Inflammatory Bowel Disease Mucosa Research Articles

Reciprocal Regulation Between Smad7 and Sirt1 in the Gut.

In inflammatory bowel disease (IBD) mucosa, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine transforming growth factor-β1, due to post-transcriptional mechanisms that enhance Smad7 acetylation status thus preventing ubiquitination-mediated proteosomal degradation of the protein. IBD-related inflammation is also marked by defective expression of Sirt1, a class III NAD+-dependent deacetylase, which promotes ubiquitination-mediated proteosomal degradation of various intracellular proteins and triggers anti-inflammatory signals. The aim of our study was to determine whether, in IBD, there is a reciprocal regulation between Smad7 and Sirt1. Smad7 and Sirt1 were examined in mucosal samples of IBD patients and normal controls by Western blotting and immunohistochemistry, and Sirt1 activity was assessed by a fluorimetric assay. To determine whether Smad7 is regulated by Sirt1, normal or IBD lamina propria mononuclear cells (LPMC) were cultured with either Sirt1 inhibitor (Ex527) or activator (Cay10591), respectively. To determine whether Smad7 controls Sirt1 expression, ex vivo organ cultures of IBD mucosal explants were treated with Smad7 sense or antisense oligonucleotide. Moreover, Sirt1 expression was evaluated in LPMC isolated from Smad7-transgenic mice given dextran sulfate sodium (DSS). Upregulation of Smad7 was seen in both the epithelial and lamina propria compartments of IBD patients and this associated with reduced expression and activity of Sirt1. Activation of Sirt1 in IBD LPMC with Cay10591 reduced acetylation and enhanced ubiquitination-driven proteasomal-mediated degradation of Smad7, while inhibition of Sirt1 activation in normal LPMC with Ex527 increased Smad7 expression. Knockdown of Smad7 in IBD mucosal explants enhanced Sirt1 expression, thus suggesting a negative effect of Smad7 on Sirt1 induction. Consistently, mucosal T cells of Smad7-transgenic mice contained reduced levels of Sirt1, a defect that was amplified by induction of DSS colitis. The data suggest the existence of a reciprocal regulatory mechanism between Smad7 and Sirt1, which could contribute to amplify inflammatory signals in the gut.

Abstract 5368: The evolutionary history of human colitis-associated colorectal cancer

Abstract Introduction: Inflammatory bowel disease (IBD) confers an increased lifetime risk of developing colorectal cancer (CRC). Our study aimed to compare the molecular and genetic features of colitis-associated CRC (CA-CRC) to the more common sporadic CRC (S-CRC), and to dissect the evolutionary history of CA-CRC using multi-region next generation sequencing. Methods: Fresh frozen colectomy specimens were collected from 12 patients with CA-CRC. For each case, whole exome sequencing was performed on multiple regions of tumor, adjacent normal mucosa and blood. Variants in key genes were validated by Sanger sequencing and BaseScope in situ hybridization, and copy numbers were validated by FISH. Copy number profiling (by SNP array or low-pass whole genome sequencing) was performed on low grade dysplasia (LGD; n=28), high grade dysplasia (HGD; n=13), mixed LGD/HGD (n=7) and CA-CRC (n=15). Phylogenetic trees were reconstructed for each case, and evolutionary analysis was used to reveal the temporal sequence of events leading to CA-CRC. Results: The majority of sequenced tumors (10/12) were microsatellite stable, and these had a median mutation rate of 3.0 single nucleotide alterations (SNAs) per Mb, ~20% higher than that of S-CRC. Mutational signatures identified ‘accelerated ageing' in the colitic bowel, likely a consequence of repeated inflammation and regeneration cycles. There was considerable mutational burden in non-dysplastic IBD mucosa (median 47 SNAs), with a median of 24% of these SNAs also clonal within the neighboring CA-CRC, indicating a mutational field. In CA-CRC the most commonly mutated gene was TP53, occurring more frequently than in S-CRC (80% vs. 58%), whereas APC mutations were significantly less common (40% vs. 75%, p=0.03). We analyzed the genetic heterogeneity of CA-CRCs and found that the number of clonal SNAs per tumor was not significantly different to S-CRCs, however CA-CRCs had significantly more SNAs that were unique to one region (p=0.04); this increase in diversity is likely due to an elevated mutation rate. CA- CRCs were often near-triploid (42%) or near-tetraploid (21%), with many other recurrent copy-number alterations that were distinct from those observed in S-CRC. Phylogenetic analysis revealed that copy number alterations (CNAs) accrue in non-dysplastic bowel, but the transition from LGD to HGD involves a punctuated ‘catastrophic' increase in CNA burden. Conclusions: Multi-region sequencing of CA-CRC has revealed a distinct pathway of colon carcinogenesis, with an increase in mutational burden and heterogeneity compared to S-CRC. Copy-number profiling indicated extensive genomic alterations, with dramatic accrual at the LGD to HGD transition. The significant mutational burden in surrounding normal mucosa indicates field cancerization, which is an encouraging prospect for screening programs; however the likelihood of punctuated evolution may offer a limited window for early detection. Citation Format: Ann-Marie Baker, William Cross, Kathleen Curtius, Chang-ho Ryan Choi, Ibrahim Al-Bakir, Daniel Temko, Pierre Martinez, Marc Williams, Hayley Davis, Sujata Biswas, Nicholas A. Wright, Morgan Moorghen, Stephen J. Hayes, Manuel Rodriguez-Justo, Andrew Silver, Lai Mun Wang, Marnix Jansen, Ailsa L. Hart, Simon J. Leedham, Trevor A. Graham. The evolutionary history of human colitis-associated colorectal cancer [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 5368.

