Colitis Colon Research Articles

Effects of water decontamination methods and bedding material on the gut microbiota.

Rodent models are invaluable to understanding health and disease in many areas of biomedical research. Unfortunately, many models suffer from lack of phenotype reproducibility. Our laboratory has shown that differences in gut microbiota (GM) can modulate phenotypes of models of colon cancer and inflammatory bowel disease. We and others have also shown that a number of factors associated with rodent research, including vendor, cage system, and bedding can alter GM. The objective of this study was to expand these studies to examine the effect of additional bedding materials and methods of water decontamination on GM diversity and composition. To this end, Crl:CD1 (ICR) mice were housed on corn cob or compressed paper chip bedding and provided water that was decontaminated by four different methods: autoclaving with reverse osmosis, autoclaving with hydrochloric acid, autoclaving with sulfuric acid, and autoclaving alone. Feces was collected at day 0, and at day 28 (endpoint), fecal and cecal samples were collected. DNA was extracted from samples, amplified by PCR using conserved bacterial primer sets and subjected to next generation sequencing. Sequence data were analyzed using Qiime and groups were compared using principal coordinate analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA). Two factor PERMANOVA of cecal GM data revealed significant changes when comparing bedding and water decontamination methods, while no significant effects were noted in the fecal GM data. Subsequent PERMANOVA and PCoA of cecal data revealed that several combinations of bedding and water decontamination methods resulted in differing GM, highlighting the complexity by which environmental factors interact to modulate GM.

Quinoa whole grain diet compromises the changes of gut microbiota and colonic colitis induced by dextran Sulfate sodium in C57BL/6 mice

A plethora of evidence highlights that the dysbiosis of gut microbiota is a critical factor for inflammatory bowel disease (IBD). Both in vivo and in vitro studies have demonstrated that quinoa possesses potential prebiotic effects. The present study aims to examine the potential in using quinoa to ameliorate the dysbiosis and colitis induced by dextran sodium sulfate (DSS). A total of 40 C57BL/6 mice were fed either an AIN-93M diet or a quinoa-based diet, separately. Colitis was induced for 10 animals/dietary group with a 5-days exposure to 2.5% DSS. The clinical symptoms were monitored every other day, and the gut microbiota was characterized by 16S rRNA gene sequencing. The results indicated that consumption of quinoa lessened clinical symptoms as indicated by the reduced disease activity index and the degree of histological damage (P < 0.05). As expected, the DSS treatment induced significant dysbiosis of gut microbiota in mice on an AIN-93M diet. However, compared to mice fed the AIN-93M diet, the consumption of quinoa alleviated the DSS-induced dysbiosis remarkably, as indicated by increased species richness and diversity, decreased abnormal expansion of phylum Proteobacteria, and decreased overgrowth of genera Escherichia/Shigella and Peptoclostridium (P < 0.05). The relative abundances of Firmicutes and Bacteroidetes were less altered in mice fed with quinoa comparing to those mice fed the AIN-93M diet. In summary, the consumption of quinoa suppressed the dysbiosis of gut microbiota and alleviated clinical symptoms induced by DSS, indicating the potential to utilize quinoa as a dietary approach to improve intestinal health.

Senescence marker protein 30 protects intestinal epithelial cells against inflammation-induced cell death by enhancing Nrf2 activity

Senescence marker protein 30 (SMP30) is a calcium-binding protein whose expression decreases during senescence. SMP30 deficiency increases susceptibility to cytokine-induced apoptosis in the liver and to radiation-induced apoptosis in the small intestine. Furthermore, colonic epithelial cell death is associated with the severity of colitis. Therefore, in the present study, we investigated the function of SMP30 during intestinal inflammation. In SMP30 deficient mice, colitis was significantly exacerbated as demonstrated by increased mortality (p = 0.001), body weight loss (p = 0.0105 at day 8), rectal bleeding (p = 0.0047 at day 8) and diarrhea (p = 0.0030 at day 8), histological scores (ulcers, p = 0.0002; edema, p = 0.0125; leukocyte infiltration, p = 0.0016) and productions of pro-inflammatory cytokines (IL-1α, p = 0.0452; IL-6, p = 0.0074; G-CSF, p = 0.0036). In addition, greater proportions of apoptotic cells and lower levels of anti-apoptotic marker proteins (total PARP-1 and Bcl-2) were observed in the inflamed intestines of SMP30 deficient mice than in wild type controls. In vitro experiments on colonic epithelial cells showed that stable SMP30 expression inhibited but that SMP30 siRNA expression increased TNF-α-induced apoptosis. SMP30 inhibition decreased Nrf2 mRNA expression levels (p < 0.0001), but SMP30 overexpression increased Nrf2 mRNA expression levels (p = 0.0495). The underlying mechanism by which SMP30 protected cells appeared to be by inhibiting Nrf2 ubiquitination and Keap1 expression, and thus enhancing Nrf2 activity. Moreover, SMP30 deficiency increased the incidence of colitis-associated colon cancer as determined by increased mortality (p = 0.0572) and average polyp number (p = 0.0277). Collectively, these findings suggest that SMP30 protects intestinal epithelial cells from apoptosis and this can contribute to amelioration of colitis and colitis-associated colon cancer.

