Ulcerative Colitis Tissues Research Articles

Expression of interleukin (IL)-19 and IL-24 in inflammatory bowel disease patients: a cross-sectional study

Interleukin (IL)-19 and IL-24 belong to the IL-20 subfamily, and are involved in host defence against bacteria and fungi, tissue remodelling and wound healing. Nevertheless, no previous studies have explored their expression in Mexican mestizo patients with inflammatory bowel disease (IBD). The aim of the study was to characterize and to enumerate peripheral and tissue IL-19- and IL-24-producing cells, as well as gene expression in patients with IBD with regard to its clinical activity. We studied a total of 77 patients with ulcerative colitis (UC), 36 Crohn's disease (CD) and 33 patients as control group (without endoscopic evidence of intestinal inflammation). Gene expression was measured by real-time-polymerase chain reaction (RT-PCR). Protein expression was detected in biopsies by immunohistochemistry and in freshly isolated peripheral blood mononuclear cells by flow cytometry. IL-19 and IL-24 gene expression was elevated significantly in patients with active IBD versus the inactive disease and non-inflammatory control groups (P < 0·05). However, IL-19- and IL-24-producing cells were only increased in active CD versus active UC and non-inflammatory tissues (P < 0·05). IL-19 was produced conspicuously by circulating B cells and monocytes in patients with inactive disease (P < 0·05). Conversely, IL-24 was noticeably synthesized by peripheral B cells, CD4(+) T cells, CD8(+) T cells and monocytes in patients with active disease. In conclusion, IL-19- and IL-24-producing cells in active CD patients were increased compared with active UC and non-inflammatory tissues. These cytokines could significantly shape and differentiate inflammatory process, severity and tolerance loss between UC and CD pathophysiology.

MicroRNA-155 Is Involved in the Pathogenesis of Ulcerative Colitis by Targeting FOXO3a

MicroRNAs (miRNAs) are important posttranscriptional regulators of gene expression. The precise role of miRNAs in ulcerative colitis (UC) is not completely understood. The purpose of this study was to identify miRNAs that are induced in patients with active UC and to assess the effect of miR-155 on improving intestinal inflammation. The miRNA profiles in patients with active UC (n = 20) and healthy subjects (n = 16) were examined using miRNA microarrays. miR-155 upregulation was confirmed by quantitative RT-PCR. Regulation of the target gene FOXO3a expression by miR-155 was assessed using luciferase reporter construct assays and miR-155 mimic or inhibitor transfections. The effects of FOXO3a or miR-155 on IκBα or IL-8 were detected by Western blot or enzyme-linked immunosorbent assay in HT29 cells, respectively. We identified 68 miRNAs that were differentially expressed (33 upregulated and 35 downregulated) in patients with active UC compared with healthy controls. One of the upregulated miRNAs in the UC tissue was miR-155 (1.22-fold, P < 0.03), which plays a key role in the regulation of inflammatory pathways. In patients with active UC, miR-155 was significantly upregulated, and the expression of FOXO3a dramatically decreased. Luciferase reporter assays demonstrated that miR-155 directly targets FOXO3a and affects the protein expression of FOXO3a in HT29 cells. Moreover, silenced FOXO3a and the overexpression of miR-155 increased the levels of IL-8 in TNF-α-treated HT29 cells by suppressing the inhibitory IκBα. miR-155 appears to play a role in the intestinal inflammation of patients with active UC by downregulating the expression of FOXO3a. This process may activate the nuclear factor kappa B signaling pathway.

