Microbiota and mucosal gene expression of fecal microbiota transplantation or placebo treated patients with chronic pouchitis.

Altered microbiota and impaired host immune function have been linked to the pathogenesis of pouchitis. We used 16S rRNA gene sequencing and RNA sequencing data from a previous randomized clinical trial (RCT) on fecal microbiota transplantation (FMT) therapy in 26 chronic pouchitis patients with one-year follow-up. We analyzed changes in both luminal and mucosal microbiota composition, as well as in host mucosal gene expression to gain insights into the host-microbiota interactions possibly underlying clinical outcomes of the patients. Antibiotic type and pattern of use were significant drivers of the luminal microbiota at baseline. Differential gene expression analysis indicated transition from ileal to colonic gene expression in the pouch, and upregulation in inflammation- and immune system-related pathways in the pouch. At 4 weeks, the non-relapsed FMT patients had a lower microbiota dissimilarity to the donor than the non-relapsed placebo patients (p = .02). While two FMT-treated patients showed a shift toward the donor's microbiota during the one-year follow-up, the overall FMT microbiota modulation effect was low. Patient's luminal and mucosal microbiota profiles were unstable in both FMT and placebo groups. Expression of the chemokine receptor CXCR4 was downregulated at 52 weeks compared to the baseline in the non-relapsed patients in both FMT and placebo groups. Microbiota modulation by FMT seems to be low in this patient group. The microbiota composition or alterations did not explain the relapse status of the patients. Some evidence for remission-related host gene expression pattern was found; specifically, CXCR4 expression may have a role in sustained remission.

Expression of MAdCAM-1 and Gut-homing T Cells in Inflamed Pouch Mucosa.

BACKGROUND AND AIMS: Pouchitis is a common complication following formation of an ileal pouch-anal anastomosis [IPAA] after proctocolectomy for ulcerative colitis [UC]. Gut-specific lymphocyte trafficking mechanisms have been identified as players in the pathogenesis of UC. In the present study, we aimed to characterise the presence of lymphocyte subsets expressing gut-homing molecules in pouches and peripheral blood of UC patients with and without pouchitis. METHODS: Biopsy samples and peripheral blood were collected from 29 patients with an IPAA [seven with active inflammation, 22 without inflammation]. Expression of adhesion molecule MAdCAM-1 was assessed using immunohistochemistry, and flow cytometry was used to characterise expression of integrin α4ß7, C-chemokine receptor 9 [CCR9], and CD103 on T cell subsets. RESULTS: MAdCAM-1 expression was significantly increased in case of active inflammation in the pouch. T cells expressing integrin α4ß7 were abundant in the pouch mucosa, but the frequency of integrin α4ß7-expressing T cells was decreased on CD4+ lymphocytes during inflammation. Co-expression of gut-homing markers CCR9 and α4ß7 was more pronounced in biopsies compared with peripheral blood, but was not enhanced upon active inflammation. CONCLUSIONS: Gut-homing T cells are abundant in pouch mucosa, but the classic hypothesis that the chronic inflammatory state is maintained by an accumulation of α4ß7-expressing effector T cells is not supported by our data.

Faecal Proteases from Pouchitis Patients Activate Protease Activating Receptor-2 to Disrupt the Epithelial Barrier.

BACKGROUND AND AIMS: The pathogenesis of pouch inflammation may involve epithelial barrier disruption. We investigated whether faecal proteolytic activity is increased during pouchitis and results in epithelial barrier dysfunction through protease activating receptor [PAR] activation, and assessed whether the intestinal microbiome may be the source of the proteases. METHODS: Faecal samples were measured for protease activity using a fluorescein isothiocyanate [FITC]-casein florescence assay. Caco-2 cell monolayers were exposed to faecal supernatants to assess permeability to FITC-dextran. Tight junction protein integrity and PAR activation were assessed by immunoblot and immunofluorescence. A truncated PAR2 protein in Caco-2 cells was achieved by stable transfection using CRISPR/Cas9 plasmid. PAR2 activation in pouch biopsies was examined using antibodies directed to the N-terminus of the protein. Microbial composition was analysed based on 16S rRNA gene sequence analysis. RESULTS: Ten pouchitis patients, six normal pouch [NP] patients and nine healthy controls [HC] were recruited. The pouchitis patients exhibited a 5.19- and 5.35-fold higher faecal protease [FP] activity [p ≤ 0.05] compared to the NP and HC participants, respectively. The genus Haemophilus was positively associated with FP activity [R = 0.718, false discovery rate < 0.1]. Faecal supernatants from pouchitis patients activated PAR2 on Caco-2 monolayers, disrupted tight junction proteins and increased epithelial permeability. PAR2 truncation in Caco-2 abrogated faecal protease-mediated permeability. Pouch biopsies obtained from pouchitis patients, but not from NP patients, displayed PAR2 activation. CONCLUSIONS: Protease-producing bacteria may increase faecal proteolytic activity that results in pouch inflammation through disruption of tight junction proteins and increased epithelial permeability in a PAR2-dependent manner. This mechanism may initiate or propagate pouch inflammation.