Fecal calprotectin correlates with active colonic inflammatory bowel disease but not with small intestinal Crohn's disease activity.

BackgroundThe utility of fecal calprotectin (FC) in small intestinal Crohn's disease (CD) is unclear. We examined how reliably FC reflects clinical and mucosal disease activity in small intestinal CD, colonic CD, and ulcerative colitis (UC).MethodsA total of 72 Inflammatory Bowel Disease (IBD) patients (23 colonic CD, 14 isolated small intestinal CD, and 35 UC) were included. Clinical activity was assessed using the Harvey–Bradshaw Index (HBI) (CD) and Mayo score (UC). Inflammatory activity was assessed through ileocolonoscopy, cross‐sectional imaging, C‐reactive protein (CRP), and FC. Clinical activity was defined as HBI > 4 or Mayo clinical score ≥ 3. Endoscopy activity was defined as Mayo endoscopic subscore ≥ 1, SES‐CD score ≥ 3, and Rutgeerts > i1.ResultsIn UC, FC was correlated with the Mayo clinical score (P < 0.0001) and was highly correlated with the total Mayo score (P < 0.0001). A cut‐off value of FC 100 μg/g provided sensitivity of 88% and specificity 100% for endoscopic activity. FC was lower for patients with endoscopic and clinical remission compared to active endoscopic disease (median 100 vs 1180 μg/g, P < 0.0001). In colonic CD, there was a significant correlation between FC and endoscopic activity (P < 0.001). For an FC cut‐off value of 100 μg/g, sensitivity was 100%, and specificity was 67%. In contrast, for isolated small intestinal CD, there was no significant correlation between FC and objective disease activity measured by either endoscopy or imaging (AUC 0.52, P = 0.58).ConclusionFC is reliable for the detection of colonic mucosal inflammation in both UC and CD but is less sensitive and reliable in the detection of small intestinal CD.

An improved real-time qPCR technique for quantification of intestinal bacteria in human fecal samples

The easy, cost-effective and rapid techniques for investigating the role of gut microbiota have become imperative owing to their importance in various diseases such as colon cancer and inflammatory bowel disease. Here we developed an absolute real-time qPCR technique for quantification of intestinal bacteria in human fecal samples for further investigation of gut microbiota composition in patients. At first, regarding bacterial species present in human fecal samples, species were cultured in anaerobic culture media and incubated at 37Cͦ in an anaerobic chamber. The number of CFU was counted. Eight fold serial dilutions of the bacterial suspension were prepared. To generate a standard curve for bacterial species in real-time PCR, DNA was extracted from different serial dilutions. Subsequently, DNA from human fecal samples was extracted. Now bacterial strains were applied as standard curve in absolute q PCR procedure for quantification of bacterial population in test samples. Based on absolute real-time analysis, intestinal bacteria were precisely quantified. The specificity of the primer pairs was checked by conventional PCR and real-time PCR regarding dissociation curve analysis. The amplification was linear in the range of 101-108. Dissociation curves had correlation coefficient values between 0.98 and 101/888. The efficiencies were between 95/073 and 99/804%. The absolute real-time PCR technique described here may be used for evaluating intestinal bacteria regarding diseases prevention.

Abstract 4082: Contrasting role for claudin-2 in colitis-associated cancer (CAC) and spontaneous colon tumorigenesis (CRC) by regulating mucosal healing

