Pouch Biopsies Research Articles

Marginal Ulcer Incidence and the Population of Gastrin Producing G cells Retained in the Gastric Pouch after Roux-en-Y Gastric Bypass: Is There a Relationship?

Marginal ulcers are the most prevalent endoscopic abnormality after RYGB. The etiology is still poorly understood; however, an increase in acid secretion has been strongly implicated as a causal agent. Although gastrin is the greatest stimulant of acid secretion, to date, the presence of gastrin producing G cells retained in the gastric pouch, related to the occurrence of marginal ulcers, has not been evaluated. Evaluate the density of G cells and parietal cells in the gastric pouch of RYGB patients with a diagnosis of marginal ulcer on the post-op EGD. We retrospectively evaluated 1104 gastric bypasses performed between 2010 and 2020. Patients with marginal ulcer who met the inclusion criteria and controls were selected from this same population. Endoscopic gastric pouch biopsies were evaluated using immunohistochemical study and HE staining to assess G cell and parietal cell density. In total, 572 (51.8%) of the patients performed endoscopic follow-up after RYGB. The incidence of marginal ulcer was 23/572 (4%), and 3 patients required revision surgery due to a recalcitrant ulcer. The mean time for ulcer identification was 24.3months (2-62). G cell count per high-power field (× 400) was statistically higher in the ulcer group (p < 0.05). There was no statistical difference in parietal cell density between groups (p 0.251). Patients with a marginal ulcer after gastric bypass present a higher density of gastrin-producing G cells retained in the gastric pouch.

P205 Impact of histologic disease activity on long-term outcomes in patients with ileal pouch-anal anastomosis for ulcerative colitis

Abstract Background Total proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) can incur in several complications like pouchitis leading to the need of immunosuppressive therapy, hospitalization and pouch failure. Unlike non-operated UC patients, therapeutic goals are an unmet need in UC-IPAA patients and the impact of the histologic activity on the development of complications is unknown. The aim of the study was to assess the association between histology and the risk of long-term negative outcomes in UC subjects with IPAA. Methods This is a single-center, retrospective study which included UC adult patients with IPAA undergoing at least one pouchscopy with biopsies in the pouch body after ileostomy closure. Pouch biopsies were blindly reviewed by a gastrointestinal pathologist with expertise in IBD. Histologic disease remission was defined as a Pouchitis Disease Activity Index (PDAI) histology subscore £ 1. Negative outcomes were: IPAA related complications (acute or chronic pouchitis, hospitalization, pouch failure) and escalation of medical therapy (start of immunosuppressive or biologics). Both descriptive and inferential statistical analyses carried out Results A total of 66 patients were included (male n=37; 56,1%) of whom 40,9% (n=27) had histological activity at the baseline pouchscopy. The characteristics of the study population are represented in table 1. The median time of follow up was 38,5 months (range 12-151). During the study period, 27 patients (40,9%) developed acute pouchitis and 10 patients (15,2%) chronic pouchitis. Thirty-nine subjects (59,1%) required antibiotic therapy while 8 subjects required systemic steroids (12,1%). Furthermore, 15 patients (22,7%) started immunomodulatory therapy with either azathioprine or biologic agents. Six patients (9,1%) needed pouch-related hospitalization and 1 patients (1,5%) experienced pouch failure. Chronic pouchitis and the need of immunosuppressive/biology therapy was observed more frequently in patients with pouch histologic activity (29,6% vs. 5,1%, and 40,7% vs. 10,3%, respectively) (P&amp;lt;0.05). After adjusting for risk factors for pouchitis, the presence of histologic activity remained an independent risk factor for developing acute pouchitis (OR 4.10, 95% CI 1.39-12.04; P= 0.010), chronic pouchitis (OR 8.51, 95% CI 1.51-47.80, P=0.015) and the need for immunosuppressive or biologic therapy (OR 6.98, 95% CI 1.69-28.80; P=0.007). Conclusion Histologic activity was associated with an increased risk of developing both acute and chronic pouchitis and with the need for immunosuppressive/biologic therapy. Future research will clarify whether histologic remission might be considered as a therapeutic target for UC-IPAA patients developing pouchitis.

Nonspecific, Acute Pouchitis in Patients With Familial Adenomatous Polyposis: Less Common Than We Think.

