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Preclinical human IBD mechanisms is part of five focus areas of the Challenges in IBD research document, which also include environmental triggers, novel technologies, precision medicine and pragmatic clinical research. The Challenges in IBD research document provides a comprehensive overview of current gaps in inflammatory bowel diseases (IBD) research and delivers actionable approaches to address them. It is the result of a multidisciplinary input from scientists, clinicians, patients, and funders, and represents a valuable resource for patient centric research prioritization. In particular, the preclinical human IBD mechanisms manuscript is focused on highlighting the main research gaps in the pathophysiological understanding of human IBD. These research gap areas include: 1) triggers of immune responses; 2) intestinal epithelial homeostasis and wound repair; 3) age-specific pathophysiology; 4) disease complications; 5) heterogeneous response to treatments; and 6) determination of disease location. As an approach to address these research gaps, the prioritization of reverse translation studies is proposed in which clinical observations are the foundation for experimental IBD research in the lab, and for the identification of new therapeutic targets and biomarkers. The use of human samples in validating basic research findings and development of precision medicine solutions is also proposed. This prioritization aims to put emphasis on relevant biochemical pathways and humanized in vitro and in vivo models that extrapolate meaningfully to human IBD, to eventually yield first-in-class and effective therapies., (© 2019 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Ulcerative colitis is an idiopathic inflammatory condition of the colon that may require surgical intervention including proctocolectomy and either ileal pouch-anal anastomosis or in the pediatric population, low ileorectal anastomosis (IRA). Often, subsequent physiologic alteration (or colonic metaplasia) occurs in the anastomosed small bowel that includes changes in mucin content, villous blunting, and increased expression of WNT5A, a marker of colonic crypt regeneration. We developed a rat low IRA model to assess and study the development of colonic metaplasia., Materials and Methods: We subjected male Sprague-Dawley rats (n = 17) to total colectomy and low IRA surgery and evaluated healing periodically by endoscopic evaluation. The ileum upstream of the anastomosis was assessed by hematoxylin and eosin staining, and the mucin content was measured by high iron diamine-Alcian blue staining. Wnt5a transcripts were quantified by reverse transcription and quantitative polymerase chain reaction at the 8-wk study end point., Results: Although no gross endoscopic evidence of inflammation was seen throughout the course of the study, colonic metaplasia in the small bowel was detected in 7 out of 10 (70%) rats at the study end point. In rats with colonic metaplasia, enhanced expression of Wnt5a was evident at the study end point compared to levels in the terminal ileum at the time of surgery., Conclusions: Within 4-8 wk, the majority of rats subjected to IRA developed colonic metaplasia defined by villous blunting, changes in mucin content, and increased expression of Wnt5a. This model provides a method to study small bowel colonic metaplasia., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: To determine if single nuclear polymorphisms (SNPs) in the TFNSF15 gene play a role in patients requiring surgery for diverticulitis., Background: A role for a genetic predisposition in diverticulitis is suggested by its association with hereditary connective tissue disorders, youthful onset in some patients, and the observation of families with multiple affected individuals. The TNFSF15 gene has been associated with other inflammatory diseases affecting the colon such as medically refractory ulcerative colitis (UC), aggressive Crohn's disease (CD), and pouchitis after restorative proctocolectomy., Methods: In the discovery phase of this study, 21 sporadic surgical diverticulitis (SD) patients (9 female, mean age = 52 ± 5) and 5 individuals from a single family with surgically managed diverticulitis [familial diverticulitis (FD), 4 female, mean age = 51.1 ± 7] were studied. SD patients were age and sex matched with 3 separate groups of healthy, CD and UC control patients. All patients were genotyped for 5 known TNFSF15-associated SNPs. The SNP discovered to be associated with diverticulitis (rs7848647) was then confirmed in a separate test group composed of 34 additional patients (20 female, mean age 57.7 ± 2) who also underwent surgical treatment for diverticulitis. These patients were age matched to a new control cohort of patients having no history of diverticulitis (26 female). Patients were genotyped using a TaqMan assay. In the discovery phase, logistical regression on matched subjects was performed to determine an association of TNFSF SNP with diverticulitis versus the control groups. In the test phase, significance for the rs7848647 SNP was assessed by the Fischer's exact test., Results: In the discovery phase, the TNFSF15 SNP rs7848647 was significantly associated with SD (p = 0.0003) versus all control groups studied. The risk allele for this SNP (G substituted for A) was found in all SD patients. The homozygous GG allele was found in 62% (13/21) of SD patients versus only 5% (1/21) of healthy controls (p = 0.001) and 24% (10/42) of all UC + CD controls (p = 0.002). All 5 members of the FD cohort were homozygous for the at-risk "G" allele. In the test group, the homozygous GG genotype was found in 56% of SD patients compared with 17% of healthy controls (p = 0.006). Risk of SD seemed to increase with number of the G alleles with 8% of SD patients having AA homozygosity, 35% of SD patients having AG heterozygosity, and 56% of SD patients having GG homozygosity., Conclusions: The SNP rs7848647 associated with the TNFSF15 gene is associated with surgical diverticulitis. This finding suggests a fundamental role for TNFSF15, a T-cell receptor gene involved in T-cell maturation, in the pathophysiology of diverticulitis requiring surgery. This SNP may be a marker of diverticular disease severity that might assist in surgical decision making.

Background: We aimed to determine if an increased incidence of incisional hernias is present in patients undergoing sigmoidectomy for diverticulitis vs cancer. The pathophysiology of diverticulitis is poorly understood, but might involve a collagen vascular abnormality that can predispose to incisional hernia., Study Design: In this IRB-approved, retrospective study, patients who underwent sigmoid colectomies for diverticulitis or cancer between January 2003 and September 2012 were studied. Exclusion criteria included the development of surgical site infections and neoadjuvant chemoradiotherapy. A multivariate logistic regression was used with covariate adjustments for known risk factors for hernia development., Results: Four hundred forty-two patients (mean age 59.3 ± 13.9 years) with a median follow-up of 30 months were analyzed. The incidence of incisional hernia was 15.1% in diverticulitis patients vs 5.8% in the cancer cohort (41 of 271 vs 10 of 171; p = 0.003). Univariate analysis of risk factors associated with postoperative incisional hernia included steroid use (p = 0.007), wound packing (p = 0.001), higher American Society of Anesthesiologists classification (p = 0.001), absorbable suture closure (p = 0.02), blood transfusion (p = 0.04), stoma formation (p = 0.02), increased body mass index (p = 0.008), and history of incisional hernia (p = 0.00008). Multivariate logistic regression demonstrated a persistent association between diverticulitis and hernia development (p = 0.01). Odds of a hernia developing after sigmoidectomy for diverticulitis were 2.82 times greater than in the cancer cohort (95% CI, 1.3-6.6)., Conclusions: The incidence of an incisional hernia developing after a sigmoid colectomy is significantly higher when performed for diverticulitis as compared with cancer. This might be due to a connective tissue disorder, which predisposes to development of both diverticula and hernias., (Copyright © 2014 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: Genetic and functional studies have associated variants in the NOD2/CARD15 gene with Crohn's disease., Aims: This study aims to replicate the association of three common NOD2 mutations with Crohn's disease, study its effect on NOD2 expression in B cells and its interaction with other IBD-associated genes., Methods: A total of 294 IBD patients (179 familial IBD, 115 sporadic IBD) and 298 unrelated healthy controls were from central Pennsylvania. NOD2 mutations were analyzed by primer-specific amplification, PCR based-RFLP, and validated with the ABI SNPlexM genotyping system. Gene-gene interaction was studied using a statistical model for epistasis analysis., Results: Three common NOD2 mutations are associated with Crohn's disease (p=5.08×10(-7), 1.67×10(-6), and 1.87×10(-2) for 1007fs, R720W, and G908R, respectively), but not with ulcerative colitis (p=0.1046, 0.1269, and 0.8929, respectively). For IBD overall, 1007finsC (p=4.4×10(-5)) and R720W (p=9.24×10(-5)) were associated with IBD, but not G908R (p=0.1198). We revealed significant interactions of NOD2 with other IBD susceptibility genes IL23R, DLG5, and OCTN1. We discovered that NOD2 was expressed in both normal human peripheral blood B cells and in EBV-transformed B cell lines. Moreover, we further demonstrated that muramyl dipeptide (MDP) stimulation of B lymphocytes up-regulated expression of NF-κB-p50 mRNA., Conclusion: NOD2 is expressed in peripheral B cells, and the up-regulation of NOD2 expression by MDP was significantly impaired by NOD2 mutations. The finding suggests a possible role of NOD2 in the immunological response in IBD pathogenesis.