DUOX2 and DUOXA2 Form the Predominant Enzyme System Capable of Producing the Reactive Oxygen Species H2O2 in Active Ulcerative Colitis and are Modulated by 5-Aminosalicylic Acid

NADPH oxidase-derived reactive oxygen species, such as H2O2, are part of the intestinal innate immune system but may drive carcinogenesis through DNA damage. We sought to identify the predominant enzyme system capable of producing H2O2 in active ulcerative colitis and assess whether it is affected by 5-aminosalicylic acid (5-ASA). We studied human mucosal biopsies by expression arrays, quantitative real-time polymerase chain reaction for NADPH oxidase family members, in situ hybridization (DUOX2 and DUOXA2) and immunofluorescence for DUOX, 8-OHdG (DNA damage), and γH2AX (DNA damage response) and sought effects of 5-ASA on ex vivo cultured biopsies and cultured rectal cancer cells. DUOX2 with maturation partner DUOXA2 forms the predominant system for H2O2 production in human colon and is upregulated in active colitis. DUOX2 in situ is exclusively epithelial, varies between and within individual crypts, and increases near inflammation. 8-OHdG and γH2AX were observed in damaged crypt epithelium. 5-ASA upregulated DUOX2 and DUOXA2 levels in the setting of active versus quiescent disease and altered DUOX2 expression in cultured biopsies. Ingenuity pathway analysis confirmed that inflammation status and 5-ASA increase expression of DUOX2 and DUOXA2. An epithelial cell model confirmed that cultured cancer cells expressed DUOX protein and produced H2O2 in response to hypoxia and 5-ASA exposure. Both DUOX2 and DUOXA2 expression are involved specifically in inflammation and are regulated on a crypt-by-crypt basis in ulcerative colitis tissues. Synergy between inflammation, hypoxia, and 5-ASA to increase H2O2 production could explain how 5-ASA supports innate defense, although potentially increasing the burden of DNA damage.

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.

Black raspberries protectively regulate methylation of Wnt pathway genes in precancerous colon tissue.

Ulcerative colitis is frequently an intermediate step to colon cancer. The interleukin-10 knockout mouse is a genetic model of this progression. We report that knockout mice fed 5% black raspberries (BRB) had significantly less colonic ulceration as compared with knockout mice that consumed the control diet. Dysfunction of the Wnt signaling pathway is a key event in ulcerative colitis-associated colon carcinogenesis. Therefore, we investigated the effects of BRBs on the Wnt pathway and found that the BRB-fed knockout mice exhibited a significantly lower level of β-catenin nuclear translocation. We followed-up this observation by evaluating the effect of BRBs on selected Wnt pathway antagonists. The mRNA expression levels of wif1, sox17, and qki were diminished in the knockout mice, whereas they were expressed at normal levels in knockout mice that were fed BRBs. The lower mRNA expression of these genes in the colon from the knockout mice correlated with hypermethylation of their promoter regions; BRBs decreased their promoter methylation and increased mRNA expression of these genes. This hypomethylation was associated with elevated protein expression of key proteins/enzymes that augment methylation, for example, dnmt3b, hdac1, hdac2, and mbd2 in the knockout mice; in addition, BRBs decreased the protein expression of these proteins/enzymes. The knockout mouse model recapitulates what occurs in human ulcerative colitis. Promoter methylation of CDH1 and SFRP1 was significantly higher in human ulcerative colitis tissues compared with their adjacent normal tissues. In conclusion, our results suggest that BRBs inhibit colonic ulceration and, ultimately, colon cancer partly through inhibiting aberrant epigenetic events that dysregulate Wnt signaling.

PTH-099 Distinctive Gene Expression Profiles in the Whole Blood of Patients with Moderate to Severe Ulcerative Colitis and Crohn’S Disease

IntroductionThe aim of this study was to define whole blood (WB) gene expression profiles in moderate to severe ulcerative colitis (UC) and Crohn’s disease (CD) patients, and to elucidate modulated...

Interleukin-17 serum levels and TLR4 polymorphisms in ulcerative colitis.

