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Inflammatory bowel diseases (IBD) are affected by dietary factors, including nondigestible carbohydrates (fibers), which are fermented by colonic microbes. Fibers are overall beneficial, but not all fibers are alike, and some patients with IBD report intolerance to fiber consumption. Given reproducible evidence of reduced fiber-fermenting microbes in patients with IBD, we hypothesized that fibers remain intact in select patients with reduced fiber-fermenting microbes and can then bind host cell receptors, subsequently promoting gut inflammation.Colonic biopsies cultured ex vivo and cell lines in vitro were incubated with oligofructose (5 g/L), or fermentation supernatants (24-hour anaerobic fermentation) and immune responses (cytokine secretion [enzyme-linked immunosorbent assay/meso scale discovery] and expression [quantitative polymerase chain reaction]) were assessed. Influence of microbiota in mediating host response was examined and taxonomic classification of microbiota was conducted with Kraken2 and metabolic profiling by HUMAnN2, using R software.Unfermented dietary β-fructan fibers induced proinflammatory cytokines in a subset of IBD intestinal biopsies cultured ex vivo, and immune cells (including peripheral blood mononuclear cells). Results were validated in an adult IBD randomized controlled trial examining β-fructan supplementation. The proinflammatory response to intact β-fructan required activation of the NLRP3 and TLR2 pathways. Fermentation of β-fructans by human gut whole microbiota cultures reduced the proinflammatory response, but only when microbes were collected from patients without IBD or patients with inactive IBD. Fiber-induced immune responses correlated with microbe functions, luminal metabolites, and dietary fiber avoidance.Although fibers are typically beneficial in individuals with normal microbial fermentative potential, some dietary fibers have detrimental effects in select patients with active IBD who lack fermentative microbe activities. The study is publicly accessible at the U.S. National Institutes of Health database (clinicaltrials.gov identification number NCT02865707).

BACKGROUND & AIMS: Dysplasia carries a high risk of cancer development, however, the cellular mechanisms for dysplasia evolution to cancer are obscure. We have previously identified two putative dysplastic stem cell (DSC) populations, CD44v6(neg)/CD133(+)/CD166(+) (DP) and CD44v6(+)/CD133(+)/CD166(+) (TP), which may contribute to cellular heterogeneity of gastric dysplasia. Here, we investigated functional roles and cell plasticity of non-cancerous Trop2(+)/CD133(+)/CD166(+) DSCs initially developed in the transition from pre-cancerous metaplasia to dysplasia in the stomach. METHODS: Dysplastic organoids established from active Kras-induced mouse stomachs were utilized for transcriptome analysis, in vitro differentiation and in vivo tumorigenicity assessments of DSCs. Cell heterogeneity and genetic alterations during clonal evolution of DSCs were examined by next-generation sequencing. Tissue microarrays were used to identify DSCs in human dysplasia. We additionally evaluated the effect of CK1α regulation on the DSC activities using both mouse and human dysplastic organoids. RESULTS: We identified a high similarity of molecular profiles between DP- and TP-DSCs, but more dynamic activities of DP-DSCs in differentiation and survival for maintaining dysplastic cell lineages through Wnt ligand-independent CK1α/β-catenin signaling. Xenograft studies demonstrated that the DP-DSCs clonally evolve towards multiple types of gastric adenocarcinomas and promote cancer cell heterogeneity by acquiring additional genetic mutations and recruiting the tumor microenvironment. Lastly, growth and survival of both mouse and human dysplastic organoids were controlled by targeting CK1α. CONCLUSIONS: These findings indicate that the DSCs are de novo gastric cancer-initiating cells responsible for neoplastic transformation and a promising target for intervention in early induction of gastric cancer.