Background: Crohn's disease is a chronic inflammatory ailment that can affect the colon and/or small intestine. A genetic basis for disease distribution is being sought, although the available data are seminal. The STAT5 gene is known to influence colonic permeability, mucosal regeneration, and interleukin 2 production, although its role in the distribution of Crohn's disease is unclear., Objective: The aim of this study was identification of single nucleotide polymorphisms associated with Crohn's distribution, with the goal of distinguishing disease subcategories and differing pathophysiologies., Design: This was a retrospective cohort study., Setting: The study was conducted in a single tertiary referral center., Patients: A total of 173 patients with Crohn's disease who were identified from our biobank were segregated by disease distribution (colitis, n = 28; ileocolic disease, n = 116; enteritis, n = 29) and were genotyped for 258 Crohn's-associated single nucleotide polymorphisms. Patients with ulcerative colitis (n = 119) were also genotyped to confirm the association of identified single nucleotide polymorphisms with small-bowel sparing, colonic pathology., Main Outcome Measures: We investigated an association between single nucleotide polymorphisms and Crohn's disease distribution., Results: Single nucleotide polymorphism rs16967637 in the STAT5 gene was associated with small-bowel sparing Crohn's disease when the enteritis group was compared with either a combined colitis/ileocolic group (p = 0.025) or those with only ileocolic disease (p = 0.04). Homozygosity for the at-risk allele (C) was present in 59% of patients with sparing of the small bowel. The association of this single nucleotide polymorphism with small-bowel sparing disease persisted when patients with ulcerative colitis were compared with the group with Crohn's enteritis (p = 0.036), as well as after combining patients with ulcerative colitis with both the Crohn's colitis group (p = 0.009) and the Crohn's ileocolitis/colitis group (p = 0.00008)., Limitations: This study was limited by the small numbers of study subjects with isolated enteritis or colitis., Conclusions: Single nucleotide polymorphism rs16967637 in the STAT5 gene was the only single nucleotide polymorphism associated with Crohn's disease without enteritis. Homozygosity for the at-risk allele demonstrated the strongest association with this phenotype. These results suggest a role for this single nucleotide polymorphism in the development of inflammatory bowel disease of the large intestine.

Introduction: Bacteria have been implicated in the development of pouchitis after ileal pouch anal anastomosis. The change in gastric pH with the use of proton pump inhibitors and H(2) antagonists may lead to alteration of enteric bacteria. We hypothesized that chronic use of these medications would decrease the incidence of pouchitis., Methods: Patients who had undergone ileal pouch anal anastomosis for ulcerative colitis were classified by history of pouchitis. Patients were further classified by their use of proton pump inhibitors, H(2) blockers, antacids, and other known risk factors for pouchitis., Results: Eighty-five patients were identified. There was a statistically significant increase in the use of daily acid suppression in patients without pouchitis. There was also a statistically significant increase in the use of antacids in patients without pouchitis. Occasional use of acid suppression did not alter the rate of pouchitis., Conclusions: Our data suggest that the daily use of proton pump inhibitors, H(2) antagonists, or antacids is associated with a decreased risk of pouchitis in ulcerative colitis. Occasional use of these agents did not seem to afford the same protection. These data suggest that altering the pH of the gastrointestinal tract may influence the development of pouchitis.

Background: Changes in the methylation status of inflammatory bowel disease (IBD)-associated genes could significantly alter levels of gene expression, thereby contributing to disease onset and progression. We previously identified seven disease-associated DNA methylation loci from intestinal tissues of IBD patients using the Illumina GoldenGate BeadArray assay., Aims: In this study, we extended this approach to identify IBD-associated changes in DNA methylation in B cells from 18 IBD patients [9 Crohn's disease (CD) and 9 ulcerative colitis (UC)]. B cell DNA methylation markers are particularly favorable for diagnosis due to the convenient access to peripheral blood., Methods: We examined DNA methylation profiles of B cell lines using the Illumina GoldenGate BeadArray assay. Disease-associated CpGs/genes with changes in DNA methylation were identified by comparison of methylation profiles between B cell lines from IBD patients and their siblings without IBD. BeadArray data were validated using a bisulfite polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) method. To verify that observed changes in DNA methylation were not due to virus transformation, we compared specific CpG DNA methylation levels of GADD45A and POMC between B cell lines and matching peripheral blood B lymphocytes from five individuals., Results: Using this approach with strict statistical analysis, we identified 11 IBD-associated CpG sites, 14 CD-specific CpG sites, and 24 UC-specific CpG sites with methylation changes in B cells., Conclusions: IBD- and subtype-specific changes in DNA methylation were identified in B cells from IBD patients. Many of these genes have important immune and inflammatory response functions including several loci within the interleukin (IL)-12/IL-23 pathway.

Background: Recurrent Clostridium difficile colitis (RCDC) is common, but data regarding recurrence rates and predisposing factors are sparse., Methods: A retrospective case-control study was performed, identifying all inpatients and outpatients ≥18 years of age with C. difficile colitis (CDC) confirmed by a positive stool sample collected at our institution from January 2008 to August 2011. Factors associated with RCDC, the number of RCDC episodes, and the need for admission for RCDC were sought., Results: A total of 739 patients (male, 47 %) were studied, of whom 527 (71 %) received inpatient treatment for their index episode of CDC. There was no difference (p = 0.53) between RCDC rates for inpatients (17.6 %) and outpatients (19.8 %). While severity score and albumin were associated with RCDC in our population, use of proton pump inhibitors (PPIs) correlated with decreased RCDC (p = 0.006) and decreased need for admission (p = 0.005). The addition of vancomycin to metronidazole therapy did not lower RCDC rates (p = 0.52) or decrease the need for admission (p = 0.78)., Conclusions: Hypoalbuminemia strongly correlated with higher recurrence rates, while PPI therapy actually reduced RCDC, representing previously underappreciated potential therapeutic targets for lowering CDC recurrence. The addition of vancomycin to metronidazole did not improve RCDC rates.

Background: Anal complications of Crohn's disease range from painless skin tags to debilitating fistulas that are imperfectly treated with tumor necrosis factor antagonists. The recent discovery of more than 190 single-nucleotide polymorphisms associated with Crohn's disease offers the opportunity to genetically define the severity of anal disease in Crohn's disease and possibly predict prognosis and anti-tumor necrosis factor response., Objectives: This study aimed to identify single nucleotide polymorphisms associated with anal disease generally, septic anal disease specifically and the responsivity to anti-tumor necrosis factor treatment., Design: All patients with ileocolonic Crohn's disease were identified from our IBD registry. One hundred ninety-six Crohn's disease-related single-nucleotide polymorphisms were analyzed by the use of a custom microarray chip. Patients' response to anti-tumor necrosis factor treatment was then assessed., Results: One hundred sixteen patients with ileocolonic Crohn's disease were identified and assigned to septic anal disease (abscesses/fistulas, n = 35), benign anal disease (skin tags/fissures/isolated pain, n = 17), and no anal disease (n = 64) cohorts. Single-nucleotide polymorphism rs212388 negatively correlated with the presence of anal disease overall and septic disease specifically. The presence of the non-wild-type allele 'G' was protective of anal sepsis with homo- and heterozygotes having a 75% chance of no anal disease (p = 0.0001). The homozygous wild-type group had the highest risk of septic disease and included 3 of 4 patients requiring diverting ileostomies. Twenty-four patients were treated with anti-tumor necrosis factors. Nine had a beneficial response (assessed at >6 months); however, no single-nucleotide polymorphism correlated with anti-tumor necrosis factor response. Rs212388 is associated with the TAGAP molecule involved in T-cell activation., Conclusions: Rs212388 most significantly correlated with the presence and severity of anal disease in ileocolonic Crohn's disease. A single copy of the risk allele was protective, whereas wild-type homozygotes had the highest risk of septic disease and stoma creation. In this select group, no single-nucleotide polymorphism was predictive of anti-tumor necrosis factor response. Mutations in TAGAP may predict a more benign form and course of anal disease in Crohn's disease.

Ulcerative colitis (UC) and Crohn's disease (CD) are two related yet different forms of chronic intestinal inflammation. We investigated the genes regulated by NKX2-3 in B cells from a UC patient by cDNA microarray and compared the results to those genes regulated by NKX2-3 in B cells from a CD patient. Genes regulated by NKX2-3 in B cells from UC were mainly involved in cell growth, inflammation, and immune response. Among the genes regulated by NKX2-3 in both UC and CD, expression of 145 genes was similarly altered and 34 genes was differentially affected by NKX2-3 knockdown. EGR1 was up-regulated in NKX2-3 knockdown B cells from UC while down-regulated in NKX2-3 knockdown B cells from CD. mRNA expressions of NKX2-3 and EGR1 were increased in diseased intestinal tissues from 19 CD patients. NKX2-3 may play different roles in UC and CD pathogenesis by differential regulation of EGR1.

Background: Severe pouchitis and Crohn's disease-like complications are 2 adverse postoperative complications that confound the success of the IPAA in patients with ulcerative colitis. To date, approximately 83 single nucleotide polymorphisms within 55 genes have been associated with IBD., Objective: The aim of this study was to identify single-nucleotide polymorphisms that correlate with complications after IPAA that could be utilized in a gene signature fashion to predict postoperative complications and aid in preoperative surgical decision making., Design: One hundred forty-two IPAA patients were retrospectively classified as "asymptomatic" (n = 104, defined as no Crohn's disease-like complications or severe pouchitis for at least 2 years after IPAA) and compared with a "severe pouchitis" group (n = 12, ≥ 4 episodes pouchitis per year for 2 years including the need for long-term therapy to maintain remission) and a "Crohn's disease-like" group (n = 26, presence of fistulae, pouch inlet stricture, proximal small-bowel disease, or pouch granulomata, occurring at least 6 months after surgery). Genotyping for 83 single-nucleotide polymorphisms previously associated with Crohn's disease and/or ulcerative colitis was performed on a customized Illumina genotyping platform. The top 2 single-nucleotide polymorphisms statistically identified as being independently associated with each of Crohn's disease-like and severe pouchitis were used in a multivariate logistic regression model. These single-nucleotide polymorphisms were then used to create probability equations to predict overall chance of a positive or negative outcome for that complication., Results: The top 2 single-nucleotide polymorphisms for Crohn's disease-like complications were in the 10q21 locus and the gene for PTGER4 (p = 0.006 and 0.007), whereas for severe pouchitis it was NOD2 and TNFSF15 (p = 0.003 and 0.011). Probability equations suggested that the risk of these 2 complications greatly increased with increasing number of risk alleles, going as high as 92% for severe pouchitis and 65% for Crohn's disease-like complications., Conclusion: In this IPAA patient cohort, mutations in the 10q21 locus and the PTGER4 gene were associated with Crohn's disease-like complications, whereas mutations in NOD2 and TNFSF15 correlated with severe pouchitis. Preoperative genetic analysis and use of such gene signatures hold promise for improved preoperative surgical patient selection to minimize these IPAA complications.

