cCPE Fusion Proteins as Molecular Probes to Detect Claudins and Tight Junction Dysregulation in Gastrointestinal Cell Lines, Tissue Explants and Patient-Derived Organoids.

Claudins regulate paracellular permeability, contribute to epithelial polarization and are dysregulated during inflammation and carcinogenesis. Variants of the claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) are highly sensitive protein ligands for generic detection of a broad spectrum of claudins. Here, we investigated the preferential binding of YFP- or GST-cCPE fusion proteins to non-junctional claudin molecules. Plate reader assays, flow cytometry and microscopy were used to assess the binding of YFP- or GST-cCPE to non-junctional claudins in multiple in vitro and ex vivo models of human and rat gastrointestinal epithelia and to monitor formation of a tight junction barrier. Furthermore, YFP-cCPE was used to probe expression, polar localization and dysregulation of claudins in patient-derived organoids generated from gastric dysplasia and gastric cancer. Live-cell imaging and immunocytochemistry revealed cell polarity and presence of tight junctions in glandular organoids (originating from intestinal-type gastric cancer and gastric dysplasia) and, in contrast, a disrupted diffusion barrier for granular organoids (originating from discohesive tumor areas). In sum, we report the use of cCPE fusion proteins as molecular probes to specifically and efficiently detect claudin expression, localization and tight junction dysregulation in cell lines, tissue explants and patient-derived organoids of the gastrointestinal tract.

Uptake of Tropheryma whipplei by Intestinal Epithelia.

BACKGROUND: Tropheryma whipplei (TW) can cause different pathologies, e.g., Whipple's disease and transient gastroenteritis. The mechanism by which the bacteria pass the intestinal epithelial barrier, and the mechanism of TW-induced gastroenteritis are currently unknown. METHODS: Using ex vivo disease models comprising human duodenal mucosa exposed to TW in Ussing chambers, various intestinal epithelial cell (IEC) cultures exposed to TW and a macrophage/IEC coculture model served to characterize endocytic uptake mechanisms and barrier function. RESULTS: TW exposed ex vivo to human small intestinal mucosae is capable of autonomously entering IECs, thereby invading the mucosa. Using dominant-negative mutants, TW uptake was shown to be dynamin- and caveolin-dependent but independent of clathrin-mediated endocytosis. Complementary inhibitor experiments suggested a role for the activation of the Ras/Rac1 pathway and actin polymerization. TW-invaded IECs underwent apoptosis, thereby causing an epithelial barrier defect, and were subsequently subject to phagocytosis by macrophages. CONCLUSIONS: TW enters epithelia via an actin-, dynamin-, caveolin-, and Ras-Rac1-dependent endocytosis mechanism and consecutively causes IEC apoptosis primarily in IECs invaded by multiple TW bacteria. This results in a barrier leak. Moreover, we propose that TW-packed IECs can be subject to phagocytic uptake by macrophages, thereby opening a potential entry point of TW into intestinal macrophages.

The combination of clinical parameters and immunophenotyping of intraepithelial lymphocytes allows to assess disease severity in refractory celiac disease.

BACKGROUND: Flow cytometry of intestinal lymphocytes is discussed to be a stronger predictor of enteropathy-associated T-cell lymphoma development in refractory celiac disease than T-cell clonality analysis. AIMS: To investigate possible associations between clinical characteristics of refractory celiac disease patients and aberrant intraepithelial lymphocytes and to evaluate the accuracy of immunophenotyping for the identification of high-risk refractory celiac disease. METHODS: Flow cytometry of isolated lymphocytes from duodenal biopsies of controls and celiac disease patients was performed and results were compared to clinical data. RESULTS: Flow cytometry analysis was performed on 42 controls, 37 non-complicated celiac disease and 30 refractory celiac disease cases with or without T-cell receptor clonality. Elevated aberrant intraepithelial lymphocyte counts were significantly associated with severe malabsorption. A 15% cut-off (aberrant lymphocytes among all lymphocytes) had the best discriminatory ability to identify high-risk patients. However, this technique failed to identify some high-risk cases (sensitivity 63%, specificity 100%). The severity of malabsorption was added to the criteria for high-risk refractory celiac disease, improving the correct patients' allocation (sensitivity 100%, specificity 96%). CONCLUSION: Immunophenotyping of aberrant intraepithelial lymphocytes is a good predictor for high-risk refractory celiac disease. Furthermore, adding the evaluation of malabsorption to the diagnostic assessment of refractory celiac disease optimizes accuracy.