Abstract A link between chronic inflammation and colon cancer has long been appreciated. Accordingly, patients with ulcerative colitis are at higher risk for developing colitis-associated cancer (CAC). Mucosal barrier dysregulation allies with inflammation and colon cancer. Accordingly, claudin-2, a tight junction protein, is upregulated in ulcerative colitis, CAC and spontaneous colon cancer (CRC). Remarkably, claudin-2 expression is restricted to the colon crypt base, among undifferentiated and proliferative colonocytes, and its increased expression is responsible for the regulation of paracellular permeability, differentiation and proliferation. However, causal role of claudin-2 in CAC and CRC remains unclear. Considering the recent data from our laboratory and of others, that in vivo genetic manipulation of claudin-2 has inverse effects on colitis, we hypothesized an adaptive role for increased claudin-2 expression in colitis by promoting mucosal healing. We further postulated that mucosal healing can manifest in contrasting phenotypes for the CAC and CRC. To examine, Villin-claudin-2 transgenic (Cldn-2) mice were subjected to CAC by exposing them to AOM/DSS-induced cancer or interbred with the APCmin mice, the mouse model of spontaneous intestinal cancer. It was intriguing that the inflammation-driven colon cancer (AOM/DSS induced colon cancer) was significantly suppressed in Cldn-2 mice compared to WT littermates (p&amp;lt;0.001). These mice also showed immune suppressive milieu ideal for spontaneous colon tumorigenesis. Accordingly, we found contrasting disparity in results from APCmin/Cldn-2 mice, generated by the cross between Cldn-2 and APCmin mice. These mice showed significant increase in intestinal tumor burden (248+40%, p&amp;lt;0.001) and colonic tumor burden [Incidence (172.50±18%, p&amp;lt;0.001) and size (521.5±34%, p&amp;lt;0.001)] compared to APCmin mice. H&amp;E analysis confirmed aggressive phenotype of APCmin-Cldn-2 mice tumors (vs APCmin mice tumors). APCmin/Cldn-2 mice also survived less than the APCmin mice. Further, inflammation induction by subjecting these mice to Dextran Sodium Sulfate (DSS) resulted in precocious colonic tumorigenesis only in APCmin mice, rejecting a role for the pro-inflammatory signaling in colon cancer progression in APCmin/Cldn-2 mice. High throughput transcriptome and oncogenic array analyses further suggested modifications of the key inflammatory and oncogenic signaling, in supporting these disparate phenotypes. Taken together, our data provide strong support for the key significance for the context and diverse immunogenic signaling in regulating CAC versus CRC. These studies also suggest that claudin-2 manipulated mice can provide the much needed mouse modelling to help identify key immunogenic signaling supportive of the colon tumorigenesis for therapeutic gains. Citation Format: Rizwan Ahmad, Balawant Kumar, Narendra Kumar, Mary K. Washington, Punita Dhawan, Amar B. Singh. Contrasting role for claudin-2 in colitis-associated cancer (CAC) and spontaneous colon tumorigenesis (CRC) by regulating mucosal healing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4082.

AlphaE Integrin Expression Is Increased in the Ileum Relative to the Colon and Unaffected by Inflammation.

BackgroundRecent findings suggest that αE expression is enriched on effector T cells and that intestinal αE+ T cells have increased expression of inflammatory cytokines. αE integrin expression is a potential predictive biomarker for response to etrolizumab, a monoclonal antibody against β7 integrin that targets both α4β7 and αEβ7. We evaluated the prevalence and localization of αE+ cells as well as total αE gene expression in healthy and inflammatory bowel disease patients.MethodsαE+ cells were identified in ileal and colonic biopsies by immunohistochemistry and counted using an automated algorithm. Gene expression was assessed by quantitative reverse-transcriptase polymerase chain reaction.ResultsIn both healthy and inflammatory bowel disease patients, significantly more αE+ cells were present in the epithelium and lamina propria of ileal compared with colonic biopsies. αE gene expression levels were also significantly higher in ileal compared with colonic biopsies. Paired biopsies from the same patient showed moderate correlation of αE expression between the ileum and colon. Inflammation did not affect αE expression, and neither endoscopy nor histology scores correlated with αE gene expression. αE expression was not different between patients based on concomitant medication use except 5-aminosalicylic acid.ConclusionαE+ cells, which have been shown to have inflammatory potential, are increased in the ileum in comparison with the colon in both Crohn’s disease and ulcerative colitis, as well as in healthy subjects. In inflammatory bowel disease patients, αE levels are stable, regardless of inflammatory status or most concomitant medications, which could support its use as a biomarker for etrolizumab.

Bisphenol-A alters microbiota metabolites derived from aromatic amino acids and worsens disease activity during colitis.

