Ulcerative Colitis Colon Research Articles

Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies.

The etiology of the inflammatory bowel diseases, including ulcerative colitis (UC), remains incompletely explained. We hypothesized that an analysis of the UC colon proteome could reveal novel insights into the disease etiology. Mucosal colon biopsies were taken by endoscopy from noninflamed tissue of 10 patients with UC and 10 controls. The biopsies were either snap-frozen for protein analysis or prepared for histology. The protein content of the biopsies was characterized by high-throughput gel-free quantitative proteomics, and biopsy histology was analyzed by light microscopy and confocal microscopy. We identified and quantified 5711 different proteins with proteomics. The abundance of the proteins calprotectin and lactotransferrin in the tissue correlated with the degree of tissue inflammation as determined by histology. However, fecal calprotectin did not correlate. Forty-six proteins were measured with a statistically significant differences in abundances between the UC colon tissue and controls. Eleven of the proteins with increased abundances in the UC biopsies were associated with neutrophils and neutrophil extracellular traps. The findings were validated by microscopy, where an increased abundance of neutrophils and the presence of neutrophil extracellular traps by extracellular DNA present in the UC colon tissue were confirmed. Neutrophils, induced neutrophil extracellular traps, and several proteins that play a part in innate immunity are all increased in abundance in the morphologically normal colon mucosa from patients with UC. The increased abundance of these antimicrobial compounds points to the stimulation of the innate immune system in the etiology of UC.

MicroRNA signatures differentiate Crohn's disease from ulcerative colitis.

BackgroundExcessive and inappropriate immune responses are the hallmark of several autoimmune disorders, including the inflammatory bowel diseases (IBD): Crohn’s disease (CD) and ulcerative colitis (UC). A complex etiology involving both environmental and genetic factors influences IBD pathogenesis. The role of microRNAs (miRNAs), noncoding RNAs involved in regulating numerous biological processes, to IBD pathology, in terms of initiation and progression, remains ill-defined. In the present study, we evaluated the relationship between colon, peripheral blood, and saliva whole miRNome expression in IBD patients and non-inflammatory bowel disease (non-IBD) controls to identify miRNAs that could discriminate CD from UC. Quantitative real-time PCR (qRT-PCR) was used to validate and assess miRNA expression.ResultsMicroarray analysis demonstrated that upwards of twenty six miRNAs were changed in CD and UC colon biopsies relative to the non-IBD controls. CD was associated with the differential expression of 10 miRNAs while UC was associated with 6 miRNAs in matched colon tissues. CD was associated with altered expression of 6 miRNAs while UC was associated with 9 miRNAs in whole blood. Expression of miR-101 in CD patients and miR-21, miR-31, miR-142-3p, and miR-142-5p in UC patients were altered in saliva.ConclusionsOur results suggest that there is specific miRNA expression patterns associated with UC versus CD in three separate tissue/body fluids (colon, blood, and saliva). Further, the aberrant miRNA expression profiles indicate that miRNAs may be contributory to IBD pathogenesis, or at least reflect the underlying inflammation. Scrutinizing miRNA expression in saliva and blood samples may be beneficial in monitoring or diagnosing disease in IBD patients. A panel of miRNAs (miR-19a, miR-21, miR-31, miR-101, miR-146a, and miR-375) may be used as markers to identify and discriminate between CD and UC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-015-0069-0) contains supplementary material, which is available to authorized users.