Nonspecific acute pouchitis is common in patients with ulcerative colitis who undergo IPAA, but there is disagreement about the rate at which this occurs in patients with familial adenomatous polyposis. This study aimed to define the rate at which nonspecific acute pouchitis occurs in patients with familial adenomatous polyposis. This study was conducted at a hereditary colorectal cancer center in a large academic medical center. This is a retrospective cohort study using prospectively gathered data. Patients with familial adenomatous polyposis who have had IPAA were included. Symptoms, pouchoscopy and pouch radiography, and pouch biopsy were evaluated. The primary outcomes measured were the presence of nonspecific acute pouchitis and the alternative diagnosis of pouch dysfunction. There were 250 patients with familial adenomatous polyposis who had undergone IPAA; 38 (15.2%) had been labeled as having "pouchitis": 23 men and 15 women. Median age was 55 years (range, 18-90 years). No patient had nonspecific, acute pouchitis as defined by our criteria. In 9 cases, the diagnosis was derived from benign ulcers common in ileal pouches. Stool frequency was the most common symptom associated with the "pouchitis" label, but actual causes for the frequency included poor pouch emptying, poor eating habits, poor bowel habits, and afferent limb syndrome. Median stool frequency was 9, ranging from 4 to 15 per day. Every patient had at least 1 pouch endoscopy, and the median number was 19 per patient (range, 1-21). Nine patients had pouch biopsies to rule out inflammation; all showed chronic active enteritis, a normal finding in ileal pouches. This study was limited by its retrospective nature. We could not confirm any case of nonspecific acute pouchitis in patients with familial adenomatous polyposis. Those caring for patients with familial adenomatous polyposis who have a pouch should seek causes for pouch-related symptoms other than "pouchitis." See Video Abstract at http://links.lww.com/DCR/B666.POUCHITIS AGUDA INESPECÍFICA EN PACIENTES CON POLIPOSIS ADENOMATOSA FAMILIAR: MENOS COMÚN DE LO QUE PENSAMOSANTECEDENTES:La pouchitis aguda e inespecífica es común en pacientes con colitis ulcerativa, sometidos a anastomosis de bolsa ileoanal, pero no hay acuerdo sobre la frecuencia en que se presenta en pacientes con poliposis adenomatosa familiar (PAF).OBJETIVO:Definir la frecuencia en la que se presenta pouchitis aguda inespecífica en pacientes con poliposis adenomatosa familiar.ENTORNO CLINICO:Centro de cáncer colorrectal hereditario en un gran centro médico académico.DISEÑO:Estudio de cohorte retrospectivo utilizando datos recopilados prospectivamente.PACIENTES:Pacientes con poliposis adenomatosa familiar sometidos a bolsa con anastomosis ileoanal.INTERVENCIONES:Síntomas, proctoscopia, radiografía y biopsia de bolsa ileoanal.PRINCIPALES MEDIDAS DE VALORACION:Presencia de pouchitis aguda inespecífica; diagnósticos alternos de disfunción de la bolsa ileal.RESULTADOS:De 250 pacientes con poliposis adenomatosa familiar sometidos a una anastomosis anal de la bolsa ileal; 38 (15,2%), habían sido etiquetados como "pouchitis", 23 hombres y 15 mujeres. La mediana de edad fue de 55 años (rango de 18 a 90 años). Ningún paciente tuvo pouchitis aguda inespecífica según lo definido por nuestros criterios. En 9 casos el diagnóstico se derivó de úlceras benignas frecuentes en bolsas ileales. La frecuencia de las evacuaciones fue el síntoma más común asociado con la etiqueta "pouchitis", pero las causas reales de la frecuencia, incluyeron un mal vaciado de la bolsa, malos hábitos alimenticios, malos hábitos intestinales y síndrome de asa aferente. La frecuencia media de las evacuaciones fue de 9, con un rango de 4 a 15 por día. A cada paciente se le realizó al menos una endoscopia de la bolsa: la mediana fue de 19 por paciente (rango de 1 a 21). A 9 pacientes se les realizó biopsia de bolsa para descartar inflamación: todos mostraron enteritis activa crónica, un hallazgo normal en las bolsas ileales.LIMITACIONES:Naturaleza retrospectiva del estudio.CONCLUSIONES:No pudimos confirmar ningún caso de pouchitis aguda e inespecífica en pacientes con PAF. Aquellos que atienden a pacientes con PAF con bolsa ileal, deben buscar otras causas para los síntomas relacionados con la bolsa que no sean "pouchitis". Consulte Video Resumen en http://links.lww.com/DCR/B666. (Traducción-Dr. Fidel Ruiz Healy).

