Enterohemorrhagic Escherichia coli colonization of human colonic epithelium in vitro and ex vivo.

Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen causing gastroenteritis and more severe complications, such as hemorrhagic colitis and hemolytic uremic syndrome. Pathology is most pronounced in the colon, but to date there is no direct clinical evidence showing EHEC binding to the colonic epithelium in patients. In this study, we investigated EHEC adherence to the human colon by using in vitro organ culture (IVOC) of colonic biopsy samples and polarized T84 colon carcinoma cells. We show for the first time that EHEC colonizes human colonic biopsy samples by forming typical attaching and effacing (A/E) lesions which are dependent on EHEC type III secretion (T3S) and binding of the outer membrane protein intimin to the translocated intimin receptor (Tir). A/E lesion formation was dependent on oxygen levels and suppressed under oxygen-rich culture conditions routinely used for IVOC. In contrast, EHEC adherence to polarized T84 cells occurred independently of T3S and intimin and did not involve Tir translocation into the host cell membrane. Colonization of neither biopsy samples nor T84 cells was significantly affected by expression of Shiga toxins. Our study suggests that EHEC colonizes and forms stable A/E lesions on the human colon, which are likely to contribute to intestinal pathology during infection. Furthermore, care needs to be taken when using cell culture models, as they might not reflect the in vivo situation.

Canonical Wnt signals combined with suppressed TGFß/BMP pathways promote renewal of the native human colonic epithelium.

BACKGROUND: A defining characteristic of the human intestinal epithelium is that it is the most rapidly renewing tissue in the body. However, the processes underlying tissue renewal and the mechanisms that govern their coordination have proved difficult to study in the human gut. OBJECTIVE: To investigate the regulation of stem cell-driven tissue renewal by canonical Wnt and TGFß/bone morphogenetic protein (BMP) pathways in the native human colonic epithelium. DESIGN: Intact human colonic crypts were isolated from mucosal tissue samples and placed into 3D culture conditions optimised for steady-state tissue renewal. High affinity mRNA in situ hybridisation and immunohistochemistry were complemented by functional genomic and bioimaging techniques. The effects of signalling pathway modulators on the status of intestinal stem cell biology, crypt cell proliferation, migration, differentiation and shedding were determined. RESULTS: Native human colonic crypts exhibited distinct activation profiles for canonical Wnt, TGFß and BMP pathways. A population of intestinal LGR5/OLFM4-positive stem/progenitor cells were interspersed between goblet-like cells within the crypt-base. Exogenous and crypt cell-autonomous canonical Wnt signals supported homeostatic intestinal stem/progenitor cell proliferation and were antagonised by TGFß or BMP pathway activation. Reduced Wnt stimulation impeded crypt cell proliferation, but crypt cell migration and shedding from the crypt surface were unaffected and resulted in diminished crypts. CONCLUSIONS: Steady-state tissue renewal in the native human colonic epithelium is dependent on canonical Wnt signals combined with suppressed TGFß/BMP pathways. Stem/progenitor cell proliferation is uncoupled from crypt cell migration and shedding, and is required to constantly replenish the crypt cell population.

Technical and Clinical Outcomes After Colorectal Stenting in Malignant Large Bowel Obstruction: A Single-Center Experience.