Relationship between dendritic cells, interleukin-23 and ulcerative colitis

Objective To compare the levels of S100+ DC,CD83 + DC and IL-23P19 between the patients with mucosa of ulcerative colitis ( UC ) and those with irritable bowel syndrome ( 0BS ).Methods Paraffin-embedded colon tissue from colonoscopic biopsy was detected in sixty UC and sixty IBS cases for the positivity of SI00 + DC,CD83 + DC and IL-23P19 using immunohistochemical SP method.Results There was no significant difference in the positive rates of S100 + DC and CD83 + DC between the two groups ( P ＞ 0.05 ),while significant difference in IL-23P19 level was found between the two groups( 85.0％ vs.70.0％ ; x2 =3.87,P ＜0.05 ).The infiltration densities of S100 + DC,CD83 + DC and expression of IL-23 P19 in the mild and severe UC patients were 24.43 ± 8.26/mm2,5.23 ± 1.66/mm2,95.03 ± 18.39/mm2,and 36.13 ± 11.36/mm2,10.13 ±3.29/mm2,133.38 ± 25.32/mm2,respectively.The difference between the mild and severe UC patients was significant ( P ＜ 0.05 ).No significant difference was found regarding the above three parameters between severity-stratified subgroups of the UC patients ( P ＞ 0.05 ).Conclusion In UC colon tissues,the infiltration density and activity of DC were increased.IL-23P19 level was elevated.The levels of S100 + DC,CD83 + DC and IL-23P19 were increased with the advancement of UC disease. Key words: Ulcerative colitis ; Irritable bowel syndrome; Dendritic cell; Interleukin-23 ; Immunohistochemistry

The route to pathologies in chronic inflammatory diseases characterized by T helper type 2 immune cells.

T helper type 2 (Th2)-characterized inflammatory responses are highly dynamic processes initiated by epithelial cell damage resulting in remodelling of the tissue architecture to prevent further harm caused by a dysfunctional epithelial barrier or migrating parasites. This process is a temporal and spatial response which requires communication between immobile cells such as epithelial, endothelial, fibroblast and muscle cells and the highly mobile cells of the innate and adaptive immunity. It is further characterized by a high cellular plasticity that enables the cells to adapt to a specific inflammatory milieu. Incipiently, this milieu is shaped by cytokines released from epithelial cells, which stimulate Th2, innate lymphoid and invariant natural killer (NK) T cells to secrete Th2 cytokines and to activate dendritic cells which results in the further differentiation of Th2 cells. This milieu promotes wound-healing processes which are beneficial in parasitic infections or toxin exposure but account for increasingly dysfunctional vital organs, such as the lung in the case of asthma and the colon in ulcerative colitis. A better understanding of the dynamics underlying relapses and remissions might lead ultimately to improved therapeutics for chronic inflammatory diseases adapted to individual needs and to different phases of the inflammation.

Role of Colonic Expression of Wnt Inhibitory Factor-1 in Ulcerative Colitis

Introduction: Wnt proteins constitute a family of molecules that are considered to have paramount importance in embryonic development. Recent data suggests that Wnt pathways play a significant role in inflammation, carcinogenesis, and bone development. Wnt inhibitor factor-1 (WIF-1) is a secreted protein that binds to Wnt proteins and inhibits their activity. Our aim was to investigate the colonic expression of WIF-1 in ulcerative colitis (UC). Methods: A total of 24 UC patients were enrolled. Four colon biopsies were obtained during routine surveillance colonoscopy from each patient--2 biopsies from normal-looking areas of colon; and 2 were sampled from inflamed colon. The degree of inflammation in multiple sections of the colon biopsies was graded as I, II, and III by an experienced pathologist. By immunohistochemistry, WIF-1 staining in the glandular region and lamina propria of the colon was graded as I, II, and III based on the percentage of positively stained cells and intensity of staining. The association between inflammation and WIF-1 staining in the multiple colon biopsy sections of UC was studied using Pearson correlation. Results: Of the 24 patients, 70% were males. Mean age was 55.2 years (26-83). WIF-1 staining significantly increased in the lamina propria (p=0.01) and glandular region (p=0.005) with increasing inflammation in UC patients (Figures). On stratification of UC colon biopsy sections in those obtained from normal-looking areas and inflamed-looking areas during colonoscopy, WIF-1 staining was significantly increased in both lamina propria (p= 0.013) and glandular region (p=0.01) in inflamed-looking areas of colon. Conclusion: WIF-1 is increasingly expressed in the inflamed areas of colon in UC patients. WIF-1 may have a crucial role in the pathogenesis of UC; and a potential novel therapeutic strategy may be identified for UC by blocking the WIF-1 in Wnt pathway.Figure 1Figure 2

Inflammatory bowel disease alters intestinal bile acid transporter expression.