Inflammatory bowel disease is a complex collection of disorders. Microbial dysbiosis as well as exposure to toxins including xenoestrogens are thought to be risk factors for inflammatory bowel disease development and relapse. Bisphenol-A has been shown to exert estrogenic activity in the colon and alter intestinal function, but the role that xenoestrogens, such as bisphenol-A , play in colonic inflammation has been previously described but with conflicting results. We investigated the ability of bisphenol-A to exacerbate colonic inflammation and alter microbiota metabolites derived from aromatic amino acids in an acute dextran sulfate sodium-induced colitis model. Female C57BL/6 mice were ovariectomized and exposed to bisphenol-A daily for 15 days. Disease activity measures include body weight, fecal consistency, and rectal bleeding. Colons were scored for inflammation, injury, and nodularity. Alterations in the levels of microbiota metabolites derived from aromatic amino acids known to reflect phenotypic changes in the gut microbiome were analyzed. Bisphenol-A exposure increased mortality and worsened disease activity as well as inflammation and nodularity scores in the middle colon region following dextran sulfate sodium exposure. Unique patterns of metabolites were associated with bisphenol-A consumption. Regardless of dextran sulfate sodium treatment, bisphenol-A reduced levels of tryptophan and several metabolites associated with decreased inflammation in the colon. This is the first study to show that bisphenol-A treatment alone can reduce microbiota metabolites derived from aromatic amino acids in the colon which may be associated with increased colonic inflammation and inflammatory bowel disease. Impact statement As rates of inflammatory bowel disease rise, discovery of the mechanisms related to the development of these conditions is important. Environmental exposure is hypothesized to play a role in etiology of the disease, as are alterations in the gut microbiome and the metabolites they produce. This study is the first to show that bisphenol-A alone alters tryptophan and microbiota metabolites derived from aromatic amino acids in a manner consistent with autoimmune diseases, specifically inflammatory bowel diseases, regardless of dextran sulfate sodium treatment. These findings indicate a potential mechanism by which bisphenol-A negatively affects gut physiology to exacerbate inflammation.

Characteristic profiles of DNA epigenetic modifications in colon cancer and its predisposing conditions\u2014benign adenomas and inflammatory bowel disease

BackgroundActive demethylation of 5-methyl-2′-deoxycytidine (5-mdC) in DNA occurs by oxidation to 5-(hydroxymethyl)-2′-deoxycytidine (5-hmdC) and further oxidation to 5-formyl-2′-deoxycytidine (5-fdC) and 5-carboxy-2′-deoxycytidine (5-cadC), and is carried out by enzymes of the ten-eleven translocation family (TETs 1, 2, 3). Decreased level of epigenetic DNA modifications in cancer tissue may be a consequence of reduced activity/expression of TET proteins. To determine the role of epigenetic DNA modifications in colon cancer development, we analyzed their levels in normal colon and various colonic pathologies. Moreover, we determined the expressions of TETs at mRNA and protein level.The study included material from patients with inflammatory bowel disease (IBD), benign polyps (AD), and colorectal cancer (CRC). The levels of epigenetic DNA modifications and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in examined tissues were determined by means of isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS). The expressions of TET mRNA were measured with RT-qPCR, and the expressions of TET proteins were determined immunohistochemically.ResultsIBD was characterized by the highest level of 8-oxodG among all analyzed tissues, as well as by a decrease in 5-hmdC and 5-mdC levels (at a midrange between normal colon and CRC). AD had the lowest levels of 5-hmdC and 5-mdC of all examined tissues and showed an increase in 8-oxodG and 5-(hydroxymethyl)-2′-deoxyuridine (5-hmdU) levels. CRC was characterized by lower levels of 5-hmdC and 5-mdC, the lowest level of 5-fdC among all analyzed tissues, and relatively high content of 5-cadC. The expression of TET1 mRNA in CRC and AD was significantly weaker than in IBD and normal colon. Furthermore, CRC and AD showed significantly lower levels of TET2 and AID mRNA than normal colonic tissue.ConclusionsOur findings suggest that a complex relationship between aberrant pattern of DNA epigenetic modification and cancer development does not depend solely on the transcriptional status of TET proteins, but also on the characteristics of premalignant/malignant cells. This study showed for the first time that the examined colonic pathologies had their unique epigenetic marks, distinguishing them from each other, as well as from normal colonic tissue. A decrease in 5-fdC level may be a characteristic feature of largely undifferentiated cancer cells.

Glucagon-like peptide 1 receptor (GLP-1R) expression by nerve fibres in inflammatory bowel disease and functional effects in cultured neurons.