Background: Early readmission after discharge from the hospital is an undesirable outcome. Ileostomies are commonly used to prevent symptomatic anastomotic complications in colorectal resections., Objective: The aim of this study was to identify factors predictive of readmission after colectomy/proctectomy and diverting loop ileostomy., Design: This study is a retrospective review., Patients: Patients were included who underwent colon and rectal resections with ileostomy at our institution. Sex, age, type of disease, comorbidities, elective vs urgent procedure, type of ileostomy, operative method, steroid use, ASA score, and the use of diuretics were evaluated as potential factors for readmission., Main Outcome Measures: The primary outcomes measured were the need for readmission and the presence of dehydration (ostomy output ≥1500 mL over 24 hours and a blood urea nitrogen/creatinine level ≥20, or physical findings of dehydration)., Results: Six hundred three loop ileostomies were created mostly in white (95.3%), male (55.6%) patients undergoing colon or rectal resections. IBD was the most common indication at 50.9%, with rectal cancer at 16.1%, and other at 31.0%. The 60-day readmission rate was 16.9% (n = 102) with the most common cause dehydration (n = 44, 43.1%). Regression analysis demonstrated that the laparoscopic approach (p = 0.02), lack of epidural anesthesia (p = 0.004), preoperative use of steroids (p = 0.04), and postoperative use of diuretics (p = 0.0001) were highly predictive for readmission. Furthermore, regression analysis for readmission for dehydration identified the use of postoperative diuretics as the sole risk factor (p = 0.0001)., Limitations: This study is limited by the retrospective analysis of data, and it does not capture patients that were treated at home or in clinic., Conclusion: Readmission after colon or rectal resection with diverting loop ileostomy was high at 16.9%. Dehydration was the major cause for readmission. Patients receiving diuretics are at increased risk for readmission for dehydration. High-risk patients should be treated more cautiously as inpatients and closely monitored in the outpatient setting to help reduce dehydration and readmission.

Background: There are no clear criteria for judging the severity of disease in patients with Crohn's disease. Yet classification of patients into low- and high-risk severity groups would benefit both medical and surgical management. At the time of this study, approximately 80 single-nucleotide polymorphisms within 55 genes had been associated with IBD., Objective: The aim of this study was to identify genetic determinants (single-nucleotide polymorphisms) that could be markers of Crohn's disease severity by the use of frequency of ileocolic surgery as a surrogate for disease severity., Design: Sixty-six patients (30 male) with ileocolonic Crohn's disease who previously underwent ileocolectomy were retrospectively studied. The severity of Crohn's disease was quantified by dividing the total number of ileocolectomy procedures by the time between IBD diagnosis and the patient's last clinic visit, the rationale being that more severe disease would be associated with a more frequent need for surgery. Genotyping for the 83 single-nucleotide polymorphisms associated with IBD was done on a customized Illumina Veracode genotyping platform. Three genetic models (general, additive, and dominant) were used to statistically quantify the genetic association of the studied single-nucleotide polymorphisms to the frequency of surgery after adjusting for covariates (age, smoking, family history, disease location, and disease behavior)., Results: For the entire group the average number of ileocolectomies per patient was 1.7 (range, 1-5) with an average duration of disease of 14.7 years. Single-nucleotide polymorphism rs4958847 in the IRGM gene (immunity-related GTPase family, M) was the most significant single-nucleotide polymorphism in all 3 models tested (p = 0.007) as being associated with ileocolectomy, and it remained significant even after a Benjamini-Hochberg false-discovery correction for multiple observations. Patients carrying the "at-risk" allele for this single-nucleotide polymorphism (n = 20) had an average of 1 surgery every 6.87 ± 1.33 years in comparison with patients carrying the wild-type genotype (n = 46) who averaged 1 surgery in 11.43 ± 1.21 years (p = 0.007, Mann-Whitney U test)., Conclusions: : Single-nucleotide polymorphism rs4958847 in the IRGM gene correlated very significantly with frequency of surgery in patients with ileocolonic Crohn's disease. IRGM is a mediator of innate immune responses and is involved in autophagy. The presence of this IRGM SNP may be a marker for disease severity and/or early recurrence after ileocolectomy and may assist in surgical and medical decision making.

PTPN2 is a risk gene for Crohn's disease (CD). We investigated whether PTPN2 genetic variants (rs2542151 and rs2542152) were associated with CD in a familial IBD registry. Both rs2542151 and rs2542152 are associated with CD, but not ulcerative colitis (UC). mRNA expression levels of PTPN2 were significantly increased in intestinal tissues (p=0.0493), and nearly significantly increased in B cells (p=0.0889) from CD patients, but not significantly altered in UC. cDNA microarray results found that PTPN2 was down-regulated by NKX2-3 knockdown in human cells. We confirmed this observation by RT-PCR analyses in NKX2-3 knockdown in B cells from IBD patients and human intestinal microvascular endothelial cells (HIMEC). In addition, we found that mRNA expression of another IBD-associated gene, NKX2-3, was increased in intestinal tissues and B cells from CD patients, but not significantly increased in UC patients. A positive correlation was observed between mRNA expression of PTPN2 and NKX2-3 in B cells and in intestinal tissues from both CD and UC patients. These results suggest that PTPN2 may have an important role in CD pathogenesis and may represent a potential diagnostic and therapeutic target for IBD.

Background and Aims: Studies have shown a decrease in key tight junction (TJ) proteins such as ZO-1 and occludin in both inflammatory bowel disease (IBD) and experimental models of inflammation. Our group has also shown an increase in claudin-1 in experimental colitis., Methods: IEC-18 cells were treated with increasing doses of tumor necrosis factor alpha (TNFα). The TJ was assessed by transepithelial resistance (TER), permeability, Western blot, PCR, and immunofluorescence. Mucosal samples from patients with ulcerative colitis (UC), Crohn's disease (CD), and without IBD (normal) were assayed for TJ proteins occludin and claudin-1 by Western blot and a ratio of claudin-1 to occludin (C:O) was calculated., Results: IEC-18 cells had increased permeability, decreased TER and an increase in claudin-1 with TNFα treatment. In human specimens, there was a decrease in occludin and an increase in claudin-1 leading to a significant increase in the C:O ratio in diseased UC colon compared to non-diseased UC colon (P < 0.001) and normal colon (P < 0.01). In CD, the C:O ratio was similar in all CD tissue irrespective of disease status., Conclusions: Treatment of IEC-18 cells with TNFα, a key inflammatory cytokine in IBD, led to a significant increase in claudin-1 expression. There was a significant increase in the C:O ratio in diseased colon in UC compared to the healthy appearing UC colon and normal controls. The C:O ratio was unchanged in CD despite presence or abscence of gross disease. This suggests that there may be an underlying difference in the TJ between UC and CD.

Background: Survival with stage III colorectal cancer (CRC) is variable and difficult to predict. Tumor size is not part of the staging system for CRC and in itself is not a predictor of survival. We hypothesize, however, that tumor size is important in determining disease free survival., Methods: Patients with stage III CRC were identified and divided into 2 categories: those who developed metastatic disease within 5 years after surgery and those who were alive and disease free at 5 years. A ratio of tumor volume to percent positive nodes was calculated for each patient (TN ratio)., Results: Sixty-three patients were identified. 35 (55%) were alive and tumor free at 5 years. The TN ratio was significantly higher in patients who were disease free. Compared to number of positive nodes, percent positive nodes, and tumor volume, the TN ratio was the most significant predictor of disease free survival at 5 years., Conclusion: Patients who survive 5 years after CRC surgery have a statistically significantly higher TN ratio than those patients who have metastatic disease within 5 years. Patients with small tumors and positive nodes have a lower TN ratio and a statistically decreased 5-year survival., (Copyright © 2011 Mosby, Inc. All rights reserved.)

