to actin has been proposed to participate in this process via unknown mechanisms. In inflammatory bowel disease, myosin light chain kinase (MLCK) is a critical mediator of actomyosin-dependent barrier function regulation, but the role of ZO-1 in this process has not been explored. We recently found that ZO-1 exchanges rapidly between tight junction and cytoplasmic pools at steady-state. The aim of this study was to determine the relationship of ZO-1 stability and tight junction barrier function and to define the role of the ZO-1 actin binding region (ABR) in actomyosin-dependent barrier regulation. METHODS: Caco-2 monolayers expressing EGFP-ZO-1, EGFP-ZO-1∆ABR in which the ABR was deleted, or EGFP-β-actin were studied by fluorescence recovery after photobleaching (FRAP) to assess protein stability at the tight junction. Barrier function was measured as transepithelial resistance (TER). Monolayers were treated with the highly specific MLCK inhibitor PIK as indicated. RESULTS: Nearly half, 45%, of ZO-1 is immobile at the tight junction in control monolayers. This ZO-1 stabilization is partially mediated via actin binding, since only 27% of ZO-1∆ABR is immobile at the tight junction. MLCK inhibition with PIK enhanced ZO-1 stability by increasing the immobile fraction at the tight junction from 45% to 80%. In contrast, PIK did not affect the stability of ZO-1∆ABR. Consistent with a relationship between ZO-1 stability at the tight junction and barrier function, PIK increased TER by 47% while ZO1∆ABR expression decreased TER by 40%. In contrast to ZO-1, continuous cycling of actin between cytosolic and tight junction-associated pools is necessary for barrier regulation. Agents that prevent actin exchange, including C. difficile toxin B (which ribosylates rho), the myosin ATPase inhibitor blebbistatin, and latrunculin A (which inhibits actin polymerization) stabilize ZO-1 but fail to enhance TER. Notably, these agents also stabilize ZO-1∆ABR, suggesting that they stabilize ZO-1 by a mechanism that does not involve the ABR. CONCLUSION: MLCK inhibition stabilizes ZO-1 at the tight junction via mechanisms that require direct interactions between actin and ZO-1 and are mediated by the ZO-1 ABR. Modulation of ZO-1 stabilization is a critical component of MLCK-dependent regulation of epithelial (tight junction) barrier function. Supported by NIDDK.
825 Reciprocal Effects of Tumor Necrosis Factor Alpha and Prohibitin in Intestinal Epithelial Cells Arianne L. Theiss, Ngozi I. Okoro, Didier Merlin, Shanthi V. Sitaraman INTRODUCTION: Expression of prohibitin (PHB), a multi-functional protein in the cell, is decreased during inflammatory bowel disease (IBD). We recently demonstrated that restoration of PHB levels decreases the severity of colitis. Since little is known regarding the regulation of PHB expression in the intestine, we examined the effect of tumor necrosis factor alpha (TNF), a cytokine known to play a central role in the pathogenesis of IBD, on PHB expression. In addition, we examined the effect of PHB overexpression on TNF-induced epithelial cell permeability. METHODS: As an In Vitro model, the Caco2-BBE and T84 human intestinal epithelial cell lines were used. The 1192 bp human PHB promoter region was cloned and the transcription start site was identified 1594 bp upstream from the translation start site due to an intervening intron. Site-directed mutagenesis was used to mutate the putative NF-kB binding site in the PHB promoter. TNF effects on PHB promoter activation, protein and mRNA expression were determined. Effect of PHB overexpression on TNF-induced increased epithelial cell permeability was assessed using FITC-dextran translocation. RESULTS: TNF decreases PHB protein and mRNA abundance in Caco2-BBE and T84 cells. TNF also inhibited PHB promoter activation as assessed by luciferase activity. Mutation of the NF-kB site in the PHB promoter or addition of NF-kB inhibitors abolished TNF-mediated inhibition of PHB promoter activation. TNF increased FITC-dextran translocation, which was inhibited in cells overexpressing PHB. CONCLUSIONS: TNF decreases PHB expression at the transcriptional level via NF-kB binding. Importantly, PHB overexpression protects against TNF-mediated epithelial barrier dysfunction. Given that PHB levels are altered in IBD, these results provide important insights into the regulation of PHB expression by TNF and suggest that restoring PHB levels in intestinal epithelial cells may combat the deleterious effects of TNF on barrier function.
AGA Abstracts
828 Glucocorticoids Stimulate Tight Junction Sealing in Intestinal Epithelial Cells By Modulating Expression of Claudin-2 and Claudin-4 Andreas Fischer, Markus Gluth, Ulrich-Frank Pape, Bertram Wiedenmann, Daniel C. Baumgart, Franz Theuring Background and aims: Tight junctions within the intestinal epithelium are key elements of the intestinal barrier, perturbations of which have been implicated in the pathogenesis of a variety of intestinal inflammatory conditions, including Crohn's disease, ulcerative colitis, celiac sprue and food allergies. Glucocorticoids have been demonstrated to induce tight junction sealing in a variety of experimental systems, however, the effect of these hormones on intestinal tight junctions has not been studied yet. Thus, we aimed to analyze the role of glucocorticoid signaling in the regulation of paracellular permeability in the intestinal
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mortality (1.6% vs 14.4%, p<0.001 χ2). CONCLUSIONS: 1) Azotemia at admission is a significant risk factor for mortality in AP 2) Among all patients, a rise in BUN predicted increased risk of mortality 3) Among patients with azotemia at admission, a reduction in BUN within 48 hours was associated with improved survival.