Appendiceal inflammation in colectomy is independently correlated with early pouchitis following ileal pouch anal anastomosis in ulcerative colitis and indeterminate colitis

BackgroundAppendiceal inflammation in colectomy is one of the histologic predictors of pouchitis in ulcerative colitis (UC) following ileal pouch anal anastomosis (IPAA). Fecal calprotectin level has been shown to increase 2 months prior to the onset of pouchitis. We evaluated whether inflammation and calprotectin expression in appendiceal specimens correlate with early-onset pouchitis in UC and indeterminate colitis (IC). Materials and methodsIPAA (2000-2018) cases with appendix blocks available in colectomy specimens were identified (n = 93, 90 UC, 3 IC). Histologic features thought to predict pouchitis were evaluated. The degree of appendiceal inflammation was scored. Calprotectin immunostain was performed on the appendix blocks and the extent of mucosal staining was quantified. Electronic medical records were reviewed for demographics, smoking history, clinical pouchitis, time of onset of pouchitis, and clinical and endoscopic components of the Pouchitis Disease Activity Index (PDAI) score. Follow-up pouch biopsies were reviewed and scored to generate histologic PDAI score, when available. ResultsAmong the patients with clinical pouchitis (n = 73), moderate to severe appendiceal inflammation independently correlated with earlier pouchitis compared to no/mild inflammation (median time to pouchitis 12.0 vs. 23.8, log rank p = 0.016). Calprotectin staining correlated with inflammatory scores of the appendix (Spearman's rho, r = 0.630, p < 0.001) but not with early pouchitis (p > 0.05). ConclusionsThe presence of moderate to severe appendiceal inflammation at the time of colectomy was associated with a shorter time to pouchitis following IPAA. Calprotectin immunostain may be used to demonstrate the presence of inflammation in the appendix but its role in predicting early pouchitis remains limited.

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

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. 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. 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. 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.

SATB2 and Hep Par 1 Immunohistochemistry Is Helpful in Distinguishing Between Inflamed and Architecturally Altered Ileal Pouch and Rectal Cuff Mucosa.

The management of patients with ulcerative colitis after proctocolectomy with ileal pouch-anal anastomosis includes independent histological assessments of inflammation in the ileal pouch and the rectal cuff. However, the distinction between pouchitis and cuffitis can be impeded both endoscopically and histologically by the combined effects of inflammation and regeneration. We investigated the use of 2 markers, hepatocyte paraffin 1 (Hep) and SATB2 (special AT-rich sequence-binding protein 2), which are expressed immunohistochemically in the small and large bowel epithelium, respectively, as ancillary methods to deal with this problem. Immunohistochemical staining was performed retrospectively on 20 consecutive pairs of post-ileal pouch-anal anastomosis biopsies with varying degrees of histological inflammation and architectural distortion, which had each been designated as "ileal pouch" or "rectal cuff" by the referring endoscopists. Expression was graded as focal (10% to 74% stained cells) or diffuse (75% to 100%). Among the ileal pouch biopsies, 20 (100%) expressed Hep either diffusely (75%) or focally (25%), whereas SATB2 staining was either negative in 15 (75%) or focal in 5 (25%), the latter group all expressing Hep diffusely. Among the rectal cuff biopsies, 14 expressed SATB2 diffusely. Of these, Hep was either negative in 11 (79%) or focally positive in 3 (21%), the latter group all expressing SATB2 diffusely. Six ostensibly rectal cuff biopsies (30%) expressed Hep diffusely and were negative for SATB2, suggesting endoscopic misidentification. None of the 40 biopsies expressed both markers diffusely. We conclude that in doubtful cases, diffuse expression of either Hep or SATB2 can be helpful in discriminating between ileal pouch and rectal cuff mucosa, respectively.

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

BackgroundInflammatory bowel disease (IBD) is associated with neuroendocrine cell hyperplasia. AimsWe investigated neuroendocrine cells in J-pouches of patients with ulcerative colitis undergoing restorative proctocolectomy and ileal pouch-anal anastomosis. MethodsSections 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. ResultsA 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. ConclusionWe here demonstrated neuroendocrine cell hyperplasia in pouch mucosa. Further studies are needed to clarify the pathophysiological implication of this finding.

Intraepithelial lymphocytosis is a frequent finding in biopsies from ileal pouch–anal anastomoses

