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BackgroundEpithelial barrier defects are well known in coeliac disease, but the mechanisms are only poorly defined. It is unclear, whether barrier disturbance reflects upregulated epithelial transcytosis or paracellular leakage.ObjectiveTo characterise the molecular structure and function of the epithelial tight junction (TJ) and mechanisms of its dysregulation.MethodsMolecular analysis of proteins involved in TJ assembly and their regulation was performed by western blotting and confocal microscopy correlated to electrophysiology.ResultsA complex alteration of the composition of epithelial TJ proteins (with more pore-forming claudins like claudin-2 and a reduction in tightening claudins like claudin-3, -5 and -7) was found for protein expression and subcellular localisation, responsible for an increase in paracellular biotin-NHS uptake. In contrast, epithelial apoptosis was only moderately elevated (accounting for a minor portion of barrier defects) and epithelial gross lesions—for example, at cell extrusion zones, were absent. This TJ alteration was linked to an altered localisation/expression of proteins regulating TJ assembly, the polarity complex protein Par-3 and the serine-/threonine phosphatase PP-1.ConclusionsChanges in cell polarity proteins Par-3 and PP-1 are associated with altered expression and assembly of TJ proteins claudin-2, -3, -5 and -7 and ZO-1, causing paracellular leakage in active coeliac disease.

Campylobacter concisus infections of the gastrointestinal tract can be accompanied by diarrhea and inflammation, whereas colonization of the human oral cavity might have a commensal nature. We focus on the pathophysiology of C. concisus and the effects of different clinical oral and fecal C. concisus strains on human HT-29/B6 colon cells. Six oral and eight fecal strains of C. concisus were isolated. Mucus-producing HT-29/B6 epithelial monolayers were infected with the C. concisus strains. Transepithelial electrical resistance (R(t)) and tracer fluxes of different molecule size were measured in Ussing chambers. Tight junction (TJ) protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29/B6 cells and impaired epithelial barrier function, characterized by a time- and dose-dependent decrease in R(t) either after infection from the apical side but even more from the basolateral compartment. TJ protein expression changes were sparse, only in apoptotic areas of infected monolayers TJ proteins were redistributed. Solely the barrier-forming TJ protein claudin-5 showed a reduced expression level to 66±8% (P<0.05), by expression regulation from the gene. Concomitantly, Lactate dehydrogenase release was elevated to 3.1±0.3% versus 0.7±0.1% in control (P<0.001), suggesting cytotoxic effects. Furthermore, oral and fecal C. concisus strains elevated apoptotic events to 5-fold. C. concisus-infected monolayers revealed an increased permeability for 332 Da fluorescein (1.74±0.13 vs. 0.56±0.17 10(-6) cm/s in control, P<0.05) but showed no difference in permeability for 4 kDa FITC-dextran (FD-4). The same was true in camptothecin-exposed monolayers, where camptothecin was used for apoptosis induction.In conclusion, epithelial barrier dysfunction by oral and fecal C. concisus strains could mainly be assigned to apoptotic leaks together with moderate TJ changes, demonstrating a leak-flux mechanism that parallels the clinical manifestation of diarrhea.

Background. Aeromonads cause a variety of infections, including gastroenteritis, sepsis, and wound necrosis.Pathogenesis of Aeromonas hydrophila and its hemolysin has been characterized, but the mechanism of the epithelial barrier dysfunction is currently poorly understood. Methods. Human colon epithelial monolayers HT-29/B6 were apically inoculated with clinical isolates of A. hydrophila or with the secreted pore-forming toxin aerolysin. Epithelial resistance and permeability for several markers were determined in Ussing chambers, using 2-path impedance spectroscopy. The subcellular distribution of tight junction (TJ) and cytoskeleton proteins was analyzed by Western blotting and confocal laser-scanning microscopy. Results. A. hydrophila infection induces chloride secretion with a small decrease in transcellular resistance.However, the major effect of A. hydrophila, mediated by its toxin aerolysin, was a sustained reduction of paracellular resistance by retracting sealing TJ proteins from the TJ strands. Aerolysin-treated monolayers showed increased paracellular permeability to FITC-dextran-4000 (0.104 ± 0.014 vs 0.047 ± 0.001 10⁻₆cm/s in control; P < .05).Moreover, restitution of epithelial lesions was impaired. The effects were myosin light chain kinase (MLCK) dependent and mediated by intracellular Ca²⁺ signaling. Conclusions. During Aeromonas infection, pore formation by aerolysin impairs epithelial integrity by promoting TJ protein redistribution and consequently affecting wound closure. Thus, Aeromonas-indnctd diarrhea is mediated by 2 mechanisms, transcellular secretion and paracellular leak flux.