Inflammatory bowel disease, an autoimmune disease, has two clinical manifestations including Crohn's disease and ulcerative colitis (UC). IL-17 has been the target of intensive research in autoimmune diseases. The influence of Toll like receptor 4 (TLR-4) gene polymorphisms on IL-17 production has also been revealed in UC patients and tissue inflammation in mice. To investigate the association between the TLR-4 gene polymorphisms, Asp299Gly and Thr399Ile and IL-17 serum levels with ulcerative colitis. Additionally, we aimed to study modulation effects of forenamed gene polymorphisms on IL-17 serum levels in UC patients and controls. A total of 256 healthy controls and 85 UC patients enrolled in our study. DNA was extracted and PCR-RFLP technique was employed to determine Asp299Gly and Thr399Ile polymorphisms in TLR-4 gene and IL-17 serum levels were measured by ELISA method. There was no significant difference between the frequency of Asp299Gly A>G and Thr399Ile C>T in UC patients and controls. While IL-17 serum levels in UC patients were significantly higher than controls (p=0.003), no significant difference in IL-17 levels between different genotypes existed. Additionally, a significant inverse relationship was observed between hemoglobin level and IL-17 serum levels in UC patients (p=0.039). Increased IL-17 serum levels in our UC patients might be explained through the synergistic activity of IL-17/IL-23 axis and pro-inflammatory cytokines, causing severe clinical outcome in patients with IBD. The prolonged excretion of blood in stool driven by inflammatory process which causes iron metabolism disorder and anemia may elucidate the inverse correlation between hemoglobin and IL-17 serum levels in UC patients. Lack of association between the TLR-4 gene polymorphisms and UC in our study was consistent with the results from other Caucasian populations.

Methylated miR-124, -137, and -34b/c as predictive biomarkers for ulcerative colitis-associated colorectal neoplasia.

e14631 Background: Accumulating evidence suggests that microRNAs (miRNAs) can be targets of methylation-induced silencing in colorectal cancer, which can occur in an age-dependent manner, and constitutes an epigenetic “field defect”. We hypothesized whether methylation of miR-124, -137 and -34b/c can predict colorectal neoplasia in patients with ulcerative colitis (UC). Methods: We examined 185 samples from 70 UC patients (45 patients without neoplasia, 12 with dysplasia and 13 with cancers) for the methylation status of miR-124, -137 and -34b/c by quantitative bisulfite pyrosequencing analysis. In addition, expression levels of these 3 miRNAs in dysplastic (n=12), cancerous (n=13) and non-neoplastic UC mucosa (n=20) were quantified by TaqMan qRT-PCR. Results: MiR-124 and -137 methylation levels were significantly higher in rectal tissues than in proximal mucosa from non-neoplastic UC tissues, and were significantly associated with age and disease duration in rectal tissues. Methylation levels of all 3 miRNAs were significantly higher in dysplasia and cancer compared to mucosa without neoplasia, and showed a “field defect” in the non-neoplastic rectal tissues from patients with cancer . Receiver operating characteristic (ROC) analysis revealed that methylation levels of all 3 miRNAs in non-neoplastic rectal tissues could discriminate patients with cancer from those without (miR-124 AUC value = 0.74; miR-137 AUC = 0.79; miR-34b/c AUC=0.72). Furthermore, methylated miR-137 in non-neoplastic rectal tissues allowed discrimination of UC patients with neoplasia from those without (AUC = 0.76), and independently predicted neoplasia (OR= 5.55; 95% CI: 1.40–22.05; P = 0.0148). Expression levels of all 3 miRNAs were significantly downregulated in dysplasia or cancer compared to non-neoplastic UC mucosa, and the expression and methylation were negatively correlated. Conclusions: Methylation of miR-124, -137 and -34b/c in rectal biopsies, particularly miR-137, may serve as novel predictive biomarkers for neoplasia in UC patients. Restoration of expression by reversing their methylation status could be an attractive strategy to slow or reverse age-related UC-associated neoplasia.