Colonic Inhibition of Phosphatase and Tensin Homolog Increases Colitogenic Bacteria, Causing Development of Colitis in Il10-/- Mice.

Phosphatase and tensin homolog (Pten) is capable of mediating microbe-induced immune responses in the gut. Thus, Pten deficiency in the intestine accelerates colitis development in Il10-/- mice. As some ambient pollutants inhibit Pten function and exposure to ambient pollutants may increase inflammatory bowel disease (IBD) incidence, it is of interest to examine how Pten inhibition could affect colitis development in genetically susceptible hosts. With human colonic mucosa biopsies from pediatric ulcerative colitis and non-IBD control subjects, we assessed the mRNA levels of the PTEN gene and the gene involved in IL10 responses. The data from the human tissues were corroborated by treating Il10-/-, Il10rb-/-, and wild-type C57BL/6 mice with Pten-specific inhibitor VO-OHpic. We evaluated the severity of mouse colitis by investigating the tissue histology and cytokine production. The gut microbiome was investigated by analyzing the 16S ribosomal RNA gene sequence with mouse fecal samples. PTEN and IL10RB mRNA levels were reduced in the human colonic mucosa of pediatric ulcerative colitis compared with non-IBD subjects. Intracolonic treatment of the Pten inhibitor induced colitis in Il10-/- mice, characterized by reduced body weight, marked colonic damage, and increased production of inflammatory cytokines, whereas Il10rb-/- and wild-type C57BL/6 mice treated with the inhibitor did not develop colitis. Pten inhibitor treatment changed the fecal microbiome, with increased abundance of colitogenic bacteria Bacteroides and Akkermansia in Il10-/- mice. Loss of Pten function increases the levels of colitogenic bacteria in the gut, thereby inducing deleterious colitis in an Il10-deficient condition.

Evaluation of interleukin-6 and its soluble receptor components sIL-6R and sgp130 as markers of inflammation in inflammatory bowel diseases