PuraStat in gastrointestinal bleeding: results of a prospective multicentre observational pilot study.

BACKGROUND: A recently developed haemostatic peptide gel for endoscopic application has been introduced to improve the management of gastrointestinal bleeding. The aim of this pilot study was to evaluate the feasibility, safety, efficacy and indication profiles of PuraStat in a clinical setting. METHODS: In this prospective observational multicentre pilot study, patients with acute non-variceal gastrointestinal bleeding (upper and lower) were included. Primary and secondary application of PuraStat was evaluated. Haemoglobin, prothrombin time, platelets and transfusion behaviour were documented before and after haemostasis. The efficacy of PuraStat was assessed during the procedure, at 3 days and 1 week after application. RESULTS: 111 patients with acute gastrointestinal bleeding were recruited into the study. 70 percent (78/111) of the patients had upper gastrointestinal bleeding and 30% (33/111) had lower gastrointestinal bleeding. After primary application of PuraStat, initial haemostatic success was achieved in 94% of patients (74/79, 95% CI 88-99%), and in 75% of the patients when used as a secondary haemostatic product, following failure of established techniques (24/32, 95% CI 59-91%). The therapeutic success rates (absence of rebleeding) after 3 and 7 days were 91% and 87% after primary use, and 87% and 81% in all study patients. Overall rebleeding rate at 30 day follow-up was 16% (18/111). In the 5 patients who finally required surgery (4.5%), PuraStat allowed temporary haemostasis and stabilisation. CONCLUSIONS: PuraStat expanded the therapeutic toolbox available for an effective treatment of gastrointestinal bleeding sources. It could be safely applied and administered without complications as a primary or secondary therapy. PuraStat may additionally serve as a bridge to surgery in order to achieve temporary haemostasis in case of refractory severe bleeding, possibly playing a role in preventing immediate emergency surgery.

Innovative Diagnostic Endoscopy in Inflammatory Bowel Diseases: From High-Definition to Molecular Endoscopy.

High-definition endoscopy is one essential step in the initial diagnosis of inflammatory bowel disease (IBD) characterizing the extent and severity of inflammation, as well as discriminating ulcerative colitis (UC) from Crohn's disease (CD). Following general recommendations and national guidelines, individual risk stratification should define the appropriate surveillance strategy, biopsy protocol and frequency of endoscopies. Beside high-definition videoendoscopy the application of dyes applied via a spraying catheter is of additional diagnostic value with a higher detection rate of intraepithelial neoplasia (IEN). Virtual chromoendoscopy techniques (NBI, FICE, I-scan, BLI) should not be recommended as a single surveillance strategy in IBD, although newer data suggest a higher comparability to dye-based chromoendoscopy than previously assumed. First results of oral methylene blue formulation are promising for improving the acceptance rate of classical chromoendoscopy. Confocal laser endomicroscopy (CLE) is still an experimental but highly innovative endoscopic procedure with the potential to contribute to the detection of dysplastic lesions. Molecular endoscopy in IBD has taken application of CLE to a higher level and allows topical application of labeled probes, mainly antibodies, against specific target structures expressed in the tissue to predict response or failure to biological therapies. First pre-clinical and in vivo data from label-free multiphoton microscopy (MPM) are now available to characterize mucosal and submucosal inflammation on endoscopy in more detail. These new techniques now have opened the door to individualized and highly specific molecular imaging in IBD in the future and pave the path to personalized medicine approaches. The quality of evidence was stated according to the Oxford Center of evidence-based medicine (March 2009). For this review a Medline search up to January 2021 was performed using the words "inflammatory bowel disease," "ulcerative colitis," "crohn's disease," "chromoendoscopy," "high-definition endoscopy," "confocal laser endomicroscopy," "confocal laser microscopy," "molecular imaging," "multiphoton microscopy."