AGA Abstracts
epithelium using an In Vitro model of the epithelial barrier. Methods: Caco-2 monolayers grown on semipermeable filter supports were stimulated with varying concentrations of endogenous and synthetic glucocorticoids and tight junction function was assessed by measuring transepithelial electrical resistance as well as permeability of fluorophore-coupled tracer compounds. Basic tight junction structure was analyzed by immunofluorescence against ZO-1 and occludin as well as phalloidin staining. Real time rt-PCR was employed to assess transcriptional changes of different claudin genes; in addition, expression of claudin2 and claudin-4 was analyzed by Western blotting. Results: Upon glucocorticoid stimulation, fully differentiated Caco-2 monolayers exhibited a dose dependent significant increase in transepithelial electrical resistance that could be completely reversed by the glucocorticoid receptor antagonist RU-486. Basic tight junction architecture as assessed by localization of ZO-1 and occludin was not altered upon steroid stimulation. Phalloidin staining of the actin cytoskeleton displayed no alterations in these cells. Using real time rt-PCR, we observed a significant down-regulation of the pore forming tight junction component claudin-2, whereas claudin-4 displayed a significant upregulation upon glucocorticoid stimulation on the RNA level. Western blotting confirmed these results on the protein level. Conclusions: Our data suggest that glucocorticoid signaling regulates intestinal barrier function by controlling claudin expression within the intestinal epithelium. We thereby identified a novel mode of action potentially contributing to the well established anti-inflammatory potency of these compounds. The elucidation of the molecular mechanisms involved in this regulation may not only enhance our understanding of glucocorticoid effects on the intestinal epithelium, but also allow for the validation of novel drug targets aiming to enhance or re-establish intestinal barrier function.
831 Prognostic Factors and Outcome of 121 Patients with Non-Surgical Therapy of Necrotizing Pancreatitis: What Is the Impact of An Infection of the Necrosis? Wolfgang Huber, Marion Neudeck, Katja Hauptvogel, Andreas Umgelter, Wolfgang Reindl, Andreas Weber, Jens T. Siveke, Christoph Lampart, Roland M. Schmid
829 Gastrointestinal Permeability and Infectious Complications in Acute Pancreatitis; a Prospective Multicenter Study Marc G. Besselink, Hjalmar C. van Santvoort, Marja A. Boermeester, Kathelijn Fischer, Willem Renooij, Martin B. de Smet, Geert A. Cirkel, Bert van Ramshorst, Bas L. Weusten, Alexander F. Schaapherder, Ben J. Witteman, Rutger J. Ploeg, Harry van Goor, Cees J. van Laarhoven, Camiel Rosman, Menno A. Brink, Erwin van der Harst, Peter J. Wahab, Casper H. van Eijck, Cornelis H. Dejong, Karel J. Van Erpecum, Louis M. Akkermans, Hein G. Gooszen, Members Dutch Acute Pancreatitis Study Group
Background: The mortality of necrotizing pancreatitis (NP) is up to 40%. Several recent studies demonstrated successful conservative (i.e. non-surgical) therapy of NP with sterile necroses. Despite the absence of convincing data, the presence of bacteria or fungi in the necroses usually leads to surgical therapy. Therefore, within the last 13 years our patients were treated conservatively regardless of the presence of an infection of necroses. Aims of the study: To investigate the prognostic factors and the outcome of 121 patients with proven NP admitted to a medical ICU between 1994 and 2006. Methods: systematic evaluation of outcome and risk factors including microbiological data from the aspirates/drainages of pancreatic necroses. Multiple regression analysis with “y = maximal increase in APACHEII score after admission on the ICU”; chi-square-test. Clinical algorithm: antibiotic therapy with imipenem or according to antibiogramm; repeated CT and puncture or drainage in case of clinical deterioration and/or deterioration of laboratory markers of inflammation (leukocytes, CRP, procalcitonin); surgery only in case of complications of the puncture or fluid collections not accessible to radiological drainage (n=3). Results: 1.) Patients characteristics: n=121; 48 female; 73male; age 56.7+-15.6 years, maximum CRP 28.4+-11.7mg/dl, max. APACHE-II-Score 20.0+-11.8, max. lipase 7375+-13193U/L. 40/98 patients (41%) required mechanical ventilation and 29/98 (30%) dialysis. 2.) Prognosis: The only independent risk factors at admission to the ICU for an unfavourable outcome were the level of serum creatinine (p=0.0007) and old age (p=0.035). The following parameters were not predictive: aetiology of pancreatitis, blood/serum levels of lipase, calcium, glucose, leukocytes and hematocrit as well as the presence of Cullen- and/or Grey-Turner-sign. 3.) Mortality: The overall mortality was 14/121 (12%). In 65 patients puncture and/or drainage of the necroses was performed. The mortality of these patients (8/65; 12%) was not different compared to the patients without puncture/drainage (6/56; 11%). In 49/65 (75%) of the patients with puncture bacteria and/or fungi were cultured in the aspirates. The mortality of these patients (6/49; 12%) was not different compared to the patients with sterile necrosis. Conclusions: 1.) The overall mortality of 12% was low with regard to severity of pancreatitis. 2.) Infection of the necroses had no impact on the patients outcome. Therefore, the presence of infected necrosis is no contraindication to conservative management of NP. 3.) The most important predictors for the outcome were serum creatinine levels and old age.
