Anton J. Kroesen

Changes in expression and distribution of claudin-2, -5 and -8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn's disease

Background: Epithelial barrier function is impaired in Crohn’s disease. Aim: To define the underlying cellular mechanisms with special attention to tight junctions. Methods: Biopsy specimens from the sigmoid colon of patients with mild to moderately active or inactive Crohn’s disease were studied in Ussing chambers, and barrier function was determined by impedance analysis and conductance scanning. Tight junction structure was analysed by freeze fracture electron microscopy, and tight junction proteins were investigated immunohistochemically by confocal laser scanning microscopy and quantified in immunoblots. Epithelial apoptosis was analysed in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling and 4′,6-diamidino-2-phenylindole staining. Results: Patients with active Crohn’s disease showed an impaired intestinal barrier function as indicated by a distinct reduction in epithelial resistance. As distribution of conductivity was even, focal epithelial lesions (eg, microerosions) did not contribute to barrier dysfunction. Instead, freeze fracture electron microscopy analysis showed reduced and discontinuous tight junction strands. Occludin and the sealing tight junction proteins claudin 5 and claudin 8 were downregulated and redistributed off the tight junction, whereas the pore-forming tight junctions protein claudin 2 was strongly upregulated, which constitute the molecular basis of tight junction changes. Other claudins were unchanged (claudins 1, 4 and 7) or not detectable in sigmoid colon (claudins 11, 12, 14, 15 and 16). Claudin 2 upregulation was less pronounced in active Crohn’s disease compared with active ulcerative colitis and was inducible by tumour necrosis factor α. As a second source of impaired barrier function, epithelial apoptosis was distinctly increased in active Crohn’s disease (mean (SD) 5.2 (0.5)% v 1.9 (0.2)% in control). By contrast, barrier function, tight junction proteins and apoptosis were unaffected in Crohn’s disease in remission. Conclusion: Upregulation of pore-forming claudin 2 and downregulation and redistribution of sealing claudins 5 and 8 lead to altered tight junction structure and pronounced barrier dysfunction already in mild to moderately active Crohn’s disease.

Mucosal and invading bacteria in patients with inflammatory bowel disease compared with controls.

Background: Endogenous intestinal bacteria and/or specific bacterial pathogens are suspected of being involved in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to investigate IBD tissues for different bacterial population groups harbouring the mucosal surface and/or invading the mucosa. Methods: Tissue sections from surgical resections from the terminal ileum and/or the colon from 24 IBD patients (12 active ulcerative colitis (UC), 12 active Crohn disease (CD)) and 14 non-IBD controls were studied by fluorescent in situ hybridization on a quantifiable basis. Results: More bacteria were detected on the mucosal surface of IBD patients than on those of non-IBD controls ( P < 0.05). Bacterial invasion of the mucosa was evident in 83.3% of colonic specimens from the UC patients, in 55.6% of the ileal and in 25% of the colonic specimens from the CD patients, but no bacteria were detected in the tissues of the controls. Colonic UC specimens were colonized by a variety of organisms, such as bacteria belonging to the gamma subdivision of Proteobacteria , the Enterobacteriaceae , the Bacteroides/Prevotella cluster, the Clostridium histolyticum/Clostridium lituseburense group, the Clostridium coccoides/Eubacterium rectale group, high G + C Gram-positive bacteria, or sulphate-reducing bacteria, while CD samples harboured mainly bacteria belonging to the former three groups. Conclusion: Pathogenic events in CD and UC may be associated with different alterations in the mucosal flora of the ileum and colon.

Through the endoscope balloon dilation of ileocolonic strictures: prognostic factors, complications, and effectiveness

Background/aimsAbout 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 methodsThirty-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.ResultsFive 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.ConclusionThrough 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.

Staging Anal Cancer: Prospective Comparison of Transanal Endoscopic Ultrasound and Magnetic Resonance Imaging

PurposeThe staging of anal cancer is extremely important for therapy and prognosis. Transanal endoscopic ultrasound and magnetic resonance imaging are routinely applied. The aim of this prospective comparative study is to evaluate whether tumor staging is concordant between these techniques.MethodsForty-five anal cancer patients underwent endoscopic ultrasound and magnetic resonance imaging. Histological confirmation was obtained in all patients. The two test methods were compared with the kappa concordance index and sensitivity for the initial method of tumor detection was calculated. For six patients who were operated upon because of tumor progression, the results were evaluated against the histological tumor stage.ResultsHigh concordance was found in the assessment of tumor size and nodal status (kappa index 0.63 and 0.77). Cancer patients were correctly identified with 100% sensitivity (45/45) by endoscopic ultrasound and with 88.9% (40/45) sensitivity by magnetic resonance imaging. In the six operated patients, T stage was correctly assessed in four of six patients by endoscopic ultrasound and in three of six patients by magnetic resonance imaging.ConclusionThe results of endoscopic ultrasound strongly coincide with those of magnetic resonance imaging. Endoscopic ultrasound may be superior to magnetic resonance imaging for detection of small superficial tumors. However, magnetic resonance imaging is needed for N staging.