Expression of tricellular tight junction proteins and the paracellular macromolecule barrier are recovered in remission of ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) has a relapsing and remitting pattern, wherein the underlying mechanisms of the relapse might involve an enhanced uptake of luminal antigens which stimulate the immune response. The tricellular tight junction protein, tricellulin, takes charge of preventing paracellular passage of macromolecules. It is characterized by downregulated expression in active UC and its correct localization is regulated by angulins. We thus analyzed the tricellulin and angulin expression as well as intestinal barrier function and aimed to determine the role of tricellulin in the mechanisms of relapse. METHODS: Colon biopsies were collected from controls and UC patients who underwent colonoscopy at the central endoscopy department of Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin. Remission of UC was defined basing on the clinical appearance and a normal Mayo endoscopic subscore. Intestinal barrier function was evaluated by electrophysiological and paracellular flux measurements on biopsies mounted in Ussing chambers. RESULTS: The downregulated tricellulin expression in active UC was recovered in remission UC to control values. Likewise, angulins were in remission UC at the same levels as in controls. Also, the epithelial resistance which was decreased in active UC was restored in remission to the same range as in controls, along with the unaltered paracellular permeabilities for fluorescein and FITC-dextran 4 kDa. CONCLUSIONS: In remission of UC, tricellulin expression level as well as intestinal barrier functions were restored to normal, after they were impaired in active UC. This points toward a re-sealing of the impaired tricellular paracellular pathway and abated uptake of antigens to normal rates in remission of UC.

Leptin Downregulates Angulin-1 in Active Crohn's Disease via STAT3.

Crohn's disease (CD) has an altered intestinal barrier function, yet the underlying mechanisms remain to be disclosed. The tricellular tight junction protein tricellulin is involved in the maintenance of the paracellular macromolecule barrier and features an unchanged expression level in CD but a shifted localization. As angulins are known to regulate the localization of tricellulin, we hypothesized the involvement of angulins in CD. Using human biopsies, we found angulin-1 was downregulated in active CD compared with both controls and CD in remission. In T84 and Caco-2 monolayers, leptin, a cytokine secreted by fat tissue and affected in CD, decreased angulin-1 expression. This effect was completely blocked by STAT3 inhibitors, Stattic and WP1066, but only partially by JAK2 inhibitor AG490. The effect of leptin was also seen at a functional level as we observed in Caco-2 cells an increased permeability for FITC-dextran 4 kDa indicating an impaired barrier against macromolecule uptake. In conclusion, we were able to show that in active CD angulin-1 expression is downregulated, which leads to increased macromolecule permeability and is inducible by leptin via STAT3. This suggests that angulin-1 and leptin secretion are potential targets for intervention in CD to restore the impaired intestinal barrier.

Leptin induces TNFα-dependent inflammation in acquired generalized lipodystrophy and combined Crohn's disease.

Leptin has been shown to modulate intestinal inflammation in mice. However, clinical evidence regarding its immune-stimulatory potential in human Crohn's disease remains sparse. We here describe a patient with the unique combination of acquired generalized lipodystrophy and Crohn's disease (AGLCD) featuring a lack of adipose tissue, leptin deficiency and intestinal inflammation. Using mass and flow cytometry, immunohistochemistry and functional metabolic analyses, the AGLCD patient was compared to healthy individuals and Crohn's disease patients regarding immune cell composition, function and metabolism and the effects of recombinant N-methionylleptin (rLeptin) were evaluated. We provide evidence that rLeptin exerts diverse pro-inflammatory effects on immune cell differentiation and function, including the metabolic reprogramming of immune cells and the induction of TNFα, ultimately aggravating Crohn's disease in the AGLCD patient, which can be reversed by anti-TNFα therapy. Our results indicate that leptin is required for human immune homeostasis and contributes to autoimmunity in a TNFα-dependent manner.

Low Sensitivity of Simtomax Point of Care Test in Detection of Celiac Disease in a Prospective Multicenter Study.