Increase in chromogranin A- and serotonin-positive cells in pouch mucosa of patients with ulcerative colitis undergoing proctocolectomy.

BACKGROUND: Inflammatory bowel disease (IBD) is associated with neuroendocrine cell hyperplasia. AIMS: We investigated neuroendocrine cells in J-pouches of patients with ulcerative colitis undergoing restorative proctocolectomy and ileal pouch-anal anastomosis. METHODS: Sections from pouch biopsies of 17 patients and ileal biopsies of 17 active IBD patients and 16 controls were processed by immunohistochemistry for chromogranin A (CgA) and serotonin. Mucosal tryptophan hydroxylase (TpH)-1 and serotonin-selective reuptake transporter (SERT) transcripts were measured by quantitative RT-PCR. TpH-1 and SERT transcripts were detected in pouch biopsies cultured with infliximab or its isotype control, while interleukin (IL)-6 and IL-8 were measured in biopsy supernatants. RESULTS: A significant increase in CgA-positive cells and serotonin-positive cells was observed in both pouch and IBD ileum compared to control ileum. Significantly raised transcripts of TpH-1, but not SERT, were found in IBD ileum in comparison to control ileum, with no significant difference between pouch and IBD ileum. Infliximab had no influence on ex vivo pouch expression of TpH-1 and SERT, nor on the production of IL-6 and IL-8. CONCLUSION: We here demonstrated neuroendocrine cell hyperplasia in pouch mucosa. Further studies are needed to clarify the pathophysiological implication of this finding.

Increase in Processing Factors Is Involved in Skewed MicroRNA Expression in Patients with Ulcerative Colitis Who Develop Small Intestine Inflammation after Pouch Surgery.

Background: A large-scale increase in microRNA (miRNA) expression was observed in patients with ulcerative colitis who underwent pouch surgery and developed inflammation of the pouch (pouchitis). In this study, we assessed miRNA expression in these patients and investigated how regulation of its expression changes in the setting of pouchitis. Methods: Autologous samples that included mucosal biopsies, peripheral blood cells, and plasma were collected from the patients. Candidate primary and mature miRNA expressions were analyzed by quantitative polymerase chain reaction. A human intestinal epithelial cell line was used to test DICER activity, and the expression of key miRNA processing factors was analyzed by Western blot. miRNA-424 and its potential target serotonin reuptake transporter (SERT) expressions were examined by quantitative reverse transcription polymerase chain reaction and Western blot in human pouch tissues and in a human intestinal epithelial cell line stimulated with inflammatory cytokines TNF-α, IL-1ß, and INF-γ. Results: Candidate miRNA expression and protein expression of DICER-1, EXPORTIN-5, and AGO-2 were increased in association with pouch inflammation. Similarly, inflammatory cytokines increased protein expression of DICER-1, EXPORTIN-5, and AGO-2 and DICER activity in the epithelial cell line. The miRNA-424 expression increased whereas SERT expression decreased in the patients' mucosa. Similarly, incubation of the epithelial cell line with inflammatory cytokines resulted in increased miRNA-424 and decreased SERT mRNA and protein expression. Conclusions: The miRNA expression and processing are augmented in the inflamed intestinal mucosa of patients with pouchitis. These alterations are accompanied by increased expression of proteins involved in miRNA processing, suggesting that pouch inflammation contributes to miRNA processing and expression.

Ileal pouch of ulcerative colitis and familial adenomatous polyposis patients exhibit modulation of autophagy markers.