Permanently increased mucosal permeability in patients with backwash ileitis after ileoanal pouch for ulcerative colitis

Objective. Backwash ileitis (BI) has not been identified as a risk factor for pouchitis. The aim of this study was to investigate the barrier function of the ileoanal pouch depending on the presence of BI. The incidence of pouchitis in a population of ulcerative colitis patients with BI is also reported. Material and methods. Biopsies were taken from 80 patients with ulcerative colitis: a) terminal ileum prior to pouch creation (pre-IAP); b) 16 months after ileostomy closure (intact pouch); and c) during pouchitis. Patients were stratified into the BI group and the non-BI (ØBI) group. Barrier function was determined in Ussing-chambers as epithelial resistance by impedance analysis and as mannitol permeability from 3H-mannitol fluxes. Na+-glucose co-transport was measured as a change in short-circuit current (ISC) after addition of glucose. Relative risk of developing pouchitis was calculated by corrected χ2 test. Results. In 13/21 (BI/ØBI) pre-IAP patients, 23/37 (BI/ØBI) with an intact pouch, and 35/7 (BI/ØBI) with pouchitis, epithelial resistance in BI/ØBI was 13.5±1.6/14.3±0.9 Ω·cm2 for pre-IAP, 12.7±1.3/16.8±1.2 Ω·cm2 (p<0.05 BI versus ØBI) for the intact pouch, and 10.1±1.1/9.9±1.8 Ω·cm2 for pouchitis (p<0.05 BI versus ØBI with an intact pouch). No differences were found for electrogenic chloride secretion and active Na+-glucose co-transport between BI/ØBI in the three groups. In patients with BI, pouchitis was more common (35 versus 7 patients, odds ratio 33.0 (95% CI 8.3–143.9; p<0.0001)). Conclusions. Ulcerative colitis patients with BI show impaired barrier function in the further course of the ileoanal pouch. Thus, BI has a long-term impact on epithelial barrier function.

Increased bacterial permeation in long‐lasting ileoanal pouches

Background and Aims: Bacterial overgrowth appears to play an important role in the pathogenesis of ileoanal pouches. Therefore, the capability of bacterial permeation and its determinants is of great interest. The aim of this study was to examine bacterial permeation in the ileoanal pouch and to correlate the results with the degree of inflammation, the epithelial resistance, the mucosal transport function, and the age of the ileoanal pouches. Materials and Methods: Biopsies were taken from 54 patients before colectomy (n = 13; preileal pouch‐anal anastomosis [IPAA]), and closure of ileostomy (n = 7; deviation), <1 year after closure of ileostomy (n = 8; intact pouch I), >1 year after closure of ileostomy (n = 16; intact pouch II), in the case of pouchitis (n = 11), and in 11 controls. Tissues were mounted in a miniaturized Ussing chamber. Escherichia coli was added to the mucosal side of the Ussing chamber, and the permeation was proven by serosal presence of E. coli. Epithelial and subepithelial resistance was determined by transmural impedance analysis. Active Na+‐glucose cotransport and active Cl− secretion were measured. Specimens were analyzed by fluorescent in situ hybridization with oligonucleotide probes targeting the bacterial 16s ribosomal RNA. The bacteria in and on the tissue were enumerated. Results: Bacterial permeation occurred in 2 of 13 pre‐IPAA, 2 of 7 deviations, 0 of 8 intact pouch I, 9 of 16 intact pouch II, 5 of 11 pouchitis specimens, and 0 of 11 ileum controls. The frequency of bacterial permeation in the intact pouch II group is higher than in the intact pouch I group (P < 0.001). Epithelial resistance, mannitol fluxes, electrogenic chloride secretion, sodium‐glucose cotransport of the bacterially permeated specimens versus nonpermeated of the intact pouch II group, and the pouchitis group and subepithelial resistance remained unchanged. Intramural bacteria could be detected by fluorescence in situ hybridization mainly in long‐lasting pouches, but there was no correlation with bacterial permeation. Conclusions: The long‐lasting ileoanal pouch is associated with increased bacterial permeability. This is not correlated with a disturbed function of the pouch mucosa but could be a precursor of pouchitis.

Complications after end-to-end vs. side-to-side anastomosis in ileocecal Crohn's disease--early postoperative results from a randomized controlled multi-center trial (ISRCTN-45665492).