Su1763 Distinctive Gene Expression Profiles in the Whole Blood of Patients With Moderate to Severe Ulcerative Colitis and Crohn's Disease

Introduction The aim of this study was to define whole blood (WB) gene expression profiles in moderate to severe ulcerative colitis (UC) and Crohn’s disease (CD) patients, and to elucidate modulated genes and pathways that are shared by and also unique to each disease. Gene expression profiling of UC and CD WB has not been compared in a study of this size, and the subset of non-overlapping genes identified could potentially lead to a means to discriminate between these two forms of IBD. A molecular diagnostic assay based on gene expression from readily accessible tissue (WB) would have great utility in differentiating between UC and CD, a common diagnostic dilemma. Methods WB samples were collected from a subset of patients in one of two clinical trials: PURSUIT-SC, a study to evaluate safety and efficacy of induction therapy with SC golimumab in patients with moderate to severe UC, and CERTIFI, a study to evaluate safety and efficacy of ustekinumab therapy in patients with moderate to severe CD. In both studies, samples (n = 69 UC, 204 CD) were collected at baseline for mRNA expression profiling using Affymetrix HG-U133+ PM arrays. Samples from healthy volunteers were obtained independently of the trials. Changes in gene expression of > 1.5-fold and false detection rate (FDR) p-value Results There was overlap in the significant changes in gene expression observed in the WB of UC and CD patients compared to normal controls. Of the 1229 differentially expressed transcripts in UC, 63% (45% relative to CD) overlapped with those in CD WB. Over-expressed genes in UC and CD included CD177, IL1R1, IL17RA, MMPs, and other genes involved in systemic inflammation, cellular cytotoxicity, and lymphocyte migration. However, significant proportions of genes (37% of UC gene changes, or 55% of CD) were uniquely expressed in either disease. Genes expressed specifically in UC included regulators of cell death and survival, eg BCL2A1, and several integrin isoforms. Differentially expressed genes specific to CD included IL23A, genes involved in ubiquitination and autophagy, eg ATG9B, and several chemokines. Pathways unique to CD involved B-cell receptor signalling and protein degradation, while oncogenic mechanisms were more predominant in pathways uniquely upregulated in UC. Conclusion Despite sharing many of the same upregulated transcripts, WB of CD and UC patients also demonstrated significant proportions of differentially expressed genes. Transcriptional profiles in circulating immune cells found in WB may serve as a surrogate for relaying the state of less-accessible luminal tissues in UC and CD patients, and have the potential to aid in differential diagnosis of these diseases. Disclosure of Interest S. Telesco Employee of: Janssen R&D, LLC, K. Li Employee of: Janssen R&D, LLC, C. Marano Employee of: Janssen R&D, LLC, C. Gasink Employee of: Janssen R&D, LLC, K. Ma Employee of: Janssen R&D, LLC, R. Strauss Employee of: Janssen R&D, LLC, C. Brodmerkel Employee of: Janssen R&D, LLC

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

DNA hypermethylation of a selective gene panel as a risk marker for colon cancer in patients with ulcerative colitis

Patients with inflammatory bowel disease (IBD) which includes ulcerative colitis (UC) and Crohn's disease (CD) of the colon are at risk of developing colorectal cancer (CRC). Here, we analyzed the methylation status of selected genes as a risk marker in UC patients. We assessed methylation frequency of 4 genes [secreted frizzled-related protein 1 (SFRP1), transcription elongation regulator 1-like (TCERG1L), fibrillin 2 (FBN2) and tissue factor pathway inhibitor 2 (TFPI2)] in biopsies of 36 UC patients. SFRP1 and TCERG1L genes showed high methylation frequencies but FBN2 and TFPI2 genes showed methylation frequencies of 50% in UC patients which suggests that our sensitive selective markers could detect half of the UC patients. We also confirmed the methylation status in UC tissues by bisulfite sequencing analysis. We compared the levels of methylation in terms of quantification between UC patients and CRC tumors. Importantly, methylation levels of these 4 genes were found to be significantly higher in CRC compared to UC patients, even though we noted a frequent methylation pattern in UC patients. Our data suggest that sensitive DNA methylation markers are able to identify UC patients and this would implicate the risk of CRC. Therefore, assessing the methylation of these 4 genes in UC patients could contribute to prevent the progression of severe disease with regular colonoscopic surveillance.