The enterohepatic circulation of bile acids (BAs) critically depends on absorption of BA in the terminal ileum and colon, which can be affected by inflammatory bowel disease (IBD). Diarrhea in IBD is believed to result in part from BA malabsorption (BAM). We explored whether IBD alters mRNA expression of key intestinal BA transporters, BA detoxifying systems, and nuclear receptors that regulate BA transport and detoxification. Using real-time polymerase chain reaction, mucosal biopsy specimens from the terminal ileum in Crohn's disease (CD) patients and from the descending colon in ulcerative colitis (UC) patients were assessed for mRNA expression. Levels were compared with healthy controls. The main ileal BA uptake transporter, the apical sodium dependent bile acid transporter, was downregulated in active CD and UC and in CD in remission. Other significant changes such as repression of breast cancer-related protein and sulphotransferase 2A1 were seen only during active disease. In UC, pancolitis (but not exclusively left-sided colitis) was associated with altered expression of major BA transporters [multidrug resistance-associated protein 3 (MRP3), MRP4, multidrug resistance gene 1, organic solute transporter α/β] and nuclear receptors (pregnane X receptor, vitamin D receptor) in the descending colon. UC pancolitis leads to broad changes and CD ileitis to selective changes in intestinal BA transporter expression. Early medical manipulation of intestinal BA transporters may help prevent BAM.

PWE-086 Determining Stem Cell And Crypt Dynamics In Inflammatory Bowel Disease

IntroductionInflammatory bowel disease (IBD) confers a high risk of development of colitis-associated colorectal cancer (CACRC) in patients with extensive colitis. It is believed that a field effect, resulting from chronic...

Paneth cell metaplasia in newly diagnosed inflammatory bowel disease in children.

BackgroundPaneth cell metaplasia (PCM) is well described in adults but little is known about the distribution of colonic Paneth cells and the occurrence of PCM in a paediatric population. The aim of this study is to determine whether Paneth cell hyperplasia or metaplasia characteristically occurs in the colons of children with newly diagnosed idiopathic inflammatory bowel disease (IBD).MethodsWe retrospectively reviewed colonic series from 28 new diagnoses of paediatric IBD at a tertiary referral centre, and from a further 14 children with IBD-like symptoms whose colonic biopsies and ancillary investigations were normal. Paneth cells were counted at 6 anatomical sites in the colon, and at each site acute and chronic inflammation were assessed semi-quantitatively and the presence or absence of crypt architectural distortion and eosinophilia was documented.ResultsIn control, ulcerative colitis (UC) and Crohn’s disease (CD) groups there was a gradient of decreasing Paneth cell numbers from caecum to rectum. Paneth cells were not seen in the distal colon in the control group, but they were present there in 11 of 13 patients with ulcerative colitis and 14 of 15 with Crohn’s disease. Only patients with IBD showed Paneth cell hyperplasia, assessed as more than 10 Paneth cells per 10 well-oriented crypts at any site. There was a statistically significant increase in Paneth cells in the caecum, ascending, transverse and descending colon in UC and in the ascending, transverse, descending and sigmoid colon in CD compared with controls. There was no significant difference between UC and CD. There was no correlation between the site of PCM and acute or chronic inflammation, crypt distortion or eosinophilia.ConclusionPaneth cells are found in the proximal but not the distal colon in otherwise normal paediatric colonic series. A high proportion of UC and CD patients show PCM in the distal colon. This is present early in the disease and does not correlate with histological features of chronicity.

