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M1952 Carcinogenesis in Ulcerative Colitis: Immunosurveillance Mechanisms
AGA Abstracts
(14%) cases. Fourteen (40%) patients had stage I or II disease and 16 (46%) had stage III or IV. Staging was not available in 5 (14%) patients. Dysplasia was found in 18 surgical specimens of SBA (51%). It was adjacent to SBA in 9 cases, distant in 2 cases and both contiguous and distant in 7 cases. Dysplasia was flat in 12 cases, elevated (DALM) in 5 cases and both flat and elevated in 1 case. High grade dysplasia was observed in 14 of 18 (78%) cases. All lesions of dysplasia without cancer were found in the ileum. It was flat dysplasia in 3 patients (low grade in 2 cases and high grade in 1 case) and one serrated lesion with low and high grade dysplasia in one patient. All carcinomatous and dysplastic lesions, except one, occurred in inflammatory areas. Conclusion: Dysplasia can be found in the vicinity or at distance from SBA in half of the cases. The dysplasia-carcinoma sequence seems valid in SBA associated with CD. Endoscopic surveillance of the small bowel aimed to detect dysplasia should be considered in patients with CD of the small bowel with risk factors associated with SBA (Am J Gastroenterol 2008;103:1). M1950 Protein Kinase D Mediates Synergistic COX-2 Expression Induced by CrossTalk Between TNF-α and G-Protein Coupled Receptor Agonists in Human Colonic Myofibroblasts James Yoo, Citlali E. Rodriguez-Perez, James Sinnett-Smith, Enrique Rozengurt
A = NK-kB gene target; B = NK-1R gene target
Background: Myofibroblasts have recently been identified as major mediators of tumor necrosis factor-α (TNF-α)-associated colitis-associated colon cancer but the precise mechanism(s) involved remains incompletely understood. Myofibroblasts are a major source of the inducible isoform of cyclooxygenase (COX-2) and of prostaglandins (e.g. PGE2) in the gastrointestinal tract. We hypothesize that TNF-α signaling cross talks with pro-inflammatory mediators, including bradykinin (BK) and lysophosphatidic acid (LPA), to elicit COX-2 expression and PGE2 production that are essential for colon cancer development. Results: Treatment of 18Co cells, a model of human colonic myofibroblasts, with TNF-α and either the G protein-coupled receptor agonists (GPCR) BK or LPA induced striking synergistic COX-2 protein expression that was paralleled by increases in the production of PGE2. Synergistic COX-2 expression was also induced by sequential exposure to TNF-α followed by BK or LPA. COX-2 expression in 18Co cells treated with BK and TNF-α was prevented by the B2 BK receptor antagonist HOE140, and the preferential protein kinase C (PKC) inhibitors Ro31-8220, GF109203X, and Go6976, suggesting that COX-2 expression in response to TNF-α and BK was mediated through a PKC/protein kinase D (PKD)-dependent pathway. Indeed, TNF-α, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation induced by BK, as measured by PKD phosphorylation at its activation loop (Ser744) and auto-phosphorylation site (Ser916). Transfection of 18Co cells with siRNA targeting PKD1 completely inhibited the synergistic increase in COX-2 protein in response to BK and TNF-α demonstrating, for the first time, a critical role of PKD1 in the pathways leading to synergistic expression of COX-2. We also found that exposure of 18Co cells to the combination of TNF-α and BK produced synergistic stimulation of ERK activation. Cell treatment with U0126, at a concentration that completely prevented ERK activation, markedly reduced COX-2 expression in response to stimulation with the combination of TNF-α and BK implying that the ERK pathway contributes to PKD signaling leading to COX-2 expression. Conclusion: Our results indicate that crosstalk between TNFα and GPCR agonists amplifies a PKC/PKD1/ERK phosphorylation cascade that mediates synergistic COX-2 expression in colonic myofibroblasts. We propose that PKD1 mediates COX-2 expression in colonic myofibroblasts to create a microenvironment that supports tumor growth.
