P. Ronan O'Connell

Fibrogenesis in Crohn's Disease

INTRODUCTION:Over one-third of patients with Crohns disease (CD) will develop an intestinal stricture and the great majority of these will require at least one surgical procedure. While the pathogenesis of inflammation in CD has been extensively investigated, knowledge of stricture pathogenesis remains limited. The aim of this review is to discuss the current understanding of fibrogenesis in CD and to outline potential directions in research and therapeutics.METHODS:The electronic literature (January 1966 to May 2006) on CD-associated fibrosis was reviewed. Further references were obtained by cross-referencing from key articles.RESULTS:CD-associated fibrosis results from chronic transmural inflammation and a complex interplay among intestinal mesenchymal cells, cytokines, and local inflammatory cells. The fibroblast is the key cell type mediating stricture formation. The cytoarchitecure of the bowel wall is altered with disruption of the muscularis mucosa, thickening of the muscularis propria, and deposition of collagen throughout. The cytokine TGF-β appears critical in this process, acting to increase growth factor and extracellular matrix (ECM) production and dysregulate ECM turnover. Potential therapeutic interventions are likely to concentrate on modulating down-stream targets of TGF-β.CONCLUSIONS:Greater understanding of the biology of fibrostenosis is likely to yield significant advances in our ability to care for patients with stricturing CD. Potential dividends of this approach include identification of novel therapeutic targets and biomarkers useful for prognostication and therapeutic monitoring.

Desulfovibrio Bacterial Species Are Increased in Ulcerative Colitis

BACKGROUND: Debate persists regarding the role of Desulfovibrio subspecies in ulcerative colitis. Combined microscopic and molecular techniques enable this issue to be investigated by allowing precise enumeration of specific bacterial species within the colonic mucous gel. The aim of this study was to combine laser capture microdissection and quantitative polymerase chain reaction to determine Desulfovibrio copy number in crypt-associated mucous gel in health and in acute and chronic ulcerative colitis. METHODS: Colonic mucosal biopsies were harvested from healthy controls (n = 19) and patients with acute (n = 10) or chronic (n = 10) ulcerative colitis. Crypt-associated mucous gel was obtained by laser capture microdissection throughout the colon. Pan-bacterial 16S rRNA and Desulfovibrio copy number/mm2 were obtained by polymerase chain reaction at each locus. Bacterial copy numbers were interrogated for correlation with location and disease activity. Data were evaluated using a combination of ordinary linear methods and linear mixed-effects models to cater for multiple interactions. RESULTS: Desulfovibrio positivity was significantly increased in acute and chronic ulcerative colitis at multiple levels within the colon, and after normalization with total bacterial signal, the relative Desulfovibrio load was increased in acute colitis compared with controls. Desulfovibrio counts did not significantly correlate with age, disease duration, or disease activity but interlevel correlations were found in adjacent colonic segments in the healthy control and chronic ulcerative colitis groups. CONCLUSION: The presence of Desulfovibrio subspecies is increased in ulcerative colitis and the data presented suggest that these bacteria represent an increased percentage of the colonic microbiome in acute ulcerative colitis.

Randomised clinical trial to assess anal sphincter function following forceps or vacuum assisted vaginal delivery

Objective To compare, in a prospective, randomised controlled trial, differences in anal sphincter function following forceps or vacuum assisted vaginal delivery in an institution practising standardised management of labour.