Total retocolectomy with ileal pouch-anal anastomosis (IPAA) is the surgery of choice for patients with ulcerative colitis (UC) that are refractory to clinical treatment. Pouchitis is one of the most common complications after this procedure. Defects in autophagy have been reported in inflammatory bowel diseases. However, there are no studies on the IP. Therefore, we studied markers for autophagy in the IP mucosa of UC and FAP patients comparing them to controls with a normal distal ileum. Sixteen patients with IP in "J" shape, asymptomatic and with endoscopically normal IP were evaluated. The control group consisted of eight patients with normal colonoscopy. There was a significant decrease in the transcriptional levels of ATG5, MAP1LC3A and BAX in the FAP group. There was also a decrease in the protein level of Beclin-1 in the UC and FAP compared to the control group. Although the LC3II levels by immunoblot were higher in the UC group, LC3/p62 co-localization were lower in the immunofluorescence analysis in the UC and FAP compared to the control group. Corroborating these results, there was an increase of p62 by immunoblot in the UC group. These findings indicated a modulation of macroautophagy markers in the IP, which may explain the mucosa inflammation predisposition.

Lactobacillus acidophilus alleviates pouchitis after ileal pouch-anal anastomosis in rats.

AIM: To assess the therapeutic potential of Lactobacillus acidophilus (LA) for the treatment of pouchitis in a rat model. METHODS: Sprague Dawley rats underwent proctocolectomy and ileal pouch-anal anastomosis followed by administration of dextran sulfate sodium (DSS) to induce pouchitis. Rats with pouchitis were randomly divided into three groups: no intervention (NI), normal saline (NS, 3 mL/d normal saline for 7 d), and LA (3 mL/d LA at 1× 1010 colony-forming units for 7 d). General body condition was recorded and pouch specimens were obtained for histological examination. mRNA expression levels of interleukin (IL)-1ß, IL-6, IL-10, and tumor necrosis factor-α were determined by RT-PCR. Zonula occludens protein 1 (ZO-1) levels were measured by immunohistochemistry. RESULTS: LA reduced weight loss associated with pouchitis (P < 0.05) and improved the symptoms of pouchitis in rats. Compared with the NI and NS groups, rats in the LA group showed earlier disappearance of hematochezia (6.17 ± 0.75, 6.50 ± 0.55, 3.17 ± 0.75, P < 0.05) and higher fecal scores (2.67 ± 0.48, 2.50 ± 0.51, 4.42 ± 0.50, respectively, P < 0.05). Histological scores were also lower in the LA group compared with the other two groups (7.17 ± 0.98, 8.00 ± 0.89, 4.00 ± 0.89, respectively, P < 0.05). mRNA expression levels of IL-1ß, IL-6, and tumor necrosis factor-α were significantly reduced, while IL-10 mRNA levels were significantly increased in the LA group (P < 0.05, respectively). ZO-1 protein levels were also significantly increased after administration of LA (P < 0.05). CONCLUSION: LA alleviates pouchitis induced by DSS after ileal pouch-anal anastomosis by decreasing pro-inflammatory factors and increasing anti-inflammatory factors, and restoring ZO-1 expression in the mucosa.

Altered expression of microRNAs in patients with pouchitis after restorative proctocolectomy.

BACKGROUND AND PURPOSE: Pouchitis is the most common long-term complication of restorative proctocolectomy with ileal pouch-anal anastomosis. We investigated alterations in the expression of microRNAs, noncoding RNAs that act as potent negative regulators of gene expression, in pouchitis. METHODS: The subjects of this study were 16 patients with diagnosed pouchitis and 48 patients without pouchitis after restorative proctocolectomy, performed for ulcerative colitis. Total RNA was extracted from biopsies and microRNAs were quantified using a real-time polymerase chain reaction. RESULTS: The expression of microRNA 21 and 223 was higher, whereas that of microRNA 192 and 196a was lower, in the inflamed mucosa from the pouchitis patients than in the mucosa from the non-pouchitis patients. The levels of 14 microRNAs were significantly lower in the mucosa from the pouchitis patients, than in the non-inflamed proximal ileal mucosal samples. The expression of microRNA 192 was remarkably reduced in pouchitis. A significant negative correlation was found between microRNA 192 and interleukin 17 receptor A mRNA levels. CONCLUSIONS: Significant alteration in miRNA expression in line with inflammatory bowel disease was evident in the mucosa from the pouchitis patients. Interleukin 17 receptor A may be involved in the pathogenesis of pouchitis through the downregulation of microRNA 192.