BACKGROUND & AIMS: Point of care tests (POCTs) might be used to identify patients with undiagnosed celiac disease who require further evaluation. We performed a large multicenter study to determine the performance of a POCT for celiac disease and assessed celiac disease prevalence in endoscopy centers. METHODS: We performed a prospective study of 1055 patients (888 adults; median age, 48 yrs and 167 children; median age, 10 yrs) referred to 8 endoscopy centers in Germany, for various indications, from January 2016 through June 2017. Patients were tested for celiac disease using Simtomax, which detects immunoglobulin (Ig)A and IgG antibodies against deamidated gliadin peptides (DGP). Results were compared with findings from histologic analyses of duodenal biopsies (reference standard). The primary aim was to determine the accuracy of this POCT for the detection of celiac disease, to identify candidates for duodenal biopsy. A secondary aim was to determine the prevalence of celiac disease in adult and pediatric populations referred for outpatient endoscopic evaluation. RESULTS: The overall prevalence of celiac disease was 4.1%. The POCT identified individuals with celiac disease with 79% sensitivity (95% CI, 64%-89%) and 94% specificity (95% CI, 93%-96%). Positive and negative predictive values were 37% and 99%. When we analyzed the adult and pediatric populations separately, we found the test to identify adults with celiac disease (prevalence 1.2%) with 100% sensitivity and 95% specificity. In the pediatric population (celiac disease prevalence 19.6%), the test produced false-negative results for 9 cases; the test therefore identified children with celiac disease with 72% sensitivity (95% CI 53%-86%). Analyses of serologic data revealed significantly lower DGP titers in the false-negative vs the true-positive group. CONCLUSIONS: In a study of more than 1000 adults and children, we found the Simtomax POCT to detect celiac disease with lower overall levels of sensitivity than expected. Although the test identifies adults with celiac disease with high levels of sensitivity and specificity, the prevalence of celiac disease was as low as 1.2% among adults. The test's lack of sensitivity might be due to the low intensity of the POCT bands and was associated with low serum DGP titers. Study ID no: DRKS00012499.

Diagnostic and therapeutic single-operator cholangiopancreatoscopy with SpyGlassDS™: results of a multicenter retrospective cohort study.

BACKGROUND AND AIMS: The aim of the study was to evaluate the usefulness and diagnostic and therapeutic outcome of the single-operator cholangiopancreatoscopy (SOC) with SpyGlassDS™. METHODS: In a retrospective multicenter study between November 2015 and January 2017, SpyGlassDS™ procedures were analyzed in participating centers. Indications, accuracy of SOC-guided biopsies, management of large bile duct stones, and complications were analyzed. Follow-up was 4 months. RESULTS: Two hundred and six patients out of 250 examinations were evaluated. Indications were biliary stones (n = 132), bile duct stenosis (n = 93), stones and stenosis combined (n = 24), and bile duct leakage (n = 1). Of the 117 cases which were suspicious of malignancy, in 99 cases the lesion could be stratified into benign (n = 55) or malignant (n = 44) indicating a sensitivity of 95.5% and a specificity of 94.5% for the indication tumor. SOC-guided biopsies revealed a sensitivity of 57.7% with a specificity of 100%. In 107 examinations, biliary stones were visualized and could be completely removed in 91.1% with a need of three procedures (range 1-6) to achieve final stone clearance. In 75 cases, lithotripsy was performed and was successful in 71 cases (95%). Four out of 45 patients (8.9%) underwent cholecystectomy with surgical bile duct revision as a final therapy. Adverse Event (AE) occurred in 33/250 patients (13.2%) and Serious Adverse Event (SAE) occurred in 1/250 patients (0.4%). Cholangitis was 1% (n = 102) after peri-interventional administration of antibiotics and 12.8% (n = 148) without antibiotic prophylaxis (p < 0.001). CONCLUSIONS: SOC with SpyGlassDS™ became a new standard for the diagnosis of indefinite biliary lesions and therapy of large bile duct stones. The diagnostic yield of SOC-guided biopsies facilitated a definite diagnosis in most cases and should be improved by standardized biopsy protocols. SOC-guided interventions allowed removal of large biliary stones by SOC-guided lithotripsy. The complication rate of 13.2% can be considerably reduced by use of a single-shot antibiotic treatment.