PH triggered delivery of curcumin from Eudragit-coated chitosan microspheres for inflammatory bowel disease: characterization and pharmacodynamic evaluation

Objective: This investigation deals with the development and evaluation (in vitro and in vivo) of pH triggered Eudragit-coated chitosan microspheres of curcumin (CUR) for treating ulcerative colitis.Methods: CUR-loaded chitosan microspheres were initially prepared by emulsion cross linking method followed by coating with Eudragit S-100. The pharmacodynamics of the developed formulation was analyzed in mice by acetic acid induced colitis model.Results: The developed microspheres were of uniform spherical shape with high entrapment efficiency. CUR-chitosan microspheres showed less intense peaks compared to free CUR confirming inclusion of drug within microspheres as revealed by X-ray diffractogram. Uncoated CUR-chitosan microspheres exhibited burst release within initial 4 h while microspheres coated with Eudragit S-100 prevented premature release of CUR and showed controlled release up to 12 h following Higuchi model. In vivo organ biodistribution study showed negligible amount of CUR in stomach and small intestine confirming integrity of microsphere in upper gastrointestinal tract (GIT). In vivo study revealed significant reduction in severity and extent of colonic damage with CUR-loaded microspheres as compared to pure CUR which was further confirmed by histopathological study.Conclusion: In vitro and in vivo studies proved the developed formulations as a promising system for pH-dependent delivery of drug to colon in ulcerative colitis.

Dramatic Changes of Matrix Metalloproteinases-7 and Lysozyme in the Ulcerative Colitis of Mice Induced by Dextran Sulfate Sodium

Ulcerative colitis (UC) is a lifelong illness with profound emotional and social impacts, and could cause serious damage to large intestine, especially in colon. However, the pathogenesis of UC remained unclear. The present study attempts to find out the role of matrix metalloproteinases-7 (MMP-7) and lysozyme in the pathogenesis of UC through a mice model induced by dextran sulfate sodium (DSS). The UC model was evaluated both by disease activity index (DAI) and the intestinal histopathology. The results show that there is a high correlation between the DAI score and the pathological changes of colon. Interleukin-6 (IL-6) serum levels and large intestinal fluids levels in UC mice are always higher than that of the control groups, which might be associated with the degree of the inflammation damage in the colon. The change tendency of the MMP-7 mRNA and protein expressions are both up-regulated firstly and then down-regulated from 1 to 5 d in the colon, but only the MMP-7 protein is up-regulated at 7 d again. The up-regulated MMP-7 levels in the early stage of UC may play a protective role through the activated defensins, while the down-regulated levels in the mid-later stage of UC may be connected with the severe lesions in the colon. However, the up-regulated MMP-7 levels in the later stage of UC in the colon may also contribute to the tissue repair or be served as a marker to CRC (colorectal cancer). The distribution of lysozyme protein indicates that there may be Paneth-like cells in the colon. Both the changes of MMP-7 and lysozyme in the small intestine may play a protective role for the safe environment of the whole gut, especially to the colon of UC.

Clinical significance of expression of tissue factor and tissue factor pathway inhibitor in ulcerative colitis.

To investigate the clinical significance of expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in ulcerative colitis (UC). Thirty UC specimens taken by colonoscopy from patients with active UC treated at the Department of Pathology, Central Hospital Affiliated to Shenyang Medical College from February 2010 to January 2012 were included in an experimental group, and 30 normal colon tissue samples taken by colonoscopy from non-UC patients were included in a control group. Expression of TF and TFPI in UC and normal colon tissue samples was detected by immunohistochemistry. The positive rate of TF in UC was significantly higher than that in normal colon tissue (63% vs 33%, χ(2) = 5.41, P < 0.05). The positive rate of TFPI in UC was also significantly higher than that in normal colon tissue (43% vs 17%, χ(2) = 5.08, P < 0.05). Positive rates of TF and TFPI expression in UC are significantly higher than those in normal colon tissue. TF and TFPI may play an important role in the pathogenesis of UC.