IntroductionGlucagon like-peptide 1 receptor (GLP-1R) agonists diminish appetite and may contribute to the weight loss in inflammatory bowel disease (IBD).ObjectivesThe aim of this study was to determine, for the first time, the expression of GLP-1R by colon nerve fibres in patients with IBD, and functional effects of its agonists in cultured rat and human sensory neurons.MethodsGLP-1R and other nerve markers were studied by immunohistochemistry in colon biopsies from patients with IBD (n = 16) and controls (n = 8), human dorsal root ganglia (DRG) tissue, and in GLP-1R transfected HEK293 cells. The morphological effects of incretin hormones oxyntomodulin, exendin-4 and glucagon were studied on neurite extension in cultured DRG neurons, and their functional effects on capsaicin and ATP signalling, using calcium imaging.ResultsSignificantly increased numbers of colonic mucosal nerve fibres were observed in IBD biopsies expressing GLP-1R (p = 0.0013), the pan-neuronal marker PGP9.5 (p = 0.0008), and sensory neuropeptide CGRP (p = 0.0014). An increase of GLP-1R positive nerve fibres in IBD colon was confirmed with a different antibody to GLP-1R (p = 0.016). GLP-1R immunostaining was intensely positive in small and medium-sized neurons in human DRG, and in human and rat DRG cultured neurons. Co-localization of GLP-1R expression with neuronal markers in colon and DRG confirmed the neural expression of GLP-1R, and antibody specificity was confirmed in HEK293 cells transfected with the GLP-1R. Treatment with oxyntomodulin, exendin-4 and GLP-1 increased neurite length in cultured neurons compared with controls, but did not stimulate calcium influx directly, or affect capsaicin responses. However, exendin-4 significantly enhanced ATP responses in human DRG neurons.ConclusionOur results show that increased GLP-1R innervation in IBD bowel could mediate enhanced visceral afferent signalling, and provide a peripheral target for therapeutic intervention. The differential effect of GLP-1R agonists on capsaicin and ATP responses in neurons suggest they may not affect pain mechanisms mediated by the capsaicin receptor TRPV1, but may enhance the effects of purinergic agonists.

Walnut phenolic extract inhibits nuclear factor kappaB signaling in intestinal epithelial cells, and ameliorates experimental colitis and colitis-associated colon cancer in mice.

Walnuts (Juglans regia) are known to have anti-cancer and immunomodulatory effects. However, little information is available on the effects of walnut phenolic extract (WPE) on intestinal inflammation and colitis-associated colon cancer. COLO205 cells were pretreated with WPE and then stimulated with tumor necrosis factor (TNF)-α. In the acute colitis model, wild type mice (C57BL/6) were administered 4% dextran sulfate sodium (DSS) for 5 days. In the chronic colitis model, interleukin (IL)-10-/- mice were administered with either the vehicle or WPE (20mg/kg) by oral gavage daily for 2 weeks. In an inflammation-associated tumor model, wild type mice were administered a single intraperitoneal injection of azoxymethane followed by three cycles of 2% DSS for 5 days and 2 weeks of free water consumption. WPE significantly inhibited IL-8 and IL-1α expression in COLO205 cells. WPE attenuated both the TNF-α-induced IκB phosphorylation/degradation and NF-κB DNA binding activity. The administration of oral WPE significantly reduced the severity of colitis in both acute and chronic colitis models, including the IL-10-/- mice. In immunohistochemical staining, WPE attenuated NF-κB signaling in the colons of both colitis models. Finally, WPE also significantly reduced tumor development in a murine model of colitis-associated colon cancer (CAC). WPE ameliorates acute and chronic colitis and CAC in mice, suggesting that WPE may have potentials for the treatment of inflammatory bowel disease.

Activation of GABAA Receptors in Colon Epithelium Exacerbates Acute Colitis.

Emerging evidence indicates that gamma-aminobutyric acid (GABA) has many beneficial effects such as ameliorating immune and inflammatory response. But, here we reported that activation of GABAA receptors (GABAA Rs) aggravated dextran sulfate sodium (DSS)-induced colitis, although the expression of pro-inflammatory cytokines was inhibited. By contrast, blocking of GABAA Rs markedly alleviated DSS-induced colitis. Notably, GABAA Rs and glutamic acid decarboxylase 65/67 were significantly increased in colon mucosa of ulcerative colitis patients and the mouse model of colitis. Further studies showed that GABA treatment resulted in an increment of serum FITC-dextran following its oral administration, a decrement of transepithelial electrical resistance, and an increment of bacterial invasion, effects which were blocked by bicuculline. In addition, GABA inhibited the expression of tight junction proteins and mucin secretion in colitis colon. GABA also decreased the expression of ki-67 and increased cleaved-caspase 3 expression in intestinal epithelia. Our data indicate that the GABAA Rs activation within colon mucosa disrupts the intestinal barrier and increases the intestinal permeability which facilitates inflammatory reaction in colon. Meanwhile, the suppression effect of GABA on pro-inflammatory cytokines leads to insufficient bacteria elimination and further aggravated the bacteria invasion and inflammatory damage.

