Conny Wallon

Corticotropin releasing hormone (CRH) regulates macromolecular permeability via mast cells in normal human colonic biopsies in vitro

Objective: Persistent stress and life events affect the course of ulcerative colitis and irritable bowel syndrome by largely unknown mechanisms. Corticotropin-releasing hormone (CRH) has been implicated as an important mediator of stress-induced abnormalities in intestinal mucosal function in animal models, but to date no studies in human colon have been reported. The aim was to examine the effects of CRH on mucosal barrier function in the human colon and to elucidate the mechanisms involved in CRH-induced hyper-permeability. Design: Biopsies from 39 volunteers were assessed for macromolecular permeability (horseradish peroxidise (HRP), 51Cr-EDTA), and electrophysiology after CRH challenge in Ussing chambers. The biopsies were examined by electron and confocal microscopy for HRP and CRH receptor localisation, respectively. Moreover, CRH receptor mRNA and protein expression were examined in the human mast cell line, HMC-1. Results: Mucosal permeability to HRP was increased by CRH (2.8±0.5 pmol/cm2/h) compared to vehicle exposure (1.5±0.4 pmol/cm2/h), p = 0.032, whereas permeability to 51Cr-EDTA and transmucosal electrical resistance were unchanged. The increased permeability to HRP was abolished by α-helical CRH (9-41) (1.3±0.6 pmol/cm2/h) and the mast cell stabiliser, lodoxamide (1.6±0.6 pmol/cm2/h). Electron microscopy showed transcellular passage of HRP through colonocytes. CRH receptor subtypes R1 and R2 were detected in the HMC-1 cell line and in lamina propria mast cells in human colon. Conclusions: Our results suggest that CRH mediates transcellular uptake of HRP in human colonic mucosa via CRH receptor subtypes R1 and R2 on subepithelial mast cells. CRH-induced macromolecular uptake in human colon mucosa may have implications for stress-related intestinal disorders.

Increased antigen and bacterial uptake in follicle associated epithelium induced by chronic psychological stress in rats

Background: Chronic stress affects the course of inflammatory bowel disease and experimental colitis, and may also initiate intestinal inflammation in rats. Aim: To investigate the effects of stress on the M cell containing follicle associated epithelium, specialised in antigen uptake. Subjects and methods: Wistar rats were submitted to acute water avoidance stress for one hour or chronic water avoidance stress for 1 hour/day for 10 consecutive days. Permeability to 51Cr-edta, horseradish peroxidase, and chemically killed Escherichia coli K-12 was studied in both villus and follicle associated epithelium in Ussing chambers. Segments were further examined by light, electron, and confocal microscopy. Results: Acute stress increased horseradish peroxidase flux in villus as well as in follicle associated epithelium. Chronic stress further increased permeability to horseradish peroxidase in villus and follicle associated epithelium, in the latter by almost fourfold. Moreover, chronic stress induced over 30 times increased E coli passage in follicle associated epithelium whereas there was no significant increase in villus epithelium. Bacterial uptake was confirmed by confocal microscopy showing fluorescent bacteria penetrating and passing through the epithelial surface. Conclusions: These results show that the barrier function of follicle associated epithelium can be modulated, and that chronic stress enhances the uptake of luminal antigens and bacteria via the follicle associated epithelium. This can increase antigen exposure in Peyer’s patches thereby having implications in the initiation of proinflammatory immune responses within the intestinal mucosa.

Laparoscopic Lavage vs Primary Resection for Acute Perforated Diverticulitis: The SCANDIV Randomized Clinical Trial