Confocal endomicroscopy in diagnosis of intestinal chronic graft-versus-host disease.

Graft-versus-host disease (GvHD) is a major complication of allogeneic stem cell transplantation. High-resolution in vivo histology of the intestine by confocal endomicroscopy (CEM) detects acute GvHD (aGvHD) with high sensitivity. This pilot study aims to evaluate the diagnostic value of CEM for intestinal chronic GvHD (cGvHD). The study included 20 patients with gastrointestinal symptoms and confirmed cGvHD in other organs as well as 20 patients with clinically suspected acute GvHD for control. Confocal endomicroscopy was performed as gastroscopy followed by sigmoidoscopy after intravenous injection of fluorescein (10%) and topical application of acriflavine (0.05%). Histopathology from H&E-stained biopsy samples throughout the intestinal tract complemented the survey. All histological features of intestinal cGvHD were predominantly mild to moderate. Stroma fibrosis detected by standard histology (16/20 patients) was not seen by CEM. Apoptosis assessed by histology in 12/20 patients was concordant with CEM (8/12 patients). Confocal endomicroscopy revealed esophageal manifestation of cGvHD in 3 patients. For each biopsy site, CEM correlated with intestinal histology (r = 0.64). Classical histology from intestinal biopsy samples taken under CEM monitoring confirmed the final diagnosis of cGvHD. The sensitivity of CEM with 40% in cGvHD was significantly lower compared to 70% in patients with aGvHD. Confocal endomicroscopy detected acute features of cGvHD and contributed to the diagnosis of esophageal cGvHD but failed to display stroma fibrosis in vivo. Although CEM represents a useful noninvasive tool in routine diagnostic of intestinal aGvHD, the method is not sufficient to fully establish the diagnosis of cGvHD within the intestinal tract. Confocal endomicroscopy allowed acquisition of targeted biopsies in patients suspected of having cGvHD.

EndoClot Polysaccharide Hemostatic System in Nonvariceal Gastrointestinal Bleeding: Results of a Prospective Multicenter Observational Pilot Study.

BACKGROUND AND STUDY AIMS: Hemostatic powders have been introduced to improve the management of gastrointestinal (GI) bleeding and to extend the variety of tools available for emergency endoscopy. The aim of the present pilot study was to evaluate the indication profiles and the short-term outcome of EndoClot. PATIENTS, MATERIALS AND METHODS: In a prospective observational pilot study patients with acute nonvariceal GI bleeding were included. Primary or secondary application of EndoClot was assessed. Hemoglobin, prothrombine time and platelets were documented before and after hemostasis. The efficacy of EndoClot was assessed 72 hours and 1 week after application. RESULTS: Seventy patients with acute GI bleeding were recruited into the study. Eighty-three percent (58/70) of the patients had upper and 17% (12/70) had lower GI bleeding. In the upper GI tract treatment success was achieved in 64% (30/47, 95% confidence interval, 50%-76%) after primary use and in all patients, when used after established techniques had failed (95% confidence interval, 70%-100%). In lower GI bleeding hemostasis was achieved in 83% of cases (10/12, 95% confidence interval 54%-97%). Rebleeding occurred in 11% (8/70), in 10% EndoClot served as a bridge to surgery (7/70). CONCLUSIONS: EndoClot expanded the therapeutic options in the management of GI bleeding. It was applicable as a monotherapy or in combination with other techniques from oozing bleeding type or lower. It was most effective in diffuse or extensive bleeding activity or when access to the bleeding vessel was difficult. EndoClot can be applied as a bridge to surgery when classical methods of hemostasis have failed.

Unintentional Long-Term Esophageal Stenting due to a Complete Response in a Patient with Stage UICC IV Adenocarcinoma of the Gastroesophageal Junction.