Following restorative proctocolectomy with an ileal pouch-anal anastomosis, the small bowel mucosa undergoes several specific histologic adaptions, which may be unrelated to the underlying disease or symptoms of pouchitis. An increase in intraepithelial lymphocytes (IELs) has not been described as part of this spectrum. Mucosal biopsies of the ileal pouch and afferent limb of 230 patients (mean age: 45.7y [18.3-74.7], gender [female/male]: 117/113) with a functioning ileal pouch-anal anastomosis (mean time since ileostomy closure: 10.8months) and associated clinically annotated outcome data were assessed for IELs/100 enterocytes. Forty-two patients (18.3%) showed an increase in IELs (≥20 IELs/100 enterocytes [range 20-39]), in pouch and/or afferent limb biopsies. Intraepithelial lymphocytosis was more commonly observed in afferent limb compared to pouch biopsies (18.8% vs 8.3%; P = .42) and in familial adenomatous polyposis compared to ulcerative colitis patients (16% vs 8%; P = 0.36), but neither difference reached statistical significance. No cases with increased IELs displayed severe villous blunting. Increased IELs were not significantly associated with age, sex, ethnicity, smoking history, time since ileostomy, use of antibiotics, biologic agents, anti-diarrheal agents or probiotics, C-reactive protein levels or differential white cell count. None of the 42 patients with increased IELs had positive celiac serology (anti-human tissue transglutaminase IgA [ELISA] with corresponding total serum IgA). Intraepithelial lymphocytosis in pouch biopsies may represent a subclinical response to an altered bacterial microenvironment. Pathologists should be aware that intraepithelial lymphocytosis is part of the spectrum of changes in pouch biopsies, and only rarely is due to celiac disease.

The Effectiveness of Cytologic Evaluation of Urinary Diversion Specimens and UroVysion Assay as Surveillance Tests in the Detection of Recurrent Bladder Cancer

Introduction: Different surveillance protocols exist for the detection of urothelial carcinoma recurrence in patients that have undergone radical cystectomy (RC) and each protocol varies in choice of test and frequency of testing. Cytologic examination of urinary diversion specimens and UroVysion assay are two surveillance tests that may be used to detect recurrent disease. The cytologic interpretation of urinary diversion specimens is suboptimal since they contain abundant degenerate glandular cells, histiocytes, bacteria, acute inflammation and non-specific debris. In this milieu recurrent disease can be obscured and identification made difficult, if not impossible. The purpose of this study is to evaluate the effectiveness of cytology and UroVysion assay in this subset of patients. Materials and Methods: A Retrospective chart review of 97 patients who underwent RC with cutaneous diversion or orthotopic neobladder reconstructions for bladder cancer andwho also had a LIS (Laboratory Information System) word search result containing “ileal conduit urine” between January 2001 and January 2012was performed. The cytopathology reports for urinary diversion specimens and any UroVysion assays from these patients were reviewed. Cytologic results were divided into positive, suspicious, atypical, negative and unsatisfactory. The atypical category did not imply malignancy or the potential for malignancy, but was used to indicate some degree of variation from cytology seen in traditional urine specimens. Results: A total of 355 urinary diversion cytology reportswere reviewedwith diagnoses as follows: positive e 0, suspicious e 3 (0.8%), atypical e 136 (38.3%), negative e 213 (60%) and unsatisfactory e 3 (0.8%). A total of 56 Urovysion molecular tests were reviewed with interpretations as follows: positive e 3 (5.3%), negative e 39 (69.6%), indeterminate e 2 (3.6%), insufficient e 12 (21.4%). Four of 97 patients (4.1%) had at least one abnormal cytologic result. One cytologic diagnosis of atypical showed no abnormality on pouch biopsy. The remaining 3 patients with abnormal cytologies had no subsequent biopsies at our institution. Two patients had positive Urovysion assays and both these patients had orthotopic neobladder reconstructions. One patient had two subsequent positive Urovysion assay results which are accounted for by the presence of residual transitional cell carcinoma in-situ (TCIS) in the urethra as this patient had a positive urethral margin at the time of his cystoprostatectomy. The second patient with a positive UroVysion assay result occurred in a patient with 5 normal urinary diversion cytologies who was ultimately diagnosed with Stage 4 urothelial cancer (urothelial carcinomametastasis to skin). Thiswas deemed to be a false positive test result based on clinical assessment. None of the four patientswith abnormal cytology results were found to have recurrent disease. Conclusion: The results of this study question the effectiveness of cytologic evaluation of urinary diversion specimens and UroVysion assay as surveillance tests for detecting recurrent bladder cancer.We did not detect any upper tract/urethral recurrence in our series. Furthermore, the UroVysion assay is not FDA-approved for testing urine specimens derived from patients

Su1117 Practice Pattern and Detection of Neoplasia by Surveillance Pouch Endoscopy After IPAA for IBD Colitis in Academic Centers in the US