PurposeInterleukin-6 (IL-6) production and signalling are increased in the inflamed mucosa in inflammatory bowel diseases (IBD). As published serum levels of IL-6 and its soluble receptors sIL-6R and sgp130 in IBD are from small cohorts and partly contradictory, we systematically evaluated IL-6, sIL-6R and sgp130 levels as markers of disease activity in Crohn’s disease (CD) and ulcerative colitis (UC).MethodsConsecutive adult outpatients with confirmed CD or UC were included, and their disease activity and medication were monitored. Serum from 212 CD patients (815 measurements) and 166 UC patients (514 measurements) was analysed, and 100 age-matched healthy blood donors were used as controls.ResultsIL-6 serum levels were significantly elevated in active versus inactive CD and UC, also compared with healthy controls. However, only a fraction of IBD patients showed increased serum IL-6. IL-6 levels ranged up to 32.7 ng/mL in active CD (> 5000-fold higher than in controls), but also up to 6.9 ng/mL in inactive CD. Increases in active UC (up to 195 pg/mL) and inactive UC (up to 27 pg/mL) were less pronounced. Associations between IL-6 serum levels and C-reactive protein concentrations as well as leukocyte and thrombocyte counts were observed. Median sIL-6R and sgp130 levels were only increased by up to 15%, which was considered of no diagnostic significance.ConclusionsOnly a minority of IBD patients shows elevated IL-6 serum levels. However, in these patients, IL-6 is strongly associated with disease activity. Its soluble receptors sIL-6R and sgp130 do not appear useful as biomarkers in IBD.

Mitochondrial DNA Is a Pro-Inflammatory Damage-Associated Molecular Pattern Released During Active IBD.

Due to common evolutionary origins, mitochondrial DNA (mtDNA) shares many similarities with immunogenic bacterial DNA. MtDNA is recognized as a pro-inflammatory damage-associated molecular pattern (DAMP) with a pathogenic role in several inflammatory diseases. We hypothesised that mtDNA is released during active disease, serving as a key pro-inflammatory factor in inflammatory bowel disease (IBD). Between 2014 and 2015, we collected plasma separated within 2 hours of sampling from 97 prospectively recruited IBD patients (67 ulcerative colitis [UC] and 30 Crohn's disease [CD]) and 40 non-IBD controls. We measured circulating mtDNA using quantitative polymerase chain reaction (amplifying mitochondria COXIII/ND2 genes) and also in mouse colitis induced by dextran sulfate-sodium (DSS). We used a mass spectometry approach to detect free plasma mitochondrial formylated peptides. Furthermore, we examined for mitochondrial damage using electron microscopy (EM) and TLR9 expression, the target for mtDNA, in human intestinal IBD mucosa. Plasma mtDNA levels were increased in UC and CD (both P < 0.0001) compared with non-IBD controls. These levels were significantly correlated to blood (C-reactive protein, albumin, white cell count), clinical and endoscopic markers of severity, and disease activity. In active UC, we identified 5 mitochondrial formylated peptides (the most abundant being fMMYALF with known chemoattractant function) in plasma. We observed mitochondrial damage in inflamed UC mucosa and significantly higher fecal MtDNA levels (vs non-IBD controls [P < 0.0001]), which supports gut mucosal mitochondrial DAMP release as the primary source. In parallel, plasma mtDNA levels increased during induction of acute DSS colitis and were associated with more severe colitis (P < 0.05). In active IBD, TLR9+ lamina propria inflammatory cells were significantly higher in UC and CD compared with controls (P < 0.05). We present the first evidence to show that mtDNA is released during active IBD. MtDNA is a potential mechanistic biomarker, and our data point to mtDNA-TLR9 as a therapeutic target in IBD. 10.1093/ibd/izy095_videoizy095.video5776747659001.

Outcomes of Endoscopic Resections of Large Laterally Spreading Colorectal Lesions in Inflammatory Bowel Disease: a Single United Kingdom Center Experience.