Roles for Intestinal Bacteria, Viruses, and Fungi in Pathogenesis of Inflammatory Bowel Diseases and Therapeutic Approaches.

Intestinal microbiota are involved in the pathogenesis of Crohn's disease, ulcerative colitis, and pouchitis. We review the mechanisms by which these gut bacteria, fungi, and viruses mediate mucosal homeostasis via their composite genes (metagenome) and metabolic products (metabolome). We explain how alterations to their profiles and functions under conditions of dysbiosis contribute to inflammation and effector immune responses that mediate inflammatory bowel diseases (IBD) in humans and enterocolitis in mice. It could be possible to engineer the intestinal environment by modifying the microbiota community structure or function to treat patients with IBD-either with individual agents, via dietary management, or as adjuncts to immunosuppressive drugs. We summarize the latest information on therapeutic use of fecal microbial transplantation and propose improved strategies to selectively normalize the dysbiotic microbiome in personalized approaches to treatment.

Tight junctions in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer.

Inflammatory bowel diseases are characterised by inflammation that compromises the integrity of the epithelial barrier. The intestinal epithelium is not only a static barrier but has evolved complex mechanisms to control and regulate bacterial interactions with the mucosal surface. Apical tight junction proteins are critical in the maintenance of epithelial barrier function and control of paracellular permeability. The characterisation of alterations in tight junction proteins as key players in epithelial barrier function in inflammatory bowel diseases is rapidly enhancing our understanding of critical mechanisms in disease pathogenesis as well as novel therapeutic opportunities. Here we give an overview of recent literature focusing on the role of tight junction proteins, in particular claudins, in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer.

Endoluminal calprotectin measurement in assessment of pouchitis and a new index of disease activity: a pilot study.

Pouchitis is the most common complication following proctocolectomy with ileal pouch-anal anastomosis for ulcerative colitis (UC). To provide a standardized definition of pouchitis clinical, endoscopic and histological markers were grouped and weighted in the pouch disease activity index (PDAI). However, the delay in the assessment of the final score due to the time requested for histological analysis remains the main obstacle to the index implementation in clinical practice so that the use of modified-PDAI (mPDAI) with exclusion of histologic subscore has been proposed. We tested the ability of calprotectin measurement in the pouch endoluminal content to mimic the histologic score as defined in the PDAI, the index that we adopted as gold standard for pouchitis diagnosis. Calprotectin was measured by ELISA in the pouch endoluminal content collected during endoscopy in 40 consecutive patients with J-pouch. In each patient PDAI and mPDAI were calculated and 15% of patients were erroneously classified by mPDAI. ROC analysis of calprotectin values vs. acute histological subscore ≥ 3 identified different calprotectin cut-off values with corresponding sensitivity and specificity allowing the definition and scoring of different range of calprotectin subscores. We incorporated the calprotectin score in the mPDAI obtaining a new score that shows the same specificity as PDAI for diagnosis of pouchitis and higher sensitivity when compared with mPDAI. The use of the proposed new score, once validated in a larger series of patients, might be useful in the early management of patients with symptoms of pouchitis.

Variable alterations of the microbiota, without metabolic or immunological change, following faecal microbiota transplantation in patients with chronic pouchitis.

Faecal microbiota transplantation (FMT) is effective in the treatment of Clostridium difficile infection, where efficacy correlates with changes in microbiota diversity and composition. The effects of FMT on recipient microbiota in inflammatory bowel diseases (IBD) remain unclear. We assessed the effects of FMT on microbiota composition and function, mucosal immune response, and clinical outcome in patients with chronic pouchitis. Eight patients with chronic pouchitis (current PDAI ≥7) were treated with FMT via nasogastric administration. Clinical activity was assessed before and four weeks following FMT. Faecal coliform antibiotic sensitivities were analysed, and changes in pouch faecal and mucosal microbiota assessed by 16S rRNA gene pyrosequencing and (1)H NMR spectroscopy. Lamina propria dendritic cell phenotype and cytokine profiles were assessed by flow cytometric analysis and multiplex assay. Following FMT, there were variable shifts in faecal and mucosal microbiota composition and, in some patients, changes in proportional abundance of species suggestive of a "healthier" pouch microbiota. However, there were no significant FMT-induced metabolic or immunological changes, or beneficial clinical response. Given the lack of clinical response following FMT via a single nasogastric administration our results suggest that FMT/bacteriotherapy for pouchitis patients requires further optimisation.