PURPOSE: Malignant large bowel obstruction is a surgical emergency that requires urgent decompression. Stents are increasingly being used, though reported outcomes are variable. We describe our multidisciplinary experience in using stents to manage malignant large bowel obstruction. METHODS: All patients undergoing colorectal stent insertion for acute large bowel obstruction in a teaching hospital were included. Outcomes, complications, and length of stay (LOS) were recorded. RESULTS: Over a 7-year period, 73 procedures were performed on 67 patients (37 male, mean age of 76 years). Interventional radiology was involved in all cases. Endoscopic guidance was required in 24 cases (32.9%). In 18 patients (26.9%), treatment intent was to bridge to elective surgery; 16 had successful stent placement; all had subsequent curative resection (laparoscopic resection, 8 of 18; primary anastomosis, 14 of 18). Overall LOS, including both index admission and elective admission, was 16.4 days. Treatment intent was palliative in 49 patients (73.1%). In this group, stents were successfully placed in 41 of 49 (83.7%). Complication rate within 30 days was 20%, including perforation (2 patients), per rectal bleeding (2), stent migration (1), and stent passage (5). Nineteen patients (38.8%) required subsequent stoma formation (6, during same admission; 13, during subsequent admission). Overall LOS was 16.9 days. CONCLUSION: In our experience colorectal stents can be used effectively to manage malignant large bowel obstruction, with only selective endoscopic input. As a bridge to surgery, most patients can avoid emergency surgery and have a primary anastomosis. In the palliative setting, the complication rate is acceptable and two-thirds avoid a permanent stoma.

Factors affecting adenoma detection rate in a national flexible sigmoidoscopy screening programme: a retrospective analysis.

BACKGROUND: A national colorectal cancer screening programme started in England in 2013, offering one-off flexible sigmoidoscopy to all men and women aged 55 years in addition to the biennial faecal occult blood testing programme offered to all individuals aged 60-74 years. We analysed data from six pilot flexible sigmoidoscopy screening centres to examine factors affecting the adenoma detection rate (ADR). METHODS: We did a retrospective analysis of flexible sigmoidoscopy screening procedures performed in individuals aged 55 years at six pilot sites in England as part of the National Health Service Bowel Scope Screening programme. ADR (number of procedures in which at least one adenoma was removed or biopsied, divided by total number of procedures) was calculated for each site and each endoscopist. Multiple regression models were used to examine the variation in ADR with withdrawal time and extent of examination, and the effect of other factors including comfort and bowel preparation on extent of examination. FINDINGS: The analysis included 8256 procedures done between May 7, 2013, and May 6, 2014. The overall ADR was 9·1% (95% CI 8·5-9·8; 755 of 8256 procedures), varying from 7·4% (6·2-8·9) to 11·0% (9·1-13·4) by screening centre. The ADR was 11·5% (95% CI 10·6-12·5; 493 of 4299 procedures) in men and 6·6% (5·9-7·4; 262 of 3957 procedures) in women (p<0·0001). On multivariate analysis, factors associated with adenoma detection were male sex (relative risk 1·69, 95% CI 1·46-1·95; p<0·0001) and a withdrawal time from the splenic flexure of at least 3·25 min in negative procedures (1·22, 1·00-1·48; p=0·045). However, increasing the withdrawal time to 4·0 min or more did not increase the likelihood of adenoma detection (1·22, 0·99-1·51; p=0·057). Procedures not reaching the splenic flexure were associated with lower chance of adenoma detection (eg, 0·77, 0·66-0·91; p=0·0015 for procedures reaching the descending colon), but there was no additional benefit associated with reaching the transverse colon (0·83, 0·67-1·02; p=0·069). Women (0·83, 0·80-0·87; p<0·0001), individuals with adequate (0·79, 0·76-0·83; p<0·0001) or poor (0·58, 0·51-0·67; p<0·0001) bowel preparation (compared with good bowel preparation), and those with mild (0·82, 0·76-0·88; p<0·0001) or moderate or severe (0·58, 0·51-0·66; p<0·0001) discomfort (compared with no discomfort) were less likely to have a procedure reaching the splenic flexure. INTERPRETATION: Key performance indicators for flexible sigmoidoscopy screening should be defined, including standards for insertion and withdrawal times, optimal depth, and bowel preparation. ADR could be improved by recommending a withdrawal time from the splenic flexure of at least 3·25 min (ideally 3·5-4·0 min). FUNDING: None.

The colon-selective spasmolytic otilonium bromide inhibits muscarinic M(3) receptor-coupled calcium signals in isolated human colonic crypts.