The SCENIC consensus statement recommends endoscopic resection of all visible dysplasia in inflammatory bowel disease, but patients with large or complex lesions may still be advised to have colectomy. This article presents outcomes for large nonpolypoid resections associated with colitis at our institution. Data including demographics, clinical history, lesion characteristics, method of resection, and postresection surveillance were collected prospectively in patients with visible lesions within colitic mucosa from January 2011 to November 2016. Resection techniques included endoscopic mucosal resection , endoscopic submucosal dissection (ESD), and hybrid ESD. Surveillance with magnification chromoendoscopy was performed at 3 months, 1-year postresection, and annually thereafter. Fifteen lesions satisfied the inclusion criteria in 15 patients. Mean lesion size was 48.3+/-21.7 (20-90) mm. All lesions were non-polypoid with distinct margins and no ulceration. 73% lesions were scarred of which 64% had undergone prior instrumentation. En bloc resection was achieved in n=6. Presumed endoscopic diagnosis was confirmed histopathologically in all resected lesions. One case of perforation and another with bleeding were both managed endoscopically. Median follow-up was 28 months (12-35) with no recurrence. This cohort series demonstrates that endoscopic resection of large non-polypoid lesions associated with colitis is feasible and safe using an array of resection methods supporting the role of advanced endoscopic therapeutics for the management of colitis associated dysplasia in a western tertiary endoscopic center.

The role of apoptosis of enterocytes in inflammatory bowel diseases

Purpose. Study the topography of cells, expressing villin, p53, Bcl-2, and PUMA in the intestinal mucosa in the non-infectious colitis. Materials and methods. We studied the morphology and localization of the immunoreactive markers of apoptosis in small intestine and bowel with ulcerative colitis and Crohn’s disease, using the material of 20 autopsies and 40 biopsies. Results. In the focus of the specific inflammation in the mucosa, we discovered numerous cells, in various degrees expressing anti- and proapoptotic molecules. In the control material, the antiapoptotic factors (villin and Bcl-2) localized mainly in the enterocytes, while the proapoptotic factors (p53 and PUMA) were expressed in the cells of the lamina propria of the mucosa. The reverse correlation between the number of cells expressing p53/PUMA and the villin/Bcl-2 positive cells had been discovered. At the same time proapoptotic molecules were discovered in the enterocytes and in the cells of the lamina propria of intestinal mucosa. Conclusion. The obtained data points to the important role of apoptosis in pathomorphogenesis of chronic non-infectious intestinal inflammation. The chronization of the process and the nature of the intestinal damage in the UC and CD are connected with balance of the regulative anti- and proapoptotic factors. The cells, expressing those factors, demonstrate the reverse number dependence.

Human Intestinal Dendritic Cells in Inflammatory Bowel Diseases.

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a serious, costly, and persistent health problem with an estimated prevalence in Western countries around 0.5% of the general population; its socioeconomic impact is comparable with that for chronic diseases such as diabetes. Conventional treatment involves escalating drug regimens with concomitant side effects followed, in some cases, by surgical interventions, which are often multiple, mainly in Crohn's disease. The goal of finding a targeted gut-specific immunotherapy for IBD patients is therefore an important unmet need. However, to achieve this goal we first must understand how dendritic cells (DC), the most potent antigen present cells of the immune system, control the immune tolerance in the gastrointestinal tract and how their properties are altered in those patients suffering from IBD. In this review, we summarize the current available information regarding human intestinal DC subsets composition, phenotype, and function in the human gastrointestinal tract describing how, in the IBD mucosa, DC display pro-inflammatory properties, which drive disease progression. A better understanding of the mechanisms inducing DC abnormal profile in IBD may provide us with novel tools to perform tissue specific immunomodulation.

Prevalence and risk factors for colorectal adenomas in patients with ulcerative colitis.

Patients with ulcerative colitis (UC) have an increased risk of colorectal cancer. Scarce data regarding the development of adenomas in these patients are available both for normal and colitic mucosa. The objective of this article is to evaluate the prevalence of adenomatous polyps and associated risk factors in patients with UC. Patients with UC were identified from the databases of two tertiary referral centers. Medical, endoscopic and histologic reports were reviewed. A total of 403 patients were included (53% male; 33% extensive colitis) and 1065 colonoscopies (median per patient, 2) were recorded and analyzed. Seventy-four adenomas in 47 patients (11.7%) and three cases of colorectal cancer were found during a median follow-up of 6.3 years. The cumulative risk of colorectal adenoma was 4.7%, 16.7%, 23.6% and 34.4% at 10, 20, 30 and 40 years from UC diagnosis, respectively. The cumulative risk of developing metachronous colorectal adenoma was 66.7%, 87.9%, and 90.9% at 5, 10, and 15 years from first adenoma detection. Older age at UC diagnosis and longer disease duration were independent risk factors for colorectal adenoma development. The prevalence of colorectal adenomas among UC patients seems to be higher than previously reported, although lower than in the background population.

