M. Brüwer

Transepithelial transport processes at the intestinal mucosa in inflammatory bowel disease

Abstract Crohns disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) of unknown etiology. Oral absorption studies have shown an increased intestinal permeability for various sugar molecules in patients with IBD and their healthy relatives as a possible pathogenetic factor. However, the various transport pathways through the mucosal barrier have not yet been examined. This study therefore investigated whether antigens pass the epithelial barrier by a transcellular or a paracellular pathway. Mucosa of freshly resected specimens from CD (n = 10) or UC (n = 10) patients was investigated by immunoelectron microscopy and compared with healthy mucosa. Epithelial transport was studied with the antigens ovalbumin and horseradish peroxidase after defined incubation. Labeling density of subunit c of ATP synthetase was determined in mitochondria of enterocytes of all specimens. In all specimens epithelial transport of OVA and HRP was principally transcellular through enterocytes with normal ultrastructure, although some tight junctions in CD and UC were dilated. Antigens were transported within vesicles to the basolateral membrane 2.5 min after incubation. The level of enterocytes with electron-lucent cytoplasm containing a high amount of antigens was higher in CD and UC than in healthy mucosa, depending on the grade of inflammation. ATP synthetase was significantly decreased in electron-lucent cytoplasm of CD and UC to normal ultrastructure of healthy mucosa. Our study shows that ovalbumin and horseradish peroxidase taken up by the apical membrane reach the paracellular space by vesicular transport in healthy and IBD enterocytes within a few minutes. Transcellular pathway is affected in both CD and UC, which is indicated by a high level of antigens within the cytosol. We speculate that increased intestinal permeability in IBD results substantially from enhanced transcellular transport.

Tight Junctions and Cell–Cell Interactions

Chronic inflammation in mucosal tissues can influence epithelial barrier function via pro-inflammatory cytokines such as interferon (IFN)-gamma and tumor necrosis factor-alpha. Increased mucosal levels of these cytokines have been observed in mucosal biopsies from patients with a chronic inflammatory condition referred to as inflammatory bowel disease. Paracellular permeability across epithelial cells is regulated by tight junctions (TJs), which are the apical most junctions in epithelial cells. Given that pro-inflammatory cytokines modulate the epithelial barrier and that TJs regulate epithelial permeability, we analyzed the influence of IFN-gamma on U function/structure. Our results suggest that IFN-gamma induced a time-dependent increase in paracellular permeability that was associated with internalization of TJ transmembrane proteins, occludin, junction adhesion molecule A, and claudin-1. In this chapter, we focus on selected methods used to investigate the influence of IFN-gamma on epithelial barrier function.

Diagnostic evaluation, surgical technique, and perioperative management after esophagectomy: consensus statement of the German Advanced Surgical Treatment Study Group

PurposeCorrect diagnosis, surgical treatment, and perioperative management of patients with esophageal carcinoma remain crucial for prognosis within multimodal treatment procedures. This study aims to achieve a consensus regarding current management strategies in esophageal cancer by questioning a panel of experts from the German Advanced Surgical Treatment Study (GAST) group, comprised of 9 centers specialized in esophageal surgery, with a combined total of >220 esophagectomies per year.Materials and methodsThe Delphi method, a systematic and interactive, evidence-based approach, was used to obtain consensus statements from the GAST group regarding ambiguities and disparities in diagnosis, patient selection, surgical technique, and perioperative management of patients with esophageal carcinoma. After four rounds of surveys, agreement was measured by Likert scales and defined as full (100% agreement), near (≥66.6% agreement), or no consensus (<66.6% agreement).ResultsFull or near consensus was obtained for essential aspects of esophageal cancer staging, proper surgical technique, perioperative management and indication for primary surgery, and neoadjuvant treatment or palliative treatment. No consensus was achieved regarding acceptability of minimally invasive technique and postoperative nutrition after esophagectomy.ConclusionThe GAST consensus statement represents a position paper for treatment of patients with esophageal carcinoma which both contributes to the development of clinical treatment guidelines and outlines topics in need of further clinical studies.

Fatal complications in fistulizing Crohn's disease: brain abscess and squamous cell carcinoma