Background: There are still no standard endoscopic surveillance guidelines to detect neoplasia (dysplasia and/or cancer) following IPAA for IBD colitis. The aim of this survey study is to investigate the practice pattern of surveillance pouchoscopy for neoplasia in academic settings. Methods: Physicians who potentially take care of patients undergoing IPAA for IBD colitis were identified by searching literatures in MEDLINE. A questionnaire including practice pattern and opinions on ileal pouch surveillance was sent by email through REDcap. Univariable and multivariable analyses were performed. Results: A total of 118 physicians (86 gastroenterologists and 32 colorectal surgeons) were surveyed and 58 (49%) of them responded. One incomplete questionnaire and 5 physicians who do not routinely followup IBD patients with ileal pouch were excluded. Of the 52 eligible participants from 32 institutions, 6 (11%) were general gastroenterologists, 31(60%) were gastroenterologists specialized in IBD and 15 (29%) were colorectal surgeons. Forty-one physicians (79%) agree that it is necessary to perform routine pouch surveillance for neoplasia arising from ileal pouch or anal transitional zone/rectal cuff in all IBD patients undergoing IPAA and 22 (55%) think that the pouchoacopy solely for neoplasia should be performed every 2-3 years. Thirtysix physicians (69%) feel that pouchoscopy with biopsy is effective for the detection of neoplasia. Altogether 41 dysplasias and 15 cancers arising from ileal pouch or anal transitional zone/rectal cuff were reported to have been found within the past 5 years by 18 physicians (35%). There were no differences in the locations of pouch observation and biopsy during pouchocsopy examination between neoplasia detection and non-detection group. Colorectal surgeons found more pouch neoplasias than both gastroenterologists specialized in IBD and general gastroenterologists (61% vs 28% vs 11%, p 3 biopsies per location, 44% vs 82%, p=0.005). The median follow-up number of IBD pouch per year was significantly higher in neoplasia detection group (50 vs 25, p=0.041).

Su1118 The Development of a Standardized Peri-Operative Steroid Management Protocol for IBD Patients

Background: There are still no standard endoscopic surveillance guidelines to detect neoplasia (dysplasia and/or cancer) following IPAA for IBD colitis. The aim of this survey study is to investigate the practice pattern of surveillance pouchoscopy for neoplasia in academic settings. Methods: Physicians who potentially take care of patients undergoing IPAA for IBD colitis were identified by searching literatures in MEDLINE. A questionnaire including practice pattern and opinions on ileal pouch surveillance was sent by email through REDcap. Univariable and multivariable analyses were performed. Results: A total of 118 physicians (86 gastroenterologists and 32 colorectal surgeons) were surveyed and 58 (49%) of them responded. One incomplete questionnaire and 5 physicians who do not routinely followup IBD patients with ileal pouch were excluded. Of the 52 eligible participants from 32 institutions, 6 (11%) were general gastroenterologists, 31(60%) were gastroenterologists specialized in IBD and 15 (29%) were colorectal surgeons. Forty-one physicians (79%) agree that it is necessary to perform routine pouch surveillance for neoplasia arising from ileal pouch or anal transitional zone/rectal cuff in all IBD patients undergoing IPAA and 22 (55%) think that the pouchoacopy solely for neoplasia should be performed every 2-3 years. Thirtysix physicians (69%) feel that pouchoscopy with biopsy is effective for the detection of neoplasia. Altogether 41 dysplasias and 15 cancers arising from ileal pouch or anal transitional zone/rectal cuff were reported to have been found within the past 5 years by 18 physicians (35%). There were no differences in the locations of pouch observation and biopsy during pouchocsopy examination between neoplasia detection and non-detection group. Colorectal surgeons found more pouch neoplasias than both gastroenterologists specialized in IBD and general gastroenterologists (61% vs 28% vs 11%, p 3 biopsies per location, 44% vs 82%, p=0.005). The median follow-up number of IBD pouch per year was significantly higher in neoplasia detection group (50 vs 25, p=0.041).

P-214 Determining the Role of TCF-4 in the Genetic Susceptibility of Crohn’s Disease of the Pouch

Crohn’s disease of the pouch is an unfortunate inflammatory complication among ulcerative colitis patients who have undergone restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) that drastically reduces quality of life. While this is a complex disease with numerous contributory factors, it is believed that genetic mutations can make certain people susceptible to CD of the pouch. The aim of our study was to evaluate whether the rs3814570 SNP found in the promoter region of TCF-4 is associated with CD of the pouch and whether this specific SNP contributes to decreased DEFA5 gene expression. DNA was isolated from blood samples and rs3814570 SNP genotyping was carried out by PCR amplification followed by restriction enzyme digest and visualization by gel electrophoresis. DEFA5 gene expression was evaluated by isolating total RNA from pouch biopsies and carrying out real time qRT-PCR. Odds ratios were calculated to detect association between the rs3814570 SNP and CD of the pouch versus normal pouch controls. Mean Ct values were calculated for DEFA5 gene expression and normalized to GADPH endogenous control and one-way ANOVA was done to detect for association of specific TCF-4 genotypes and DEFA5 expression. Subjects with 1 copy of the rs3814570 SNP T allele (heterozygous) or 2 copies of the T-allele (minor allele homozygous) had OR (95% CI) of 1.02 (0.59–1.76) and 1.67 (0.71–3.89), respectively when tested for association with CD of the pouch compared to normal pouch controls. Major allele homozygous (CC) had a DEFA5 mean Ct value (Std Error) of -3.28 (0.13), heterozygous (CT) -1.87 (0.14) and minor allele homozygous (TT) -1.44 (0.24) with one-way ANOVA showing F (2, 108) = 44.7, P < 0.001. Our study is consistent with other studies that found that having 1 or 2 copies of the TCF-4 rs3814570 T allele is associated with decreased DEFA5 expression. There is no significant association with rs3814570 and CD of the pouch, which could either be real or could be due to an inadequate sample size and power to detect an association at the SNP level.