Background & aims Despite advances in gene editing technologies, generation of tissue-specific knockout mice is time-consuming. We used CRISPR/Cas9-mediated genome editing to disrupt genes in livers of adult mice in just a few months, which we refer to as somatic liver knockouts. Methods In this system, Fah–/– mice are given hydrodynamic tail vein injections of plasmids carrying CRISPR/Cas9 designed to excise exons in Hpd; the Hpd-edited hepatocytes have a survival advantage in these mice. Plasmids that target Hpd and a separate gene of interest can therefore be used to rapidly generate mice with liver-specific deletion of nearly any gene product. Results We used this system to create mice with liver-specific knockout of argininosuccinate lyase, which develop hyperammonemia, observed in humans with mutations in this gene. We also created mice with liver-specific knockout of ATP binding cassette subfamily B member 11, which encodes the bile salt export pump. We found that these mice have a biochemical phenotype similar to that of Abcb11–/– mice. We then used this system to knock out expression of 5 different enzymes involved in drug metabolism within the same mouse. Conclusions This approach might be used to develop new models of liver diseases and study liver functions of genes that are required during development.

Background & Aims Chronic pancreatitis is characterized by alternating phases of acute inflammation and quiescent disease. Involvement of T-cell responses has been suggested, but pancreatitis-specific T cells have not been described. Methods We characterized T-cell responses against pancreatitis, pancreatic carcinoma-associated antigens, and tetanus toxoid in the bone marrow, blood, and/or pancreatitis lesions of patients with pancreatitis, pancreatic cancer, and healthy individuals. T cells were functionally characterized by antigen-dependent secretion of interferon (IFN)-γ, interleukin (Il)-4, and IL-10, which indicate type 1, type 2, or regulatory T-cell responses, respectively. Regulatory T cells were characterized by multicolor flow cytometry. Isolated regulatory T cells were tested for their capacity to recognize pancreatitis-associated antigens and to suppress conventional T cells in an antigen-dependent manner. T cell-derived cytokines in tissue lesions were quantified by enzyme-linked immunosorbent assay. Results Chronic pancreatitis patients showed similar to pancreatic cancer patients and healthy individuals type 1 T-cell responses against tetanus toxoid; however, they exhibited strong IL-10-based T-cell responses against pancreatitis-associated but not pancreatic carcinoma-associated antigens. T cells from pancreatic cancer patients responded to pancreatic cancer-associated but not pancreatitis-associated antigens with IFN-γ secretion. Pancreatitis-specific IL-10 responses were mediated by IL-10+IFN-γ−FoxP3+ regulatory T cells, which were expanded in the blood, bone marrow, and pancreatitis lesions and possessed the potential to suppress the proliferation of autologous conventional T cells in an antigen-specific manner. Pancreatitis lesions, in comparison with pancreatic carcinomas, contained increased concentrations of IL-10 and reduced levels of IFN-γ, suggesting pancreatitis-specific activity of regulatory T cells in situ. Conclusions Chronic pancreatitis is associated with disease-specific regulatory T-cell responses.

Background & Aims This study aimed to identify the predictors for early hepatitis B e antigen (HBeAg) seroconversion following acute exacerbation (AE) in patients with HBeAg-positive chronic hepatitis B (CHB). Methods A total of 151 patients with HBeAg-positive CHB and AE were consecutively enrolled. AE was defined as an elevation of alanine aminotransferase level to more than 5 times the upper limit of normal and more than twice the patients' baseline value. Early HBeAg seroconversion was defined as HBeAg loss and the appearance of hepatitis B e antibody within 6 months of AE. Clinical and laboratory data were compared between an early HBeAg seroconversion group and a nonearly HBeAg seroconversion group. Results All patients had genotype C hepatitis B virus (HBV). Early HBeAg seroconversion occurred in 35.5% (39/110). Under univariate analysis, age, transmission mode, disease status, and serum HBV DNA level were associated with early HBeAg seroconversion. Multivariate analysis showed that nonvertical transmission mode (RR, 3.681; 95% CI, 1.279–10.592; P = .016) and low serum HBV DNA levels (≤ log [copies/mL]; RR, 6.891; 95% CI, 2.165–21.935; P = .001) were independent predictors. Conclusions Patients with CHB with genotype C may have a higher chance of early HBeAg seroconversion after AE in the context of nonvertical transmission and/or had low serum HBV DNA levels (≤7 log [copies/mL]) at AE. Therefore, we should take into account transmission modes and serum HBV DNA levels when choosing appropriate management strategies for patients who exhibit AE of HBeAg-positive CHB with genotype C.