M1952 Carcinogenesis in Ulcerative Colitis: Immunosurveillance Mechanisms Marco Scarpa, Marina Bortolami, Diego Faggian, Andromachi Kotsafti, Cesare Ruffolo, Filippo Navaglia, Anna Pozza, Renata D'Incà, Mario Plebani, Giacomo C. Sturniolo, Imerio Angriman Background The inconsistency between dysplasia rate and incidence of cancer in ulcerative colitis (UC)suggests the presence of an immunosurveillance mechanism. The aim of our study was to analyse the expression of costimulatory molecules CD80 and CD86, the lamina propria mononuclear cells populations and the cytokines network at the different stages of carcinogenesis in UC compared to the non inflammatory omologues. Patients and methods Five groups of patients affected by UC (19 pts), UC with colonic dysplasia (10 pts), UC and cancer (7 pts), colonic adenoma (11 pts), sporadic colonic cancer (15 pts) and a group of healthy control (11 pts) were enrolled. Mucosal CD80 and CD86 mRNA levels were quantified with Real Time PCR. Mucosal expression of CD80, CD86, CD3, CD20, CD68 and p53 was analysed by immunohistochemistry. Mucosal levels of IL-1β, IL-2 and TNFα were measured with immunometric assays. Kruskal-Wallis ANOVA, Mann-Whitney U test and Kendall's tau correlation test were used. Results In UC, CD80 expression was higher in patients with dysplasia (p=0.017). This difference was not evident in the non inflammatory carcinogenesis (fig 1). CD80 expression directly correlated with IL-2 expression and with lamina propria T and B lymphocytes and monocytes populations. In UC, CD86 expression resulted significantly higher than in non inflammatory carcinogenesis without any differences between the steps of the carcinogenesis. CD86 expression correlated with inflammation severity and with IFNgamma expression. Conclusion In UC, CD80 is significantly more expressed in the dysplastic colonic tissue and it seems to be down regulated in cancer. CD80 mucosal levels correlate with T cell population and with IL-2 expression suggesting its involvement in the immunosurveillance mechanism in UC that prevent that all the dysplasia progress to cancer.
M1951 Gene Expression of the Truncated Neurokinin-1 Receptor as Well as Two NFkB Subunits is Altered in the Transition of Colonic Epithelial Cells From Quiescent Colitis to High Grade Dysplasia to Carcinoma in Colitis Associated Colorectal Cancer Earl Gillespie, Jeremy T. Hetzel, Jennifer Coukos, Francis A. Farraye, Sandra R. Cerda, Michael J. O'Brien, Susan E. Leeman, Arthur F. Stucchi, James M. Becker INTRODUCTION Patients with long-standing ulcerative colitis (UC) have an increased risk of developing colorectal cancer (CRC). Although chronic inflammation is a key risk factor, the mechanisms that initiate the transition of normal epithelia to dysplasia to cancer remain unclear. It is known that the neurokinin-1 receptor (NK-1R) is pro-inflammatory and is overexpressed in many cancers. The NK-1R exists in two forms: A full-length and truncated form, but the differences in their expression in colitis-associated CRC have not been previously studied. The aim of this study was to examine changes in gene expression in the progression of colitis-associated CRC. METHODS Archived formalin-fixed, paraffin-embedded (FFPE) samples were obtained from 5 colectomy specimens of CRC patients with pre-existing UC. From each colon, samples were identified and graded by pathologists (MO, SC) as carcinoma (CA), high-grade dysplasia (HGD), or quiescent colitis with no evidence of dysplasia or cancer (QC). Colonic crypt epithelial cells were microdissected using laser capture microscopy, and RNA was extracted and purified using MDS Analytical Technologies' Paradise Plus kit. Reverse transcription and pre-amplification of 88 target genes related to cancer and inflammation was performed using RT2 FFPE Pre-Amp kit; and gene expression was assayed by RT2 Profiler PCR Array (SABiosciences). The data was analyzed by paired t-test using R (HTqPCR Bioconductor package). RESULTS The expression of several genes downstream of the NK1R and NF-kB is significantly changed in the progression from QC to HGD to CA in colitisassociated CRC. Of particular interest are the increased expression of the truncated NK-1R gene, TACR1S, the lack of change in the full-length NK-1R gene, and the increased expression of the NF-kB subunits REL and NFKB2 (see table). CONCLUSION The genes identified in this study, especially the truncated NK-1R, further our understanding of the mechanisms by which chronic inflammation promotes the progression to CRC.
