Mike G. Laukoetter

JAM-A regulates permeability and inflammation in the intestine in vivo

Recent evidence has linked intestinal permeability to mucosal inflammation, but molecular studies are lacking. Candidate regulatory molecules localized within the tight junction (TJ) include Junctional Adhesion Molecule (JAM-A), which has been implicated in the regulation of barrier function and leukocyte migration. Thus, we analyzed the intestinal mucosa of JAM-A–deficient (JAM-A−/−) mice for evidence of enhanced permeability and inflammation. Colonic mucosa from JAM-A−/− mice had normal epithelial architecture but increased polymorphonuclear leukocyte infiltration and large lymphoid aggregates not seen in wild-type controls. Barrier function experiments revealed increased mucosal permeability, as indicated by enhanced dextran flux, and decreased transepithelial electrical resistance in JAM-A−/− mice. The in vivo observations were epithelial specific, because monolayers of JAM-A−/− epithelial cells also demonstrated increased permeability. Analyses of other TJ components revealed increased expression of claudin-10 and -15 in the colonic mucosa of JAM-A−/− mice and in JAM-A small interfering RNA–treated epithelial cells. Given the observed increase in colonic inflammation and permeability, we assessed the susceptibility of JAM-A−/− mice to the induction of colitis with dextran sulfate sodium (DSS). Although DSS-treated JAM-A−/− animals had increased clinical disease compared with controls, colonic mucosa showed less injury and increased epithelial proliferation. These findings demonstrate a complex role of JAM-A in intestinal homeostasis by regulating epithelial permeability, inflammation, and proliferation.

Interferon-γ Regulates Intestinal Epithelial Homeostasis through Converging β-Catenin Signaling Pathways

Inflammatory cytokines have been proposed to regulate epithelial homeostasis during intestinal inflammation. We report here that interferon-gamma (IFN-gamma) regulates the crucial homeostatic functions of cell proliferation and apoptosis through serine-threonine protein kinase AKT-beta-catenin and Wingless-Int (Wnt)-beta-catenin signaling pathways. Short-term exposure of intestinal epithelial cells to IFN-gamma resulted in activation of beta-catenin through AKT, followed by induction of the secreted Wnt inhibitor Dkk1. Consequently, we observed an increase in Dkk1-mediated apoptosis upon extended IFN-gamma treatment and reduced proliferation through depletion of the Wnt coreceptor LRP6. These effects were enhanced by tumor necrosis factor-alpha (TNF-alpha), suggesting synergism between the two cytokines. Consistent with these results, colitis in vivo was associated with decreased beta-catenin-T cell factor (TCF) signaling, loss of plasma membrane-associated LRP6, and reduced epithelial cell proliferation. Proliferation was partially restored in IFN-gamma-deficient mice. Thus, we propose that IFN-gamma regulates intestinal epithelial homeostasis by sequential regulation of converging beta-catenin signaling pathways.

Regulation of the intestinal epithelial barrier by the apical junctional complex.

Purpose of review Epithelial intercellular junctions are important components of the epithelial barrier and are compromised in disorders such as Crohns disease. We will highlight recent progress in understanding the role of an intercellular junction referred to as the apical junctional complex in regulating small intestinal epithelial permeability in health and disease. Recent findings Recent studies have implicated aberrant regulation of the AJC as an underlying factor contributing to a leaky epithelial barrier in Crohns patients. Consequences of increased epithelial permeability include exposure of intestinal tissue to luminal antigens/pathogens which in turn influence disease activity. Furthermore, proinflammatory cytokines released into the milieu of the epithelium in patients with Crohns disease influence apical junctional complex and epithelial barrier function. Such cytokines induce disassembly of the apical junctional complex by promoting differential endocytosis of component proteins. Additionally, apical junctional complex proteins are targeted by pathogens that use the epithelium as a portal of entry to establish disease in the host. Summary The epithelial apical junctional complex is important in determining epithelial barrier properties. Recent studies have highlighted contribution of proinflammatory cytokines and endocytosis of apical junctional complex proteins to the epithelial barrier defect. Continued advances in understanding of this field will yield new therapeutic targets for intestinal disorders.

Annexin A1 Regulates Intestinal Mucosal Injury, Inflammation, and Repair

During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (−/−) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (−/−) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.