MLN3126, an antagonist of the chemokine receptor CCR9, ameliorates inflammation in a T cell mediated mouse colitis model

C-C chemokine receptor 9 (CCR9) is the homing receptor for C-C motif chemokine ligand 25 (CCL25), and contributes to the maintenance of mucosal immunity and pathogenesis of inflammatory bowel disease (IBD) through the recruitment of T cells into the gut mucosa. Recent reports suggest that the interaction of CCR9 and CCL25 in the large intestine correlate with disease severity of colonic IBD. MLN3126 is an orally available small molecular compound with potent and selective CCR9 antagonist activity. MLN3126 inhibited CCL25-induced calcium mobilization in human CCR9 transfected cells and CCL25-induced chemotaxis of mouse primary thymocytes in a dose-dependent manner. The potential effect of MLN3126 in an activated T cell transfer mouse colitis model was compared with that of an anti-tumor necrosis factor (TNF)-α antibody. CCL25 protein was detected in the colon of mucosal epithelial cells and CCR9+ CD4+ T cells were observed in the lamina propria of the colon of mice with colitis. Dietary administration of MLN3126 to the mice maintained sufficient concentration of the compound in the plasma and dose-dependently inhibited progression of colitis compared to the vehicle control group. Anti-TNF-α antibody, a surrogate for a standard of care for IBD treatment, was also efficacious in the colitis model. These results suggest that MLN3126 would be a promising orally available CCR9 antagonist to treat colonic IBD.

Induction of Nitric-Oxide Metabolism in Enterocytes Alleviates Colitis and Inflammation-Associated Colon Cancer

SummaryNitric oxide (NO) plays an established role in numerous physiological and pathological processes, but the specific cellular sources of NO in disease pathogenesis remain unclear, preventing the implementation of NO-related therapy. Argininosuccinate lyase (ASL) is the only enzyme able to produce arginine, the substrate for NO generation by nitric oxide synthase (NOS) isoforms. Here, we generated cell-specific conditional ASL knockout mice in combination with genetic and chemical colitis models. We demonstrate that NO derived from enterocytes alleviates colitis by decreasing macrophage infiltration and tissue damage, whereas immune cell-derived NO is associated with macrophage activation, resulting in increased severity of inflammation. We find that induction of endogenous NO production by enterocytes with supplements that upregulate ASL expression and complement its substrates results in improved epithelial integrity and alleviation of colitis and of inflammation-associated colon cancer.

The nuclear translocation of the kinases p38 and JNK promotes inflammation-induced cancer.

The stimulated nuclear translocation of signaling proteins, such as MAPKs, is a necessity for the initiation and regulation of their physiological functions. Previously, we determined that nuclear translocation of the MAPKs p38 and JNK involves binding to heterodimers comprising importin 3 and either importin 7 or importin 9. Here, we identified the importin-binding region in p38 and JNK and developed a myristoylated peptide targeting this site that we called PERY. The PERY peptide specifically blocked the interaction of p38 and JNK with the importins, restricted their nuclear translocation, and inhibited phosphorylation of their nuclear (but not cytoplasmic) substrates. Through these effects, the PERY peptide reduced the proliferation of several (but not all) cancer cell lines in culture and inhibited the growth of a human breast cancer xenograft in mice. In addition, the PERY peptide substantially inhibited inflammation in mice, as manifested in models of colitis and colitis-associated colon cancer. The PERY peptide more effectively prevented colon cancer development than did a commercial p38 inhibitor. In vivo analysis further suggested that this effect was mediated by PERY peptide-induced prevention of the nuclear translocation of p38 in macrophages. Together, these results support the use of the nuclear translocation of p38 and JNK as a novel drug target to treat various cancers and inflammation-induced diseases.

Symptoms Do Not Correlate With Findings From Colonoscopy in Children With Inflammatory Bowel Disease and Primary Sclerosing Cholangitis