Background: Infectious complications and organ failure are major determinants for mortality in acute pancreatitis. Increase in gastrointestinal permeability is thought to be an early step in the pathophysiological process leading up to these events. To date, no study has reported a relationship between increased gastrointestinal permeability and the risk of infectious complications. Methods: We conducted a prospective study in 159 patients with acute pancreatitis in 14 hospitals in the Netherlands. Within 72 hours after admission a mixture of 4 polyethylene glycols (PEGs) with varying molecular weights: 5g PEG 400, 1.5g PEG 1500, 5g PEG 4000 and 10g PEG 10000 dissolved in 100 ml water was orally administered. PEG 400 can freely pass the intact intestinal mucosal barrier, whereas larger PEGs can only pass if intestinal permeability is compromised. Twenty-four hours urine output was collected and PEG-recovery determined by high performance liquid chromatography. We investigated whether the intestinal permeability, as measured with PEGs, is associated with bacteremia, and infected pancreatic necrosis, organ failure and mortality. Onset of organ failure, bacteremia and infected necrosis is presented in days after admission (mean, ± SD). When there was a significant difference in recovery of PEGs of several molecular weights, only for the largest PEG the p-value is given. Results: Overall mortality rate was 2.5%. Recovery of PEG 10000 in urine was higher in deceased patients (p=0.01). Organ failure at the time of the PEG test (9 patients; 6%) was associated with higher PEG 4000 recovery (p=0.02). The 15 patients who developed organ failure during admission (9.4%; mean 6.3 days ± 9.1 after admission) had higher recovery of PEG 10000 than patients without organ failure during admission (p=0.03). The 22 patients with bacteremia during admission (13.8%; mean 9.0 days ± 10.6 after admission) had higher recovery of PEG 4000 than patients without bacteremia (p=0.001). In patients without organ failure during the PEG test, bacteremia remained associated with higher recovery of PEG 4000 (p=0.005). Twelve patients with infected necrosis during admission (7.5%; mean 38.8 days ± 25.1) had non-significantly higher recovery of PEG 1500 than patients without infected necrosis (p=0.11). Conclusions: Increased intestinal permeability in the first 72 hours of acute pancreatitis is associated with mortality, organ failure (early and late) and bacteremia. Strategies aiming at a reduction of infectious complications by fortifying the gastrointestinal mucosal barrier should start as early as possible in the course of acute pancreatitis.
832 Prospective Randomized Trial Comparing Early Versus Delayed Oral Refeeding in Moderate Acute Pancreatitis Pierre H. Deprez, Andre P. Geubel, Etienne Danse, Yves Horsmans INTRODUCTION: In mild to moderate acute pancreatitis (AP) oral refeeding can be started when pain is controlled and the pancreatic enzymes return to normal (ESPEN guidelines 2002). Unfortunately, not enough information is available on the optimal timing of refeeding. AIMS & METHODS: To compare early vs. delayed oral refeeding in a prospective randomised trial. Inclusion criteria: AP of mild to moderate severity defined as Ranson's score <3, CT scan severity index CTSI <5, necrosis <30% and CRP < 12mg/dl, delay between start of pain and admission less than 48h, inform consent. Timing of refeeding: early (in the 24h after pain disappearance) vs. delayed (after normalisation of pancreatic enzymes or 72h after admission). Progressive intake of 1000, 1400,1800 kcal on 1st, 2nd and 3rd day, respectively. Resting energy expenditure (REE) was measured by indirect calorimetry. Objectives: comparison of length of stay, tolerance and pain relapse, nutritional and inflammatory parameters. Results are expressed as mean ± 95%CI and statistical analysis was done by one-way analysis of variance. RESULTS: 34 pts were included (16 W,18 M; mean age 47±6, BMI 25±2), 14 in the early and 11 in the delayed refeeding group. Results are summarized in table. Mean CRP level at 48h was 8.7±3.1mg/dl, CTSI 2,3±0,4, Ranson's score 1±0,3 and length of stay 149,7±21,9 hours. Delay between start of pain and admission was 15±6h and total duration of pain 71±16 h. There were no differences between groups concerning patients characteristics, etiology (42% biliary, 36% ethanol, 22% misc.), tolerance to refeeding, CRP or pancreatic enzymes rises after refeeding. Daily caloric intake was 851±109 kcal (12.1% proteins, 45% glucids, 30% lipids) increasing from 417 to 818 and 1378 kcal on 1st, 2nd and 3rd days, respectively, sign. below measured REE. Early refeeding however reduced significantly length of stay compared to delayed oral refeeding (P=0,03). Higher CRP levels, CTSI scores were significantly correlated to length of stay. CONCLUSION: Oral early refeeding defined as intake of food in the 24h following disappearance of pain resulted in a significant shortening