Consecutive Monitoring of Fecal Calprotectin and Lactoferrin for the Early Diagnosis and Prediction of Pouchitis after Restorative Proctocolectomy for Ulcerative Colitis.

OBJECTIVES: This prospective study was conducted to evaluate the significance of consecutive monitoring of fecal calprotectin and lactoferrin for the early diagnosis and prediction of pouchitis after restorative proctocolectomy for ulcerative colitis (UC). METHODS: Sixty patients who had ileostomy closure following total proctocolectomy and ileal pouch-anal anastomosis for UC were included. Stool samples were collected for the measurement of calprotectin and lactoferrin every 2 months up to 12 months after the ileostomy closure. When patients had symptoms suggestive of pouchitis, endoscopic examination was immediately undertaken. All asymptomatic patients underwent endoscopy at 12 months. Pouchitis was defined as a pouchitis disease activity index score of ≥7. RESULTS: During the 12 months, 10 patients (17%) developed pouchitis. In patients with pouchitis, fecal calprotectin and lactoferrin levels were elevated already 2 months before the diagnosis of pouchitis. In contrast, these fecal biomarkers remained at low levels, and they did not change significantly in patients without pouchitis. A cutoff value of 56 µg/g for calprotectin had a sensitivity of 100% and a specificity of 84% to predict pouchitis, whereas a cutoff value of 50 µg/g for lactoferrin had a sensitivity of 90% and a specificity of 86%. At the time of endoscopy, the median calprotectin and lactoferrin levels were significantly higher in patients with pouchitis than those without pouchitis. CONCLUSIONS: Elevated fecal calprotectin and lactoferrin levels appeared to be significant predictors of pouchitis after restorative proctocolectomy for UC. Consecutive monitoring of these fecal biomarkers is useful for the early diagnosis of pouchitis.

FK506-Binding Protein 5 mRNA Levels in Ileal Mucosa Are Associated with Pouchitis in Patients with Ulcerative Colitis.

BACKGROUND: Although the pathogenesis of pouchitis is incompletely understood, steroid and FK506 therapy are significantly associated with pouchitis. These medical treatments are regulated by the FK506-binding protein (FKBP) 4 and FKBP5 genes. AIM: This study aimed to evaluate the relationship between pouchitis and FKBP4 and FKBP5 mRNA expression in ileal mucosa at the time of colectomy. METHODS: Ileal mucosa specimens were collected from 71 patients who underwent ileal pouch-anal anastomosis for ulcerative colitis. FKBP4 and FKBP5 mRNA expression was evaluated. The relationship between mRNA expression and clinicopathological factors, including developed pouchitis, was investigated. RESULTS: Of these 71 patients, 25 (35.2 %) patients developed pouchitis in a mean duration of 20.2 months (range 0-68 months). FKBP4 mRNA levels in patients who received an immunomodulator were significantly higher than those in untreated patients (0.167 ± 0.060 vs 0.131 ± 0.065, p = 0.009). However, FKBP5 mRNA levels in patients who received a three-stage operation were significantly lower than those in the other patients (1.97 ± 1.15 vs 2.70 ± 1.12, p = 0.02). A total dose of prednisolone >9.4 g (HR 2.84, p = 0.02) before colectomy and FKBP5 mRNA level higher than the median (HR 4.49, p = 0.01) were identified as factors related to pouchitis. CONCLUSIONS: FKBP5 mRNA levels in ileal mucosa at the time of colectomy are significantly associated with pouchitis and may be a predictive factor for developing pouchitis.

Faecal metabolite profiling identifies medium-chain fatty acids as discriminating compounds in IBD.