BackgroundRecurrent Crohn’s disease activity at the site of anastomosis after ileocecal resection is of great surgical importance. This prospective randomized multi-center trial with an estimated case number of 224 patients was initially planned to investigate whether stapled side-to-side anastomosis, compared to hand-sewn end-to-end anastomosis, results in a decreased recurrence of Crohn’s disease following ileocolic resection (primary endpoint). The secondary endpoint was to focus on the early postoperative results comparing both surgical methods. The study was terminated early due to insufficient patient recruitment and because another large study investigated the same question, while our trial was ongoing.Methods and study designPatients with stenosing ileitis terminalis in Crohn’s disease who underwent an ileocolic resection were randomized to side-to-side or end-to-end anastomosis. Due to its early discontinuation, our study only investigated the secondary endpoints, the early postoperative results (complications: bleeding, wound infection, anastomotic leakage, first postoperative stool, duration of hospital stay).ResultsFrom February 2006 until June 2010, 67 patients were enrolled in nine participating centers. The two treatment groups were comparable to their demographic and pre-operative data. BMI and Crohn’s Disease Activity Index were 22.2 (±4.47) and 200.5 (±73.66), respectively, in the side-to-side group compared with 23.3 (±4.99) and 219.6 (±89.03) in the end-to-end group. The duration of surgery was 126.7 (±42.8) min in the side-to-side anastomosis group and 137.4 (±51.9) min in the end-to-end anastomosis group. Two patients in the end-to-end anastomosis group developed an anastomotic leakage (6.5%). Impaired wound healing was found in 13.9% of the side-to-side anastomosis group, while 6.5% of the end-to-end anastomosis group developed this complication. The duration of hospital stay was comparable in both groups with 9.9 (±3.93) and 10.4 (±3.26) days, respectively.ConclusionsBecause of the early discontinuation of the study, it is not possible to provide a statement about the perianastomotic recurrence rates regarding the primary endpoint. With regard to the early postoperative outcome, we observed no difference between the two types of anastomosis.

Sacral Nerve Stimulation Induces Changes in the Pelvic Floor and Rectum that Improve Continence and Quality of Life

PurposeSacral nerve stimulation (SNS) can improve fecal incontinence, though the exact mechanism is not known. This study examines the following hypotheses: SNS leads to contraction of the pelvic floor, influences rectal perception, and improves continence and quality of life.MethodsFourteen patients with sacral nerve stimulators implanted for fecal incontinence were examined prospectively. Morphological and functional assessment was done by endosonography, manometry, and volumetry with the stimulator turned on and off in direct succession. Questionnaires were used to determine incontinence and quality of life.ResultsWith the stimulator turned on, rectal filling conditions were perceived only at higher volumes; in particular, the defecation urge was sensed only at higher volumes. There was also a reduction in the diameters of the external and internal anal sphincters and a decrease in the distance between the anal mucosa and the symphysis as a sign of pelvic floor elevation. Six months after surgery, continence and quality of life were markedly better than before the operation.ConclusionsWe were able to confirm the hypotheses given above. The improvements of pelvic floor contraction and rectal perception are rapid adjustment processes in response to stimulation of sacral nerves S3/S4 when turning on the stimulator.

Rectal Anatomy After Rectopexy: Cinedefecography Versus MR-Defecography

BACKGROUND Conventional defecography can reveal abnormalities in patients with evacuatory disorders. With fast dynamic MR imaging systems, MR-defecography has become possible, which does not expose patients to ionizing radiation. The purpose of this study was to assess the correlation of both methods after rectopexy. MATERIALS AND METHODS Twenty-one consecutive patients underwent abdominal sigmoidectomy and rectopexy due to evacuatory disorders. Postoperatively, all patients were investigated by cineradiographic defecography. Fourteen patients underwent MR-defecography additionally. The results were screened for anorectal angle and pelvic floor position (rest, squeezing, and evacuation). The findings were depicted in Box plot analysis and compared with the Friedman-test. Descent of pelvic organs was also assessed. RESULTS In MR-defecography, anorectal angle at rest was smaller than in conventional defecography, but there was no difference during squeezing and defecation. Concerning pelvic floor position, during squeezing, MR-defecography illustrated a lower perineum and a broader range of pelvic settings, but no difference at rest and during evacuation. In four patients, MR-defecography visualized a descent of the bladder. However, in four patients with complete evacuation in cineradiography and with no clinical complaints about incomplete evacuation, MR imaging showed deficient evacuation. Overall continence of patients was significantly improved through surgery, but there was no change in sphincter pressure, radial asymmetry, or sphincter length. CONCLUSIONS In general, with respect to anorectal angle and perineal motility, both methods revealed consistent results. The concomitant depiction of structures in MR-defecography is helpful in the assessment of descent of pelvic organs and permits visualization of enteroceles. However, in 30% of patients, MR-defecography wrongly showed incomplete evacuation.

NALP expression in Paneth cells provides a novel track in IBD signaling.

PurposePaneth cells are part of the innate mucosal immunity of the gut with possible regulatory function. This study intends to identify the gene expression pattern of the orthotopic and metaplastic Paneth cells, searching for differences between metaplastic occurrence between Crohns disease and ulcerative colitis.MethodsPaneth cells were collected in RNAse-free conditions via micro dissection. RNA isolation and super amplification was followed by microarray analysis of whole genome expression activity of the orthotopic and metaplastic Paneth cells. Immunohistology of β-catenin and Frizzled-5 receptor was performed.ResultsHistological analysis showed no morphological or secretory change (Frizzled-5 receptor and β-catenin) in orthotopic and metaplastic Paneth cells. Microarray analysis indicated an increased, but not mutant activation of Wnt/β-catenin signaling and firstly showed expression of NALP 1, 7, 8 and 11 in metaplastic Paneth cells.ConclusionsPaneth cells might play a NALP-mediated role in the pathogenesis of IBD.