830 Correction of Azotemia Is Associated with Reduced Mortality in Acute Pancreatitis Bechien U. Wu, Richard S. Johannes, Xiaowu Sun, Darwin Conwell, Peter A. Banks INTRODUCTION: While an increase in BUN >5mg/dL at 48 hours is one of the Ranson criteria of severity in AP, the impact of correcting azotemia on mortality is unknown. AIMS: 1) To confirm whether azotemia at admission (BUN>25 mg/dL)is a risk factor for in-hospital mortality in AP 2) To determine whether serial BUN measurements can help predict inhospital mortality 3) To assess whether correction of azotemia is associated with reduced mortality. METHODS: A retrospective cohort study was performed on patient data obtained from the Cardinal Health Outcomes Research Database; a large population-based dataset. Cases from 72 participating U.S. hospitals were identified from 2003 thru 2006 by ICD9CM 577.0 (AP). Eligible cases were further restricted to those with ≥3 BUN measurements within 48 hours of admission. Linear regression was used to identify individual patient laboratory trajectories. RESULTS: There were a total of 15,153 AP cases with 173 (1.1%) deaths. Among these, 6,613 patients had ≥ 3 BUN measurements within 48-hours. There were 93 (1.4%) deaths in this cohort of patients. Azotemia at admission was a significant risk factor for mortality (0.8% vs. 4.1%, p<0.001 χ2). Among all patients, an increase in BUN was associated with significantly increased mortality (p<0.001 χ2, see figure). Among the 1,301 patients with azotemia at admission, there were 53 (4.1%) deaths. In these patients, correction of azotemia within the first 48 hours was associated with significantly reduced
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AGA Abstracts
the role of myosin light chain kinase (MLCK) in epithelial cell shedding and whether the tight junction potentially contributes to maintenance of barrier function at these sites. Methods: Transgenic mice were generated that expressed ZO-1 or occludin as fusion proteins with fluorescent proteins, with expression in intestinal epithelia driven by the villin promoter. In some experiments, these mice were crossed with long myosin light chain kinase (MLCK) knockout mice. After injection with TNF-α (7.5µg, i.p.) a laparotomy was performed on the anesthetised mouse and a segment of small intestine opened so that the shedding of epithelial cells could be evaluated by video confocal microscopy. Results: TNF-α increased shedding by 27-fold (p<0.007) versus non-injected controls, beginning 60 min after TNFα exposure. The fluorescent fusion proteins showed normal cellular distribution compared to immunostained wildtype mice. However during stimulated cell shedding a redistribution of both ZO-1 and occludin was observed. Over the 10 min during which shedding of a single cell occurred, both ZO-1 and occludin migrated downward along lateral membranes and condensed beneath shedding nuclei to form a funnel that moved apically with the extruding cell. Extracellular dye probes (e.g. Alexa 488 in perfusate solution) approached but did not cross the line of ZO-1 and occludin, demonstrating maintenance of the mucosal barrier at these sites. MLCK knockout mice had similar ZO-1 remodelling, suggesting this process may not require MLCK. Conclusion: We conclude that ZO-1 and occludin undergo novel MLCK-independent redistribution at sites of barrier maintenance during TNF-induced cell shedding. Supported by NIH.
826 T403/404d Mutation of Occludin Attenuates PKCη-Mediated Regulation of Epithelial Tight Junctions (TJS) Bertha Elias, Sudhir Aggarwal, Le Shen, Jerrold R. Turner, Radhakrishna (RK) Rao Occludin, an integral transmembrane protein of TJs, plays diverse roles in the regulation of epithelial cell morphology. In the intact TJs, occludin is hyper phosphorylated on Ser/ Thr residues, which undergoes dephosphorylation during the disruption by inflammatory mediators. Previous studies showed that PKCη phosphorylates occludin on T403 and T404, and its activity is required for the assembly of TJs in Caco-2 and MDCK cells. In the present study, we investigated the possible link between PKCη activity and phosphorylation of T403 and T404 in the assembly of occludin into TJs. Methods: MDCK and Caco-2 cell monolayers were transfected with GFP-OccludinWT, GFP-OccludinT403/404A or GFP-OccludinT403/ 404D. The assembly of GFP-OccludinWT and mutants into TJs was analyzed by calcium switch method. The localization of GFP-Occludin was analyzed by confocal microscopy for GFP. PKCη activity was inhibited by treatment with a PKCη-selective pseudo substrate (PKCηPS). Association of occludin with the actin cytoskeleton was determined by immunoblot analysis of detergent-insoluble fractions. Interaction between GFP-Occludin and ZO-1 was evaluated by co-immunoprecipitation. Integration of GFP-Occludin into plasma membrane was determined by biotinylation of cell surface proteins at the extracellular surface. Results: Expression of GFP-OccludinWT in both Caco-2 and MDCK cells showed its translocation into the intercellular junctions. GFP-OccludinT403/404A mutant failed to localize at the intercellular junctions with predominant localization in the intracellular compartment. GFPOccludinT403/404D mutant, on the other hand, was translocated to the intercellular junctions, which is similar to wild type occludin. PKCηPS prevented the calcium-induced assembly of GFP-OccludinWT into the intercellular junctions. On the other hand, PKCηPS failed to affect calcium-induced junctional localization of GFP-OccludinT403/404D mutant, indicating that PKCη-mediated assembly of occludin into the TJs requires its phosphorylation on T403 and T404 residues. PKCηPS induced redistribution of GFP-OccludinWT in intact cell monolayers, while it showed in effect on the junctional organization of GFP-OccludinT403/ 404D. Extracellular biotinylation studies showed that both GFP-OccludinWT and GFP-OccludinT403/404A were at least associated with the plasma membrane, thus ruling out the possibility that loss of T403/404 phosphorylation results in defective occludin trafficking to the plasma membrane. Conclusion: Therefore, phosphorylation of occludin on T403/404 is required for the assembly of occludin into TJs and PKCη activity may play a role in the phosphorylation of occludin.