NKX2-3 SNP rs11190140 is associated with inflammatory bowel disease (IBD). The T allele is over-transmitted in IBD and the C allele represents a potential CpG methylation site. We hypothesize that genetic variation and/or methylation of SNP rs11190140 may play a role in NKX2-3 gene expression by affecting transcription factor binding. We studied 233 IBD cases and 250 unrelated healthy individuals from an IBD population from central Pennsylvania and performed genotype analyses of the genetic variation and methylation status analysis using PCR-based RFLP. For transcription factor binding, nuclear extracts from human B cells were incubated with biotin-labeled oligonucleotide sequences of the NKX2-3 promoter region containing the genetic variation of T, non-methylated C or methylated C at rs11190140, followed by biotin pull-down and Western blot analysis for transcription factors SP1, NFAT1, NF-κB, and ETS-1. In case-control analysis, the genetic variation was significantly associated with IBD (OR=0.503, 95% CI=0.330-0.764, p<0.001). Methylation status analyses revealed that the C allele is subject to modification by DNA methylation. transcription factor binding assay indicated distinct differential binding of NFAT1 to the NKX2-3 promoter sequence, with higher binding to those with non-methylated and methylated C than to T. The binding of NFAT1 to the NKX2-3 promoter region with rs1190140 was confirmed by ChIP assay. We speculate that the rs11190140 may regulate NKX2-3 expression and have a role in IBD pathogenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Surfactant protein-D (SP-D) is expressed on mucosal surfaces and functions in the innate immune response to microorganisms. We studied the genetic association of the two nonsynonymous SP-D single nucleotide polymorphisms (SNPs) rs721917 and rs2243639 in 256 inflammatory bowel disease (IBD) cases (123 CD and 133 UC) and 376 unrelated healthy individuals from an IBD population from Central Pennsylvania. Case-control analysis revealed a significant association of rs2243639 with susceptibility to Crohn's disease (CD) (p= 0.0036), but not ulcerative colitis (UC) (p= 0.883), and no association of rs721917 with CD (p= 0.328) or UC (p= 0.218). Using intestinal tissues from 19 individuals heterozygous for each SNP, we compared allelic expression of these two SNPs between diseased and matched normal tissues. rs2243639 exhibited balanced biallelic (BB) expression; while rs721917 exhibited differential allelic expression (BB 37%, imbalanced biallelic [IB] 45%, and dominant monoallelic [DM] 18%). Comparison of allelic expression pattern between diseased and matched normal tissues, 13 of 19 individuals (14 UC, 5 CD) showed a similar pattern. The six patients exhibiting a different pattern were all UC patients. The results suggest that differential allelic expression may affect penetrance of the SNP rs721917 disease-susceptibility allele in IBD. The potential impact of SP-D monoallelic expression on incomplete penetrance is discussed., (© 2011 The Authors Annals of Human Genetics © 2011 Blackwell Publishing Ltd/University College London.)

Overwhelming evidence supports the theory that inflammatory bowel disease (IBD) is caused by a complex interplay between genetic predispositions of multiple genes, combined with an abnormal interaction with environmental factors. It is becoming apparent that epigenetic factors can have a significant contribution in the pathogenesis of disease. Changes in the methylation state of IBD-associated genes could significantly alter levels of gene expression, potentially contributing to disease onset and progression. We have explored the role of DNA methylation in IBD pathogenesis. DNA methylation profiles (1505 CpG sites of 807 genes) of matched diseased (n = 26) and non-diseased (n = 26) intestinal tissues from 26 patients with IBD [Crohn's disease (CD) n = 9, ulcerative colitis (UC) n = 17] were profiled using the GoldenGate™ methylation assay. After an initial identification of a panel of 50 differentially methylated CpG sites from a training set (14 non-diseased and 14 diseased tissues) and subsequent validation with a testing set (12 non-diseased and 12 diseased tissues), we identified seven CpG sites that are differentially methylated in intestinal tissues of IBD patients. We have also identified changes in DNA methylation associated with the two major IBD subtypes, CD and UC. This study reports IBD-associated changes in DNA methylation in intestinal tissue, which may be disease subtype-specific., (© 2010 John Wiley & Sons A/S.)

Background: Surgical site infections (SSIs) are a well known complication of gastrointestinal surgery and associated with an increased morbidity, mortality and overall cost. Diabetes mellitus (DM) is a risk factor for SSI. However, there is no clear consensus as to which other risk factors play a significant role. The goal of this study was to identify risk factors associated with SSI in patients with DM undergoing colorectal resection., Study Design: A retrospective review was conducted of DM patients who underwent colorectal resection from June 2000 to June 2009 at Milton S Hershey Medical Center, Division of Colorectal Surgery. Individual measures were analyzed using chi-square, t-test, and Mann-Whitney U tests, and statistical significance was confirmed using a multiple logistical regression model., Results: There were 183 DM patients included in the study, 28 (15%) of whom developed SSI. Glucose levels were significantly higher in the SSI group for each time interval, 0 to 6 hours (211 mg/dL, p = 0.03), 0 to 48 hours (176 mg/dL, p = 0.001), and 48 to 96 hours (167 mg/dL, p = 0.012) postoperatively. Other measures significantly associated with SSI included the use of drains (p = 0.05) and the use of prophylactic antibiotics for more than 24 hours (p = 0.02). Body mass index and stoma creation approached statistical significance (p = 0.08, 0.07, respectively). The type of hypoglycemic regimen, immunosuppression, and emergency surgery were not associated with an increased rate of SSI., Conclusions: Higher than normal glucose control at all postoperative time intervals was associated with SSI. The majority of glucose levels were below the American Diabetes Association recommended level of 200 mg/dL, but patients still developed SSI. Type of perioperative glucose control did not affect the incidence of SSI. These data suggest that DM patients undergoing colectomy should have glucose tightly controlled, avoid placement of drains, and receive antibiotics for less than 24 hours., (Copyright © 2011 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: NKX2-3 is associated with inflammatory bowel disease (IBD). NKX2-3 is expressed in microvascular endothelial cells and the muscularis mucosa of the gastrointestinal tract. Human intestinal microvascular endothelial cells (HIMECs) are actively involved in the pathogenesis of IBD and IBD-associated microvascular dysfunction. To understand the cellular function of NKX2-3 and its potential role underlying IBD pathogenesis, we investigated the genes regulated by NKX2-3 in HIMEC using cDNA microarray., Methodology/principal Findings: NKX2-3 expression was suppressed by shRNA in two HIMEC lines and gene expression was profiled by cDNA microarray. Pathway Analysis was used to identify gene networks according to biological functions and associated pathways. Validation of microarray and genes expression in intestinal tissues was assessed by RT-PCR. NKX2-3 regulated genes are involved in immune and inflammatory response, cell proliferation and growth, metabolic process, and angiogenesis. Several inflammation and angiogenesis related signaling pathways that play important roles in IBD were regulated by NKX2-3, including endothelin-1 and VEGF-PI3K/AKT-eNOS. Expression levels of NKX2-3, VEGFA, PI3K, AKT, and eNOS are increased in intestinal tissues from IBD patients and expression levels of EDN1 are decreased in intestinal tissues from IBD patients. These results demonstrated the important roles of NKX2-3, VEGF, PI3K, AKT, eNOS, and EDN1 in IBD pathogenesis. Correlation analysis showed a positive correlation between mRNA expression of NKX2-3 and VEGFA and a negative correlation between mRNA expression of NKX2-3 and EDN1 in intestinal tissues from IBD patients., Conclusion/relevance: NKX2-3 may play an important role in IBD pathogenesis by regulating endothelin-1 and VEGF signaling in HIMECs.

Purpose: Ileal pouch-anal anastomosis continues to be confounded by Crohn's disease-like complications after surgery. Such patients experience significant morbidity and often require either pouch excision or diversion. This study evaluated the effectiveness in our hands of infliximab and/or azathioprine/6-mercaptopurine in treating this patient population., Methods: We conducted a retrospective chart review of all patients who underwent IPAA who experienced Crohn's disease-like complications (pouch fistulas, stricturing small-bowel disease, or pouchitis unresponsive to antibiotics) after ileostomy closure. Patients were segregated according to treatment (azathioprine/6-mercaptopurine only, infliximab only, or both azathioprine/6-mercaptopurine and infliximab) and evaluated for clinical response defined by significant symptomatic improvement and avoidance of stoma., Results: Of 382 IPAAs, 32 (8.4%) patients developed Crohn's disease-like complications a mean of 17 months after stoma closure. Of these, 22 were treated with azathioprine/6-mercaptopurine and/or infliximab with one lost to follow-up. Overall mean follow-up was 97 ± 11.8 months. Failure rate (requiring stomas) was highest in the fistula group treated with infliximab and azathioprine/6-mercaptopurine (6/13, 46%). Patients with stricturing disease (6) or severe pouchitis (2) were all effectively treated with azathioprine/6-mercaptopurine (5/6) or infliximab (1 patient allergic to azathioprine/6-mercaptopurine) and none of these patients required stomas. In the group not receiving stomas, bowel frequency improved from 8.3 ± 1 to 5.7 ± 0.5 per day (P < .05)., Conclusion: Fistulizing disease after IPAA has the highest failure/stoma rate (46%) despite treatment with infliximab and/or azathioprine/6-mercaptopurine. IPAA patients with stricturing disease and/or antibiotic resistant pouchitis were successfully treated without stomas and all had resolution of symptoms, which suggests that fistulous disease after IPAA should be treated with infliximab, but stricturing disease and antibiotic resistant pouchitis may be effectively treated with azathioprine/6-mercaptopurine only. Such a protocol will potentially minimize the risks associated with infliximab in this difficult group of patients.