BACKGROUND: Bacteria play a role in the onset and perpetuation of intestinal inflammation in IBD. Compositional alterations may also change the metabolic capacities of the gut bacteria. OBJECTIVE: To examine the metabolic activity of the microbiota of patients with Crohn's disease (CD), UC or pouchitis compared with healthy controls (HC) and determine whether eventual differences might be related to the pathogenesis of the disease. METHODS: Faecal samples were obtained from 40 HC, 83 patients with CD, 68 with UC and 13 with pouchitis. Disease activity was assessed in CD using the Harvey-Bradshaw Index, in UC using the UC Disease Activity Index and in pouchitis using the Pouchitis Disease Activity Index. Metabolite profiles were analysed using gas chromatography-mass spectrometry. RESULTS: The number of metabolites identified in HC (54) was significantly higher than in patients with CD (44, p<0.001), UC (47, p=0.042) and pouchitis (43, p=0.036). Multivariate discriminant analysis predicted HC, CD, UC and pouchitis group membership with high sensitivity and specificity. The levels of medium-chain fatty acids (MCFAs: pentanoate, hexanoate, heptanoate, octanoate and nonanoate), and of some protein fermentation metabolites, were significantly decreased in patients with CD, UC and pouchitis. Hexanoate levels were inversely correlated to disease activity in CD (correlation coefficient=-0.157, p=0.046), whereas a significant positive correlation was found between styrene levels and disease activity in UC (correlation coefficient=0.338, p=0.001). CONCLUSIONS: Faecal metabolic profiling in patients with IBD relative to healthy controls identified MCFAs as important metabolic biomarkers of disease-related changes. TRIAL REGISTRATION NO: NCT 01666717.

Gene expression alterations in ulcerative colitis patients after restorative proctocolectomy extend to the small bowel proximal to the pouch.

OBJECTIVES: To evaluate molecular profiles in the small bowel (SB) mucosa proximal to the pouch in ulcerative colitis (UC) patients after pouch surgery. DESIGN: Patients were prospectively recruited and stratified according to disease behaviour: normal pouch (NP), chronic pouchitis (CP), and Crohn's-like disease of the pouch (CLDP). Biopsies obtained from the pouch and the normal-appearing proximal SB (40 cm proximal to the anal verge) were compared to ileal biopsies from normal controls (NC). A histopathological score based on the degree of polymorphonuclear and mononuclear infiltrates was used to assess inflammation in the pouch and the proximal SB. Gene expression analysis was performed using microarrays, and validated by real-time PCR. Gene ontology and clustering were evaluated by bioinformatics. RESULTS: Thirty-six subjects were recruited (age 18-71 years, 16 males). Histopathology scores demonstrated minimal differences in the normal-appearing proximal SB of all groups. Nonetheless, significant (fold change ≥2, corrected p [FDR] ≤ 0.05) molecular alterations in the proximal SB were detected in all groups (NP n=9; CP n=80; and CLDP n=230) compared with NC. The magnitude of DUOX2 alteration in the proximal SB was highest. An increase of 6.0, 9.8 and 21.7 folds in DUOX2 expression in NP, CP, CLDP, respectively was observed. This was followed by alterations in MMP1, SLC6A14 and PGC. Gene alterations in the proximal SB overlapped with alterations within the pouch (76% and 97% overlap in CP and CLDP, respectively). Gene ontology analysis in the proximal SB and pouch were comparable. CONCLUSIONS: Significant gene expression alterations exist in an apparently unaffected proximal SB. Alterations in the pouch and the proximal SB were comparable, suggesting that inflammation may not be limited to the pouch, but that it extends to the proximal SB.

Innate immune factors in the development and maintenance of pouchitis.

BACKGROUND: Tight junction proteins (TJPs) and dendritic cells (DC) are critical in the pathogenesis of inflammatory bowel diseases. The ileal pouch formed by restorative proctocolectomy provides a unique human model for studying the pathogenesis of inflammatory bowel diseases. Data implicate the microbiota in the pathogenesis of pouchitis, while the role of innate immune factors remains unclear. We performed longitudinal and cross-sectional studies of patients after restorative proctocolectomy and assessed TJP and DC characteristics in the ileal pouch. METHODS: Mucosal biopsies were taken from the ileal pouch of patients with ulcerative colitis (UC) and familial adenomatous polyposis (n = 8). Of patients with UC, one group (n = 5) was followed longitudinally over the first year after ileostomy closure, another group had pouchitis (n = 15), and another group no inflammation (n = 18). Dendritic cell phenotype and epithelial cell TJP expression were assessed using flow cytometric analysis. RESULTS: Increased epithelial expression of the "pore-forming" TJP claudin 2, and DC expression of gut-homing markers CCR 9 and integrin ß7, occurred early after ileostomy closure. In patients with UC with pouchitis, epithelial expression of ZO-1 and claudin 1 were reduced, DC were activated with increased CD40, and Toll-like receptor 4 expression increased. In pouchitis, DC expressing CCR 9 were decreased, whereas DC expressing ß7 increased. CONCLUSIONS: Abnormalities were found in TJP expression in the pouch of patients with UC, in particular, increased expression of the pore-forming claudin 2 as an early event in the development of pouch inflammation and an aberrant DC phenotype was characterized in the ileal pouch of patients with UC.