Formyl Peptide Receptor-1 Activation Enhances Intestinal Epithelial Cell Restitution through Phosphatidylinositol 3-Kinase-Dependent Activation of Rac1 and Cdc42

Inflammatory disorders of the gastrointestinal tract result in the breakdown of the intestinal epithelial barrier in the form of erosion and ulceration. To reestablish the epithelial barrier, the epithelium must efficiently migrate to reseal wounds. Numerous signaling cascades are involved in the induction and regulation of this complex process. N-formyl peptide receptors comprise a group of Gi-coupled receptors that regulate innate immune responses. Previously, we identified the expression of functional N-formyl peptide receptors in model SK-CO15 intestinal epithelial cells and observed a role for activation of these receptors in regulating cellular invasive behavior. In these studies, we performed formyl peptide receptor-1 (FPR) localization and evaluated its role in regulating intestinal epithelial cell wound closure. Immunolocalization studies using a recently developed specific monoclonal anti-FPR Ab demonstrated its localization along the lateral membrane of crypt epithelial cells in normal human colonic epithelium. In vitro studies using the classical FPR agonist fMLF showed that FPR activation significantly enhances model intestinal epithelial cell restitution and that FPR localized along actin filaments in lamellipodial and filopodial extrusions. The increase in cell migration was associated with activation of PI3K, Rac1, and Cdc42. Pharmacologic inhibition of PI3K activity abrogated the fMLF-induced increase in wound closure and activation of both Rac1 and Cdc42. Inhibition of Rac1 and Cdc42 using pharmacologic inhibitors and dominant negative mutants also inhibited the fMLF-induced increase in cell migration. Taken together, theses results support a novel role for FPR stimulation in enhancing intestinal epithelial cell restitution through PI3K-dependent activation of Rac1 and Cdc42.

Preoperative Steroid Administration: Effect on Morbidity among Patients Undergoing Intestinal Bowel Resection for Crohn’s Disease

Long-term steroid therapy may predispose to increased perioperative morbidity in patients undergoing surgery with bowel anastomoses. The aim of our study was to review our data to determine if the steroid dosage is associated with the incidence of early complications after bowel resection in patients with prolonged steroid therapy for Crohn’s disease (CD). Altogether, 397 patients underwent bowel resection with primary intestinal anastomoses for CD between 1982 and 2000 in our institution. The mortality and morbidity rates, anastomotic leakage, wound infections, intraabdominal abscesses, reoperation rate, and length of postoperative hospitalization in patients who were having high-dose (≥ 20 mg of prednisolone per day, n = 73) and low-dose (< 20 mg prednisolone per day, n = 146) steroid therapy for more than 1 month before surgery were compared with those of patients (n = 177) who were not receiving steroids. Statistical analysis was performed using Fisher’s exact test and Student’s t-test, with p < 0.05 considered significant. The three groups were similar in terms of gender, duration since first diagnosis, American Society of Anesthesiologists classification, and obesity. Mortality, morbidity, anastomotic leakage, wound infections, intraabdominal abscesses, reoperation rate, and average postoperative stay were not statistically different in patients with high-dose, low-dose, or no steroid therapy. The only factor associated with increased morbidity was a low preoperative hemoglobin level. Our results demonstrate that, in patients who are undergoing bowel resection for CD, even high-dose prolonged preoperative systemic steroid therapy is not associated with increased postoperative complications.

Novel treatment options for perforations of the upper gastrointestinal tract: Endoscopic vacuum therapy and over-the-scope clips

Endoscopic management of leakages and perforations of the upper gastrointestinal tract has gained great importance as it avoids the morbidity and mortality of surgical intervention. In the past years, covered self-expanding metal stents were the mainstay of endoscopic therapy. However, two new techniques are now available that enlarge the possibilities of defect closure: endoscopic vacuum therapy (EVT), and over-the-scope clip (OTSC). EVT is performed by mounting a polyurethane sponge on a gastric tube and placing it into the leakage. Continuous suction is applied via the tube resulting in effective drainage of the cavity and the induction of wound healing, comparable to the application of vacuum therapy in cutaneous wounds. The system is changed every 3-5 d. The overall success rate of EVT in the literature ranges from 84% to 100%, with a mean of 90%; only few complications have been reported. OTSCs are loaded on a transparent cap which is mounted on the tip of a standard endoscope. By bringing the edges of the perforation into the cap, by suction or by dedicated devices, such as anchor or twin grasper, the OTSC can be placed to close the perforation. For acute endoscopy associated perforations, the mean success rate is 90% (range: 70%-100%). For other types of perforations (postoperative, other chronic leaks and fistulas) success rates are somewhat lower (68%, and 59%, respectively). Only few complications have been reported. Although first reports are promising, further studies are needed to define the exact role of EVT and OTSC in treatment algorithms of upper gastrointestinal perforations.