IL-22 is related to development of human colon cancer by activation of STAT3

BackgroundIt has been previously reported that IL-22, one of the cytokines secreted by Th17 cells, demonstrates both a protective and inflammatory promotion effect in inflammatory bowel disease (IBD) through STAT3 signaling activation. We sought to investigate the role of IL-22 expression in colon cancer (CC).MethodsThe expression of IL-22 and related molecules were detected in human CC, the detail function and mechanism of IL-22 were investigated by in vivo and in vitro model.ResultsOur results demonstrated significant upregulation of IL-22 in human CC tumor infiltrated leukocytes (TILs) compared to peripheral lymphocytes. Moreover, our findings demonstrated that IL-22 expression was significantly higher in ulcerative colitis (UC) tissues versus normal colon tissues. Both IL-22 receptor α1 (IL-22RA1) and IL-23 were highly expressed in CC and UC tissues compared to normal controls. TILs exhibiting various IL-22 expression levels isolated from CC patients were demonstrated to enhance tumor growth and metastasis co-transplanted with Hct-116 cells underwent subcutaneous transplantation in mice model. Tumor growth and metastasis was promoted by STAT3 phosphorylation and upregulation of its downstream genes such as Bcl-xl, CyclinD1, and VEGF. In vitro studies confirmed the anti-apoptotic and pro-proliferation effect of IL-22 according to the BrdU cooperation assay and peroxide induced apoptosis analysis with or without the presence of IL-22.ConclusionIn this study we demonstrated that excessive IL-22 in the CC and UC microenvironment leads to tumor growth, inhibition of apoptosis, and promotion of metastasis depend on STAT3 activation.

Expression of interleukin-22/STAT3 signaling pathway in ulcerative colitis and related carcinogenesis

To investigate the expression of interleukin (IL)-22 and its related proteins in biopsy specimens from patients with ulcerative colitis (UC) and UC-related carcinogenesis. Biopsy specimens were obtained from patients with inactive (n = 10), mild-to-moderately active (n = 30), severely active (n = 34), initial (n = 30), and chronic UC (n = 44), as well as UC patients with dysplasia (n = 10). Specimens from patients without colonic abnormalities (n = 20) served as controls. Chronic colitis in experimental mice was induced by 2.5% dextran sodium sulfate. The expression levels of IL-22, IL-23, IL-22R1 and phosphorylated STAT3 (p-STAT3) were determined by immunohistochemistry. Bcl-2, cyclin D1 and survivin expression was detected by Western blotting. Patients with active UC had significantly more IL-22, IL-23, IL-22R1 and p-STAT3-positive cells than the patients with inactive UC and normal controls. Furthermore, IL-22 and related proteins were closely related to the severity of the colitis. The expression of IL-22 and IL-22R1 in the tissue of initial UC was stronger than in that of chronic UC, whereas the expression of p-STAT3 was significantly increased in chronic UC tissues. In dysplasia tissues, the expression level of IL-22 and related proteins was higher compared with controls. Mouse colitis model showed that expression of IL-22, IL-22R1 and IL-23 was increased with time, p-STAT3 and the downstream gene were also remarkably upregulated. IL-22/STAT3 signaling pathway may be related to UC and UC-induced carcinogenesis and IL-22 can be used as a biomarker in judging the severity of UC.

Tu1325 Association Between Common Functional Genetic Polymorphisms and CpG Island Methylation Status in Colonic Mucosa in Ulcerative Colitis