824 Acetaldehyde Disrupts Tight Junctions (TJS) in CACO-2 Cell Monolayers By Pp2a Translocation and Dephosphorylation of Occludin On Threonine Residues M. Dunagan, Radhakrishna (RK) Rao, Ankur Seth Elevated intestinal permeability and endotoxemia play a crucial role in the pathogenesis of alcoholic liver disease. Acetaldehyde produced in colonic lumen via ethanol metabolism by intestinal flora increases intestinal permeability to endotoxins. In the present study we evaluated the role of PP2A in the acetaldehyde-induced disruption of TJ in Caco-2 cell monolayers. METHODS: Caco-2 cell monolayers were exposed to 400 µM acetaldehyde for varying times, and the epithelial barrier function was evaluated by measuring transepithelial electrical resistance (TER) and inulin permeability. The integrity of TJs was assessed by confocal microscopy for occludin and ZO-1. The role of PP2A was determined by evaluating the effects of fostriecin (a PP2A-selective inhibitor) and knockdown of PP2A by siRNA on acetaldehyde-induced TJ disruption. PP2A translocation to TJ was evaluated by coimmunoprecipitation and colocalization of PP2A with occludin. PP2A was detected by immunoblot analysis and its activity measured by immunocomplex PP2A-phosphatase assay. The phosphorylation state of occludin was determined by immunoprecipitation of p-Thr from the detergent-insoluble fraction followed by immunoblot analysis and densitometry. RESULTS: Acetaldehyde treatment resulted in a time-dependent increase in inulin permeability without affecting the cell viability. Increase in inulin permeability was associated with a redistribution of occludin and ZO-1 from the intercellular junction into the intracellular compartment. Preincubation of cells with 50 nM fostriecin significantly reduced acetaldehydeinduced increase in inulin permeability and redistribution of occludin and ZO-1. Selective knockdown of PP2A by siRNA also significantly attenuated acetaldehyde-induced paracellular permeability and TJ disruption. Co-immunoprecipitation studies showed that interaction of PP2A with occludin was dramatically increased following acetaldehyde treatment. PP2Aphosphatase activity associated with the occludin-immunocomplex was also increased by acetaldehyde. Acetaldehyde treatment rapidly reduced Thr-phosphorylation of occludin, but the presence of fostriecin significantly preserved Thr-phosphorylation. Acetaldehyde also reduced Thr-phosphorylation of ZO-1, but fostriecin showed no significant influence on it. Thr-phosphorylation of E-cadherin and β-catenin was unaffected by acetaldehyde. Fostriecin also attenuated acetaldehyde-induced reduction in the levels of occludin in the detergentinsoluble fractions. CONCLUSIONS: These results demonstrate that acetaldehyde-induced disruption of TJ is mediated by translocation of PP2A to the TJ and dephosphorylation of TJ on Thr residues.
827 Actomyosin-Dependent ZO-1 Stabilization At the Tight Junction Regulates Barrier Function. Dan Yu, Jerrold R. Turner The epithelial tight junction is a highly-organized structure supported by a perijunctional ring of F-actin and myosin II. Actin depolymerization disrupts tight junction structure and barrier function. The tight junction protein ZO-1, which binds directly to actin has been proposed to participate in this process via unknown mechanisms. In inflammatory bowel disease, myosin light chain kinase (MLCK) is a critical mediator of actomyosin-dependent barrier function regulation, but the role of ZO-1 in this process has not been explored. We recently found that ZO-1 exchanges rapidly between tight junction and cytoplasmic pools at steady-state. The aim of this study was to determine the relationship of ZO-1 stability and tight junction barrier function and to define the role of the ZO-1 actin binding region (ABR) in actomyosin-dependent barrier regulation. METHODS: Caco-2 monolayers expressing EGFP-ZO-1, EGFP-ZO-1∆ABR in which the ABR was deleted, or EGFP-β-actin were studied by fluorescence recovery after photobleaching (FRAP) to assess protein stability at the tight junction. Barrier function was measured as transepithelial resistance (TER). Monolayers were treated with the highly specific MLCK inhibitor PIK as indicated. RESULTS: Nearly half, 45%, of ZO-1 is immobile at the tight junction in control monolayers. This ZO-1 stabilization is partially mediated via actin binding, since only 27% of ZO-1∆ABR is immobile at the tight junction. MLCK inhibition with PIK enhanced ZO-1 stability by increasing the immobile fraction at the tight junction from 45% to 80%. In contrast, PIK did not affect the stability of ZO-1∆ABR. Consistent with a relationship between ZO-1 stability at the tight junction and barrier function, PIK increased TER by 47% while ZO1∆ABR expression decreased TER by 40%. In contrast to ZO-1, continuous cycling of actin between cytosolic and tight junction-associated pools is necessary for barrier regulation. Agents that prevent actin exchange, including C. difficile toxin B (which ribosylates rho), the myosin ATPase inhibitor blebbistatin, and latrunculin A (which inhibits actin polymerization) stabilize ZO-1 but fail to enhance TER. Notably, these agents also stabilize ZO-1∆ABR, suggesting that they stabilize ZO-1 by a mechanism that does not involve the ABR. CONCLUSION: MLCK inhibition stabilizes ZO-1 at the tight junction via mechanisms that require direct interactions between actin and ZO-1 and are mediated by the ZO-1 ABR. Modulation of ZO-1 stabilization is a critical component of MLCK-dependent regulation of epithelial (tight junction) barrier function. Supported by NIDDK.