DCC and UNC5C, Netrin-1 dependence receptors, perform an important role in intestinal epithelial biology. Both receptors frequently are down-regulated in colorectal cancer (CRC). Although CRCs frequently lose DCC owing to deletions at 18q, the mechanism for the UNC5C loss is poorly understood. We hypothesized that UNC5C is silenced epigenetically in CRC, and that there are interactions between losses of UNC5C and DCC in colorectal tumorigenesis.Gene expression and epigenetic analysis of UNC5C was examined in 8 CRC cell lines, 147 sporadic CRCs with corresponding normal mucosa, and 52 adenomatous polyps (APs). Allelic imbalances at DCC were determined in CRCs. The molecular analyses were compared with genetic and clinicopathologic features.All CRC cell lines showed UNC5C methylation and an associated loss of gene expression. Treatment with 5-Aza-2'-deoxycytidine resulted in restoration of gene transcription. UNC5C methylation was significantly higher in CRCs (76.2%) and APs (63.5%) than in corresponding normal mucosa (6%; P.0001). Allelic imbalance at DCC was observed in 61% of CRCs. Overall, 89.3% of CRCs had alterations of one of the dependence receptors. UNC5C methylation occurred predominantly in the earlier lesions (APs and early CRCs), whereas DCC losses were more often in advanced CRCs.The majority of CRCs harbor defects in Netrin-1 receptors, emphasizing the importance of this growth regulatory pathway in cancer. Furthermore, the timing of the molecular alterations in the Netrin-1 receptors is not random because UNC5C inactivation occurs early, whereas DCC losses occurs in later stages of multistep colorectal carcinogenesis.

Background & Aims:Clostridium difficile toxin A causes acute inflammation and fluid secretion in experimental animals and patients with C difficile infection. We previously reported that toxin A increased cyclooxygenase-2/prostaglandin E2 (PGE2) expression and apoptosis in human colonocytes. Here, we assessed the role of secreted PGE2 in inflammation and enterocyte apoptosis in toxin A enteritis. Methods: Effects of PGE2 and PGE2 blockade on toxin A-induced apoptosis of human colonocytes (NCM460) and of PGE2 or toxin A on the Fas ligand (FasL) induction were analyzed by flow cytometry and Western blot. Functional activity of elevated FasL on colonocytes was assessed by coculture of colonocytes with Fas bearing Jurkat T cells. The involvement of PGE2-dependent Fas/FasL activation in toxin A enteritis was further assessed in either scid or FasL and Fas deficient mice. Results: Inhibition of cyclooxygenase-2 by NS-398 and of PGE2 using a blocking antibody markedly attenuated apoptosis in colonocytes exposed to toxin A. Enhanced expression and release of FasL followed PGE2 or toxin A exposure in vivo and in vitro and also was significantly attenuated by treatment with NS-398 and PGE2 blocking antibody. PGE2 acting through an EP1 receptor activated nuclear factor-κB, which induced transcription of FasL. Toxin A enteritis was accompanied by increased cellular infiltration, fluid secretion, and mucosal damage in control mice, but this response was markedly reduced in both Fas−/− and FasL−/− mice. Conclusions: Toxin A enteritis involves release of PGE2, which activates the Fas/FasL system, causing enterocyte apoptosis and inflammation.

Background & Aims: Clostridium difficile toxin A causes marked apoptosis of colonocytes in vivo and in vitro, which contributes to the formation of ulcers and pseudomembranes. We investigated the role of p53-dependent pathways and p38 mitogen-activated protein kinase (p38) in toxin A–induced colonocyte apoptosis. Methods: The effects of the activation of p53 and p53-dependent pathways including p21(WAF1/CIP1) were assessed in nontransformed human colonic NCM460 epithelial cells exposed to toxin A. Phosphorylation of p53 protein by p38 was measured by in vitro kinase assay, whereas p21 induction by activated p53 was determined by gel shift assays and RNA silencing (small interfering RNA). The relationship between colonocyte apoptosis and p38/p53-dependent pathways was studied in intact mice. Results: Toxin A stimulated p38 and p53 activation and induced cell cycle arrest (G2–M) with persistent expression of p21(WAF1/CIP1). Blockage of p38 by SB203580 inhibited p53 phosphorylation and induction of p21(WAF1/CIP1). In intact mice, p38 blockade suppressed toxin A–mediated destruction of intestinal villi, p21(WAF1/CIP1) expression, and enterocyte apoptosis. In addition, toxin A–mediated p21(WAF1/CIP1) and Bak induction, cytochrome c release, and caspase-3 activation were markedly attenuated in p53-silenced colonocytes, despite active p38. Overexpression of p21(WAF1/CIP1) triggered apoptosis and increased toxin A–associated colonocyte apoptosis. Conclusions:: The signaling pathway for colonocyte apoptosis following toxin A exposure involves p38-dependent activation of p53 and subsequent induction of p21(WAF1/CIP1), resulting in cytochrome c release and caspase-3 activation through Bak induction.