Approximately 75% of children with primary sclerosing cholangitis (PSC) also have inflammatory bowel disease (IBD). IBD in patients with PSC (PSC-IBD) often has a unique phenotype, including a mild clinical course, yet it is associated with an increased risk of colorectal cancer compared with colonic IBD without PSC. We investigated whether subclinical endoscopic and histologic inflammation could account for the increased risk of colorectal cancer in patients with PSC-IBD, and whether these patients have increased fecal levels of calprotectin, a marker of inflammation. We performed a prospective study of children (age, <18 y) with colonic IBD with and without PSC who underwent colonoscopy from February 1, 2016, through March 31, 2017, at the Hospital for Sick Children in Toronto, Canada. We collected pediatric ulcerative colitis activity index (PUCAI) scores (to measure symptoms) and fecal levels of calprotectin from 37 children with PSC-IBD and 50 children with only IBD (controls; UC or IBD-unclassified). Colonoscopies were scored using the Mayo endoscopic subscore and the UC Endoscopic Index of Severity (UCEIS) scores, and histologic activity was graded. Among patients in clinical remission, endoscopic scores and the odds of active endoscopic disease (based on a UCEIS score ≥1) were compared between patients with and without PSC in univariate and multivariable analyses. Correlations between activity markers were compared between groups. The ability of fecalcalprotectin to identify mucosal healing in patients with PSC-IBD was assessed using receiver operating characteristic curve analyses. Analogous analyses were performed for histologic activity. Patients with PSC-IBD in clinical remission had higher endoscopic scores and greater odds of active endoscopic disease than controls (odds ratio, 5.9; 95% CI, 1.6-21.5). There was a higher degree of correlation between PUCAI and UCEIS scores in controls (r= 0.82) than in patients with PSC-IBD (r= 0.51; P= .01). Fecal levels of calprotectin correlated with UCEIS in patients with PSC-IBD (r= 0.84) and controls (r= 0.82; P= .80). Fecal levels of calprotectin identified mucosal healing in patients with PSC-IBD with an area under the receiver operating characteristic curve of 0.94 (optimal cut-point, 93 μg/g; 100% sensitivity and 92% specificity). Histologic activity scores and the odds of active histologic disease were also greater in patients in clinical remission with PSC-IBD than controls. Children with PSC-IBD in clinical remission, based on PUCAI scores, have a significantly higher risk of active endoscopic and histologic disease than children with colitis without PSC. Fecal levels of calprotectin correlate with endoscopic findings in pediatric patients with PSC-IBD; levels below 93 μg/g are associated with mucosal healing.

High Risk of Advanced Colorectal Neoplasia in Patients With Primary Sclerosing Cholangitis Associated With Inflammatory Bowel Disease

Patients with inflammatory bowel disease (IBD) and primary sclerosing cholangitis (PSC, termed PSC-IBD) are at increased risk for colorectal cancer, but their risk following a diagnosis of low-grade dysplasia (LGD) is not well described. We aimed to determine the rate of advanced colorectal neoplasia (aCRN), defined as high-grade dysplasia and/or colorectal cancer, following a diagnosis of indefinite dysplasia or LGD in this population. We performed a retrospective, longitudinal study of 1911 patients with colonic IBD (293 with PSC and 1618 without PSC) who underwent more than 2 surveillance colonoscopies from 2000 through 2015 in The Netherlands or the United States (9265 patient-years of follow-up evaluation). We collected data on clinical and demographic features of patients, as well as data from each surveillance colonoscopy and histologic report. For each surveillance colonoscopy, the severity of active inflammation was documented. The primary outcome was a diagnosis of aCRN during follow-up evaluation. We also investigated factors associated with aCRN in patients with or without a prior diagnosis of indefinite dysplasia or LGD. Patients with PSC-IBD had a 2-fold higher risk of developing aCRN than patients with non-PSC IBD. Mean inflammation scores did not differ significantly between patients with PSC-IBD (0.55)vs patients with non-PSC IBD (0.56) (P= .89), nor did proportions of patients with LGD (21% of patients with PSC-IBD vs 18% of patients with non-PSC IBD) differ significantly (P= .37). However, the rate of aCRN following a diagnosis of LGD was significantly higher in patients with PSC-IBD (8.4 per 100 patient-years) than patients with non-PSC IBD (3.0 per 100 patient-years; P= .01). PSC (adjusted hazard ratio [aHR], 2.01; 95% CI, 1.09-3.71), increasing age (aHR 1.03; 95% CI, 1.01-1.05), and active inflammation (aHR, 2.39; 95% CI, 1.63-3.49) were independent risk factors for aCRN. Dysplasia was more often endoscopically invisible in patients with PSC-IBD than in patients with non-PSC IBD. In a longitudinal study of almost 2000 patients with colonic IBD, PSC remained a strong independent risk factor for aCRN. Once LGD is detected, aCRN develops at a higher rate in patients with PSC and is more often endoscopically invisible than in patients with only IBD. Our findings support recommendations for careful annual colonoscopic surveillance for patients with IBD and PSC, and consideration of colectomy once LGD is detected.

Colonic lesion characterization in inflammatory bowel disease: A systematic review and meta-analysis.