Lost therapeutic potential of monocyte-derived dendritic cells through lost tissue homing: stable restoration of gut specificity with retinoic acid

Human monocyte-derived dendritic cells (DC) (MoDC) are utilized for immunotherapy. However, in-vitro immunological effects are often not mirrored in vivo. We studied the tissue-homing potential of MoDC. Circulating monocytes and DC expressed different tissue-homing markers and, during in-vitro development of MoDC, homing marker expression was lost resulting in a ‘homeless’ phenotype. Retinoic acid (RA) induced gut-homing markers (β7 and CCR9) and a regulatory phenotype and function [decreased human leucocyte antigen D-related (HLA-DR) and increased ILT3 and fluorescein isothiocyanate (FITC-dextran uptake) in MoDC]. RA-MoDC were less stimulatory and primed conditioned T cells with a gut-homing profile (β7+CLA−). Unlike the normal intestinal microenvironment, that from inflamed colon of ulcerative colitis (UC) patients did not induce regulatory properties in MoDC. However, RA-MoDC maintained their regulatory gut-specific properties even in the presence of UC microenvironment. Therefore, MoDC may be ineffectual for immunotherapy because they lack tissue-homing and tissue-imprinting specificity. However, MoDC rehabilitation with gut-homing potential by RA could be useful in promoting immunotherapy in pathologies such as UC.

Interleukin‐17 Immunity in Pediatric Crohn Disease and Ulcerative Colitis

The present understanding of inflammatory bowel disease pathogenesis mainly relies on studies of adult patients. Therefore, we studied the balance between T-effector and regulatory cells in pediatric inflammatory bowel disease. Quantitative polymerase chain reaction and immunohistochemistry served to quantify the expression of immunological markers in mucosal biopsies and flow cytometry analysis was used in peripheral blood mononuclear cells. Colonic interleukin (IL)-17+, IL-22, and IL-6 mRNA upregulation and increase in the number of colonic IL-17 cells were demonstrated in both Crohn disease (CD) and ulcerative colitis (UC). Likewise, colonic forkhead box P3 (FOXP3+) mRNA expression and the number of colonic FOXP3 cells were increased both in CD and in UC and were accompanied in CD also with increased numbers of FOXP3+CD25 High CD4 cells in peripheral blood. Ileal relation of IL-17/CD4 cells was increased only in CD. We showed activation of colonic IL-17/IL-22 axis and upregulation of FOXP3 to occur both in pediatric CD and in UC, indicating shared immunological characteristics. Upregulation of IL-17 was restricted to colon in UC, but existed in the ileum and in the colon in active CD.

Abstract 2885: Features of innate immunity dominate serum and tissue protein and cytokine profiles in both mouse and human inflammatory bowel disease.

Abstract Inflammatory bowel disease (IBD) is a chronic and relapsing intestinal inflammatory disease that arises through unknown genetic, environmental, and bacterial origins. Ulcerative colitis (UC) and Crohn's disease (CD) are the two main forms of IBD, and their incidence is increasing in industrialized countries. Further, IBD is a significant risk factor for the development of colon cancer. Though the specific determinants remain elusive, persistent inflammation is believed to play a significant role in colon cancer carcinogenesis. To better define the molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in mouse and human IBD. Helicobacter hepaticus-infected Rag2-/- mice emulate many aspects of human IBD, and our recent work with this model highlights the importance of neutrophils in the pathology of colitis and colon cancer. Analysis of neutrophil- and macrophage-derived damage products revealed accumulation of 3-chlorotyrosine (CTyr) and 3-nitrotyrosine (NTyr) in inflamed mice colons that increased with disease duration. These results were further corroborated in mouse studies by histological evaluation, which demonstrated strong infiltration of neutrophils and macrophages to the site of inflammation. Human studies revealed an increase in CTyr in the colon of UC and CD tissues relative to serum levels. The nucleic acid chlorination damage product, 5-chloro-2′-deoxycytidine (5-Cl-dC), was quantified in human colon and found to be present at similar levels to that of inflamed mice colons. Multivariate analysis of these markers along with serum proteins and cytokines revealed a general signature of activated innate immunity in human IBD. UC sera were strongly suggestive of neutrophil activity while CD and mouse sera were suggestive of macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into disease activity in IBD. Citation Format: Charles G. Knutson, Aswin Mangerich, Yu Zeng, Arkadiusz R. Raczynski, Rosa G. Liberman, Pilsoo Kang, Wenjie Ye, Guanyu Gong, Erin Prestwich, Kun Lu, John S. Wishnok, Joshua R. Korzenik, Gerald N. Wogan, James G. Gox, Peter C. Dedon, Steven R. Tannenbaum. Features of innate immunity dominate serum and tissue protein and cytokine profiles in both mouse and human inflammatory bowel disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2885. doi:10.1158/1538-7445.AM2013-2885