The natural history of anal transition zone inflammation and possible relationship to pouchitis: a long-term longitudinal study

Reservoir ileitis (pouchitis) is the most common complication after pelvic pouch surgery for ulcerative colitis and the aetiology remains largely unknown. The anal transition zone (ATZ) contains the only remaining colonic epithelium after ileal pouch anal anastomosis (IPAA) and may provide important clues as to whether ulcerative colitis and pouchitis share a common pathogenesis. The aim of this study was to evaluate longitudinally the long-term histological changes in the ATZ and their relationship to the incidence of pouchitis. Patients with a double-stapled IPAA for ulcerative colitis at an academic medical centre with at least 10years of clinical and histological follow-up were identified from a prospective database. Annual ATZ and pouch biopsies were taken and interpreted by two expert gastrointestinal pathologists. ATZ histological variability score, the incidence of pouchitis, and function were correlated over time. ATZ biopsies were scored from one to three based on the extent of inflammation. Sixteen of the 114 patients having IPAA fulfilled the criteria for admission to the study. There were 179 biopsies of the ATZ. All exhibited variability in ATZ histology over time and 81% had a 2-unit change in their inflammatory score. There was no correlation between pouchitis and histological severity score of the ATZ. Similarly, function over time did not vary with the intensity of ATZ inflammation. ATZ inflammation varies substantially over time in most patients. But these changes from year to year did not correlate with function or the occurrence of pouchitis.

Chronic Pouchitis Versus Recurrent Crohn’s Disease: A Diagnostic Challenge

Chronic Pouchitis Versus Recurrent Crohn’s Disease: A Diagnostic Challenge

Activation of sterol regulatory element binding protein and NLRP3 inflammasome in atherosclerotic lesion development in diabetic pigs.

BackgroundAberrantly elevated sterol regulatory element binding protein (SREBP), the lipogenic transcription factor, contributes to the development of fatty liver and insulin resistance in animals. Our recent studies have discovered that AMP-activated protein kinase (AMPK) phosphorylates SREBP at Ser-327 and inhibits its activity, represses SREBP-dependent lipogenesis, and thereby ameliorates hepatic steatosis and atherosclerosis in insulin-resistant LDLR−/− mice. Chronic inflammation and activation of NLRP3 inflammasome have been implicated in atherosclerosis and fatty liver disease. However, whether SREBP is involved in vascular lipid accumulation and inflammation in atherosclerosis remains largely unknown.Principal FindingsThe preclinical study with aortic pouch biopsy specimens from humans with atherosclerosis and diabetes shows intense immunostaining for SREBP-1 and the inflammatory marker VCAM-1 in atherosclerotic plaques. The cleavage processing of SREBP-1 and -2 and expression of their target genes are increased in the well-established porcine model of diabetes and atherosclerosis, which develops human-like, complex atherosclerotic plaques. Immunostaining analysis indicates an elevation in SREBP-1 that is primarily localized in endothelial cells and in infiltrated macrophages within fatty streaks, fibrous caps with necrotic cores, and cholesterol crystals in advanced lesions. Moreover, concomitant suppression of NAD-dependent deacetylase SIRT1 and AMPK is observed in atherosclerotic pigs, which leads to the proteolytic activation of SREBP-1 by diminishing the deacetylation and Ser-372 phosphorylation of SREBP-1. Aberrantly elevated NLRP3 inflammasome markers are evidenced by increased expression of inflammasome components including NLPR3, ASC, and IL-1β. The increase in SREBP-1 activity and IL-1β production in lesions is associated with vascular inflammation and endothelial dysfunction in atherosclerotic pig aorta, as demonstrated by the induction of NF-κB, VCAM-1, iNOS, and COX-2, as well as by the repression of eNOS.ConclusionsThese translational studies provide in vivo evidence that the dysregulation of SIRT1-AMPK-SREBP and stimulation of NLRP3 inflammasome may contribute to vascular lipid deposition and inflammation in atherosclerosis.