A Novel Topical PPARγ Agonist Induces PPARγ Activity in Ulcerative Colitis Mucosa and Prevents and Reverses Inflammation in Induced Colitis Models

Peroxisome proliferator-activated receptor-gamma (PPARγ) exerts anti-inflammatory effects and is therefore a potential target in ulcerative colitis (UC). A novel PPARγ agonist (AS002) developed for local action was evaluated ex vivo in biopsies from UC patients and in vivo in mice with low-grade dextran sodium sulfate (DSS)- and trinitrobenzene sulfonic acid (TNBS)-induced colitis. Colonic biopsies from UC patients (n = 18) and healthy controls (n = 6) were incubated with AS002 or rosiglitazone (positive control) to measure mRNA expression of the PPARγ-responsive gene ADIPOPHILIN and protein levels of UC-related cytokines (enzyme-linked immunosorbent assay). AS002 absorption was determined in the colonic mucosa of UC patients. DSS-colitis mice received PPARγ agonists or vehicle daily by intrarectal administration starting 2 days before induction of colitis (preventive) or from days 3 to 8 (curative). Myeloperoxidase (MPO) and cytokine levels in colonic mucosa were determined. In addition, AS002 effects were studied in TNBS colitis. AS002 displayed an absorption pattern of a lipophilic drug totally metabolized in the mucosa. AS002 and rosiglitazone increased ADIPOPHILIN mRNA expression (3-fold) and decreased TNF-α, IL-1β, and IL-13 levels in human UC biopsies. In DSS, in both preventive and curative treatment and in TNBS colitis, AS002 protected against macroscopic and histological damage and lowered MPO and TNF-α, IL-1β, and IL-13 levels. AS002 triggers anti-inflammatory PPARγ activity in the human colonic mucosa of UC patients and prevents and reverses colitis in mice. Our data suggest that AS002 has potential for topical maintenance treatment of UC, which warrants further studies in vivo in patients.

Dysplasia can be a pain in the gut

In the gastrointestinal tract, the term 'dysplasia' is used to refer to non-invasive neoplastic epithelium. Although we recognise adenomas of the gut as dysplastic, we don't use the term 'dysplasia' in reporting them. The use of the term 'dysplasia' for pre-invasive epithelium gradually came to replace other terms in studies attempting to identify epithelial changes that were cancer precursors in surveillance biopsies of patients with chronic colitides. The two-tier system of classification, dividing dysplasias into low-grade or high-grade lesions is conceptually straightforward; however, difficulties exist in the distinction of regenerative epithelium from low-grade dysplasia, of low-grade from high-grade dysplasia, and in identifying superficially invasive carcinoma in a dysplastic mucosa. The category 'indefinite for dysplasia' is an honest recognition of the difficulties in distinguishing reactive or regenerative epithelium from low-grade dysplasia, since these epithelia share many cytological features. In surveillance biopsies in ulcerative colitis and Barrett's mucosa, for each epithelial category from non-dysplastic through indefinite, low-grade and high-grade dysplasia, there are specific management recommendations that vary from no change in surveillance to increased surveillance to definitive therapy that is often a major resection. The recommendation for referral of high-grade dysplasias to consultants reflects the concern pathologists have about making such clinically significant diagnoses with limited experience. Pathologists should use the accepted terminology, share cases to expand their experience, and seek consultation in selected cases. This paper follows the evolution of the dysplasia concept, details the difficult areas of diagnosis, and discusses the importance of interaction between clinicians and pathologists in dealing with dysplasias.

Peroxiredoxin 1 expression in active ulcerative colitis mucosa identified by proteome analysis and involvement of thioredoxin based on immunohistochemistry.