IMPORTANCE Perforated colonic diverticulitis usually requires surgical resection, which is associated with significant morbidity. Cohort studies have suggested that laparoscopic lavage may treat perforated diverticulitis with less morbidity than resection procedures. OBJECTIVE To compare the outcomes from laparoscopic lavage with those for colon resection for perforated diverticulitis. DESIGN, SETTING, AND PARTICIPANTS Multicenter, randomized clinical superiority trial recruiting participants from 21 centers in Sweden and Norway from February 2010 to June 2014. The last patient follow-up was in December 2014 and final review and verification of the medical records was assessed in March 2015. Patients with suspected perforated diverticulitis, a clinical indication for emergency surgery, and free air on an abdominal computed tomography scan were eligible. Of 509 patients screened, 415 were eligible and 199 were enrolled. INTERVENTIONS Patients were assigned to undergo laparoscopic peritoneal lavage (n = 101) or colon resection (n = 98) based on a computer-generated, center-stratified block randomization. All patients with fecal peritonitis (15 patients in the laparoscopic peritoneal lavage group vs 13 in the colon resection group) underwent colon resection. Patients with a pathology requiring treatment beyond that necessary for perforated diverticulitis (12 in the laparoscopic lavage group vs 13 in the colon resection group) were also excluded from the protocol operations and treated as required for the pathology encountered. MAIN OUTCOMES AND MEASURES The primary outcome was severe postoperative complications (Clavien-Dindo score >IIIa) within 90 days. Secondary outcomes included other postoperative complications, reoperations, length of operating time, length of postoperative hospital stay, and quality of life. RESULTS The primary outcome was observed in 31 of 101 patients (30.7%) in the laparoscopic lavage group and 25 of 96 patients (26.0%) in the colon resection group (difference, 4.7% [95% CI, -7.9% to 17.0%]; P = .53). Mortality at 90 days did not significantly differ between the laparoscopic lavage group (14 patients [13.9%]) and the colon resection group (11 patients [11.5%]; difference, 2.4% [95% CI, -7.2% to 11.9%]; P = .67). The reoperation rate was significantly higher in the laparoscopic lavage group (15 of 74 patients [20.3%]) than in the colon resection group (4 of 70 patients [5.7%]; difference, 14.6% [95% CI, 3.5% to 25.6%]; P = .01) for patients who did not have fecal peritonitis. The length of operating time was significantly shorter in the laparoscopic lavage group; whereas, length of postoperative hospital stay and quality of life did not differ significantly between groups. Four sigmoid carcinomas were missed with laparoscopic lavage. CONCLUSIONS AND RELEVANCE Among patients with likely perforated diverticulitis and undergoing emergency surgery, the use of laparoscopic lavage vs primary resection did not reduce severe postoperative complications and led to worse outcomes in secondary end points. These findings do not support laparoscopic lavage for treatment of perforated diverticulitis. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01047462.

Endoscopic biopsies in Ussing chambers evaluated for studies of macromolecular permeability in the human colon

Objective Studies of mucosal permeability to protein antigens in humans are limited to in vitro techniques. The use of surgical specimens for such studies has major shortcomings. Endoscopic biopsies in Ussing chambers have been introduced as a means of studying secretion and transepithelial permeability, but have not been evaluated for studies of protein antigen uptake in human intestine. Material and methods Standard forceps biopsies from the sigmoid colon of 24 healthy volunteers were mounted in Ussing chambers with an exposed tissue area of 1.76 mm2. 51Cr-EDTA (paracellular probe) and horseradish peroxidase (HRP; 45 kDa protein antigen) were used as permeability markers. Mucosal permeability, electrophysiology, histology and energy contents of the biopsies were studied over time. To evaluate the ability of the technique to detect permeability changes, the mucosa was modulated with capric acid, a medium-chain fatty acid, known to affect tight junctions. Results In the Ussing chamber the mucosal biopsies were viable for 160 min with stable levels of ATP and lactate, and only minor changes in morphology. Steady-state permeability with low variability was seen for both markers during the 30–90 min period. Exposure to capric acid induced a rapid decrease in short-circuit current (Isc) and a slower reversible decrease in transepithelial resistance (TER), as well as an increased permeability to 51Cr-EDTA and HRP. Conclusions Endoscopic biopsies of human colon are viable in Ussing chambers and are reliable tools for studies of mucosal permeability to protein antigens. The technique offers a broad potential for studies of mucosal function in the pathophysiology of human gastrointestinal diseases.

Characterization of antigen and bacterial transport in the follicle-associated epithelium of human ileum.

The follicle-associated epithelium (FAE), covering Peyers patches, provides a route of entry for antigens and microorganisms. Animal studies showed enhanced antigen and bacterial uptake in FAE, but no study on barrier function of human FAE has been reported. Our aim was to characterize the normal barrier properties of human FAE. Specimens of normal ileum were taken from 30 patients with noninflammatory colonic disease. Villus epithelium (VE) and FAE were identified and mounted in Ussing chambers. Permeability to 51Cr-EDTA, transmucosal flux of the protein antigen, horseradish peroxidase (HRP), and transport of fluorescent Escherichia coli (chemically killed K-12 and live HB101) were measured. Uptake mechanisms were studied by confocal- and transmission electron microscopy, and by using pharmacological inhibitors in an in vitro coculture model of FAE and in human ileal FAE. HRP flux was substantially higher in FAE than in VE, and was reduced by an amiloride analog. Electron microscopy showed HRP-containing endosomes. Transport of E. coli K-12 and HB101 was also augmented in FAE and was confirmed by confocal microscopy. In vitro coculture experiments and electron microscopy revealed actin-dependent, mainly transcellular, uptake of E. coli K-12 into FAE. 51Cr-EDTA permeability was equal in FAE and VE. Augmented HRP flux and bacterial uptake but similar paracellular permeability, suggest functional variations of transcellular transport in the FAE. We show for the first time that FAE of human ileum is functionally distinct from regular VE, rendering the FAE more prone to bacterial–epithelial cell interactions and delivery of antigens to the mucosal immune system.

Is a dissection balloon beneficial in totally extraperitoneal endoscopic hernioplasty (TEP)? A randomized prospective multicenter study.