Is Pyloric Gland Metaplasia in Ileal Pouch Biopsies a Marker for Crohn’s Disease?

Approximately 5-10 % of ulcerative colitis (UC) patients who undergo ileal pouch-anal anastomosis (IPAA) will develop postoperative complications such as refractory pouchitis or a change in diagnosis to Crohn's disease (CD). Serological markers and histologic aspects of the pouch such as pyloric gland metaplasia (PGM) have been associated with a risk for these complications. Twenty-eight IPAA patients with either CD of the pouch or chronic pouchitis (cases) and 36 IPAA controls who experienced a normal postoperative course were originally consented. Of these 64 subjects, 22 cases and 17 controls had histopathologic and serologic data available and were subsequently enrolled. Demographic and clinical data were entered into a database, blood analyzed for serological markers (Prometheus Labs, San Diego, CA) and biopsies of the pouch and the afferent limb reviewed by two GI pathologists. Of the cases, 55 % (12/22) had evidence of PGM in their pouch and/or small bowel biopsies, as compared to 12 % (2/17) of the controls (p = 0.006). Of 13 subjects with CD, 77 % (10/13) were found to have PGM versus subjects with chronic pouchitis in which 22 % (2/9) were found to have PGM (p = 0.03). There was a trend of ASCA positivity (both IgG and IgA, p = 0.20) and of higher ASCA titer levels (p = 0.07) with postoperative complications. This study suggests that the presence of ileal pouch PGM is associated with postoperative complications and favors a diagnosis of CD over UC with chronic pouchitis.

Increased Crypt Apoptosis Is a Feature of Autoimmune-Associated Chronic Antibiotic Refractory Pouchitis

Restorative proctocolectomy with IPAA is the surgical treatment of choice in patients with ulcerative colitis who require colectomy. Pouchitis is the most common long-term complication in patients with IPAA. While immune-mediated disorders frequently coexist with IBD, they appear to be associated with an increased risk for chronic antibiotic-refractory pouchitis. This study aimed to evaluate histologic features of autoimmune-associated chronic antibiotic-refractory pouchitis in comparison with controls and to assess their possible diagnostic utility. Clinical definition for autoimmune-associated chronic pouchitis included 1) chronic antibiotic-refractory pouchitis with response only to corticosteroids, immunomodulators, or biologics; 2) positive serum autoantibodies, including antinuclear antibody, rheumatoid factor, and antimicrosomal antibody; and 3) concurrent immune-mediated disorders. Various histologic features of pouch biopsy specimens were evaluated. The investigation was conducted at a tertiary referral center. From our Pouchitis Registry, all eligible patients with autoimmune-associated pouchitis (n = 17) were included. The control groups included 16 patients with nonautoimmune-associated chronic antibiotic-refractory pouchitis, 39 with antibiotic-responsive pouchitis, and 19 patients with normal pouches. Various histologic features of pouch biopsy specimens were evaluated. In comparison with the control groups, the autoimmune-associated pouchitis group showed a significant increase in deep crypt apoptosis (p < 0.001). It also showed more pyloric gland metaplasia in comparison with antibiotic-responsive pouchitis and normal pouches. With the use of apoptosis score which we developed as a diagnostic marker for autoimmune-associated chronic antibiotic-refractory pouchitis, we constructed a receiver operating curve and obtained an area-under-curve value of 0.89 (95% CI: 0.79, 0.99). Increased deep crypt apoptosis is a distinctive histologic feature of autoimmune-associated chronic antibiotic-refractory pouchitis, and this feature may aid in the diagnosis and differential diagnosis in pouchitis.

Matrix metalloproteinases in the restorative proctocolectomy pouch of pediatric ulcerative colitis