ICAM-1 and VCAM-1 antisense oligonucleotides attenuate in vivo leucocyte adherence and inflammation in rat inflammatory bowel disease

Background: Recruitment of circulating cells to the inflamed intestine is modulated by adhesion molecules expressed on the surface of both leucocytes and endothelial cells. Aims: The objective of this study was to test whether 2`-O-methoxyethyl chimeric antisense oligonucleotides directed against endothelial intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) can downregulate leucocyte-endothelial interactions and thereby attenuate inflammation in rat experimental ileitis. Methods: Indomethacin (7.5 mg/kg ) was injected subcutaneously into Sprague-Dawley rats 48 and 24 hours prior to intravital microscopy. Animals were treated with either ICAM-1 (ISIS 17470), VCAM-1 (ISIS 18155), or scrambled control antisense oligonucleotides administered subcutaneously or intravenously in parallel with indomethacin. Leucocyte trafficking was observed in ileal submucosal collecting venules. Macroscopic and histological grades of inflammation were measured 48 hours after the first indomethacin application. ICAM-1 and VCAM-1 expression in ileal submucosal venules was detected by immunohistochemistry. Results: Intravenous administration of ICAM-1 oligonucleotides 2 mg/kg (rolling leucocytes 5.7 (2.4)/0.01 mm2 endothelial surface, adherent leucocytes 0.8 (1.1)) and VCAM-1 oligonucleotides 8 mg/kg (9.2 (4.4), 0.6 (0.8)) significantly reduced leucocyte adhesion compared with diseased controls (27.8 (5.3), 14 (4.4)) in a dose dependent manner whereas subcutaneous treatment did not. Correspondingly, macroscopic and histological inflammation was significantly decreased. ICAM-1 oligonucleotides markedly reduced endothelial ICAM-1 expression while VCAM-1 oligonucleotides clearly diminished endothelial VCAM-1 expression. Conclusions: Both ICAM-1 and VCAM-1 2`-O-methoxyethyl chimeric antisense oligonucleotides attenuate rat ileitis by downregulation of leucocyte adherence and thus are potential candidates for anti-inflammatory treatment in inflammatory bowel disease.

Is Colonoscopy Alone Sufficient to Screen for Ulcerative Colitis-associated Colorectal Carcinoma?

Patients with ulcerative colitis (UC) are at increased risk for colorectal carcinoma (CAC). Despite the fact that patients at risk are followed closely by colonoscopy to screen for dysplasia, the prevalence of CAC is still unacceptably high. The aim of this study was to evaluate the prevalence of risk factors for CAC, such as dysplasia, and to determine the relevance of colonoscopic surveillance in the group who went on to develop cancer. A series of 24 patients with UC were diagnosed with CAC. The patients’ records were analyzed retrospectively for duration of UC, prevalence of preoperative dysplasia, and other cancer risk factors (CRFs) (e.g., pancolitis, primary sclerosing cholangitis, early onset of UC, and backwash ileitis). The mean age of the patients at the time of cancer diagnosis was 43 years with an average UC duration of 15 years (6 patients had had UC less than 8 years). CAC was identified preoperatively by colonoscopy in 15 of 24 patients, with an additional 7 of 15 showing flat dysplasia. Five of nine patients without preoperatively diagnosed CAC had flat dysplasia. Overall, 19 patients had additional CRFs, most of them with at least two more CRFs. Despite a regular colonoscopic follow-up for most patients with UC, flat dysplasia was missed in 12 patients preoperatively. Therefore we suggest that patient information should also always include surgical options in each case where significant cancer risk factors are found.

Pre-emptive endoscopic vacuum therapy for treatment of anastomotic ischemia after esophageal resections

Background and study aims Endoscopic vacuum therapy (EVT) is a promising new approach for the treatment of anastomotic leakage in the gastrointestinal tract. Here, we present the first case series demonstrating successful use of EVT for the treatment of post-esophagectomy anastomotic ischemia prior to development of leakage. Patients and methods Between 2012 and 2015, intraluminal EVT was performed in eight patients with anastomotic ischemia following esophagectomy. The primary outcome measure was successful mucosal recovery. Secondary outcome measures were duration of treatment, number of sponge changes, septic course, and associated complications. Results Complete mucosal recovery was achieved in six patients (75 %) with different degrees of anastomotic ischemia. In two patients (25 %), small anastomotic leaks developed, which resolved by continuing the EVT treatment. Median duration of EVT treatment until mucosal recovery was 16 days (range 6 - 35), with a median of 5 sponge changes per patient (range 2 - 11). No EVT-associated complications were noted. Three patients developed anastomotic stenoses, which were treated by endoscopic dilation therapy. Conclusion This is the first case series to demonstrate that the early use of EVT potentially modulates clinical outcomes and infection parameters in patients with anastomotic ischemia following esophagectomy. Further studies are needed to define the indications and patients who are most likely to benefit from early EVT.