Background & Aims: There has been evidence that dysregulation of apoptosis is involved in the pathogenesis of cancer development. Caspase-8 is an initiation caspase that activates the caspase cascade during apoptosis. The aim of this study was to explore the possibility that mutation of the caspase-8 gene might be involved in the development of colorectal cancer. Methods: We analyzed the entire coding region of the caspase-8 gene for the detection of somatic mutations in 180 colorectal tumors (98 invasive carcinomas and 82 adenomas) by polymerase chain reaction, single-strand conformation polymorphism, and DNA sequencing. Results: Overall, we detected a total of 5 somatic mutations in 98 invasive carcinomas (5.1%), but no mutations were detected in 82 adenomas (0%). The frequency of caspase-8 mutation in the carcinomas was significantly higher than that in adenomas (P < 0.05). The 5 mutations consisted of 1 frameshift, 1 nonsense mutation, and 3 missense mutations. We expressed the 5 tumor-derived caspase-8 mutants and found that 3 of the 5 mutations markedly decreased apoptosis activity of caspase-8. Furthermore, expression of the inactivating caspase-8 mutants interfered with apoptosis by death receptor overexpression, indicating that these mutants have dominant-negative inhibition of the death receptor-induced apoptosis. Conclusions: The presence of caspase-8 mutation in colon carcinomas suggests that caspase-8 gene mutation might lead to the loss of its apoptotic function and contribute to the pathogenesis of colorectal carcinomas, especially at the late stage of colorectal carcinogenesis.

Background & Aims: To understand the molecular etiology of Cowden disease‐associated gastrointestinal polyps, we analyzed the mutational status of PTEN/ MMAC1, a recently identified Cowden disease gene located at 10q23, in gastric hamartomas, colonic adenoma, and juvenile polyps of 3 patients with Cowden disease. Methods: Messenger RNA expression, gene deletion, and sequence alteration of PTEN/MMAC1 were evaluated by quantitative polymerease chain reaction (PCR), PCR‐single-strand conformation polymorphism, and sequencing analysis. Results: Germline missense mutation at codon 289 (AAA to GAA, Lys to Glu) and deletion of the wild-type allele were detected in the polyps of 2 patients with Cowden disease in the same family. Germline allelic deletion and transcriptional silencing of the remaining allele, probably caused by abnormal methylation, were also observed in a gastric hamartoma of 1 patient. Conclusions: The germline mutation and alteration of the remaining allele observed in this study strongly support that PTEN/MMAC1 functions as a tumor suppressor in Cowden disease. This study is the first to show that the mutational abrogation of PTEN/MMAC1 plays a causal role in the genesis of gastrointestinal polyps in Cowden disease, providing molecular genetic evidence that colonic adenoma, juvenile polyp, and gastric hamartoma could be included in the manifestations of Cowden disease.