BackgroundLaparoscopic hernioplasty has been criticized because of its technical complexity and increased costs. Disposable dissection balloons can be used to facilitate the creation of the initial working space in totally extraperitoneal endoscopic hernioplasty (TEP), but their use adds to the cost of the operation.MethodsA total of 322 men with unilateral, primary, or recurrent inguinal hernias were randomized to undergo TEP with or without a dissection balloon.ResultsIn the group with the balloon, three of 161 patients (2.5%) required conversion to transabdominal preperitoneal hernioplasty (TAPP), or open herniorraphy, whereas 17 of 161 patients (10.6%) were converted to TAPP or open herniorraphy in the group without the balloon (p = 0.002). The mean operation time was 55 min in the group with the balloon and 63 min in the group without the balloon (p=0.004). There was no difference between them in postoperative morbidity, and there were no major complications in either group. The recurrence rate was 3.1% in the group with the balloon and 3.7 % in the group without the balloon (p = 0.8).ConclusionThe use of a dissection balloon in TEP reduces the conversion rate and may be especially beneficial early in the learning curve.

The effects of probiotics on barrier function and mucosal pouch microbiota during maintenance treatment for severe pouchitis in patients with ulcerative colitis

A total of 10–15% of patients with an ileoanal pouch develop severe pouchitis necessitating long‐term use of antibiotics or pouch excision. Probiotics reduce the risk of recurrence of pouchitis, but mechanisms behind these effects are not fully understood.

Corticotropin-Releasing Hormone and Mast Cells in the Regulation of Mucosal Barrier Function in the Human Colon

Corticotropin‐releasing hormone (CRH) is an important neuro‐endocrine mediator of the stress response. Local effects of CRH in the intestinal mucosa have become evident in recent years. We showed that CRH activates CRH receptor subtypes R1 and R2 on subepithelial mast cells, thereby inducing increased transcellular uptake of protein antigens in human colonic biopsies in Ussing chambers. Ongoing studies also implicate local cholinergic signaling in regulation of macromolecular permeability in the human colon. Since increased uptake of antigenic molecules is associated with mucosal inflammation, our findings may have implications for understanding stress‐related intestinal disorders.

Dynamics of mucosal permeability and inflammation in collagenous colitis before, during, and after loop ileostomy

Collagenous colitis has become a more frequent diagnosis but the aetiology of this disease is still unknown. We describe a female patient with intractable collagenous colitis who was treated with a temporary loop ileostomy. She was followed clinically, histopathologically, and functionally by measuring mucosal permeability before surgery, after ileostomy, and after bowel reconstruction. In our case report, active collagenous colitis was combined with increased transcellular and paracellular mucosal permeability. Diversion of the faecal stream decreased inflammation of the mucosa and normalised epithelial degeneration and mucosal permeability. After restoration of bowel continuity, mucosal permeability was altered prior to the appearance of a collagenous layer.

Eosinophils Express Muscarinic Receptors and Corticotropin-Releasing Factor to Disrupt the Mucosal Barrier in Ulcerative Colitis

BACKGROUND & AIMS Altered intestinal barrier function has been implicated in the pathophysiology of ulcerative colitis (UC) in genetic, functional, and epidemiological studies. Mast cells and corticotropin-releasing factor (CRF) regulate the mucosal barrier in human colon. Because eosinophils are often increased in colon tissues of patients with UC, we assessed interactions among mast cells, CRF, and eosinophils in the mucosal barrier of these patients. METHODS Transmucosal fluxes of protein antigens (horseradish peroxidase) and paracellular markers ((51)Cr-EDTA, fluorescein isothiocyanate-dextran 4000) were studied in noninflamed, colonic mucosal biopsy samples collected from 26 patients with UC and 53 healthy volunteers (controls); samples were mounted in Ussing chambers. We also performed fluorescence and electron microscopy of human tissue samples, assessed isolated eosinophils, and performed mechanistic studies using in vitro cocultured eosinophils (15HL-60), mast cells (HMC-1), and a colonic epithelial cell line (T84). RESULTS Colon tissues from patients with UC had significant increases in permeability to protein antigens compared with controls. Permeability was blocked by atropine (a muscarinic receptor antagonist), α-helical CRF(9-41) (a CRF receptor antagonist), and lodoxamide (a mast-cell stabilizer). Eosinophils were increased in number in UC tissues (compared with controls), expressed the most M2 and M3 muscarinic receptors of any mucosal cell type, and had immunoreactivity to CRF. In coculture studies, carbachol activation of eosinophils caused production of CRF and activation of mast cells, which increased permeability of T84 epithelial cells to macromolecules. CONCLUSIONS We identified a neuroimmune intercellular circuit (from cholinergic nerves, via eosinophils to mast cells) that mediates colonic mucosal barrier dysfunction in patients with UC. This circuit might exacerbate mucosal inflammation.