Endoscopic stent implantation is a common short-treatment option in palliative settings in patients with esophageal cancer. Advanced disease is associated with low survival rates; therefore, data on the long-term outcome are limited. So far, cases of long-term remission or even cure of metastasized adenocarcinoma of the gastroesophageal junction or stomach (AGS) have only been reported from Asia. A 51-year-old male patient primarily diagnosed with metastasized adenocarcinoma of the gastroesophageal junction (GEJ) [type I, cT3cN+cM1 (hep), CEA positive, UICC stage IV] received palliative esophageal stenting with a self-expandable metal stent. As disease progressed after four cycles with epirubicin, oxaliplatin, and capecitabin, treatment was changed to 5-FU and Irinotecan. The patient did not return after 5 cycles of FOLFIRI, but presented 4 years later with mild dysphagia. Endoscopy surprisingly revealed no relevant stenosis or stent migration. Repeated histological analyses of a residual mass at the GEJ did not detect malignancy. Since the initially diagnosed hepatic metastases were no longer detectable by computed tomography, cure from esophageal cancer was assumed. Dysphagia was ascribed to esophageal motility disorder by a narrowed esophageal lumen after long-term stenting. Thus, endoscopic stent implantation is an important method in palliative treatment of dysphagia related to AGS. New systemic treatment strategies like trastuzumab in Her2neu positive cases or new VEGF-inhibitors like ramucirumab will lead to more long-time survivors with AGS. In conclusion, future endoscopic treatment strategies in AGS represent a challenge for the development of new stent techniques in either extraction or programmed complete dissolution.

Mucosal Inducible NO Synthase-Producing IgA+ Plasma Cells in Helicobacter pylori-Infected Patients.

The mucosal immune system is relevant for homeostasis, immunity, and also pathological conditions in the gastrointestinal tract. Inducible NO synthase (iNOS)-dependent production of NO is one of the factors linked to both antimicrobial immunity and pathological conditions. Upregulation of iNOS has been observed in human Helicobacter pylori infection, but the cellular sources of iNOS are ill defined. Key differences in regulation of iNOS expression impair the translation from mouse models to human medicine. To characterize mucosal iNOS-producing leukocytes, biopsy specimens from H. pylori-infected patients, controls, and participants of a vaccination trial were analyzed by immunohistochemistry, along with flow cytometric analyses of lymphocytes for iNOS expression and activity. We newly identified mucosal IgA-producing plasma cells (PCs) as one major iNOS(+) cell population in H. pylori-infected patients and confirmed intracellular NO production. Because we did not detect iNOS(+) PCs in three distinct infectious diseases, this is not a general feature of mucosal PCs under conditions of infection. Furthermore, numbers of mucosal iNOS(+) PCs were elevated in individuals who had cleared experimental H. pylori infection compared with those who had not. Thus, IgA(+) PCs expressing iNOS are described for the first time, to our knowledge, in humans. iNOS(+) PCs are induced in the course of human H. pylori infection, and their abundance seems to correlate with the clinical course of the infection.

ENaC Dysregulation Through Activation of MEK1/2 Contributes to Impaired Na+ Absorption in Lymphocytic Colitis.

BACKGROUND: Lymphocytic colitis (LC) causes watery diarrhea. We aimed to identify mechanisms of altered Na absorption and regulatory inputs in patients with LC by examining the epithelial Na channel (ENaC) function as the predominant Na transport system in human distal colon. METHODS: Epithelial Na channel function and regulation was analyzed in biopsies from sigmoid colon of patients with LC and in rat distal colon in Ussing chambers. ENaC-subunit expression was measured by real-time PCR and RNA sequencing. Correction factors for subepithelial resistance contributions were determined by impedance spectroscopy. Upstream regulators in LC were determined by RNA sequencing. RESULTS: Epithelial Na channel-mediated electrogenic Na transport was inhibited despite aldosterone stimulation in human sigmoid colon of patients with LC. The increase in γ-ENaC mRNA expression in response to aldosterone was MEK1/2-dependently reduced in LC, since it could be restored toward normal by MEK1/2 inhibition through U0126. Parallel experiments for identification of signaling in rat distal colon established MEK1/2 to be activated by a cytokine cocktail of TNFα, IFNγ, and IL-15, which were identified as the most important regulators in the upstream regulator analysis in LC. CONCLUSIONS: In the sigmoid colon of patients with LC, the key effector cytokines TNFα, IFNγ, and IL-15 inhibited γ-ENaC upregulation in response to aldosterone through a MEK1/2-mediated pathway. This prevents ENaC to reach its maximum transport capacity and results in Na malabsorption which contributes to diarrhea.