Background: Nkx2-3 has been reported to be up-regulated in B cell lines and intestinal tissues from Crohn's disease patients and down-regulated in colorectal cancer., Aims: The purpose of the current study is to determine genes regulated by Nkx2-3 in sporadic (CRS61) and inflammatory bowel disease-associated (CRS4) colorectal cancer cell lines., Methods: Small interfering RNA-mediated knockdown of Nkx2-3 in both cell lines was generated and high-density cDNA microarrays representing over 25,000 genes were performed. Microarray results were validated by RT-PCR and immunofluorescence. Pathway analysis was used to identify gene networks associated with Nkx2-3 knockdown in these cell lines., Results: A total of 1,677 genes were regulated by Nkx2-3 in CRS4 cells; 1,375 genes were regulated by Nkx2-3 in CRS61 cells. Among those genes regulated by Nkx2-3, 254 genes were similarly regulated by Nkx2-3 knockdown in both cell lines; 159 genes were differentially regulated by Nkx2-3 knockdown between the two lines. Genes regulated by Nkx2-3 were grouped primarily within the following two functional categories: (1) immune and inflammatory response; and (2) cell proliferation, growth, and oncogenesis. Among the genes with similarly changed expression in the two cell lines, the top affected pathways included antigen presentation and cell-cell signaling. Among the genes with differentially changed expression between the two cell lines, ingenuity pathway analysis indicated that the top affected pathway included genes directly involved in Wnt signaling., Conclusions: Nkx2-3 may contribute to the pathogenesis of IBD-associated CRC and sporadic CRC by regulating the Wnt signaling pathway.

Purpose: Pouchitis and Crohn's-like complications can plague patients after IPAA. NOD2 is an intracellular sensor for bacterial cell wall peptidoglycan. NOD2 mutations compromise host response to enteric bacteria and are increased in Crohn's disease. We hypothesize that IPAA patients with complications (Crohn's disease-like/pouchitis) have a higher rate of NOD2 mutations compared with asymptomatic IPAA patients., Methods: Patients were retrospectively subclassified into the following groups: 1) IPAA with Crohn's-like complications (n = 28, perianal fistula, pouch inlet stricture/upstream small-bowel disease, or biopsies showing granulomata) occurring at least 6 months after ileostomy closure; 2) IPAA with mild pouchitis (n = 33, ≤3 episodes/y for 2 consecutive years); 3) IPAA with severe pouchitis (n = 9, ≥4 episodes/y for 2 consecutive years or need for continuous antibiotics); 4) IPAA without complications or pouchitis (n = 37); 5) patients with Crohn's disease with colitis undergoing total proctocolectomy/ileostomy (n = 11); and 6) healthy controls (n = 269). The 3 NOD2 single-nucleotide polymorphism mutations (rs2066844, rs2066845, and rs2066847) previously identified as associated with Crohn's disease were genotyped using polymerase chain reaction. Groups were compared by use of χ with Yates continuity correction., Results: NOD2 mutations were found in 8.5% of healthy controls. NOD2 mutations were significantly higher in the severe pouchitis group (67%) compared with both asymptomatic IPAA (5.4%, P < .001) and IPAA with Crohn's disease-like complications (14.3%, P = .008) groups., Conclusions: 1) Asymptomatic IPAA patients have a low incidence of NOD2 mutations not significantly different from patients with mild pouchitis or healthy controls. 2) Patients with severe pouchitis had the highest incidence of NOD2 mutations, suggesting that this group may have a compromised host defense mechanism to enteric bacteria. 3) Patients with Crohn's-like complications after IPAA have a significantly lower incidence of NOD2 mutations than patients with severe pouchitis, suggesting a different genetic makeup in these 2 patient groups. Preoperative assessment of NOD2 in the equivocal IPAA candidate may predict severe pouchitis and might assist in preoperative surgical decision making.

Background: The Interleukin-4 signal transducer and activator of transcription 6 (IL-4-STAT6) signaling pathway plays a pivotal role in regulation of gene transcription. We have previously identified a defective STAT6 activational phenotype in response to IL-4 in patients from our familial Inflammatory Bowel Disease registry. This has been termed Stat6(null) and Stat6(high) is the normal phenotype. The purpose of this study was to investigate the defect in Stat6 activation in Stat6(null) cells., Methods: Stat6(null) and Stat6(high) Epstein Barr virus transformed cell lines were stimulated with 10 ng/mL of IL-4 for 0, 10, 30, or 60 min and cytoplasmic and nuclear proteins harvested. Western blot for STAT6, phosphorylated STAT6 (pSTAT6), Janus Kinase (Jak)1 and Jak3 was performed. Cells were also cultured for 48 h and interferon gamma (IFNgamma) measured in the supernatant. Additional cells were cultured with 20 ng/mL of IFNgamma for 90 min or 5 ug of antibody to IFNgamma for 48 h, and then stimulated with IL-4., Results: There were no differences in cytoplasmic STAT6 in Stat6(null)versus Stat6(high) cells. In Stat6(high) cells, STAT6 was rapidly phosphorylated and translocated to the nucleus. In Stat6(null) cells there was minimal phosphorylation and translocation of pSTAT6 to the nucleus. Spontaneous secretion of IFNgamma by Stat6(null) cells was significantly higher than Stat6(null) cells. Addition of IFNgamma decreased pSTAT6 in Stat6(high) cells to Stat6(null) levels while blocking IFNgamma increased baseline pSTAT6 in Stat6(null) cells to levels similar to Stat6(high) cells., Conclusion: This suggests that the spontaneously produced IFNgamma in the Stat6(null) cell lines suppresses STAT6 function and creates the Stat6(null) phenotype., (Copyright 2010 Elsevier Inc. All rights reserved.)

Nkx2-3 gene variants are strongly associated with inflammatory bowel disease (IBD) and its expression is up-regulated in Crohn's disease (CD). However, the nature of its role underlying IBD pathogenesis is unknown. We investigated the genes regulated by Nkx2-3 using cDNA microarray. A small interfering RNA (siRNA)-mediated knockdown of Nkx2-3 in a B cell line from a CD patient was generated. Gene expression was profiled on high-density cDNA microarrays representing over 25,000 genes. Ingenuity pathway analysis (IPA) was used to identify gene networks according to biological functions and associated pathways. Expression profiling analysis by cDNA microarray showed that 125 genes were regulated by Nkx2-3 knockdown (fold change >or=3.0, p<0.01), among which 51 genes were immune and inflammatory response genes. Microarray results were validated by RT-PCR and further confirmed in a B cell line expressing siRNA of Nkx2-3 from an additional CD patient. The results showed that Nkx2-3 was up-regulated (p<0.05) and EDN1 was down-regulated (p<0.05) in B cell lines from CD patients. mRNA expression levels of Nkx2-3 were negatively correlated with those of EDN1 (r=-0.6044, p<0.05). EDN1 was also down-regulated in intestinal tissues from UC patients (p<0.05). Our present results demonstrate that a decrease in Nkx2-3 gene expression level can profoundly alter the expression of genes and cellular functions relevant to the pathogenesis and progression of IBD, such as EDN1., ((c) 2010 Elsevier Inc. All rights reserved.)

Purpose: Inflammatory bowel disease (IBD)-associated colorectal carcinogenesis involves dysregulation of multiple cellular pathways, including p53 signaling and cytokine action. The purpose of the current study was to evaluate the effects of tumor necrosis factor alpha (TNF-alpha) on p53 and p53 up-regulated modulator of apoptosis (PUMA), a downstream effector of p53 in the apoptotic pathway in colorectal cancer cells., Methods: The cell lines HT29 (which express mutant p53) and HCT116 (which express wild-type p53) were treated with TNF-alpha (0, 50, 100, or 500 ng/mL) for 1, 12, 24, or 48 hours. Protein expression and subcellular localization of p53 and PUMA were determined by immunoblot and immunofluorescence. Changes in p53 and PUMA mRNA expression were determined by quantitative real time polymerase chain reaction., Results: Nuclear p53 expression was increased in TNF-alpha-treated HT29 cells; in contrast, expression was decreased or minimally changed in TNF-alpha-treated HCT116 cells, as determined by immunoblot and immunofluorescence. At 24 hours, p53 mRNA transcript levels were minimally increased in HT29 cells, whereas PUMA increased 34-fold., Conclusions: TNF-alpha increased nuclear p53 expression in HT29 cells, which express p53 mutation, but not in HCT116 cells, which are wild type for p53. In addition, TNF-alpha markedly up-regulated PUMA mRNA levels in HT29 cells. Our findings suggest that TNF-alpha may be a factor in carcinogenesis in IBD in cells carrying a p53 mutation.

Cultured cell lines have played an integral role in the study of tumor biology since the early 1900's. The purpose of this study is to evaluate colorectal cancer (CRC) cell lines with respect to progenitor tumors and assess whether these cells accurately and reliably represent the cancers from which they are derived. Primary cancer cell lines were derived from fresh CRC tissue. Tumorigenicity of cell lines was assessed by subcutaneous injection of cells into athymic mice and calculation of tumor volume after 3 weeks. DNA ploidy was evaluated by flow cytometry. Invasive ability of the lines was tested with the MATRIGEL invasion assay at 24 or 48 hours. Cells were assessed for the presence of Kirsten-Ras (K-Ras), p-53, deleted in colon cancer (DCC), and Src. Protein profiling of cells and tissue was performed by surface enhanced laser desorption/ionization-time of flight/mass spectroscopy. microRNA expression in cell and tumor tissue samples was evaluated by FlexmiR MicroRNA Assays. Four cell lines were generated from tumor tissue from patients with CRC and confirmed to be tumorigenic (mean tumor volume 158.46 mm(3)). Two cell lines were noted to be diploid; two were aneuploid. All cell lines invaded the MATRIGEL starting as early as 24 hours. K-Ras, p53, DCC, and Src expression were markedly different between cell lines and corresponding tissue. Protein profiling yielded weak-to-moderate correlations between cell samples and respective tissues of origin. Weak-to-moderate tau correlations for levels of expression of human microRNAs were found between cells and respective tissue samples for each of the four pairings. Although our primary CRC cell lines vary in their expression of several tumor markers and known microRNAs from their respective progenitor tumor tissue, they do not statistically differ in overall profiles. Characteristics are retained that deem these cell lines appropriate models of disease; however, data acquired through the use of cell culture may not always be a completely reliable representation of tumor activity in vivo.