Transforming Growth Factor-β Promotes Pro-fibrotic Behavior by Serosal Fibroblasts via PKC and ERK1/2 Mitogen Activated Protein Kinase Cell Signaling

Objective:To assess the role of fibroblasts, transforming growth factor (TGF)-β, and cell signal pathways in promoting fibrosis in Crohns disease (CD). Summary Background Data:Intestinal strictures are a major source of morbidity in CD. Fibroblasts found at sites of stricture promote fibrogenesis. The mechanisms underlying this pro-fibrotic behavior remain elusive. Methods:Fibroblasts were isolated from strictured and macroscopically normal serosa in patients with CD and from normal serosa in patients with colorectal cancer. Whole cell connective tissue growth factor (CTGF) and fibronectin expression were determined by Western blot analysis. Fibroblast type I collagen expression was evaluated by real-time PCR, while fibroblast contractile activity was measured using fibroblast populated collagen lattices. Cells were stimulated with TGF-β1 and inhibitors of the protein kinase C (PKC) and ERK 1/2 mitogen activated protein (MAP) kinase cell signaling pathways. Results:Stricture fibroblasts displayed enhanced constitutive expression of fibronectin. TGF-β promoted fibroblast CTGF, fibronectin, and type I collagen expression and enhanced fibroblast contractile activity. Inhibition of PKC reduced basal collagen expression and contractile activity in Crohns fibroblasts and attenuated the effect of TGF-β on fibroblast CTGF, fibronectin, and collagen I expression as well as fibroblast contractility. ERK 1/2 inhibition had a similar effect on TGF-β-induced CTGF and fibronectin expression. Conclusions:TGF-β is a critical pro-fibrotic growth factor in CD, and its effects are mediated via PKC and ERK 1/2 MAP kinase cell signaling. These pathways may represent novel therapeutic targets for patients with CD characterized by recurrent intestinal stricture formation.

Pathogenesis of and unifying hypothesis for idiopathic pouchitis.

Ileal pouch-anal anastomosis is the procedure of choice in the surgical management of refractory ulcerative colitis. Pouchitis affects up to 60% of patients following ileal pouch-anal anastomosis for ulcerative colitis. It overlaps significantly with ulcerative colitis such that improvements in our understanding of one will impact considerably on the other. The symptoms are distressing and impinge significantly on patients’ quality of life. Despite 30 years of scientific and clinical investigation, the pathogenesis of pouchitis is unknown; however, recent advances in molecular and cell biology make a synergistic hypothesis possible. This hypothesis links interaction between epithelial metaplasia, changes in luminal bacteria (in particular sulfate-reducing bacteria), and altered mucosal immunity. Specifically, colonic metaplasia supports colonization by sulfate-reducing bacteria that produce hydrogen sulfide. This causes mucosal depletion and subsequent inflammation. Although in most cases antibiotics lead to bacterial clearance and symptom resolution, immunogenetic subpopulations can develop a chronic refractory variant of pouchitis. The aims of this paper are to discuss proposed pathogenic mechanisms and to describe a novel mechanism that combines many hypotheses and explains several aspects of pouchitis. The implications for the management of both pouchitis and ulcerative colitis are discussed.

Patterns of abnormal pudendal nerve function that are associated with postpartum fecal incontinence

OBJECTIVE The purpose of this study was to assess patterns of abnormal pudendal nerve function in women who complain of postpartum fecal incontinence. STUDY DESIGN During a 12-month period, a cohort of 83 women underwent neurophysiologic assessment as part of an evaluation of fecal incontinence after vaginal delivery. Pudendal nerve assessment consisted of the measurement of the clitoral-anal reflex and quantitative electromyography of the external anal sphincter. Endoanal ultrasound examination and anal manometry were also performed in each patient. RESULTS Thirty of 83 women (38%) with fecal incontinence were found to have abnormal neurophysiologic condition, among whom four identifiable patterns of abnormality emerged. Five women (17%) had evidence of pudendal nerve demylenation with a prolonged sensory threshold of the clitoral-anal reflex (>5.2 mA), although electromyography studies were normal. Eight women (27%) had abnormal electromyography results that were consistent with axonal neuropathy with or without reinervation, in whom the clitoral-anal reflex was normal. Thirteen women (43%) demonstrated a mixed demyelinating and axonal pudendal neuropathy, with evidence of reinervation. Four women (13%) had abnormal patterns of neurophysiologic condition that was not attributable directly to past obstetric trauma but to coincident medical problems. CONCLUSION Four abnormal patterns of pudendal nerve function may be identified, three of which (demyelinating, axonal, and mixed demyelinating/axonal) can be attributed to specific past obstetric events, although a fourth radicular pattern is due to coincident medical or orthopedic problems. Assessment of pudendal nerve function is important in women with postpartum fecal incontinence because particular patterns of abnormality correlate with different symptoms and can influence treatment options.