The type of a nucleic acid and the type of the cell to be transfected generally affect the efficiency of electroporation, the versatile method of choice for gene regulation studies or for recombinant protein expression. We here present a combined square pulse electroporation strategy to reproducibly and efficiently transfect eukaryotic cells. Cells suspended in a universal buffer system received an initial high voltage pulse that was continuously combined with a subsequent low voltage pulse with independently defined electric parameters of the effective field and the duration of each pulse. At comparable viable cell recoveries and transfection efficiencies of up to 95% of all cells, a wide variety of cells especially profited from this combined pulse strategy by high protein expression levels of individual cells after transfection. Long-term silencing of gene expression by transfected small interfering RNA was most likely due to the uptake of large nucleic acid amounts as shown by direct detection of fluorochromated small interfering RNA. The highly efficient combined pulse electroporation strategy enables for external regulation of the number of naked nucleic acid molecules taken up and can be easily adapted for cells considered difficult to transfect.

Osteopontin (OPN) is characterized as a major amplifier of Th1‐immune responses. However, its role in intestinal inflammation is currently unknown. We found considerably raised OPN levels in blood of wild‐type (WT) mice with dextran sodium sulfate (DSS)‐induced colitis. To identify the role of this mediator in intestinal inflammation, we analysed experimental colitis in OPN‐deficient (OPN−/−) mice. In the acute phase of colitis these mice showed more extensive colonic ulcerations and mucosal destruction than WT mice, which was abrogated by application of soluble OPN. Within the OPN−/– mice, infiltrating macrophages were not activated and showed impaired phagocytosis. Reduced mRNA expression of interleukin (IL)‐1 β and matrix metalloproteinases was found in acute colitis of OPN−/– mice. This was associated with decreased blood levels of IL‐22, a Th17 cytokine that may mediate epithelial regeneration. However, OPN–/– mice showed increased serum levels of tumour necrosis factor (TNF)‐α, which could be due to systemically present lipopolysaccharide translocated to the gut. In contrast to acute colitis, during chronic DSS‐colitis, which is driven by a Th1 response of the lamina propria infiltrates, OPN−/– mice were protected from mucosal inflammation and demonstrated lower serum levels of IL‐12 than WT mice. Furthermore, neutralization of OPN in WT mice abrogated colitis. Lastly, we demonstrate that in patients with active Crohn's disease OPN serum concentration correlated significantly with disease activity. Taken together, we postulate a dual function of OPN in intestinal inflammation: During acute inflammation OPN seems to activate innate immunity, reduces tissue damage and initiates mucosal repair whereas during chronic inflammation it promotes the Th1 response and strengthens inflammation.

Barrett's metaplasia is an aquired condition resulting from longstanding gastroesophageal reflux disease. Approximately 10% of esophagitis patients develop Barrett's esophagus. There is increasing evidence that duodenogastroesophageal reflux plays a role in the progression of disease. We further analyzed the correlation of acid and biliary reflux with reflux esophagitis and Barrett's esophagus and tested the effects of proton pump inhibitor therapy. Patients with either reflux esophagitis (group 1) or Barrett's esophagus (group 2) prospectively underwent simultaneous 24-h esophageal pH and bile reflux testing without any therapy affecting acid secretion or GI motility. A total of 16 patients in group 1 and 18 patients in group 2 were tested again under proton pump inhibitor therapy. Acid and bile exposure were significantly increased in Barrett's patients (n = 23) compared to 20 esophagitis patients (median percentage of time that pH was <4 was 24.6% vs 12.4%, p = 0.01, median percentage of time that bilirubin absorbance was >0.2 was 34.7% vs 12.8%, p < 0.05). During therapy, both acid and bile reflux decreased significantly in both groups. Median percentage of time that pH was <4 and bilirubin absorbance was >0.2 before and during therapy was 18.2%/2.3% and 29.8%/0.7% (p = 0.001 and p = 0.001) in Barrett's esophagus patients versus 14.5%/3.6% and 21.5%/0.9% (p = 0.002 and p = 0.011) in esophagitis patients. There was no significant difference between the groups. In two esophagitis patients, bile reflux increased during therapy. There is a good correlation of the duration of esophageal exposure to acid and bile with the severity of pathological change in the esophagus. Both acid and bile reflux is significantly suppressed by proton pump inhibitor therapy with exceptions among individual esophagitis patients. The prolonged simultaneous attack of bile and acid may play a key role in the development of Barrett's metaplasia.