Aim: To replicate the association of Nkx2-3 rs10883365 SNP with Crohn's disease in patients from a familial IBD registry from the central Pennsylvania area and study mRNA and protein expression of Nkx2-3 in CD patients., Materials and Methods: We genotyped the Nkx2-3 rs10883365 SNP in 75 CD patients,137 non-CD family members and 118 unrelated healthy controls from EBV-transformed B cell lines of a familial IBD registry in central Pennsylvania. mRNA and protein expression levels of Nkx2-3 were measured by RT-PCR and Western blot, respectively., Results: rs10883365 was found to be associated with CD. A significant difference between the homozygous variant genotype (GG) compared to the wild type sequence (AA) was observed between CD and individuals without IBD, including both non-IBD family members from the familial IBD registry and unrelated healthy controls. However, there was not a significant difference between CD and non-IBD related family members. mRNA and protein expression levels of Nkx2-3 were increased in CD compared with non-CD sibling and healthy controls. A total of 16 sibling pairs were examined, and the mRNA and protein expression levels of Nkx2-3 from 12 of the sibling pairs (75%) were increased in the CD individual compared with the non-CD sibling. mRNA expression levels of Nkx2-3 were increased in diseased tissues compared with adjacent normal tissues in 7 of 9 patients (77.8%)., Conclusions: Nkx2-3 is genetically associated with CD and is up-regulated in CD, suggesting that Nkx2-3 is involved in the pathogenesis of CD.

Background/objectives: Despite multiple options for operative repair of parastomal hernia, results are frequently disappointing. We review our experience with parastomal hernia repair., Methods: A retrospective chart review was performed on all patients with parastomal hernia who underwent LAP or open repair at our institution between 1999 and 2006. Information collected included demographics, indication for stoma creation, operative time, length of stay, postoperative complications, and recurrence., Results: Twenty-five patients who underwent laparoscopic or open parastomal hernia repair were identified. Laparoscopic repair was attempted on 12 patients and successfully completed on 11. Thirteen patients underwent open repair. Operative time was 172+/-10.0 minutes for laparoscopic and 137+/-19.1 minutes for open cases (P=0.14). Lengths of stay were 3.1+/-0.4 days (laparoscopic) and 5.1+/-0.8 days (open), P=0.05. Immediate postoperative complications occurred in 4 laparoscopic patients (33.3%) and 2 open patients (15.4%), P=0.38. Parastomal hernia recurred in 4 laparoscopic patients (33.3%) and 7 open patients (53.8%) after 13.9+/-4.5 months and 21.4+/-4.3 months, respectively, P=0.43., Conclusion: Laparoscopic modified Sugarbaker technique in the repair of parastomal hernia affords an alternative to open repair for treating parastomal hernia.

Background: Pyoderma gangrenosum (PG) occurs in about 1% to 5% of patients with inflammatory bowel disease (IBD). Peristomal pyoderma gangrenosum (PPG) is particularly difficult to manage., Study Design: A retrospective chart review was performed on all patients with IBD in whom PPG developed from 1997 to 2007 at the Milton S Hershey Medical Center., Results: Sixteen patients (11 women) were identified. Seven had Crohn's disease (CD), seven had ulcerative colitis (UC), and two had indeterminate colitis. Six patients underwent total proctocolectomy, six patients had total abdominal colectomy (TAC), and four patients had diverting loop stomas. PPG occurred an average of 18.4+/-7.5 months after stoma creation. Twelve patients had active IBD when PPG developed. Two patients had stoma revisions and both had recurrence of the PPG with the new stoma. Medical therapy was successful in eight patients. Five patients had their stomas closed, with active PPG, and all five resolved their lesions. In four of five, surgical management was altered because of PPG (one early stoma closure, two ileal pouches without stomas, one ileal pouch with high body mass index). Of the seven and six patients treated with cyclosporine or infliximab, respectively, there were only two successes with each., Conclusions: PPG is more common in the presence of active IBD. Surgical closure of the stoma was successful in resolving PPG in all patients. Cure rate of PPG was poor with cyclosporine and only marginally better with infliximab. Medical treatment of PPG is imperfect, and the best therapy is stoma closure when possible.

Background: The claudins are tight junction (TJ) proteins. Claudin-2 has been found to negatively affect the TJ, causing a decrease in transepithelial resistance. Patients with inflammatory bowel disease have altered intestinal permeability, suggesting a TJ disruption. Interferon-gamma (IFNgamma) and interleukin-4 (IL-4) negatively regulate each other and may have opposing roles in inflammatory bowel disease., Hypothesis: IFNgamma and IL-4 will have opposing effects on the expression of claudin-2., Methods: Confluent T84 monolayers were apically incubated with IFNgamma or IL-4. The monolayers were immunofluorescently stained or lysed for Western blot with anti-claudin-2 or -4. Additional monolayers were grown on transwell plates, treated with IFNgamma or IL-4, measured for changes in transepithelial resistance, and assayed for changes in permeability using FITC-dextran-4000. Statistics were calculated by analysis of variance., Results: Addition of IFNgamma to T84 monolayers resulted in decreased claudin-2 and addition of IL-4 resulted in increased claudin-2 by Western blot. By immunofluorescence, there was a loss of claudin-2 from the membrane in cells treated with IFNgamma. Transepithelial resistance across T84 monolayers increased with IFNgamma and decreased with IL-4. T84 monolayer permeability increased with IL-4 but not with IFNgamma., Conclusions: Incubation of T84 cells with IL-4 leads to increased claudin-2 with a corresponding decrease in transepithelial resistance and increase in permeability. Incubation of T84 cells with IFNgamma leads to decreased claudin-2 and increased transepithelial resistance. These cytokines have opposite effects on the expression of claudin-2 and the physiology of the TJ.

Background: Inflammatory bowel disease (IBD) is associated with increased intestinal permeability and decreased expression of tight junction (TJ) proteins in the inflamed mucosa. Whether this alteration in TJ expression is a prerequisite for the development of intestinal inflammation or a secondary result of that inflammation is unknown. This study looked at the expression of the TJ protein ZO-1 and the corresponding permeability changes in dextran sulfate sodium (DSS) induced colitis in a mouse model., Materials and Methods: BALB/c mice were fed 3% DSS or water for 1, 3, 5, or 7 days. The animals were weighed, stool was checked for blood, and the colon length measured. Segments of the colon were used for histology, immunohistochemistry for ZO-1, or Western blot for TJ proteins. Colonic permeability was measured using Evan's Blue dye., Results: DSS treated animals had heme positive stools, colitis by histology, significant weight loss, and colon shortening. There was an absence of ZO-1 by Western blot in the 7-day DSS treated animals, double the amount of claudin-1 and normal cytokeratin. The loss of ZO-1 started after 1 d of DSS treatment and was followed by a significant increase in permeability to Evan's blue by day 3., Conclusions: The loss of ZO-1 and increased permeability preceded the development of significant intestinal inflammation suggesting that in DSS colitis alterations in the TJ complex occur before the intestinal inflammation and not as a consequence of it. These changes in the TJ complex may facilitate the development of the inflammatory infiltrate seen in colitis.

Purpose: Low molecular mass polypeptide 2 is an inducible immunoproteasome subunit. The expression of low molecular mass polypeptide 2 has not been examined in the intestine of patients with inflammatory bowel disease. This study was designed to determine whether the intestinal expression of low molecular mass polypeptide 2 was enhanced in a group of patients with inflammatory bowel disease compared with a group of control patients without inflammatory bowel disease. Moreover, we examined the association between low molecular mass polypeptide 2 expression and histologic pathology in these patients., Methods: Twenty-one patients participated in the study. These included six control subjects without inflammatory bowel disease, eight patients with ulcerative colitis, and seven patients with Crohn's disease. Intestinal low molecular mass polypeptide 2 expression was evaluated by immunohistochemistry, as well as by Western blot. Histology scores (0-40 severity scale) were determined on the same sections of intestine as those used for low molecular mass polypeptide 2 histochemistry., Results: By immunohistochemistry, low molecular mass polypeptide 2 expression was significantly enhanced (P < 0.05 vs. control subjects) throughout visibly diseased areas of colon, rectum, and ileum from patients with inflammatory bowel disease. Low molecular mass polypeptide 2 expression also was increased in macroscopically normal intestine from patients with inflammatory bowel disease compared with normal tissue from control subjects. There was a significant correlation (P < 0.0001) between low molecular mass polypeptide 2 expression and histologic pathology in our patients. Western blot results confirmed that low molecular mass polypeptide 2 expression was enhanced in patients with ulcerative colitis (3.1-fold) and in patients with Crohn's disease (3.5-fold)., Conclusions: Intestinal low molecular mass polypeptide 2 expression is significantly increased in inflammatory bowel disease. The association between intestinal low molecular mass polypeptide 2 expression and histologic pathology suggests that this proteasome subunit plays a role in the pathogenesis of inflammatory bowel disease.