1. Otilonium bromide (OB) is a smooth muscle relaxant used in the treatment of irritable bowel syndrome. Otilonium bromide has been shown to interfere with the mobilization of calcium in intestinal smooth muscle, but the effects on other intestinal tissues have not been investigated. We identified the muscarinic receptor subtype coupled to calcium signals in colonic crypt derived from the human colonic epithelium and evaluated the inhibitory effects of OB. 2. Calcium signals were monitored by fluorescence imaging of isolated human colonic crypts and Chinese hamster ovary cells stably expressing the cloned human muscarinic M(3) receptor subtype (CHO-M(3)). Colonic crypt receptor expression was investigated by pharmacological and immunohistochemical techniques. 3. The secretagogue acetylcholine (ACh) stimulated calcium mobilization from intracellular calcium stores at the base of human colonic crypts with an EC(50) of 14 micro M. The muscarinic receptor antagonists 4-DAMP, AF-DX 384, pirenzepine and methroctamine inhibited the ACh-induced calcium signal with the following respective IC(50) (pK(b)) values: 0.78 nM (9.1), 69 nM (7.2), 128 nM (7.1), and 2510 nM (5.8). 4. Immunohistochemical analyses of muscarinic receptor expression demonstrated the presence of M(3) receptor subtype expression at the crypt-base. 5. Otilonium bromide inhibited the generation of ACh-induced calcium signals in a dose dependent manner (IC(50)=880 nM). 6. In CHO-M(3) cells, OB inhibited calcium signals induced by ACh, but not ATP. In addition, OB did not inhibit histamine-induced colonic crypt calcium signals. 7. The present studies have demonstrated that OB inhibited M(3) receptor-coupled calcium signals in human colonic crypts and CHO-M(3) cells, but not those induced by stimulation of other endogenous receptor types. We propose that the M(3) receptor-coupled calcium signalling pathway is directly targeted by OB at the level of the colonic epithelium, suggestive of an anti-secretory action in IBS patients suffering with diarrhoea.

Dynamic and differential regulation of NKCC1 by calcium and cAMP in the native human colonic epithelium.

The capacity of the intestine to secrete fluid is dependent on the basolateral Na(+)-K(+)-2Cl(-) co-transporter (NKCC1). Given that cAMP and Ca(2+) signals promote sustained and transient episodes of fluid secretion, respectively, this study investigated the differential regulation of functional NKCC1 membrane expression in the native human colonic epithelium. Tissue sections and colonic crypts were obtained from sigmoid rectal biopsy tissue samples. Cellular location of NKCC1, Na(+)-K(+)-ATPase, M3 muscarinic acetylcholine receptor (M(3)AChR) and lysosomes was examined by immunolabelling techniques. NKCC1 activity (i.e. bumetanide-sensitive uptake), intracellular Ca(2+) and cell volume were assessed by 2',7'-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), Fura-2 and differential interference contrast/calcein imaging. Unstimulated NKCC1 was expressed on basolateral membranes and exhibited a topological expression gradient, predominant at the crypt base. Cholinergic Ca(2+) signals initiated at the crypt base and spread along the crypt axis. In response, NKCC1 underwent a Ca(2+)-dependent 4 h cycle of recruitment to basolateral membranes, activation, internalization, degradation and re-expression. Internalization was prevented by the epidermal growth factor receptor kinase inhibitor tyrphostin-AG1478, and re-expression was prohibited by the protein synthesis inhibitor cylcoheximide; the lysosome inhibitor chloroquine promoted accumulation of NKCC1 vesicles. NKCC1 internalization and re-expression were accompanied by secretory volume decrease and bumetanide-sensitive regulatory volume increase, respectively. In contrast, forskolin (i.e. cAMP elevation)-stimulated NKCC1 activity was sustained, and membrane expression and cell volume remained constant. Co-stimulation with forskolin and acetylcholine promoted dramatic recruitment of NKCC1 to basolateral membranes and prolonged the cycle of co-transporter activation, internalization and re-expression. In conclusion, persistent NKCC1 activation by cAMP is constrained by a Ca(2+)-dependent cycle of co-transporter internalization, degradation and re-expression; this is a novel mechanism to limit intestinal fluid loss.