825 Reciprocal Effects of Tumor Necrosis Factor Alpha and Prohibitin in Intestinal Epithelial Cells Arianne L. Theiss, Ngozi I. Okoro, Didier Merlin, Shanthi V. Sitaraman INTRODUCTION: Expression of prohibitin (PHB), a multi-functional protein in the cell, is decreased during inflammatory bowel disease (IBD). We recently demonstrated that restoration of PHB levels decreases the severity of colitis. Since little is known regarding the regulation of PHB expression in the intestine, we examined the effect of tumor necrosis factor alpha (TNF), a cytokine known to play a central role in the pathogenesis of IBD, on PHB expression. In addition, we examined the effect of PHB overexpression on TNF-induced epithelial cell permeability. METHODS: As an In Vitro model, the Caco2-BBE and T84 human intestinal epithelial cell lines were used. The 1192 bp human PHB promoter region was cloned and the transcription start site was identified 1594 bp upstream from the translation start site due to an intervening intron. Site-directed mutagenesis was used to mutate the putative NF-kB binding site in the PHB promoter. TNF effects on PHB promoter activation, protein and mRNA expression were determined. Effect of PHB overexpression on TNF-induced increased epithelial cell permeability was assessed using FITC-dextran translocation. RESULTS: TNF decreases PHB protein and mRNA abundance in Caco2-BBE and T84 cells. TNF also inhibited PHB promoter activation as assessed by luciferase activity. Mutation of the NF-kB site in the PHB promoter or addition of NF-kB inhibitors abolished TNF-mediated inhibition of PHB promoter activation. TNF increased FITC-dextran translocation, which was inhibited in cells overexpressing PHB. CONCLUSIONS: TNF decreases PHB expression at the transcriptional level via NF-kB binding. Importantly, PHB overexpression protects against TNF-mediated epithelial barrier dysfunction. Given that PHB levels are altered in IBD, these results provide important insights into the regulation of PHB expression by TNF and suggest that restoring PHB levels in intestinal epithelial cells may combat the deleterious effects of TNF on barrier function.
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828 Glucocorticoids Stimulate Tight Junction Sealing in Intestinal Epithelial Cells By Modulating Expression of Claudin-2 and Claudin-4 Andreas Fischer, Markus Gluth, Ulrich-Frank Pape, Bertram Wiedenmann, Daniel C. Baumgart, Franz Theuring Background and aims: Tight junctions within the intestinal epithelium are key elements of the intestinal barrier, perturbations of which have been implicated in the pathogenesis of a variety of intestinal inflammatory conditions, including Crohn's disease, ulcerative colitis, celiac sprue and food allergies. Glucocorticoids have been demonstrated to induce tight junction sealing in a variety of experimental systems, however, the effect of these hormones on intestinal tight junctions has not been studied yet. Thus, we aimed to analyze the role of glucocorticoid signaling in the regulation of paracellular permeability in the intestinal
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mortality (1.6% vs 14.4%, p<0.001 χ2). CONCLUSIONS: 1) Azotemia at admission is a significant risk factor for mortality in AP 2) Among all patients, a rise in BUN predicted increased risk of mortality 3) Among patients with azotemia at admission, a reduction in BUN within 48 hours was associated with improved survival.
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epithelium using an In Vitro model of the epithelial barrier. Methods: Caco-2 monolayers grown on semipermeable filter supports were stimulated with varying concentrations of endogenous and synthetic glucocorticoids and tight junction function was assessed by measuring transepithelial electrical resistance as well as permeability of fluorophore-coupled tracer compounds. Basic tight junction structure was analyzed by immunofluorescence against ZO-1 and occludin as well as phalloidin staining. Real time rt-PCR was employed to assess transcriptional changes of different claudin genes; in addition, expression of claudin2 and claudin-4 was analyzed by Western blotting. Results: Upon glucocorticoid stimulation, fully differentiated Caco-2 monolayers exhibited a dose dependent significant increase in transepithelial electrical resistance that could be completely reversed by the glucocorticoid receptor antagonist RU-486. Basic tight junction architecture as assessed by localization of ZO-1 and occludin was not altered upon steroid stimulation. Phalloidin staining of the actin cytoskeleton displayed no alterations in these cells. Using real time rt-PCR, we observed a significant down-regulation of the pore forming tight junction component claudin-2, whereas claudin-4 displayed a significant upregulation upon glucocorticoid stimulation on the RNA level. Western blotting confirmed these results on the protein level. Conclusions: Our data suggest that glucocorticoid signaling regulates intestinal barrier function by controlling claudin expression within the intestinal epithelium. We thereby identified a novel mode of action potentially contributing to the well established anti-inflammatory potency of these compounds. The elucidation of the molecular mechanisms involved in this regulation may not only enhance our understanding of glucocorticoid effects on the intestinal epithelium, but also allow for the validation of novel drug targets aiming to enhance or re-establish intestinal barrier function.