Background: Historical studies have shown that percutaneous drainage alone for intraabdominal abscess secondary to Crohn's disease is successful in avoiding surgery in only approximately 50% of patients. Failure, however, can require urgent surgery and is then associated with increased morbidity, extended hospital stays, and increased risk for stoma creation. Because of this, our current protocol is initial percutaneous drainage of the abscess, 5-7 days of broad spectrum IV antibiotics with simultaneous high-dose steroids and hyperalimentation, followed by planned one stage resection with primary anastomosis. The aim of the present study was to evaluate the success of this protocol with regard to length of stay, complications associated with the protocol, and its ability to avoid stoma creation., Methods: A retrospective chart review was performed for all Crohn's disease patients with intraabdominal abscess who underwent the above protocol from 1992 to the present., Results: Nineteen patients (11 male) were identified. Sixteen underwent ileocolectomy with primary anastomosis while only three patients required an upstream diverting ileostomy in addition to resection due to incompletely drained abscesses. The mean length of hospital stay was 13.9 +/- 0.6 days including 6.4 +/- 0.4 postoperative days. Four patients had post-op complications that did not require surgery (two self-limited anastomotic bleeds, one wound infection, and one pelvic abscess treated with a percutaneous drain). One patient needed reoperation for a small bowel obstruction., Conclusions: Crohn's disease patients with intraabdominal abscess can safely undergo planned resection with primary anastomosis if initially treated with successful percutaneous drainage and aggressive antibiotic and steroid management. Such a protocol provides a standard of care against which nonsurgical management can be compared and judged.

In the current medical climate, there is increased pressure on colon and rectal surgeons to generate clinical revenue and decreased availability of time and resources to do research. For faculty who want to pursue research as part of an academic career, this article reviews some of the issues involved. The first half of the article discusses key issues in choosing the right job, such as institutional/departmental support and resources. The second half of the article discusses what is needed to make a research program successful, including funding mechanisms, mentors, collaborators, and the different requirements for basic science versus clinic research.

Purpose: Intravenous cyclosporine often is used to treat patients with severe steroid refractory colitis secondary to ulcerative colitis in an attempt to avoid urgent total abdominal colectomy. The purpose of this study was to evaluate the success and cost of cyclosporine., Methods: A retrospective, chart review of all patients from 1996 to 2002 who were treated with cyclosporine and/or had a three-stage ileal pouch-anal anastomosis for severe steroid refractory colitis at our institution was performed. Patients were divided into three groups: TAC and CyA: patients who failed cyclosporine and had urgent total abdominal colectomy on the same admission; TAC no CyA: patients who had an urgent total abdominal colectomy without cyclosporine; and CyA only: patients treated successfully with cyclosporine and discharged without surgery. A subgroup of patients who had an ileal pouch-anal anastomosis was identified from each group. Cost data were obtained from the hospital's financial records., Results: Forty-one patients (25 males) were identified. Twenty-nine patients received cyclosporine for severe steroid refractory colitis. Of these, 18 (62 percent) failed and underwent total abdominal colectomy on the same admission. Eleven (38 percent) responded to the cyclosporine and were discharged. Of the 11, 4 never had surgery, 1 had a three-stage ileal pouch-anal anastomosis, 5 had a two-stage ileal pouch-anal anastomosis, and 1 had a total abdominal colectomy only. Only 14 percent of patients avoided colectomy in the long-term. Complications of cyclosporine occurred in 8 patients (28 percent), and surgical complications occurred in 12 patients., Conclusions: The highest costs, highest length of stay, and highest number of overall complications were found in the group of patients who failed intravenous cyclosporine and required colectomy during the same hospitalization.

Purpose: A three-stage operative approach to ileal pouch-anal anastomosis is usually undertaken in patients presenting with severe colitis. Increasingly, however, we have performed a two-stage modified ileal pouch-anal anastomosis (colectomy followed by ileal pouch-anal anastomosis without ileostomy). The present study sought to evaluate the safety, results, cost, and length of hospital stay using this modified approach compared to that of the traditional three-stage ileal pouch-anal anastomosis., Methods: Clinical and financial data were gathered by retrospective review of patients undergoing ileal pouch-anal anastomosis at our institution since 1995. Complications were defined as any event prolonging hospitalization or requiring readmission and were included in the analysis up to six months after final surgery. Functional performance was assessed as of the last clinic visit. Data were compared with Student's t-test and chi-squared analysis. Multivariate analysis was also used to assess risk factors., Results: A total of 23 patients who underwent the two-stage modified procedure and 31 patients who had the three-stage procedure were identified. The two groups were found to be statistically comparable in terms of patient age, gender, duration of illness, and preoperative hematocrit. Follow-up was shorter in the modified group because of its more recent introduction (9.7 months vs. 30.5 months mean follow-up). Ninety-five percent of patients were on immunosuppressive medication before colectomy, but all were off it before the reconstruction. clinical outcomes after ileal pouch-anal anastomosis were equivalent in terms of the number of bowel movements, prevalence of fecal incontinence, and the use of hypomotility medications. No patients with the two-stage modified procedure had anastomotic complications requiring stoma creation. One patient in the three-stage group required re-creation of a stoma after stoma closure for perianal complications suggesting Crohn's disease. Total hospital cost was significantly less in the modified group: USD $27,270 vs. $38,184 (P = 0.0119). Length of stay was also shorter in the two-stage modified group although missing absolute statistical significance (21.0 days vs. 26.0 days, P = 0.0882)., Conclusions: Interval ileal pouch-anal anastomosis reconstruction without a stoma (two-stage modified procedure) after colectomy is functionally equivalent to the traditional three-stage protocol in terms of clinical outcome. However, it has the advantage of overall lower hospital costs and probably a shorter length of hospital stay.

Background: Inflammatory bowel disease (IBD) is associated with altered apoptosis and increased levels of Th1 cytokines (IL-12, TNF-alpha, and IFN-gamma). These proinflammatory events may result from dysfunctional IL-4/Stat6 signal transduction that normally promotes Th2 lymphocyte differentiation and consequential down-regulation of the immune response. The goal of the present study was to measure apoptosis, levels of relevant cytokines, and the effects of cytokine manipulation on apoptosis in cell lines derived from IBD patients that express dysfunctional Stat6 (Stat6(null phenotype)) and wild-type Stat6 (Stat6(high phenotype))., Materials and Methods: Lymphocytes with Stat6(null phenotype) (n = 5) or wild-type (n = 5) status were cultured with and without the addition of exogenous cytokines or neutralizing antibodies (IL-12, TNF-alpha, and IFN-gamma). Apoptosis was determined by flow cytometry using Annexin V-PE dual staining. Cytokine levels were determined by ELISA., Results: Stat6(null phenotype) cells exhibited increased apoptosis compared with wild-type cell lines (13.3% +/- 2.9 versus 4.5% +/- 0.4, P < 0.0001). Four of five Stat6(null phenotype) cell lines expressed 5- to 10-fold elevations in IL-12 and IFN-gamma. Addition of exogenous cytokines or neutralizing antibodies had no effect on apoptosis., Conclusions: Apoptotic cell death is elevated in Stat6(null phenotype) cell lines suggesting a role for Stat6 in apoptosis regulation, a previously unrecognized observation. Increased levels of IL-12 and IFN-gamma were found in the Stat6(null phenotype) cell lines; however, the apoptosis observed is not the consequence of increased IL-12, IFN-gamma, or TNF-alpha. Stat6(null phenotype) cell lines exhibit variably increased levels of these Th1 cytokines, consistent with their human source, and may be a valid source for investigations into IBD pathophysiology.

Background and Aims: Fistulous disease is common in Crohn's disease, and entero- and colocutaneous fistulae are particularly debilitating and difficult to manage. We present the results of surgical management of these fistulas., Patients and Methods: Retrospective chart review of all 51 patients with Crohn's disease (56 surgical procedures) undergoing surgery for cutaneous fistulae between 1983 and 2000., Results: Previous surgery for Crohn's disease had been carried out in 43 patients (84%). The fistula site was enterocutaneous in 36 patients (64%), colocutaneous in 12 (21%), and anastomotic in 8 (14%); 9 patients (16%) also had associated enteroenteric fistulas. The onset of the fistula followed abscess drainage in 15 (27%) and occurred at the site of recurrent disease in 41 (73%). Forty patients (71%) initially underwent conservative management prior to surgery; 16 (28%) underwent surgery directly. Surgical procedures were: 25 ileocolic resections, 8 stoma revisions with resection, 8 small bowel resections 7 subtotal colectomies, 4 partial colectomies, 3 proctocolectomies, and one fistula tract excision. Mean total length of stay was 18 days (postoperative 10.7 days). Six (11%) patients had eight postoperative complications. Mean follow-up was 48.6 months (range 3-187). Recurrence as defined by either clinical examination or reoperation was documented in nine fistulas (16%), with a mean time to recurrence of 27 months., Conclusion: Entero-and colocutaneous fistulae usually occur from a site of active disease. Surgical management with bowel resection, including the fistula, is the preferred method of treatment. Morbidity has been low and recurrence rate lower than expected., (Copyright 2004 Springer-Verlag)