α-Haemolysin of Escherichia coli in IBD: a potentiator of inflammatory activity in the colon.

OBJECTIVE: α-Haemolysin (HlyA) influences host cell ionic homeostasis and causes concentration-dependent cell lysis. As a consequence, HlyA-producing Escherichia coli is capable of inducing 'focal leaks' in colon epithelia, through which bacteria and antigens translocate. This study addressed the role of HlyA as a virulence factor in the pathogenesis of colitis according to the 'leaky gut' concept. DESIGN: To study the action of HlyA in the colon, we performed oral administration of HlyA-expressing E coli-536 and its isogenic α-haemolysin-deficient mutant (HDM) in three mouse models: wild type, interleukin-10 knockout mice (IL-10(-/-)) and monoassociated mice. Electrophysiological properties of the colonised colon were characterised in Ussing experiments. Inflammation scores were evaluated and focal leaks in the colon were assessed by confocal laser-scanning microscopy. HlyA quantity in human colon biopsies was measured by quantitative PCR. RESULTS: All three experimental mouse models infected with HlyA-producing E coli-536 showed an increase in focal leak area compared with HDM. This was associated with a decrease in transepithelial electrical resistance and an increase in macromolecule uptake. As a consequence, inflammatory activity index was increased to a higher degree in inflammation-prone mice. Mucosal samples from human colon were E coli HlyA-positive in 19 of 22 patients with ulcerative colitis, 9 of 9 patients with Crohn's disease and 9 of 12 healthy controls. Moreover, focal leaks were found together with 10-fold increased levels of HlyA in active ulcerative colitis. CONCLUSIONS: E coli HlyA impairs intestinal barrier function via focal leak induction in the epithelium, thereby intensifying antigen uptake and triggering intestinal inflammation in vulnerable mouse models. Therefore, HlyA-expressing E coli strains should be considered as potential cofactors in the pathogenesis of intestinal inflammation.

Risk factors in the development of esophageal adenocarcinoma.

OBJECTIVES: It is assumed that esophageal adenocarcinoma is the end result of a stepwise disease process that transitions through gastroesophageal reflux disease (GERD) and Barrett's esophagus. The aim of this study was to examine at what stage known risk factors exert their influence toward the progression to cancer. METHODS: We enrolled 113 consecutive outpatients without GERD, 188 with GERD, 162 with Barrett's esophagus, and 100 with esophageal adenocarcinoma or high-grade dysplasia (HGD). All patients underwent a standard upper endoscopy and completed a standardized questionnaire about their social history, symptoms, dietary habits, and prescribed medications. We used adjusted logistic regression analysis to assess risk factors between each two consecutive disease stages from the absence of reflux disease to esophageal adenocarcinoma. RESULTS: Overall, male gender, smoking, increased body mass index (BMI), low fruit and vegetable intake, duration of reflux symptoms, and presence of a hiatal hernia were risk factors for cancer/HGD. However, different combinations of risk factors were associated with different disease stages. Hiatal hernia was the only risk factor to be strongly associated with the development of GERD. For GERD patients, male gender, age, an increased BMI, duration of reflux symptoms, and presence of a hiatal hernia were all associated with the development of Barrett's esophagus. Finally, the development of cancer/HGD among patients with Barrett's esophagus was associated with male gender, smoking, decreased fruit and vegetable intake, and a long segment of Barrett's esophagus, but not with age, BMI, or a hiatal hernia. CONCLUSIONS: While some risk factors act predominantly on the initial development of reflux disease, others appear to be primarily responsible for the development of more advanced disease stages.