Prognostic significance of tumor budding in rectal cancer biopsies before neoadjuvant therapy

Tumor budding is an increasingly important prognostic feature for pathologists to recognize. The aim of this study was to correlate intra-tumoral budding in pre-treatment rectal cancer biopsies with pathological response to neoadjuvant chemoradiotherapy and with long-term outcome. Data from a prospectively maintained database were acquired from patients with locally advanced rectal cancer who underwent neoadjuvant chemoradiotherapy. Pre-treatment rectal biopsies were retrospectively reviewed for evidence of intra-tumoral budding. Multivariate logistic regression was used to identify factors contributing to cancer-specific death, expressed as hazard ratios with 95% confidence intervals. Of the 185 patients with locally advanced rectal cancer, 89 patients met the eligibility criteria, of whom 18 (20%) exhibited budding in a pre-treatment tumor biopsy. Intra-tumoral budding predicted a poor pathological response to neoadjuvant chemoradiotherapy (higher ypT stage, P=0.032; lymph node involvement, P=0.018; lymphovascular invasion, P=0.004; and residual poorly differentiated tumors, P=0.005). No patient with intra-tumoral budding exhibited a tumor regression grade 1 or complete pathological response, providing a 100% specificity and positive predictive value for non-response to neoadjuvant chemoradiotherapy. Intra-tumoral budding was associated with a lower disease-free 5-year survival rate (33 vs 78%, P<0.001), cancer-specific 5-year survival rate (61 vs 87%, P=0.021) and predicted cancer-specific death (hazard ratio 3.51, 95% confidence interval 1.03–11.93, P=0.040). Intra-tumoral budding at diagnosis of rectal cancer identifies those who will poorly respond to neoadjuvant chemoradiotherapy and those with a poor prognosis.

Transcriptomic analysis of intestinal fibrosis‐associated gene expression in response to medical therapy in Crohn's disease

Background: Glucocorticoids and monoclonal antibodies to tumor necrosis factor reduce inflammation in Crohns disease (CD). Rapid luminal healing, however, may promote intestinal stricture formation. The aim of this study was to examine fibrosis‐associated gene expression in the intestine of patients with CD and correlate expression levels with prior medical therapies. Methods: In all, 37 patients with stricturing CD and 18 non‐CD controls underwent a transmural biopsy at the time of elective intestinal resection. Quantitative real‐time polymerase chain reaction (PCR) was conducted to determine differential mRNA expression of TGF‐&bgr;1, Smad‐7, CTGF, collagen‐1&agr;, fibronectin, BMP‐7, and MIF. Intestinal fibroblasts were treated in vitro with dexamethasone. Results: Relative to control, strictured CD intestinal tissue expressed increased TGF‐&bgr;1, CTGF, collagen‐1&agr;, and BMP‐7 (all P < 0.05). TGF‐&bgr;1 gene expression positively correlated with the expression of its downstream targets (all P < 0.001). Preoperative infliximab exposure was not associated with increased expression in any of the target genes nor did the number of infliximab infusions correlate with gene expression. The number of cycles of corticosteroid treatment preoperatively was positively associated with CTGF (r = 0.486, P = 0.016) and MIF (r = 0.524, P = 0.009) expression. Intestinal fibroblasts treated in vitro with dexamethasone upregulated CTGF expression (P = 0.023). Conclusions: Exposure to infliximab does not appear to induce a profibrotic transcriptional response in the CD intestine. Previous corticosteroid treatment is associated with increased expression of CTGF and MIF. Treating intestinal fibroblasts in vitro with steroids upregulates CTGF expression.