Novel Pharmacologic Approaches to the Prevention and Treatment of Ulcerative Colitis

Ulcerative colitis (UC) is a chronic, relapsing inflammatory disorder of the colonic mucosa followed by poor quality of healing and recurring lesions. Recent studies demonstrated that the poor healing and chronic inflammation in colon of UC could be the result of microvascular dysfunction and endothelial barrier defect, resulting in sustained tissue hypoperfusion and ischemia in the colon. Long before angiogenesis became a popular research topic, our laboratory was the first to postulate that stimulation of angiogenesis alone might be sufficient to accelerate ulcer healing in the gastrointestinal tract. Our earlier studies demonstrated that therapy with genes or peptides of angiogenic growth factors, e.g., bFGF, PDGF and VEGF significantly accelerated healing of experimental duodenal ulcers (DU), while blockade of these angiogenic factors resulted in impaired healing of DU. However, unlike the angiogenesis in DU, increasing evidences from us and others indicate that angiogenesis plays a pathogenic role in UC, e.g., VEGF induces an abnormal "pathologic" angiogenesis which interferes with UC healing. Recently, another angiogenic factor, placental growth factor (PlGF), has also been suggested to be a marker of pathologic angiogenesis and may play a critical role in pathogenesis of UC. Although inhibition of pathologic angiogenesis by, e.g., anti-VEGF or -PlGF, was demonstrated to be a new approach to attenuate UC development, additional data of our and others showed that stimulating angiogenesis by administration of PDGF or bFGF significantly accelerated healing of UC. Also, activation of Rac1, a small GTPase, markedly improved VEGF-induced neovessel architecture defect and reduced vascular permeability (VP) in an angiogenic model. Thus, it seems that both angiogenic and anti-angiogenic therapies may be used in various stages of UC. More recently, we demonstrated that increased VP in colonic mucosa is an early and essential element in the initiation and progression of UC. The increased VP is initiated by early release of histamine and maintained/aggravated by VEGF, leading to perivascular edema, vascular stasis, hypoxia, inflammatory cell infiltration, and colonic erosions/ulcers. Inhibition of increased VP prevents or reduces development and progression of UC. In this review, we discuss novel pharmacologic approaches to prevent UC, differential actions of angiogenic growth factors in UC pathogenesis and blocking the early increase in VP in UC development, these new findings may provide new insights into the regulation of angiogenesis in UC and may lead to development of VP-related drugs to accelerate the healing of UC.

Novel Pharmacologic Approaches to the Prevention and Treatment of Ulcerative Colitis