Introduction/Aims: Lubiprostone enhances intestinal fluid secretion by activation of ClC-2, and likely CFTR chloride channels, and is used clinically to treat constipation. It has also been shown to stimulate recovery of mucosal barrier function in ischemic porcine intestine and modify tight junction (TJ) proteins. A defect in barrier function is thought to contribute to both the pathogenesis of inflammatory bowel disease (IBD) and to disease relapse. The aim of this study was to investigate if lubiprostone improves barrier function in colonic biopsies from IBD patients. Methods: Mucosal biopsies were obtained from the sigmoid colon of normal subjects undergoing screening colonoscopy, Crohn's disease (CD) and ulcerative colitis (UC) patients in remission with approval of UCSD IRB. Biopsies were mounted in Ussing chambers, and bathed in Ringer's solution at 37oC. After stabilization for 30 minutes, the tissues were treated with lubiprostone (0.01, 0.1 or 1.0 μM), or vehicle. Paracellular permeability to macromolecules was assessed with the fluorescent marker, FITCdextran (4 kD). Transepithelial electrical resistance (TER) was monitored over 120 minutes. Ion transport responses, measured as short-circuit current (Isc), were recorded for lubiprostone, the cAMP agonist forskolin, and the calcium agonist carbachol (CCh). Statistical analysis was performed using ANOVA or Student's t-test. Results: Lubiprostone increased Isc across human colon in a concentration-dependent manner in all tissues (p<0.05; n=1517). Peak responses to lubiprostone were significantly lower in CD and UC vs. controls (p<0.05; n=15-17). The Isc response to forskolin was similar in controls, CD and UC tissues, while lubiprostone reduced the Isc response to forskolin in a dose-dependent manner. The Isc response to CChwas similar in all three subject groups and was unaffected by lubiprostone pre-treatment. Lubiprostone (1.0 μM) increased the diminished TER in CD biopsies vs. untreated by 8 ± 3 % (n=17, p<0.05) but had no significant effect on UC or normal biopsies. FITC-dextran permeability was not increased in CD or UC vs. controls and was unaffected by lubiprostone. Conclusion: Lubiprostone restored TER in CD biopsies indicating a possible role for lubiprostone as an enhancer of barrier function in CD vs. UC. Moreover, the Isc response to lubiprostone was lower in biopsies from IBD patients vs. normal controls whereas responses to forskolin or CCh were unchanged by disease status. This suggests that lubiprostone could be used to improve barrier function in CD without a risk of excessive fluid secretion. Support provided by Takeda Pharmaceuticals North America, Inc. & NIH DK080506.

Epstein-Barr Virus Infection Is Common in Inflamed Gastrointestinal Mucosa

Epstein-Barr virus (EBV) is present in the malignant epithelial cells of 10% of all gastric adenocarcinomas; however, localization of the virus in normal gastrointestinal mucosa is largely unexplored. In the present study, we measured EBV DNA and localized viral gene products in gastritis specimens (n = 89), normal gastric and colonic mucosa (n = 14), Crohn's disease (n = 9), and ulcerative colitis (n = 11) tissues. A battery of sensitive and specific quantitative polymerase chain reactions targeted six disparate regions of the EBV genome: BamH1 W, EBNA1, LMP1, LMP2, BZLF1, and EBER1. EBV infection was localized by EBV-encoded RNA (EBER) in situ hybridization and by immunohistochemical stains for viral latent proteins LMP1 and LMP2 and for viral lytic proteins BMRF1 and BZLF1. B lymphocytes were identified using CD20 immunostains. EBV DNA was essentially undetectable in normal gastric mucosa but was present in 46% of gastritis lesions, 44% of normal colonic mucosa, 55% of Crohn's disease, and 64% of ulcerative colitis samples. Levels of EBV DNA exceeded what would be expected based on the numbers of B lymphocytes in inflamed tissues, suggesting that EBV is preferentially localized to inflammatory gastrointestinal lesions. Histochemical staining revealed EBER expression in lymphoid cells of some PCR-positive lesions. The viral lytic viral proteins, BMRF1 and BZLF1, were expressed in lymphoid cells of two ulcerative colitis tissues, both of which had relatively high viral loads by quantitative PCR. EBV-infected lymphocytes are frequently present in inflamed gastric and colonic mucosa. Active viral replication in some lesions raises the possibility of virus-related perpetuation of gastrointestinal inflammation.