Dear Editor: Crohns disease (CD), as one of the entities of inflammatory bowel disease, is associated with chronically relapsing inflammatory episodes. Although the terminal ileum and proximal colon are most frequently affected, the whole gastrointestinal tract can be involved. Additionally to the development of intestinal symptoms, CD typically affects the perianal or perineal region, where a variety of manifestations further complicates the course of the disease. Predominantly, abscess and fistula formations dominate perianal disease and most severely influence the quality of life of the patients. Here, we report the case of a male patient with highly complicated fistulizing disease that further was impaired by the development of severe treatment and disease-associated complications. In January 2009, a 41-year-old male patient was administered to the emergency room of the University Clinic of Muenster after having experienced an epileptic seizure with unconsciousness for 10 min. On physical exam, he had subfebrile body temperature, but did not show any neurologic deficit. His Glasgow Coma Scale score was 14 (range, 3–15). His family reported that he previously suffered from headache, vomiting, and impaired vision. Furthermore, he was aggressive and irascible. A CT scan and MRI of his brain revealed a brain abscess in the left temporal lobe. Analysis of the patients cerebrospinal fluid showed signs of acute bacterial meningitis (total cell count 1,638/μl, granulocytes 1,314/μl, lymphocytes 258/μl, protein 3,080 mg/l, glucose quotient 0.5, and lactate 4.3 mmol/l). He underwent trepanation and aspiration of the abscess. Microbiologic diagnostics of its content showed combined infection with Streptococcus intermedius and anaerobic bacteria. Systemic antibiotic treatment with ceftriaxone, metronidazole, meronem, and phosphomycin was then started. Prior to this incident, the patient had been suffering from CD for a period of over 25 years beginning in the late 1980s. The disease affected the stomach, small intestine as well as the colorectum. In 1994, he suffered from an acute flare, which needed surgical treatment of the colon and stomach. In 1998, he first developed fistulizing disease with recurrent perianal fistula formations that needed local surgical therapy and creation of a protective loop ileostomy for 6 months. However, despite repeated excisions and drainages, in 2003, he presented a complicated fistula formation that involved the right perianal side and stretched out perirectal to infiltrate the pelvic diaphragm. In 2004, this complicated fistulizing formations were joined by a gluteal fistula on the right side. Additionally, a hemicolectomy of the left colon had to be performed due to another inflammatory flare in 2005. Ethical statement The presented patient has been treated according to the standard treatment regimes of our hospital. The requirements of the patient anonymity are according to the rules of the institutional board of the University of Muenster, Germany. An institutional review board approval was not required following local customs and practice. C. Haane : E. Rijcken :M. Brüwer : P.-A. Neumann (*) Department of General and Visceral Surgery, University Hospital Muenster, Waldeyerstr. 1, 48149 Muenster, Germany e-mail: philipp-alexander.neumann@ukmuenster.de

Chirurgische Therapiestrategien beim Ösophaguskarzinom

ZusammenfassungDie chirurgische Resektion des Ösophaguskarzinoms stellt auch bei multimodalen Therapieansätzen den entscheidenden Prognosefaktor dar. Wesentliche Ziele des präoperativen Stagings sind daher die Identifikation von komplett resezierbaren Tumoren, aber auch die Vorhersage des perioperativen Risikos. Bei der chirurgischen Resektion gilt in den westlichen Ländern derzeit der rechtsseitige transthorakale Zugang mit einer Zweifeld-Lymphadenektomie mediastinal und abdominal als Standard. Die bevorzugte Rekonstruktion der Nahrungspassage erfolgt durch Bildung eines Magenschlauchs mit intrathorakaler Anastomose. Postoperative Komplikationen können durch ein standardisiertes perioperatives Management reduziert werden. Entscheidend zur Senkung der perioperativen Mortalität ist neben der interdisziplinären präoperativen Risikoanalyse, Tumorkonferenz und postoperativen Morbiditäts-/Mortalitätskonferenz der entsprechende individuelle „caseload“ des Operateurs.AbstractSurgical treatment of esophageal carcinoma remains crucial for the prognosis even within multimodal treatment procedures. Important goals of preoperative staging consist therefore not only of the identification of curative surgery but also of prediction of the perioperative risk. In the case of curative treatment surgical success highly correlates with the extent of lymphadenectomy. In western countries a two-field lymphadenectomy by right-sided transthoracic and transabdominal access is regarded as the standard. Gastric tube formation with intrathoracic anastomosis represents the preferred approach for reconstruction. A standardized perioperative management is crucial for reduction of postoperative complications. The corresponding individual caseload of the surgeon and the centre experience, including preoperative risk analysis, tumor board and regular interdisciplinary conferences are crucial to reduce perioperative mortality to below 5%.

Probiotika erhalten die epitheliale Barrierefunktion in der akuten DSS-Kolitis der Maus