To investigate matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in pouch mucosa of pediatric onset ulcerative colitis (UC). In this cross-sectional study, 28 patients with pediatric onset UC underwent ileal pouch biopsy 13 years (median) after proctocolectomy. Expression of MMPs-3, -7, -8, -9, -12 and -26 and TIMPs-1, -2 and -3 in samples was examined using immunohistochemichal methods, and another biopsy was used to evaluate the grade of histological inflammation. Two investigators independently graded the immunohistochemical specimens in a semiquantitative fashion, using a scale marking staining intensity as follows: 0 = less than 20 positive cells; 1 = 20-50 positive cells; 2 = 50-200 positive cells; 3 = over 20 positive cells. Fecal calprotectin and blood inflammatory markers [serum C-reactive protein (CRP) and erythrocyte sedimentation rate] were determined during a follow-up visit to examine correlations between these markers and the expression of MMPs and TIMPs. Of the 28 patients with pediatric onset UC, nine had not experienced pouchitis, whereas thirteen reported a single episode, and six had recurrent pouchitis (≥ 4 episodes). At the time of the study, six patients required metronidazole. In all of the others, the most recent episode of pouchitis had occurred over one month earlier, and none were on antibiotics. Only four samples depicted no sign of inflammation, and these were all from patients who had not had pouchitis. Two samples were too small to determine the grade of inflammation, but both had suffered pouchitis, the other recurrent. No sample depicted signs of colonic metaplasia. Most pouch samples showed expression of epithelial (e) and stromal (s) MMP-3 (e, n = 22; s, n = 20), MMP-7 (e, n = 28; s, n = 27), MMP-12 (e, n = 20; s, n =24), TIMP-2 (e, n = 23; s, n = 23) and MMP-3 (e, n = 23; s, n = 28) but MMP-8 (e, n = 0; s, n = 1), MMP-9 (e, n = 0; s, n = 9) and MMP-26 (e, n = 0; s, n = 3) and TIMP-1 (n = 0, both) were lacking. In samples with low grade of inflammatory activity, the epithelial MMP-3 and MMP-7 expression was increased (r = -0.614 and r = -0.472, respectively, P < 0.05 in both). MMPs and TIMPs did not correlate with the markers of inflammation, fecal calprotectin, erythrocyte sedimentation rate, or CRP, with the exception of patients with low fecal calprotectin (< 100 μg/g) in whom a higher expression of epithelial MMP-7 was found no differences in MMP- or TIMP-profiles were seen in patients with a history of pouchitis compared to ones with no such episodes. Anastomosis with either straight ileoanal anastomosis or ileoanal anastomosis with J-pouch did depict differences in MMP- or TIMP-expression. The expression of MMPs pediatric UC pouch in the long-term shares characteristics with inflammatory bowel disease, but inflammation cannot be classified as a reactivation of the disease.

Clinical Significance of Indefinite for Dysplasia on Pouch Biopsy in Patients with Underlying Inflammatory Bowel Disease

Background“Indefinite for dysplasia” (IND) on pouch mucosal biopsy is occasionally reported during routine histopathological evaluation. The natural history and implication of this histologic entity in ileal pouch-anal anastomosis (IPAA) has not been studied. AimThe aim of this study is to characterize cumulative probability, natural history, and clinical outcome of pouch IND in a cohort of patients with inflammatory bowel disease (IBD). MethodsAll 932 patients with restorative proctocolectomy and IPAA for IBD were included. Patients with or without IND were classified into the study and control groups. Demographic, clinical, endoscopic, and histologic variables were analyzed. ResultsThe mean duration from IBD diagnosis to colectomy and from pouch construction to data entry was 8.4±8.5 and 9.7±6.2 years, respectively. A total of 2,250 surveillance or diagnostic pouchoscopies with biopsies were performed for the cohort. Twenty-one patients (2.3%) were diagnosed with anal transitional zone and/or pouch IND, for whom subsequent pouchoscopies were performed with the mean procedure number being 3.4±2.2 per patient during a mean of follow-up of 19.3±16.1 months. One patient with IND developed low-grade dysplasia and one had high-grade dysplasia in a separate endoscopy. Cox model showed the presence of primary sclerosing cholangitis was an independent risk factor for pouch IND [hazard ratio = 6.76 (95% CI 2.56–17.88)]. Interobserver agreement (kappa score) for diagnosing pouch IND between GI pathologists ranged from 0.67 to 0.76. ConclusionsSubsequent dysplasia was uncommon in pouch patients with IND. Natural history of pouch IND warrants further long-term investigation.

Are we reporting ileal pouch biopsies correctly?

The accuracy of ileo-anal pouch biopsy reporting was assessed. The pathology reports of 100 consecutive pouch biopsies were reviewed to assess the accuracy and consistency with which the St Mark's histological scoring criteria were applied. The quality of pouch biopsy sampling and provision of clinical and endoscopic information on pathology request forms was also assessed. In 27% of cases no relevant endoscopic or clinical information was provided with the pathology request form. Separately labelled biopsies from the prepouch ileum, pouch and columnar cuff were submitted in only 4% of cases. In 32% of pathology reports, no acute or chronic St Mark's score was included. In 2% of cases the St Mark's scoring criteria were applied inappropriately. Twenty per cent of cases histologically diagnosed as pouchitis did not include a numerical score. In 30% of cases diagnosed histologically as pouchitis, an acute inflammatory score of < 4 (i.e. insufficient for this diagnosis) was included in the report. Pouchitis is a combined clinical, endoscopic and histological diagnosis. The correct interpretation and application of the St Mark's histological scoring criteria for pouch biopsies is an important part of this diagnostic process.