Serum leucine-rich alpha-2 glycoprotein is a disease activity biomarker in ulcerative colitis

Reliable biomarkers for monitoring disease activity have not been clinically established in ulcerative colitis (UC). This study aimed to investigate whether levels of serum leucine-rich alpha-2 glycoprotein (LRG), identified recently as a potential disease activity marker in Crohn's disease and rheumatoid arthritis, correlate with disease activity in UC. Serum LRG concentrations were determined by enzyme-linked immunosorbent assay (ELISA) in patients with UC and healthy controls (HC) and were evaluated for correlation with disease activity. Expression of LRG in inflamed colonic tissues from patients with UC was analyzed by western blotting and immunohistochemistry. Interleukin (IL)-6-independent induction of LRG was investigated using IL-6-deficient mice by lipopolysaccharide (LPS)-mediated acute inflammation and dextran sodium sulfate (DSS)-induced colitis. Serum LRG concentrations were significantly elevated in active UC patients compared with patients in remission (P < 0.0001) and HC (P < 0.0001) and were correlated with disease activity in UC better than C-reactive protein (CRP). Expression of LRG was increased in inflamed colonic tissues in UC. Tumor necrosis factor alpha (TNF-α), IL-6, and IL-22, serum levels of which were elevated in patients with active UC, could induce LRG expression in COLO205 cells. Serum LRG levels were increased in IL-6-deficient mice with LPS-mediated acute inflammation and DSS-induced colitis. Serum LRG concentrations correlate well with disease activity in UC. LRG induction is robust in inflamed colons and is likely to involve an IL-6-independent pathway. Serum LRG is thus a novel serum biomarker for monitoring disease activity in UC and is a promising surrogate for CRP.

Proteomic Analysis of Colonic Mucosal Tissue from Tuberculous and Ulcerative Colitis Patients

Changes in the expression profiles of specific proteins leads to serious human diseases, including colitis. The proteomic changes related to colitis and the differential expression between tuberculous (TC) and ulcerative colitis (UC) in colon tissue from colitis patients has not been defined. We therefore performed a proteomic analysis of human TC and UC mucosal tissue. Total protein was obtained from the colon mucosal tissue of normal, TC, and UC patients, and resolved by 2-dimensional electrophoresis (2-DE). The results were analyzed with PDQuest using silver staining. We used matrix-assisted laser desorption ionization time-of-flight/time-of-flight spectrometry (MALDI TOF/TOF) to identify proteins differentially expressed in TC and UC. Of the over 1,000 proteins isolated, three in TC tissue and two in UC tissue displayed altered expression when compared to normal tissue. Moreover, two proteins were differentially expressed in a comparative analysis between TC and UC. These were identified as mutant β-actin, α-enolase and Charcot-Leyden crystal protein. In particular, the expression of α-enolase was significantly greater in TC compared with normal tissue, but decreased in comparison to UC, implying that α-enolase may represent a biomarker for differential diagnosis of TC and UC. This study therefore provides a valuable resource for the molecular and diagnostic analysis of human colitis.

Comparative proteomics analysis of serum proteins in ulcerative colitis patients

In the present study, we investigated the differentially expressed proteins associated with ulcerative colitis (UC) using proteomic methods. Two-dimensional electrophoresis (2-DE) technology was performed to separate the total proteins of ulcerative tissues from those of the normal tissues of UC patients. PDQuest software was applied to analyze the obtained 2-DE images. Candidate protein spots between the two groups were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and bioinformatics analysis. The well resolution and reproducible 2-DE patterns of UC and normal tissues were established. Of the 12 differentially expressed proteins, 9 were successfully identified, of which 6 proteins were up-regulated including apolipoprotein C-III, haptoglobin, receptor tyrosine kinase, aldehyde reductase, pericentriolar material 1, and heat shock factor protein 2, and 3 were down-regulated including keratin, filamin A-interacting protein 1, and tropomyosin 3. These identified proteins were related to hormonal modulation, immune response, oxidative stress, and signal conduction. The 2-DE protein expression profile of the UC tissues displays an obvious difference from that of the normal controls. Various proteins may be involved in the occurrence of UC.