Diabetes is a risk factor for colorectal cancer. We studied the association between markers of glucose metabolism and metabolic syndrome and the presence of colorectal adenomas in a large number of asymptomatic men and women attending a health screening program in South Korea. We also investigated whether these associations depend on adenoma location.In a cross-sectional study, we measured fasting levels of glucose, insulin, hemoglobin A1c, and C-peptide and calculated homeostatic model assessment (HOMA) values (used to quantify insulin resistance) for 19,361 asymptomatic South Korean subjects who underwent colonoscopy examinations from January 2006 to June 2009. Participants completed a standardized self-administered health questionnaire and a validated semiquantitative food frequency questionnaire. Blood samples were collected on the day of the colonoscopy; fasting blood samples were also collected. Robust Poisson regression was used to model the associations of glucose markers with the prevalence of any adenoma.Using detailed multivariable-adjusted dose-response models, the prevalence ratios (aPR, 95% confidence interval [CI]) for any adenoma, comparing the 90th with the 10th percentile, were 1.08 (1.00-1.16; P = .04) for fasting glucose, 1.07 (0.99-1.15; P = .10) for insulin, 1.09 (1.02-1.18, P = .02) for HOMA, 1.09 (1.01-1.17; P = .02) for hemoglobin A1c, and 1.14 (1.05-1.24; P = .002) for C-peptide. The corresponding ratios for nonadvanced adenomas were 1.11 (0.99-1.25; P = .08), 1.10 (0.98-1.24; P = .12), 1.15 (1.02-1.29; P = .02), 1.14 (1.01-1.28; P = .03), and 1.20 (1.05-1.37; P = .007), respectively. The corresponding ratios for advanced adenomas were 1.32 (0.94-1.84; P = .11), 1.23 (0.87-1.75; P = .24), 1.30 (0.92-1.85; P = .14), 1.13 (0.79-1.61; P = .50), and 1.67 (1.15-2.42; P = .007), respectively. Metabolic syndrome was associated with the prevalence of any adenoma (aPR, 1.18; 95% CI, 1.13-1.24; P .001), nonadvanced adenoma (aPR, 1.30; 95% CI, 1.20-1.40; P.001), and advanced adenoma (aPR, 1.42; 95% CI, 1.14-1.78; P = .002). Associations were similar for adenomas located in the distal versus proximal colon.Increasing levels of glucose, HOMA values, levels of hemoglobin A1c and C-peptide, and metabolic syndrome are significantly associated with the prevalence of adenomas. Adenomas should be added to the list of consequences of altered glucose metabolism.

Background & Aims Immunoglobulin transcription factor 2 (ITF2) was believed to promote neoplastic transformation via activation of β-catenin. However, ITF2 recently was reported to suppress colon carcinogenesis. We investigated the roles of ITF2 in colorectal cancer cell lines and tumor formation and growth in mice. Methods Levels of ITF2, β-catenin, and c-Myc were measured in 12 human colorectal tumor samples and by immunohistochemistry. ITF2 regulation of β-catenin and T-cell factor (TCF) were analyzed using luciferase reporter, reverse-transcription quantitative polymerase chain reaction, flow cytometry, and immunoblot analyses. Mice were given subcutaneous injections of human colorectal cancer cell lines that stably express ITF2, small hairpin RNAs to reduce levels of ITF2, or control plasmids; xenograft tumor growth was assessed. Human colorectal carcinoma tissue arrays were used to associate levels of ITF2 expression and clinical outcomes. Results Levels of β-catenin, cMyc, and ITF2 were increased in areas of human colon adenomas and carcinomas, compared with nontumor areas of the same tissues. ITF2 levels were reduced and cMyc levels were increased in areas of carcinoma, compared with adenoma. In human colorectal cancer cell lines, activation of the β-catenin–TCF4 complex and expression of its target genes were regulated negatively by ITF2. ITF2 inhibited formation of the β-catenin–TCF4 complex by competing with TCF4 for β-catenin binding. Stable transgenic expression of ITF2 in human colorectal cancer cell lines reduced their proliferation and tumorigenic potential in mice, whereas small hairpin RNA knockdown of ITF2 promoted growth of xenograft tumors in mice. In an analysis of colorectal tumor tissue arrays, loss of ITF2 from colorectal tumor tissues was associated with poor outcomes of patients. A gene set enrichment analysis supported the negative correlation between the level of ITF2 and activity of the β-catenin–TCF4 complex. Conclusions In human colorectal cancer cell lines and tissue samples, ITF2 appears to prevent activation of the β-catenin–TCF4 complex and transcription of its gene targets. Loss of ITF2 promotes the ability of colorectal cancer cells to form xenograft tumors, and is associated with tumor progression and shorter survival times of patients.