Background: In inflammatory bowel diseases (IBD) intestinal permeability is increased. The effect of VSL#3, a mixture of 8 probiotic bacteria, on the epithelial barrier was studied in a murine model of colitis. Material and Methods: Three groups, each with 14 balb/c mice, were studied: healthy controls, acute colitis + placebo, and acute colitis + 15 mg VSL#3 daily (induction of colitis with Dextrane-Sodium-Sulphate (DSS) 3.5 % in drinking water for 7 days; placebo/VSL#3 via gastric tube once daily). The inflammation was assessed by a daily disease activity index (DAI) and a histological inflammation score. Colonic permeability to Evans Blue in vivo was measured (extinction/gram colonic tissue). Epithelial apoptotic ratio was assessed by immunofluorescence for cleaved caspase 3. The expression of epithelial tight junction and adherens junction proteins was studied by immunofluorescence and Western blot. Results: VSL#3 reduced the DAI on days 5–7 vs. placebo (day 7: healthy controls 0 ± 0, DSS + placebo 8.4 ± 0.4, DSS + VSL#3 5.4 ± 0.9; p = 0.012), and histological inflammation scores (healthy controls 0.9 ± 0.3, DSS + placebo 14.6 ± 2.5, DSS + VSL#3 8.4 ± 1.8; p = 0.011). In DSS colitis + placebo, colonic permeability was increased compared to controls (5.7 ± 1.7 vs. 0.4 ± 0.1; p < 0.001), whereas this increase was prevented by VSL#3 (0.3 ± 0.1; p = 0.003 vs. DSS + placebo, N.S. vs. healthy controls). VSL#3 prevented the DSS induced increase of epithelial apoptotic ratio (healthy controls 1.58 ± 0.01/1000 cells, DSS + placebo 13.35 ± 1.29/1000 cells, DSS + VSL#3 1.67 ± 0.10/1000 cells; p = 0.012 vs. DSS + placebo, N.S. vs. controls). In DSS colitis + placebo, immunofluorescence revealed a reduced apical expression of tight junction proteins occludin, ZO-1, claudin-1, and -5, whereas VSL#3 therapy preserved the physiological expression pattern. No changes were observed for claudin-3, and for adherens junction proteins β-catenin and E-cadherin in the three groups. Western blots showed a quantitatively reduced expression of occludin, claudin-1, -2 and -4 for DSS + placebo. VSL#3 prevented this decrease in expression. Conclusion: The probiotic mixture VSL#3 preserves the epithelial barrier by preventing the redistribution and the decrease in expression of tight junction proteins, and by reducing the epithelial apoptotic ratio. This may lead to the anti-inflammatory effect observed in this IBD model.

Reply to the letter by L. Bonavina regarding our article "Effect of pyloric drainage procedures on gastric passage and bile reflux after esophagectomy with gastric conduit reconstruction"

We would like to thank Prof. Bonavina for his interest and comments on our paper. Our paper deals with the effect of pyloric drainage procedures on gastric passage and bile reflux after esophagectomy with gastric conduit reconstruction. Omitting pyloric drainage is not only safe after esophagectomy, it also avoids bile reflux and esophagitis in the long term as gastric outlet obstruction due to pyloric spasm only represents a transient phenomena that either spontaneously disappears or can be successfully treated by endoscopic intervention [1, 2]. Alternatively, additional intraoperative mechanical dilatation of the pylorus has been shown to be as effective and safe [3]. However, until now, the pathomechanism of the spontaneously declining pylorospasm has not been completely understood. We agree that the size of the gastric conduit might contribute to a faster passage due to an increased wall stress, e.g., by inducing peristaltic waves, which possibly reduce pylorus spasm respectively lead to a reflectory opening of the pylorus. Therefore, in our institution, we always create a 4to 5-cm wide tubularized stomach, and the anastomosis is placed above the arch of the azygos vein to reduce reflux esophagitis. It is interesting to observe that by laparoscopic conditioning of the stomach, the incidence of clinically significant gastric outlet obstruction seems to be further reduced (3.6 vs 6.1% [4]). On the other hand, it is also surprising that anastomotic leakage rate seems to be not reduced by ischemic conditioning of the stomach. Theoretically, after partial devascularization, the gastric conduit should recover, and gastric tissue perfusion should be improved prior to pull-up and anastomosis to the esophagus resulting in better prerequisites for anastomotic healing. Perhaps, the size of the leakage and severity of symptoms can be reduced by ischemic conditioning as all patients with leakages of the esophagogastrostomy (6.1%) had only minor clinical symptoms, and endoscopy showed very well-vascularized resection lines of the esophagus and stomach [4]. However, information whether ischemic conditioning of the stomach before esophagectomy affects local tumor recurrence, and therefore, long-term survival would be desirable. Further controlled randomized studies on these topics are necessary and worthwhile for patients with esophageal carcinoma, as normal gastrointestinal function after esophagectomy is still rare and improving the quality of life represents one of the main goals in these patients [5]. Langenbecks Arch Surg (2008) 393:119–120 DOI 10.1007/s00423-007-0241-y

Interferon-gamma induziert Myosin vermittelte Endozytose von Tight Junction Proteinen