Ulcerative colitis (UC) is a chronic, relapsing inflammatory disorder of the colonic mucosa followed by poor quality of healing and recurring lesions. Recent studies demonstrated that the poor healing and chronic inflammation in colon of UC could be the result of microvascular dysfunction and endothelial barrier defect, resulting in sustained tissue hypoperfusion and ischemia in the colon. Long before angiogenesis became a popular research topic, our laboratory was the first to postulate that stimulation of angiogenesis alone might be sufficient to accelerate ulcer healing in the gastrointestinal tract. Our earlier studies demonstrated that therapy with genes or peptides of angiogenic growth factors, e.g., bFGF, PDGF and VEGF significantly accelerated healing of experimental duodenal ulcers (DU), while blockade of these angiogenic factors resulted in impaired healing of DU. However, unlike the angiogenesis in DU, increasing evidences from us and others indicate that angiogenesis plays a pathogenic role in UC, e.g., VEGF induces an abnormal “pathologic” angiogenesis which interferes with UC healing. Recently, another angiogenic factor, placental growth factor (PlGF), has also been suggested to be a marker of pathologic angiogenesis and may play a critical role in pathogenesis of UC. Although inhibition of pathologic angiogenesis by, e.g., anti-VEGF or –PlGF, was demonstrated to be a new approach to attenuate UC development, additional data of our and others showed that stimulating angiogenesis by administration of PDGF or bFGF significantly accelerated healing of UC. Also, activation of Rac1, a small GTPase, markedly improved VEGF-induced neovessel architecture defect and reduced vascular permeability (VP) in an angiogenic model. Thus, it seems that both angiogenic and anti-angiogenic therapies may be used in various stages of UC. More recently, we demonstrated that increased VP in colonic mucosa is an early and essential element in the initiation and progression of UC. The increased VP is initiated by early release of histamine and maintained/aggravated by VEGF, leading to perivascular edema, vascular stasis, hypoxia, inflammatory cell infiltration, and colonic erosions/ulcers. Inhibition of increased VP prevents or reduces development and progression of UC. In this review, we discuss novel pharmacologic approaches to prevent UC, differential actions of angiogenic growth factors in UC pathogenesis and blocking the early increase in VP in UC development, these new findings may provide new insights into the regulation of angiogenesis in UC and may lead to development of VP-related drugs to accelerate the healing of UC. Keywords: Angiogenesis, angiogenic growth factors, bFGF, PDGF, VEGF, PlGF, Rac-1, pathologic angiogenesis, histamine, Src kinase, VE-cadherin, vascular permeability, ulcer healing, ulcerative colitis

PTU-130 Mucosal MRNA expression profiling from the terminal ileum and colon reveals under expression of claudin 8, a tight junction molecule, as potentially causal in ulcerative colitis

IntroductionIntestinal barrier dysfunction plays an important role in the pathogenesis of ulcerative colitis (UC). We investigated mRNA profiles of mucosa from the colon and terminal ileum, in patients with UC...

PTU-114 Expression of toll-like receptor (TLR)-2 and -4 in the intestinal crypt epithelial cells in inflammatory bowel disease (IBD)

IntroductionTLRs are pattern recognition receptors which detect conserved molecular patterns on commensal and pathogenic bacteria. Studies in mice have shown that TLR signalling protects against the development of colitis but...

Tu1353 Mucosal mRNA Expression Profiling From the Terminal Ileum and Colon Reveals Under Expression of Claudin 8, a Tight Junction Molecule, as Potentially Causal in Ulcerative Colitis

Introduction Intestinal barrier dysfunction plays an important role in the pathogenesis of ulcerative colitis (UC). We investigated mRNA profiles of mucosa from the colon and terminal ileum, in patients with UC and controls (HC) to identify genes that might be implicated in the pathogenesis of the disease. Methods Mucosal biopsies were taken from 24 quiescent UC patients (Mayo score <3) and 33 HCs undergoing colonoscopy. Patients were on no treatment or on 5-aminosalicylates ± azathioprine. HCs were patients without organic disease. Parallel biopsies were taken for RNA extraction and histology from macroscopically non-inflamed mucosa in the terminal ileum (TI), ascending, descending and sigmoid colon, and rectum. cRNA was hybridised to Illumina HumanHT v12.0 Expression Beadchips. Expression data were log transformed and normalised. Probes with a detection p value <0.01 were analysed. Comparing 85 biopsies from HCs and 68 biopsies from UC patients across the colon, the data for each bowel location were adjusted to the mean HC rectal expression level. Where multiple biopsies were taken from the same individual, the adjusted data across all biopsies for that individual were averaged. T tests between groups and outlier analysis (p<0.005, fold change (FC) ≥1.5) were performed using proprietary software. Results Of the ∼30K probes analysed, the two most significantly under-expressed in UC in the colon were those of claudin 8 (CLDN8) with FCs 2.94 (p=1.29×10−5) and 3.45 (p=3.92×10−5). The expression of claudin 8 increased distally in the colon, whereas claudins 3, 7 and 23 were highly, and uniformly, expressed throughout the colon and were normal in UC. Outlier analysis between HC and UC showed CLDN8 to be significantly under-expressed in 25%>40% of UC patients at all 4 colonic sites. There were no CLDN8 UC outliers in the TI. Most of these outlier patients demonstrated consistent levels of under-expression throughout the colon, and their histology revealed microscopic inflammation. Other patients with histologically active disease had normal CLDN8 expression. Conclusion CLDN8 is significantly under-expressed in the UC colon. Outlier analysis has also identified a group of patients in whom CLDN8 is grossly under-expressed. Low expression of CLDN8 in UC could be secondary to inflammation, although the evidence presented here is against this. Reduced levels of CLDN8 could lead to a weak and permeable mucosa predisposing to UC by reducing barrier resistance and allowing penetration by microbes. Competing interests None declared.