Ulcerative colitis (UC) is a chronic, relapsing, inflammatory bowel disease, and patients with long-standing UC are at high risk of developing colorectal cancer as a typical case of the organ-specific chronic inflammation-carcinoma sequence. Interactions between epithelial and stromal cells and alterations in a variety of stromal microenvironments have been demonstrated to have important roles in the carcinogenesis of UC-associated carcinoma. Therefore, the identification of proteins in the inflammatory microenvironment is important not only in the epithelium, however also in the stroma of UC inflammatory foci. To identify proteins associated with UC-associated carcinoma, the present study used proteomic analysis with two-dimensional electrophoresis and mass spectrometry. Differentially expressed proteins were assessed between active and inactive UC biopsy specimens. Results were verified by immunohistochemistry. Peroxiredoxin 1 (PRDX1) was among the proteins identified to have increased expression in active compared with inactive UC. Immunohistochemical analysis indicated that the expression of both PRDX1 and thioredoxin (TRX) increased with increasing inflammation grade in epithelial cells in UC mucosal crypts. PRDX1-positive stromal cells in the lower half of the lamina propria increased along with colitis severity. Furthermore, the expression of both PRDX1 and TRX proteins was increased in UC-associated neoplastic lesions compared with normal mucosa. A stepwise increase in PRDX1 expression was clear with increasing tumor progression in UC-associated tumorigenesis. Since PRDX1 and TRX overexpression was a unique characteristic of UC activity and UC-associated neoplastic lesions, PRDX1 and TRX expression may reflect oxidative stress along with the severity of colitis activity and UC-associated tumorigenesis in patients with UC.

Expression and Clinical Significance of Elafin in Inflammatory Bowel Disease.

The expression of elafin in inflammatory bowel disease (IBD) is controversial. Here, we detected the expression of elafin in the peripheral blood and colonic mucosa of patient with IBD and then explored its role and value in assessing the activity and severity of IBD. Sixty-eight patients with IBD were selected as an experimental group. The control group included 38 healthy individuals. The expression of elafin mRNA in peripheral blood leukocytes and in serum was detected by qRT-PCR and enzyme-linked immunosorbent assay, respectively. The inflamed and noninflamed tissues were collected by colonoscopy. The expression of elafin in the intestinal mucosa was determined by immunohistochemistry staining and qRT-PCR. The expression of elafin between groups and among each stage of IBD was compared. The correlations of elafin expression with erythrocyte sedimentation rate and C-reactive protein were determined by Spearman's correlation analysis and with clinical disease activity indices (Best Crohn's Disease Activity Index and modified Mayo scores) by Pearson's correlation analysis. Elafin mRNA levels decreased significantly in active ulcerative colitis (UC) but increased in remission UC. However, in Crohn's disease (CD), we did not detect the aforementioned significant differences. Although serum IL-8 levels increased, serum elafin concentrations decreased both in UC and in CD, but the differences among stages were not significant. The expression of elafin in the inflamed colonic mucosa in both CD and UC was lower than that in the normal mucosa in controls and lower than that in the noninflamed mucosa in IBD. Moreover, the relative expression of elafin mRNA in peripheral blood leukocytes in UC was negatively correlated with erythrocyte sedimentation rate, C-reactive protein, and modified Mayo scores, and in CD, it was negatively correlated with Best Crohn's Disease Activity Index scores. Elafin decreased in active patients with IBD and was negatively correlated with disease activity, suggesting that elafin may play a protective role and could be used as an index to evaluate disease activity in IBD.

Expression of human cathelicidin peptide LL-37 in inflammatory bowel disease.