Patients suffering from ulcerative colitis (UC) bear an increased risk for colorectal cancer. Due to the sparsity of colitis-associated cancer (CAC) and the long duration between UC initiation and overt carcinoma, elucidating mechanisms of inflammation-associated carcinogenesis in the gut is particularly challenging. Adequate murine models are thus highly desirable. For human CACs a high frequency of chromosomal instability (CIN) reflected by aneuploidy could be shown, exceeding that of sporadic carcinomas. The aim of this study was to analyze mouse models of CAC with regard to CIN. Additionally, protein expression of p53, beta-catenin and Ki67 was measured to further characterize murine tumor development in comparison to UC-associated carcinogenesis in men. The AOM/DSS model (n = 23) and IL-10(-/-) mice (n = 8) were applied to monitor malignancy development via endoscopy and to analyze premalignant and malignant stages of CACs. CIN was assessed using DNA-image cytometry. Protein expression of p53, beta-catenin and Ki67 was evaluated by immunohistochemistry. The degree of inflammation was analyzed by histology and paralleled to local interferon-γ release. CIN was detected in 81.25% of all murine CACs induced by AOM/DSS, while all carcinomas that arose in IL-10(-/-) mice were chromosomally stable. Beta-catenin expression was strongly membranous in IL-10(-/-) mice, while 87.50% of AOM/DSS-induced tumors showed cytoplasmatic and/or nuclear translocation of beta-catenin. p53 expression was high in both models and Ki67 staining revealed higher proliferation of IL-10(-/-)-induced CACs. AOM/DSS-colitis, but not IL-10(-/-) mice, could provide a powerful murine model to mechanistically investigate CIN in colitis-associated carcinogenesis.

The endothelium plays an important role in the exchange of molecules, but also of immune cells between blood and the underlying tissue. The endothelial molecule S-Endo 1 antigen (CD146) is preferentially located at endothelial junctions and has been claimed to support endothelial integrity. In this study we show that the monoclonal antibody ME-9F1 recognizes the extracellular portion of murine CD146. Making use of ME-9F1 we found CD146 highly expressed and widely spread on endothelial cells in the analyzed murine tissues. In contrast to humans that express CD146 also on T cells or follicular dendritic cells, murine CD146 albeit at low levels was only found on a subset of NK1.1 + cells. The antibody against murine CD146 is useful for immunomagnetic sorting of primary endothelial cells not only from the liver but from various other organs. In vitro, no evidence was seen that the formation and integrity of endothelial monolayers or the transendothelial migration of T cells was affected by antibody binding to CD146 or by crosslinking of the antigen. This makes the antibody ME-9F1 an excellent tool especially for the ex vivo isolation of murine endothelial cells intended to be used in functional studies.

Background/aims About half of all Crohn’s disease (CD) patients undergo surgery at some point, many because of strictures. An alternative possibility is to dilate strictures endoscopically. However, little is known about prognostic factors. Patients and methods Thirty-two patients with primary CD ( n = 2), radiogenic strictures ( n = 1), or postoperative strictures (27 because of CD; 2 after resection because of cancer), were planned to undergo colonoscopic dilatation of which 25 patients were dilated (10 men; 15 women; median age 48). Length of stenosis, diameter of stricture, balloon size, smoking status, ulcer in the stricture, passage postdilatation, hemoglobin level, complications, redilatation, and subsequent surgery were recorded. Only patients with at least 6 months follow up were included. Results Five out of 32 patients had no stenosis, marked inflammation, or fistulas adjacent to the stricture. One patient each had a long stricture (8cm) or a filiform stenosis ruling out dilatation [technical success, 25/27 (92.6%)]. Among these 25 patients, 39 colonoscopies with 51 dilatations were performed. After a single dilatation, 52% were asymptomatic while 48% needed another intervention, half of them surgery. Bleeding without need for transfusion occurred in 3 out of 39 colonoscopies and one perforation required surgery. Significant prognostic factors were smoking and ulcers in the stricture ( P < 0.05 each). Some ulcers led to intussusception requiring surgery in spite of good dilatation results. Conclusion Through the endoscope balloon stricture dilatation is a relatively safe and often effective treatment modality in ileocolonic strictures. The presence of ulcers in the stricture have a worse outcome as do smokers.

BackgroundUlcerative colitis (UC) is a complex disease in which the interaction of genetic, environmental and microbial factors drives chronic intestinal inflammation that finally leads to extensive tissue fibrosis.DiscussionThe present review discusses the current knowledge on genetic susceptibility, especially of the IL-12/IL-23 pathway, the pathophysiologic role of the involved cytokines (e.g. IL-13, IL-23, TGFβ1) and immune cells (e.g. T cells, epithelial cells, fibroblasts) in UC followed by an overview on actual therapeutic considerations. These future therapies will target selectively the involved cell types by blocking their activation and its downstream signalling, by inhibiting their migration to the inflamed site and by anti-cytokine strategies. This may avoid–when initiated in time–the perpetuation of the inflammatory mechanisms thus preventing fibrosis. With respect to animal models that have guided the most productive efforts for understanding human inflammatory bowel disease, these will be shortly discussed in the respective context.