Based on Stat6 gene knockout animal models, the Stat6 signaling pathway has been suggested to play a role in carcinogenesis and Th1/Th2 cytokine balance. Using a semiquantitative EMSA assay and EBV-transformed human B lymphoblast cell lines, we have previously identified three Stat6 activational phenotypes, termed as Stat6high, Stat6low, and Stat6null. A genetic mechanism has been proposed which determines the IL-4-induced activation of the human Stat6 signaling. With respect to the contribution of variant phenotypes to human disease, we further hypothesize that the Stat6null phenotype may result from a partial defect in Stat6 signaling which resembles Stat6 knockout animals in several functional aspects. The characterization of the human Stat6null phenotype stably displayed by the EBV-B cell lines is easily assailable and possesses important implications with respect to Th1/Th2 cytokine imbalance in diseases such as cancer development/metastasis and inflammatory diseases. In this study, we have extended our investigation to the downstream regulatory consequences associated with these Stat6 phenotypes. Production of three important proinflammatory cytokines, IL-12, TNFalpha and IFNgamma was examined in spontaneous EBV-B cell culture using ELISA methodology. Individual cell lines defined as Stat6null produced significantly higher levels of IL-12, TNFalpha and IFNgamma on day 4 in spontaneous culture in comparison with cell lines characterized as Stat6high and Stat6low. These observations of the human Stat6null phenotype, together with those accruing from Stat6 knockout mouse model studies, suggest that the Stat6 signaling pathway may play a role in maintaining the Th1/Th2 cytokine balance by directly and indirectly down-regulating the production of proinflammatory cytokines, a regulatory process which appears to go awry in inflammatory diseases. Moreover, observations from signal transduction studies in our human B lymphocyte model may be compatible with those in the chosen mouse B lymphocyte for establishing signaling networks by the Alliance for Cellular Signaling (AfCS).

Background: We have previously shown an increase in intestinal permeability and a corresponding decrease in the expression of tight junction (TJ) proteins in the in testines of patients with Crohn's disease (CD). Tumor necrosis factor-alpha (TNFalpha) has been implicated in the inflammatory process of CD and its suppression has therapeutic benefit. ZO-1, occludin, and the claudins are key proteins in the TJ., Hypothesis: TNFalpha disrupts the TJ., Methods: MDCK cells were incubated with TNFalpha (0-100 ng/ml) for 5 days. Qualitative evaluation of the TJ was done with monoclonal antibody to ZO-1 detected by an immunofluorescence. Duplicate cells were lysed and ZO-1, occludin, and claudin-1 amount determined by western blot., Results: Immunofluorescent staining of MDCK cells for ZO-1 showed TJ structural disruption with increasing amount of TNFalpha characterized by fragmented staining of ZO-1. There were no significant differences in quantitation of ZO-1 or occludin in the MDCK cells for all TNFalpha concentrations. There was a significant decrease in the amount of claudin-1 with increasing concentration of TNFalpha., Conclusions: (1) MDCK TJs are qualitatively disrupted by TNFalpha. (2) This disruption is not because of a decrease in cell number, lack of cell layer confluency, or a decrease in the amount of ZO-1 or occludin. (3) The amount of claudin-1 present in the cell is decreased with increasing amounts of TNFalpha suggesting that the lack of claudin-1 may cause a relocation of ZO-1 away from the TJ. (4) This rearrangement may play a role in the increased intestinal permeability seen in CD and other diseases.

Introduction: The surgical management of ulcerative colitis in the patient with primary sclerosing cholangitis is controversial., Methods: This study was designed as a retrospective chart review of all patients with primary sclerosing cholangitis who were surgically treated for ulcerative colitis., Results: Sixteen patients with primary sclerosing cholangitis and ulcerative colitis were identified. The indication for ulcerative colitis surgery was dysplasia in 7 patients (44 percent), cancer in 2 (13 percent), intractability in 4 (25 percent), and unknown in 1. Final colon pathology demonstrated cancer in three patients and dysplasia in four. Two patients had biliary cancer discovered at the time of orthotopic liver transplantation. Thirteen patients were known to have primary sclerosing cholangitis when they underwent surgery for ulcerative colitis; two patients with severe primary sclerosing cholangitis underwent simultaneous orthotopic liver transplantation/total abdominal colectomy and did well with subsequent ileal pouch reconstruction. Two patients had orthotopic liver transplantation first and then ileal pouch-anal anastomosis (1 patient) or total abdominal colectomy (1 patient) and did well. Seven patients had well-controlled primary sclerosing cholangitis on medication and underwent ileal pouch-anal anastomosis or total abdominal proctocolectomy without significant hepatic compromise. One patient with moderate primary sclerosing cholangitis underwent ileorectal anastomosis and had severe liver failure postoperatively but survived. Another patient with worsening primary sclerosing cholangitis after total abdominal colectomy has since developed persistent bleeding from peristomal varices., Conclusions: The overall cancer/premalignant lesion rate was high (50 percent in this study) in patients with primary sclerosing cholangitis and ulcerative colitis. Complications associated with the surgical management of ulcerative colitis are largely dictated by the degree of liver disease present at the time of surgery. Patients with significant primary sclerosing cholangitis that requires colectomy can undergo simultaneous orthotopic liver transplantation/total abdominal colectomy and then be candidates for subsequent ileal pouch-anal anastomosis reconstruction once liver function has improved. Patients with well-controlled primary sclerosing cholangitis can undergo ileal pouch-anal anastomosis surgery safely.

The operative treatment of elderly patients with inflammatory bowel disease (IBD) has often been avoided in favor of medical management because of a perceived increase in surgical risk. This study sought to define the following in the elderly IBD patient population: (1) the risk of surgical management and (2) those factors affecting risk. Thirty patients with IBD, aged 60 years or more, who were surgically managed by a single surgeon over a 10-year period, were retrospectively matched to 75 patients with IBD who were less than 60 years of age; patients were matched according to sex, date of surgery, and type of surgery performed. Regression analysis using generalized estimating equation methodology to account for the matched clusters of patients was performed to evaluate the effect of age group on the complication rate, operating room time, and length of hospital stay. Presence of comorbid conditions, surgical indications, prior surgery for IBD, and the use of immunosuppressive medications were studied in multivariate models, adjusting for age group. By means of univariate analysis, the odds of complications in elderly IBD patients were shown to be statistically higher than the odds seen in younger patients (47% vs. 20%, P = 0.01). Also observed in the elderly group were a longer length of hospital stay (11.5 days vs. 7.1 days, P = 0.001) and longer operating room time (249 minutes vs. 212 minutes, P = 0.02). Multivariate analysis revealed that the effect of age remained statistically significant, even when adjusted for potential confounding variables such as comorbidity, medications, date of diagnosis of IBD, and indications for surgery. The complication outcome was significantly associated with the surgical indication, with obstruction, fistula, and bleeding having increased odds of complications as compared with other indications (odds ratio = 1.7 vs. 4.2 vs. 7.2, respectively, P = 0.02). The length of hospital stay similarly was significantly associated with the surgical indication (fistula, 10.5 days vs. bleeding, 9.8 days vs. obstruction, 7.4 days vs. other, 9.3 days; P = 0.04) and a history of prior surgery. A significant interaction for length of hospital stay was present between age group and prior surgery status (with prior surgery: old, 18 days vs. young, 6.4 days, P = 0.0001; without prior surgery: old, 9.5 days vs. young 7.3 days, P = 0.10). Elderly patients with IBD have an increased rate of postoperative complications along with an increased length of hospital stay and increased operating room time. This effect of age persists when adjusted for comorbidity and immunosuppressive therapy. Complications are most dependent on surgical indications, with obstruction being the least and bleeding the worst predictive factors. The longest hospital stay is associated with patients who require surgery for fistulous disease and patients who have undergone previous surgery. The fact that the higher complication rate seen in older patients with IBD is associated with disease-defined surgical indications suggests that IBD in elderly patients may be more aggressive than what is observed in younger individuals.

Purpose: Tumor necrosis factor antagonist therapy in the form of infliximab has been shown to promote significant healing in fistulizing Crohn's disease and therefore is often considered as a possible alternative to surgery. Our aim was to evaluate the role of infliximab in supplanting surgery for fistulizing Crohn's disease., Methods: We performed a retrospective chart review of all adult patients who received infliximab for fistulizing Crohn's disease at one institution between September 1998 and October 2000., Results: Twenty-six patients (14 male; mean age, 38 years; range, 19-80 years) received a mean of three (range, one to six) doses of infliximab (5 mg/kg) with the intent to cure fistulizing Crohn's disease. Nine patients (35 percent) had perianal, 6 (23 percent) enterocutaneous, 3 (12 percent) rectovaginal, 4 (15 percent) peristomal, and 4 (15 percent) intra-abdominal fistulas. Nineteen (73 percent) of the patients had had prior surgery for Crohn' s disease. Six patients (23 percent) had a complete response to infliximab with fistula closure, 12 (46 percent) had a partial response, and 8 (31 percent) had no response to infliximab. Fourteen (54 percent) patients still required surgery for their fistulizing Crohn's disease after infliximab therapy (10 bowel resections, 4 perianal procedures), whereas half (6/12) of the patients treated with infliximab who still had open fistulas after treatment declined surgical intervention. Five of six patients with fistula closure on infliximab had perianal or rectovaginal fistulas. None of the patients with either enterocutaneous or peristomal fistulas were healed with infliximab., Conclusions: Although it was associated with a 61 percent complete or partial response rate, infliximab therapy did not supplant the need for surgical intervention in the majority of our patients with fistulizing Crohn's disease. Seventy-three percent of the patients either required surgery or still had open fistulas after infliximab therapy. Infliximab was much more effective in treating perianal disease than abdominal enterocutaneous disease.