Cathelicidin peptide LL-37 plays an important role in the early host response against invading pathogens via its broad-spectrum anti-microbial activity. In this study, we investigated LL-37 expression in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Furthermore, the regulatory mechanism of LL-37 induction was investigated in human colonic subepithelial myofibroblasts (SEMFs). LL-37 mRNA expression and protein secretion were analysed using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Intracellular signalling pathways were analysed using immunoblotting and specific small interference RNA (siRNA). The expression of LL-37 mRNA was increased significantly in the inflamed mucosa of ulcerative colitis and Crohn's disease. The Toll-like receptor (TLR)-3 ligand, polyinosinic-polycytidylic acid (poly(I:C), induced LL-37 mRNA expression and stimulated LL-37 secretion in colonic SEMFs. The transfection of siRNAs specific for intracellular signalling proteins [Toll/IL-1R domain-containing adaptor-inducing interferon (IFN) (TRIF), tumour necrosis factor receptor-associated factor (TRAF)6, transforming growth factor β-activated kinase (TAK)1] suppressed the poly(I:C)-induced LL-37 mRNA expression significantly. Poly(I:C)-induced phosphorylation of mitogen-activated protein kinases (MAPKs) and activated nuclear factor kappa B (NF-κB) and activating factor protein (AP)-1. siRNAs specific for NF-κB and c-Jun inhibited poly(I:C)-induced LL-37 mRNA expression. LL-37 suppressed lipopolysaccharide (LPS)-induced interleukin (IL)-6 and IL-8 expression significantly in colonic SEMFs. The expression of LL-37 was up-regulated in the inflamed mucosa of IBD patients. LL-37 was induced by TLR-3 stimulation and exhibited an anti-microbial effect via interaction with lipopolysaccharide (LPS).

Cytokine production profile in intestinal mucosa of paediatric inflammatory bowel disease.

In the recent years, the incidence of inflammatory bowel disease (IBD) has dramatically increased in young subjects, however, the pathogenesis of paediatric IBD is poorly investigated. In this study we aimed to evaluate the cytokine pattern and the phenotype of cytokine producing cells in the intestinal mucosa of paediatric patients affected by Crohn’s disease (CD) or ulcerative colitis (UC) and of non-IBD healthy controls (HC). Cytokine (IL-15, TNF-α, INF-γ) production was analyzed at basal condition and after mitogen stimulation either intracellularly by flow cytometry or in intestinal cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). A higher frequency of enterocytes (EpCam+ cells) was observed in UC patients compared to CD or HC. An expansion of enterocytes producing IL-15 and TNF-α were found in IBD patients compared to HC. A marked expression of IL-15 in the intestinal epithelium of IBD patients was further confirmed by immunohistochemistry. Myeloid dendritic (CD11c+) cells producing TNF-α and INF-γ were increased in IBD biopsies. Unexpectedly, only after a strong mitogen stimulus, as phytohaemagglutinin, the frequency of CD3+ cells producing IFN-γ was increased in IBD compared to control intestinal mucosa. Interestingly, functional studies performed on organ cultures of intestinal biopsies with neutralizing anti-IL-15 monoclonal antibody showed a marked reduction of mononuclear cell activation, proliferation of crypt enterocytes, as well as a reduction of TNF-α release in organ culture supernatants. In conclusion, we found that in the gut mucosa of IBD children both enterocytes and dendritic cells produce proinflammatory cytokines. The over-expression of IL-15 by enterocytes in IBD intestine and the reduced IBD inflammation by IL-15 blockage suggests that this cytokine could be a therapeutic target in IBD.

Usefulness of Different Pathological Scores to Assess Healing of the Mucosa in Inflammatory Bowel Diseases: A Real Life Study

The concept of remission for patients with inflammatory bowel diseases has recently evolved, and should also include histological healing of the mucosa, difficult to evaluate since there is no agreement on pathological scores and those available are quite complex to use in the daily routine. We evaluated the possible usefulness of a simplified pathological score to assess histological healing of the mucosa in inflammatory bowel diseases patients compared with four commonly proposed pathological scores. Slides from 24 patients (12 Crohn’s disease, 12 ulcerative colitis, age range 24–62 years), pre- and post-treatment with biological agents and displaying endoscopic remission were assessed by two pathologists. Pre- and post-treatment results and the time employed to calculate the various scores were obtained. All scores were useful to document highly significant post-treatment decreases of histological activity. However, the simplified score needed significant less time to be calculated for each slide, had high inter-rater agreement, and avoided subjectivity from the pathologists. The simplified score is easy to calculate and seems apt to document histological healing of the mucosa, in a manner similar to the more complex scores. It remains to be established whether this score could simplify the daily routinary practice in this context.