Tight junctions (TJ) are critical regulators of epithelial paracellular permeability, which is compromised in intestinal mucosa of patients with inflammatory bowel disease. Intestinal inflammation was modeled in vitro by incubating a model intestinal epithelial cell line, T84 with the pro-inflammatory cytokine, interferon-gamma (IFN-γ). We previously found that IFN-γ disrupts epithelial barrier function in model polarized intestinal epithelial cells in an apoptosis independent manner by inducing endocytosis of transmembrane TJ proteins. These proteins were endocytosed into early and recycling endosomes by a macropinocytosis-like process. In the present study, we investigated the role of the actin/myosin cytoskeleton in regulation of TJ proteins endocytosis. Internalized TJ proteins were observed in large vacuoles coated with F-actin and markers of the apical plasma membrane that resemble the vacuolar apical compartment (VAC). Both depolymerization and stabilization of F-actin did not inhibit TJ protein endocytosis suggesting an actin filament turnover independent machinery. Transmembrane TJ protein endocytosis and formation of VACs were blocked by blebbistatin, an inhibitor of non-muscle myosin II A. Phosphorylated myosin light chain, activated form of myosin II A, colo calized with VACs. We conclude that IFN-γ induces endocytosis of TJ transmembrane proteins, occludin, JAM-A, claudin-1 into a vacuolar apical compartment driven by myosin II A mediated formation of VACs. Einleitung

Störung des HLA-vermittelten Antigentransports in Enterozyten von Patienten mit chronisch-entzündlichen Darmerkrankungen

Introduction: The pathogenesis of inflammatory bowel disease (IBD; e.g. ulcerative colitis (UC) and Crohn‘s disease, CD) is still unknown. Recent reports suggest a possible disorder of oral tolerance caused by a dysfunctional MHC regulated antigen presentation of enterocytes to lymphocytes of the mucosa-associated lymphatic system (MALT). Therefore the objective of our study was to investigate the intracellular MHC distribution of enterocytes of patients with IBD and compare them with enterocytes of a healthy control group (CG). In IBD-patients both regular enterocytes (NE) and enterocytes with cytosolic antigen intake (RACE, rapid antigen uptake into the cytosol enterocytes) were examined in this case. Method: Fresh mucosal specimens of MC (ileum [il]), CU (colon [co]) patients and controls (CG-ileum, CG-colon, each n = 5) were incubated with egg albumin antigen (OVA). Following MHC-DP, -DQ, -DR (MHC-II) and OVA as well as β 2-microglobulin, a structure protein of MHC-I and OVA were localized using an immunogold double-labeling method with mono- and polyclonal antibodies by electron microscopy. Distinguished between antigen-loaded and antigen-free MHC-positive vesicles the number of this vesicle per cut cell face were determined. As a measure of the concentration of MHC-I being in the rough endoplasmic reticulum (RER) the number of gold points were counted per membrane length. Statistical analysis was performed using χ2- or Fischer‘s exact test with p < 0.05 considered significant. Results: Compared to control group normal enterocytes of patients with MC and CU showed a significantly stronger enrichment of antigen-loaded (MC: 22.7 ± 8.8 vs. 2.1 ± 1.9; CU: 70.8 ± 15.3 vs. 0) and antigen-free (MC: 32.1 ± 10.9 vs. 14.6 ± 6.5; CU: 54.1 ± 17.5 vs. 6.2 ± 5.0) vesicles. In contrast to that the MHC-II-positive vesicles were significantly reduced at RACE of MC (2.8 ±2.4) and CU (25.5 ±8.1) compared with NE as well as compared to CG-enterocytes. In the RER of RACE a significant stronger expression of MHC-I (MC: 30.3 ±6.1; CU: 24.3 ± 4.7) were found compared to control enterocytes (MC: 6.9 ± 2.1; CU: 11.2 ± 3.5) and NE (MC: 2.1 ± 0.6; CU: 1.5 ± 0.5) of MC and CU. Conclusion: The decrease of antigen presentation provided by MHC-II proteins in RACE cells at simultaneous activation of MHC-I proteins in the RER possibly represents a morphological correlative for a changed oral tolerance in patients with IBD.

Kolorektales Karzinom bei chronisch entzündlichen Darmerkrankungen

Patienten mit langjähriger Dauer einer chronisch entzündlichen Darmerkrankung (CED) – Colitis ulcerosa (CU) oder Morbus Crohn (MC) mit Dickdarmbeteiligung – haben ein erhöhtes Risiko, an einem kolorektalen Karzinom zu erkranken. Doch trotz koloskopisch – bioptischer Überwachungsprogramme zur Erkennung prämaligner Veränderungen (Dysplasien), erkranken immer wieder Patienten mit CED an einem manifesten Dickdarmkarzinom. Ziel dieser Studie war die Evaluation eines Patientenkollektivs mit CU oder MC und einem assoziierten kolorektalen Karzinom hinsichtlich klinischem Erscheinungsbild und Stellenwert koloskopisch–bioptischer Überwachungsprogramme.