S100A4 expression is increased in stricture fibroblasts from patients with fibrostenosing Crohn's disease and promotes intestinal fibroblast migration

Fibroblasts represent the key cell type in fibrostenosing Crohns disease (FCD) pathogenesis. S100A4 is an EF-hand calcium-binding protein family member, implicated in epithelial-mesenchymal transition and as a marker of activated T lymphocytes and fibroblasts in chronic tissue remodeling. The aim of this study was to examine the expression profile of S100A4 in the resected ileum of patients with FCD. Mucosa, seromuscular explants, and transmural biopsies were harvested from diseased and proximal, macroscopically normal margins of ileocecal resections from patients with FCD. Samples were processed for histochemistry, immunohistochemistry, real-time RT-PCR, Western blotting, and transmission electron microscopy. Primary explant cultures of seromuscular fibroblasts were exposed to transforming growth factor (TGF)-beta1 (1 ng/ml), and S100A4 expression and scratch wound-healing activity were assessed at 24 h. CCD-18Co fibroblasts were transfected with S100A4 small interfering RNA, treated with TGF-beta1 (1 ng/ml) for 30 min or 24 h, and then assessed for S100A4 and Smad3 expression and scratch wound-healing activity. S100A4 expression was increased in stricture mucosa, in the lamina propria, and in CD3-positive intraepithelial CD3-positive T lymphocytes. Fibroblastic S100A4 staining was observed in seromuscular scar tissue. Stricture fibroblast explant culture showed significant upregulation of S100A4 expression. TGF-beta1 increased S100A4 expression in cultured ileal fibroblasts. In CCD-18Co fibroblasts, S100A4 small interfering RNA inhibited scratch wound healing and modestly inhibited Smad3 activation. S100A4 expression is increased in fibroblasts, as well as immune cells, in Crohns disease stricture and induced by TGF-beta1. Results from knockdown experiments indicate a potential role for S100A4 in mediating intestinal fibroblast migration.

N‐cadherin is overexpressed in Crohn's stricture fibroblasts and promotes intestinal fibroblast migration

Background: Intestinal fibroblasts mediate stricture formation in Crohns disease (CD). Transforming growth factor‐&bgr;1 (TGF‐&bgr;1) is important in fibroblast activation, while cell attachment and migration is regulated by the adhesion molecule N‐cadherin. The aim of this study was to investigate the expression and function of N‐cadherin in intestinal fibroblasts in patients with fibrostenosing CD. Methods: Intestinal fibroblasts were cultured from seromuscular biopsies from patients undergoing resection for terminal ileal fibrostenosing CD (n = 14) or controls patients (n = 8). N‐cadherin expression was assessed using Western blot and quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR). Fibroblasts were stimulated with TGF‐&bgr;1 and selective pathway inhibitors Y27632, PD98050, and LY294002 were used to examine the Rho/ROCK, ERK‐1/2, and Akt signaling pathways, respectively. Cell migration was assessed using a scratch wound assay. N‐cadherin was selectively overexpressed using a plasmid. Results: Fibroblasts from fibrostenosing CD express increased constitutive N‐cadherin mRNA and protein and exhibit enhanced basal cell migration relative to those from directly adjacent normal bowel. Control fibroblasts treated with TGF‐&bgr;1 induced N‐cadherin in a dose‐dependent manner which was inhibited by Rho/ROCK and Akt pathway modulation. Control fibroblasts exhibited enhanced cell migration in response to treatment with TGF‐&bgr;1 or transfection with an N‐cadherin plasmid. Conclusions: Fibroblasts from strictures in CD express increased constitutive N‐cadherin and exhibit enhanced basal cell migration. TGF‐&bgr;1 is a potent inducer of N‐cadherin in intestinal fibroblasts resulting in enhanced cell migration. The TGF‐&bgr;1‐mediated induction of N‐cadherin may potentiate Crohns stricture formation. (Inflamm Bowel Dis 2011;)