Targeting S1P in Inflammatory Bowel Disease: New Avenues for Modulating Intestinal Leukocyte Migration.

Sphingosine 1 phosphate [S1P] is a bioactive lipid mediator involved in the regulation of several cellular processes though the activation of a G protein-coupled receptor family known as the S1P receptors [S1PRs]. Advances in the understanding of the biological activities mediated by S1PRs have sparked great interest in the S1P/S1PRs axes as new therapeutic targets for the modulation of several cellular processes. In particular, the S1P/S1PR1 axis has been identified as key regulator for lymphocyte migration from lymph nodes. The blockade of this axis is emerging as a new therapeutic approach to control the aberrant leukocyte migration into the mucosa in inflammatory bowel disease [IBD]. This review briefly summarises the current evidence coming from clinical studies, and discusses the future prospects of S1P inhibitors for treatment of inflammatory bowel disease.

Potential link between Fusobacterium enrichment and DNA methylation accumulation in the inflammatory colonic mucosa in ulcerative colitis.

BACKGROUND AND AIMFusobacterium enrichment has been associated with colorectal cancer development. Ulcerative colitis (UC) associated tumorigenesis is characterized as high degree of methylation accumulation through continuous colonic inflammation. The aim of this study was to investigate a potential link between Fusobacterium enrichment and DNA methylation accumulation in the inflammatory colonic mucosa in UC.METHODSIn the candidate analysis, inflamed colonic mucosa from 86 UC patients were characterized the methylation status of colorectal a panel of cancer related 24 genes. In the genome-wide analysis, an Infinium HumanMethylation450 BeadChip array was utilized to characterize the methylation status of >450,000 CpG sites for fourteen UC patients. Results were correlated with Fusobacterium status.RESULTSUC with Fusobacterium enrichment (FB-high) was characterized as high degree of type C (for cancer-specific) methylation compared to other (FB-low/neg) samples (P<0.01). Genes hypermethylated in FB-high samples included well-known type C genes in colorectal cancer, such as MINT2 and 31, P16 and NEUROG1. Multivariate analysis demonstrated that the FB high status held an increased likelihood for methylation high as an independent factor (odds ratio: 16.18, 95% confidence interval: 1.94-135.2, P=0.01). Genome-wide methylation analysis demonstrated a unique methylome signature of FB-high cases irrespective of promoter, outside promoter, CpG and non-CpG sites. Group of promoter CpG sites that were exclusively hypermethylated in FB-high cases significantly codified the genes related to the catalytic activity (P=0.039).CONCLUSIONOur findings suggest that Fusobacterium accelerates DNA methylation in specific groups of genes in the inflammatory colonic mucosa in UC.

H. pylori attenuates TNBS-induced colitis via increasing mucosal Th2 cells in mice.

There is an epidemiological inverse relationship between Helicobacter pylori (H. pylori) infection and Crohn’s disease (CD). However, whether H. pylori plays a protective role against CD remains unclear. Since 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis is thought to resemble CD, we investigated whether H. pylori can attenuate TNBS-induced colitis in mice. Here we show that H. pylori can attenuate the severity of TNBS-induced colitis. In addition, H. pylori not only down-regulates Th17 and Th1 cytokine expression, but can up-regulate Th2 cytokine expression and increase the Th2:Th17 ratio of CD4+ T in the colonic mucosa of TNBS-induced colitis. Our results indicate that H. pylori attenuates TNBS-induced colitis mainly through increasing Th2 cells in murine colonic mucosa. Our finding offers a novel view on the role of H. pylori in regulating gastrointestinal immunity, and may open a new avenue for development of therapeutic strategies in CD by making use of asymptomatic H. pylori colonization.

Mo1921 - Ulcerative Colitis-Associated Escherichia Coli Colonize the Intestinal Mucosa of Susceptible Host and Promote Colitis via Hemolysin Production

Mo1921 - Ulcerative Colitis-Associated Escherichia Coli Colonize the Intestinal Mucosa of Susceptible Host and Promote Colitis via Hemolysin Production