831 Prognostic Factors and Outcome of 121 Patients with Non-Surgical Therapy of Necrotizing Pancreatitis: What Is the Impact of An Infection of the Necrosis? Wolfgang Huber, Marion Neudeck, Katja Hauptvogel, Andreas Umgelter, Wolfgang Reindl, Andreas Weber, Jens T. Siveke, Christoph Lampart, Roland M. Schmid
829 Gastrointestinal Permeability and Infectious Complications in Acute Pancreatitis; a Prospective Multicenter Study Marc G. Besselink, Hjalmar C. van Santvoort, Marja A. Boermeester, Kathelijn Fischer, Willem Renooij, Martin B. de Smet, Geert A. Cirkel, Bert van Ramshorst, Bas L. Weusten, Alexander F. Schaapherder, Ben J. Witteman, Rutger J. Ploeg, Harry van Goor, Cees J. van Laarhoven, Camiel Rosman, Menno A. Brink, Erwin van der Harst, Peter J. Wahab, Casper H. van Eijck, Cornelis H. Dejong, Karel J. Van Erpecum, Louis M. Akkermans, Hein G. Gooszen, Members Dutch Acute Pancreatitis Study Group
Background: The mortality of necrotizing pancreatitis (NP) is up to 40%. Several recent studies demonstrated successful conservative (i.e. non-surgical) therapy of NP with sterile necroses. Despite the absence of convincing data, the presence of bacteria or fungi in the necroses usually leads to surgical therapy. Therefore, within the last 13 years our patients were treated conservatively regardless of the presence of an infection of necroses. Aims of the study: To investigate the prognostic factors and the outcome of 121 patients with proven NP admitted to a medical ICU between 1994 and 2006. Methods: systematic evaluation of outcome and risk factors including microbiological data from the aspirates/drainages of pancreatic necroses. Multiple regression analysis with “y = maximal increase in APACHEII score after admission on the ICU”; chi-square-test. Clinical algorithm: antibiotic therapy with imipenem or according to antibiogramm; repeated CT and puncture or drainage in case of clinical deterioration and/or deterioration of laboratory markers of inflammation (leukocytes, CRP, procalcitonin); surgery only in case of complications of the puncture or fluid collections not accessible to radiological drainage (n=3). Results: 1.) Patients characteristics: n=121; 48 female; 73male; age 56.7+-15.6 years, maximum CRP 28.4+-11.7mg/dl, max. APACHE-II-Score 20.0+-11.8, max. lipase 7375+-13193U/L. 40/98 patients (41%) required mechanical ventilation and 29/98 (30%) dialysis. 2.) Prognosis: The only independent risk factors at admission to the ICU for an unfavourable outcome were the level of serum creatinine (p=0.0007) and old age (p=0.035). The following parameters were not predictive: aetiology of pancreatitis, blood/serum levels of lipase, calcium, glucose, leukocytes and hematocrit as well as the presence of Cullen- and/or Grey-Turner-sign. 3.) Mortality: The overall mortality was 14/121 (12%). In 65 patients puncture and/or drainage of the necroses was performed. The mortality of these patients (8/65; 12%) was not different compared to the patients without puncture/drainage (6/56; 11%). In 49/65 (75%) of the patients with puncture bacteria and/or fungi were cultured in the aspirates. The mortality of these patients (6/49; 12%) was not different compared to the patients with sterile necrosis. Conclusions: 1.) The overall mortality of 12% was low with regard to severity of pancreatitis. 2.) Infection of the necroses had no impact on the patients outcome. Therefore, the presence of infected necrosis is no contraindication to conservative management of NP. 3.) The most important predictors for the outcome were serum creatinine levels and old age.