AIMTo perform a systematic review and meta-analysis for the diagnostic accuracy of in vivo lesion characterization in colonic inflammatory bowel disease (IBD), using optical imaging techniques, including virtual chromoendoscopy (VCE), dye-based chromoendoscopy (DBC), magnification endoscopy and confocal laser endomicroscopy (CLE).METHODSWe searched Medline, Embase and the Cochrane library. We performed a bivariate meta-analysis to calculate the pooled estimate sensitivities, specificities, positive and negative likelihood ratios (+LHR, -LHR), diagnostic odds ratios (DOR), and area under the SROC curve (AUSROC) for each technology group. A subgroup analysis was performed to investigate differences in real-time non-magnified Kudo pit patterns (with VCE and DBC) and real-time CLE.RESULTSWe included 22 studies [1491 patients; 4674 polyps, of which 539 (11.5%) were neoplastic]. Real-time CLE had a pooled sensitivity of 91% (95%CI: 66%-98%), specificity of 97% (95%CI: 94%-98%), and an AUSROC of 0.98 (95%CI: 0.97-0.99). Magnification endoscopy had a pooled sensitivity of 90% (95%CI: 77%-96%) and specificity of 87% (95%CI: 81%-91%). VCE had a pooled sensitivity of 86% (95%CI: 62%-95%) and specificity of 87% (95%CI: 72%-95%). DBC had a pooled sensitivity of 67% (95%CI: 44%-84%) and specificity of 86% (95%CI: 72%-94%).CONCLUSIONReal-time CLE is a highly accurate technology for differentiating neoplastic from non-neoplastic lesions in patients with colonic IBD. However, most CLE studies were performed by single expert users within tertiary centres, potentially confounding these results.

L-Glutamine Attenuates DSS-Induced Colitis via Induction of MAPK Phosphatase-1.

Inflammatory bowel disease (IBD), encompassing ulcerative colitis and Crohn’s disease, is a multifactorial inflammatory disease of the small intestine and colon. Many investigators have reported that l-glutamine (Gln) therapy improves outcomes of experimental colitis models, although the mechanism is not fully understood. Regarding the anti-inflammatory properties of Gln, we have shown that Gln can effectively deactivate cytosolic phospholipase A2 (cPLA2) by rapid induction of MAPK phosphatase (MKP)-1. In this study, we explore the possibility that Gln ameliorates dextran sulfate sodium (DSS)-induced colitis via MKP-1 induction, resulting in inhibition of cPLA2, which has been reported to play a key role in the pathogenesis of IBD. Oral Gln intake attenuated DSS-induced colitis. Gln inhibited cPLA2 phosphorylation, as well as colonic levels of TNF-α and leukotriene (LT)B4. Gln administration resulted in early and enhanced MKP-1 induction. Importantly, MKP-1 small interfering RNA (siRNA), but not control siRNA, significantly abrogated the Gln-mediated (1) induction of MKP-1; (2) attenuation of colitis (colon length, histological abnormality, and inflammation; and (3) inhibition of cPLA2 phosphorylation and colonic levels of TNF-α and LTB4. These data indicated that Gln ameliorated DSS-induced colitis via MKP-1 induction.

Layperson's knowledge and perceptions of irritable bowel syndrome as potential barriers to care.

The aim of this study was to describe the layperson's knowledge and perceptions regarding the aetiology, pathogenesis, prevalence, medical evaluation, diagnosis and treatment of irritable bowel syndrome. Diagnosis acceptance and adherence to treatment is influenced by the views of the patient's social networks. Little is known how these networks influence those with irritable bowel syndrome. Cross-sectional study of two-hundred four laypersons, ages 18-80years without an irritable bowel syndrome diagnosis. Data were collected May 2016-March 2017. Laypersons without a diagnosis of IBS self-reported their knowledge and perceptions about IBS. Participants were able to identify many symptoms associated with irritable bowel syndrome however held misconceptions regarding the development of irritable bowel syndrome as noted by the endorsement of genetics, environment and diet or alcohol/smoking behaviours as specific causes. Further misconceptions held included the belief that irritable bowel syndrome was associated with an increased risk for the development of colon cancer and inflammatory bowel disease. Contrary to current guidelines, many thought a gastroenterologist was the only person appropriate to diagnose irritable bowel syndrome and objective testing, such as colonoscopy, was necessary to establish a diagnosis. Laypersons have an understanding of the symptoms associated with IBS; however, hold numerous misconceptions regarding the aetiology, role of the healthcare provider, necessary testing and risks associated with irritable bowel syndrome. These misconceptions are inconsistent with current guidelines and practices. Establishing partnerships and educating social networks in addition to patients may enhance outcomes for those with IBS.