Relationship between pouch microbiota and pouchitis following restorative proctocolectomy for ulcerative colitis.

Restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) has become the surgical treatment of choice for many patients with medically refractory ulcerative colitis (UC) and familial adenomatous polyposis (FAP). UC patients with IPAA (UC-IPAA) are, nevertheless, susceptible to inflammatory and noninflammatory sequelae such as pouchitis, which is only rarely noted in FAP patients with IPAA. Pouchitis is the most frequent long-term complication of UC-IPAA patients, with a cumulative prevalence of up to 50%. Although the aetiology of pouchitis remains unclear, accumulating evidence suggests that a dysbiosis of the pouch microbiota and an abnormal mucosal immune response are implicated in its pathogenesis. Studies using culture and molecular techniques have detected a dysbiosis of the pouch microbiota in patients with pouchitis. Risk factors, genetic associations, and serological markers suggest that interactions between the host immune response and the pouch microbiota underlie the aetiology of this idiopathic inflammatory condition. This systematic review focuses on the dysbiosis of the microbiota that inhabit the pouch in UC and FAP patients and its interaction with the mucosal immune system. A meta-analysis was not attempted due to the highly heterogeneous microbiota composition and the different detection methods used by the various studies. Although no specific bacterial species, genus, or family has as yet been identified as pathogenic, there is evidence that a dysbiosis characterized by decreased gut microbiota diversity in UC-IPAA patients may, in genetically predisposed subjects, lead to aberrant mucosal immune regulation triggering an inflammatory process.

Trace elements and rat pouchitis.

The procedure of restorative proctocolectomy is associated with a complete removal of the colon and slight reduction of ileum length, which together can lead to systemic shortages of trace elements. Inflammatory changes in the pouch mucosa may also have some impact. However, there is no data on trace elements in pouchitis. Therefore, in the present study we aimed to assess the effect of acute pouchitis on the status of selected trace elements in rats. Restorative proctocolectomy with the construction of intestinal J-pouch was performed in twenty-four Wistar rats. Three weeks after the surgery, pouchitis was induced. Eight untreated rats created the control group. Liver concentrations of selected micronutrients (Zn, Cu, Co, Mn, Se) were measured in both groups six weeks later, using inductively coupled plasma mass spectrometry. Liver concentrations of trace elements did not differ between the study and the control groups. However, copper, cobalt and selenium concentrations [µg/g] were statistically lower (p<0.02, p<0.05 and p<0.04, respectively) in rats with severe pouchitis (n=9) as compared with rats with mild pouchitis (n=7) [median (range): Cu--7.05 (3.02-14.57) vs 10.47 (5.16-14.97); Co--0.55 (0.37-0.96) vs 0.61 (0.52-0.86); Se--1.17 (0.69-1.54) vs 1.18 (0.29-1.91)]. In conclusion, it seems that acute pouchitis can lead to a significant deficiency of trace elements.

Ki-67 and p53 expression in ileal pouches.

Development of dysplastic lesions that may potentially lead to cancer is sometimes reported within the ileal pouches. Dysplasia is in turn associated with increased expression of proliferation indices. The goal of this study was to evaluate the mitotic activity and possible expression of p53 in the epithelium within the ileal pouches in patients with chronic ulcerative colitis. The study involved archive material consisting of ileal pouches surgically removed from 17 patients diagnosed with ulcerative colitis. Several specimens were collected from each pouch. The immunohistochemistry (Ki-67 and p53 protein) control group (14 cases) consisted of the resection line specimens of colons removed due to colorectal adenocarcinoma. Intensity of the expression of the markers under study within the inflammatory infiltrates was assessed using a 5-point scale proposed by Berstein et al. Ki-67 expression was observed in all studied patients with marked intensity (Bernstein scale score +3, +4). Protein p53 expression was observed only in eight patients, and was mostly of low intensity (Bernstein scale score +1, +2). Immunohistochemical results confirmed the histopathological results that revealed dysplastic lesions, which are often an indication for radical procedures in ulcerative colitis patients. Our results suggest the usefulness of these examinations, also in the ileal pouch material.