Background: Infectious complications and organ failure are major determinants for mortality in acute pancreatitis. Increase in gastrointestinal permeability is thought to be an early step in the pathophysiological process leading up to these events. To date, no study has reported a relationship between increased gastrointestinal permeability and the risk of infectious complications. Methods: We conducted a prospective study in 159 patients with acute pancreatitis in 14 hospitals in the Netherlands. Within 72 hours after admission a mixture of 4 polyethylene glycols (PEGs) with varying molecular weights: 5g PEG 400, 1.5g PEG 1500, 5g PEG 4000 and 10g PEG 10000 dissolved in 100 ml water was orally administered. PEG 400 can freely pass the intact intestinal mucosal barrier, whereas larger PEGs can only pass if intestinal permeability is compromised. Twenty-four hours urine output was collected and PEG-recovery determined by high performance liquid chromatography. We investigated whether the intestinal permeability, as measured with PEGs, is associated with bacteremia, and infected pancreatic necrosis, organ failure and mortality. Onset of organ failure, bacteremia and infected necrosis is presented in days after admission (mean, ± SD). When there was a significant difference in recovery of PEGs of several molecular weights, only for the largest PEG the p-value is given. Results: Overall mortality rate was 2.5%. Recovery of PEG 10000 in urine was higher in deceased patients (p=0.01). Organ failure at the time of the PEG test (9 patients; 6%) was associated with higher PEG 4000 recovery (p=0.02). The 15 patients who developed organ failure during admission (9.4%; mean 6.3 days ± 9.1 after admission) had higher recovery of PEG 10000 than patients without organ failure during admission (p=0.03). The 22 patients with bacteremia during admission (13.8%; mean 9.0 days ± 10.6 after admission) had higher recovery of PEG 4000 than patients without bacteremia (p=0.001). In patients without organ failure during the PEG test, bacteremia remained associated with higher recovery of PEG 4000 (p=0.005). Twelve patients with infected necrosis during admission (7.5%; mean 38.8 days ± 25.1) had non-significantly higher recovery of PEG 1500 than patients without infected necrosis (p=0.11). Conclusions: Increased intestinal permeability in the first 72 hours of acute pancreatitis is associated with mortality, organ failure (early and late) and bacteremia. Strategies aiming at a reduction of infectious complications by fortifying the gastrointestinal mucosal barrier should start as early as possible in the course of acute pancreatitis.
832 Prospective Randomized Trial Comparing Early Versus Delayed Oral Refeeding in Moderate Acute Pancreatitis Pierre H. Deprez, Andre P. Geubel, Etienne Danse, Yves Horsmans INTRODUCTION: In mild to moderate acute pancreatitis (AP) oral refeeding can be started when pain is controlled and the pancreatic enzymes return to normal (ESPEN guidelines 2002). Unfortunately, not enough information is available on the optimal timing of refeeding. AIMS & METHODS: To compare early vs. delayed oral refeeding in a prospective randomised trial. Inclusion criteria: AP of mild to moderate severity defined as Ranson's score <3, CT scan severity index CTSI <5, necrosis <30% and CRP < 12mg/dl, delay between start of pain and admission less than 48h, inform consent. Timing of refeeding: early (in the 24h after pain disappearance) vs. delayed (after normalisation of pancreatic enzymes or 72h after admission). Progressive intake of 1000, 1400,1800 kcal on 1st, 2nd and 3rd day, respectively. Resting energy expenditure (REE) was measured by indirect calorimetry. Objectives: comparison of length of stay, tolerance and pain relapse, nutritional and inflammatory parameters. Results are expressed as mean ± 95%CI and statistical analysis was done by one-way analysis of variance. RESULTS: 34 pts were included (16 W,18 M; mean age 47±6, BMI 25±2), 14 in the early and 11 in the delayed refeeding group. Results are summarized in table. Mean CRP level at 48h was 8.7±3.1mg/dl, CTSI 2,3±0,4, Ranson's score 1±0,3 and length of stay 149,7±21,9 hours. Delay between start of pain and admission was 15±6h and total duration of pain 71±16 h. There were no differences between groups concerning patients characteristics, etiology (42% biliary, 36% ethanol, 22% misc.), tolerance to refeeding, CRP or pancreatic enzymes rises after refeeding. Daily caloric intake was 851±109 kcal (12.1% proteins, 45% glucids, 30% lipids) increasing from 417 to 818 and 1378 kcal on 1st, 2nd and 3rd days, respectively, sign. below measured REE. Early refeeding however reduced significantly length of stay compared to delayed oral refeeding (P=0,03). Higher CRP levels, CTSI scores were significantly correlated to length of stay. CONCLUSION: Oral early refeeding defined as intake of food in the 24h following disappearance of pain resulted in a significant shortening
830 Correction of Azotemia Is Associated with Reduced Mortality in Acute Pancreatitis Bechien U. Wu, Richard S. Johannes, Xiaowu Sun, Darwin Conwell, Peter A. Banks INTRODUCTION: While an increase in BUN >5mg/dL at 48 hours is one of the Ranson criteria of severity in AP, the impact of correcting azotemia on mortality is unknown. AIMS: 1) To confirm whether azotemia at admission (BUN>25 mg/dL)is a risk factor for in-hospital mortality in AP 2) To determine whether serial BUN measurements can help predict inhospital mortality 3) To assess whether correction of azotemia is associated with reduced mortality. METHODS: A retrospective cohort study was performed on patient data obtained from the Cardinal Health Outcomes Research Database; a large population-based dataset. Cases from 72 participating U.S. hospitals were identified from 2003 thru 2006 by ICD9CM 577.0 (AP). Eligible cases were further restricted to those with ≥3 BUN measurements within 48 hours of admission. Linear regression was used to identify individual patient laboratory trajectories. RESULTS: There were a total of 15,153 AP cases with 173 (1.1%) deaths. Among these, 6,613 patients had ≥ 3 BUN measurements within 48-hours. There were 93 (1.4%) deaths in this cohort of patients. Azotemia at admission was a significant risk factor for mortality (0.8% vs. 4.1%, p<0.001 χ2). Among all patients, an increase in BUN was associated with significantly increased mortality (p<0.001 χ2, see figure). Among the 1,301 patients with azotemia at admission, there were 53 (4.1%) deaths. In these patients, correction of azotemia within the first 48 hours was associated with significantly reduced
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AGA Abstracts