Matriptase Protects Against Experimental Colitis and Promotes Intestinal Barrier Recovery

Matriptase is a membrane-anchored serine protease encoded by suppression of tumorigenicity-14 (ST14) that is required for epithelial barrier homeostasis. However, its functional role in inflammatory bowel disease (IBD) is unexplored. Matriptase expression in control, Crohn's disease, and ulcerative colitis tissue specimens was studied by quantitative polymerase chain reaction (qPCR) and immunostaining. Matriptase function was investigated by subjecting St14 hypomorphic and control littermates to dextran sodium sulfate (DSS)-induced colitis and by siRNA silencing in cultured monolayers. Mice were analyzed for clinical, histological, molecular, and cellular effects. Matriptase protein and ST14 mRNA levels are significantly downregulated in inflamed colonic tissues from Crohn's disease and ulcerative colitis patients. Matriptase-deficient St14 hypomorphic mice administered DSS for 7 days followed by water without DSS for 3 days develop a severe colitis, with only 30% of the St14 hypomorphic mice surviving to day 14, compared with 100% of control littermates. Persistent colitis in surviving St14 hypomorphic mice was associated with sustained cytokine production, an inability to recover barrier integrity, and enhanced claudin-2 expression. Cytokines implicated in barrier disruption during IBD suppress matriptase expression in T84 epithelial monolayers and restoration of matriptase improves barrier integrity in the cytokine-perturbed monolayers. These data demonstrate a critical role for matriptase in restoring barrier function to injured intestinal mucosa during colitis, which is suppressed by excessive activation of the immune system. Strategies to enhance matriptase-mediated barrier recovery could be important for intervening in the cycle of inflammation associated with IBD.

Abstract B33: Molecular targets in early detection and differentiation of inflammatory bowel disease associated colon-rectal-anal cancer disparities.

Abstract The inflammatory bowel disease (IBD), Crohn's (CC) and ulcerative colitis (UC), affect approximately 1–2 of every 1000 people in developed countries. These chronic inflammatory diseases result in significant morbidity and mortality. All IBD-associated colorectalanal cancers (CRAC) occurred in segments of colitis and are frequently diagnosed at an advanced stage. This presentation is a continuation of our work that investigates potential molecules that could define a unique classifier between CC and UC and early detection of CRAC. There are multiple challenges to identifying protein classifiers suggesting outcome prediction and differentiation for patients with IBD and/or IBD-CRAC from molecular interpretation standpoints is complex. While there are technical approach disagreements, the goal is clear: to produce scientific evidence which can provide personalized expert care to patients. We have developed an amenable proteomic methodology that supports the diagnostic feasibility to discriminate molecularly, different inflammatory colitis. The histologic layers of colectomy samples from patients with confirmed UC and CC tissues were analyzed using matrix-assisted laser de-sorption/ionization mass spectrometry (MALDI MS) for proteomic profiling. Our previous findings1 prompted further sample collection resulting in an increased sample size that would allow a more robust analysis. The samples from colon tissues collected in 2008 and samples collected in 2010 were re-randomized into training and independent test sets in order to avoid systematic differences between new and old data sets, typical for MALD-ToF spectra acquired in situ from tissue at different times. MALDI-ToF spectra were included in the analysis only for samples containing more than 3 unique spectra. The results have successfully identified 11 highly significant mass-to-charge ratio (m/z) signals (m/z 5045, 6139, 9245, 8413, 3666, 3595, 4122, 8774, 2778, 9232 and 9519) that distinguish CC from UC. These features are independent of the tissue of origin and represent disease specific markers. Some of these signatures were only found in the colonic mucosa (m/z 8413, 3666 &amp; 3595) or submucosa (m/z 4122, 8774, 2778, 9232 &amp; 9519) while others were found in both two layers (m/z 5045, 6139 &amp; 9245). This information may provide new avenues for the development of novel diagnostic, prognostic and therapeutic targets. We will analyze CRAC in IBD segments2,3 to look for these proteins that may help in studying their biological mechanisms in cancer transformation.. Support: 3U54CA091408-09S1 (to MMC-VICC Partnership: SE Adunyah &amp; HL Moses)