Tu1850 Expression of Toll-Like Receptor (TLR)-2 and −4 in the Intestinal Crypt Epithelial Cells in Inflammatory Bowel Disease (IBD)

Introduction TLRs are pattern recognition receptors which detect conserved molecular patterns on commensal and pathogenic bacteria. Studies in mice have shown that TLR signalling protects against the development of colitis but may exacerbate established colitis. Epithelial stem cells and their progeny are located in crypts of the small and large intestine. We have investigated TLR2 and TLR4 expression in ulcerative colitis (UC) and Crohn9s disease (CD) crypt intestinal epithelial cells (IEC). Methods Ileal and colonic mucosal samples were obtained from operation resection specimens and were histologically normal (>5 cm from cancer; normal controls) or inflamed (CD or UC). Additionally, paired samples from histologically normal (proximal colon) and inflamed (distal colon) mucosa were obtained from five colectomy specimens with isolated left-sided UC. Crypt IECs were isolated using EDTA and pancreatin. Expression of mRNA transcripts was studied by conventional and real-time RT-PCR. Cell surface TLR protein was studied by flow cytometry (expressed as median fluorescent intensity, MFI). TLR expression was also studied by immunofluorescent (IF) staining of tissue sections. Data are expressed as median (IQR). Results Relative to normal controls, TLR2 mRNA expression in IEC was significantly elevated in inflamed colonic UC [3.18-fold (1.03–10.40), p=0.003] and inflamed colonic CD [3.45 (0.80–5.40), p=0.012]. TLR4 mRNA was significantly elevated in normal colonic UC [1.90 (1.69–4.31], p=0.017), inflamed colonic UC [2.33 (1.15–4.45), p=0.024], inflamed colonic CD [1.71 (1.00–4.32), p=0.042] and inflamed ileal CD [1.84 (1.43–4.66), p=0.03]. In paired analyses, IEC TLR2 and TLR4 mRNA expression was not significantly different in normal UC mucosa compared to inflamed UC mucosa. Compared to IEC from normal control colon, IEC from colonic CD showed significantly greater expression of cell surface TLR2 protein [MFI 10.1 (2.4–29.0) vs 33.1 (17.7–107.5), p=0.05] and TLR4 protein [MFI 12.1 (4.9–25.3) vs 40.9 (23.2–103.5), p=0.04]. IF staining confirmed TLR2 and TLR4 protein expression on the luminal surface of crypt IEC. Conclusion IEC expression of TLR4 mRNA was up-regulated in inflamed and un-inflamed colonic and ileal IBD. TLR2 mRNA was up-regulated in inflamed colonic IBD. Interestingly, TLR2 and TLR4 mRNA expression was similar in IECs isolated from un-inflamed, histologically normal mucosa as it was in IECs isolated from inflamed mucosa in UC. This may suggest TLR2 and TLR4 up-regulation is a primary rather than secondary event in UC. Moreover, TLR2 and TLR4 protein was expressed in greater amounts on the surface of IECs in CD. Competing interests None declared.