AGA Abstracts
M1953 JC Virus T-Antigen Expression in Gastrointestinal Mucosa of Immunosuppressed Patients - A Prospective, Controlled Study Alex Vilkin, Doron Boltin, Zohar Levi, Ori Elkayam OBJECTIVES: JC virus (JCV), a polyoma virus, is the etiological agent of progressive multifocal leukoencephalopathy in immunosuppressed patients. JCV T-Ag has proven oncogenic potential and is expressed in colonic polyps and carcinomas. We proposed that the prevalence of JCV T-Ag is higher in the normal gastrointestinal (GI) mucosa of immunosuppressed patients compared to their immune competent counterparts. METHODS: Morphologically normal samples of upper and lower GI mucosa were obtained from 38 immunosuppressed patients. A control group included samples from 19 immune competent inflammatory bowel disease (IBD) and 29 non-IBD cases. DNA was extracted and PCR was performed using primers specific for T-Ag. RESULTS: JCV T-Ag was found in 9 of the immunosuppressed patients (23.7%) and in 3 of the controls (6.3%), P=0.02. Transplant recipients had a particularly high prevalence of JCV T-Ag (35.3%). Patients with IBD receiving immunosuppressive drugs had a higher prevalence of JCV T-Ag in comparison with IBD patients who did not receive immunosuppression, 22.2% versus 10.5% respectively, but this difference was not statistically significant (P=0.574). CONCLUSION: JCV T-Ag is more prevalent in
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(14%) cases. Fourteen (40%) patients had stage I or II disease and 16 (46%) had stage III or IV. Staging was not available in 5 (14%) patients. Dysplasia was found in 18 surgical specimens of SBA (51%). It was adjacent to SBA in 9 cases, distant in 2 cases and both contiguous and distant in 7 cases. Dysplasia was flat in 12 cases, elevated (DALM) in 5 cases and both flat and elevated in 1 case. High grade dysplasia was observed in 14 of 18 (78%) cases. All lesions of dysplasia without cancer were found in the ileum. It was flat dysplasia in 3 patients (low grade in 2 cases and high grade in 1 case) and one serrated lesion with low and high grade dysplasia in one patient. All carcinomatous and dysplastic lesions, except one, occurred in inflammatory areas. Conclusion: Dysplasia can be found in the vicinity or at distance from SBA in half of the cases. The dysplasia-carcinoma sequence seems valid in SBA associated with CD. Endoscopic surveillance of the small bowel aimed to detect dysplasia should be considered in patients with CD of the small bowel with risk factors associated with SBA (Am J Gastroenterol 2008;103:1). M1950 Protein Kinase D Mediates Synergistic COX-2 Expression Induced by CrossTalk Between TNF-α and G-Protein Coupled Receptor Agonists in Human Colonic Myofibroblasts James Yoo, Citlali E. Rodriguez-Perez, James Sinnett-Smith, Enrique Rozengurt
A = NK-kB gene target; B = NK-1R gene target
Background: Myofibroblasts have recently been identified as major mediators of tumor necrosis factor-α (TNF-α)-associated colitis-associated colon cancer but the precise mechanism(s) involved remains incompletely understood. Myofibroblasts are a major source of the inducible isoform of cyclooxygenase (COX-2) and of prostaglandins (e.g. PGE2) in the gastrointestinal tract. We hypothesize that TNF-α signaling cross talks with pro-inflammatory mediators, including bradykinin (BK) and lysophosphatidic acid (LPA), to elicit COX-2 expression and PGE2 production that are essential for colon cancer development. Results: Treatment of 18Co cells, a model of human colonic myofibroblasts, with TNF-α and either the G protein-coupled receptor agonists (GPCR) BK or LPA induced striking synergistic COX-2 protein expression that was paralleled by increases in the production of PGE2. Synergistic COX-2 expression was also induced by sequential exposure to TNF-α followed by BK or LPA. COX-2 expression in 18Co cells treated with BK and TNF-α was prevented by the B2 BK receptor antagonist HOE140, and the preferential protein kinase C (PKC) inhibitors Ro31-8220, GF109203X, and Go6976, suggesting that COX-2 expression in response to TNF-α and BK was mediated through a PKC/protein kinase D (PKD)-dependent pathway. Indeed, TNF-α, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation induced by BK, as measured by PKD phosphorylation at its activation loop (Ser744) and auto-phosphorylation site (Ser916). Transfection of 18Co cells with siRNA targeting PKD1 completely inhibited the synergistic increase in COX-2 protein in response to BK and TNF-α demonstrating, for the first time, a critical role of PKD1 in the pathways leading to synergistic expression of COX-2. We also found that exposure of 18Co cells to the combination of TNF-α and BK produced synergistic stimulation of ERK activation. Cell treatment with U0126, at a concentration that completely prevented ERK activation, markedly reduced COX-2 expression in response to stimulation with the combination of TNF-α and BK implying that the ERK pathway contributes to PKD signaling leading to COX-2 expression. Conclusion: Our results indicate that crosstalk between TNFα and GPCR agonists amplifies a PKC/PKD1/ERK phosphorylation cascade that mediates synergistic COX-2 expression in colonic myofibroblasts. We propose that PKD1 mediates COX-2 expression in colonic myofibroblasts to create a microenvironment that supports tumor growth.
M1952 Carcinogenesis in Ulcerative Colitis: Immunosurveillance Mechanisms Marco Scarpa, Marina Bortolami, Diego Faggian, Andromachi Kotsafti, Cesare Ruffolo, Filippo Navaglia, Anna Pozza, Renata D'Incà, Mario Plebani, Giacomo C. Sturniolo, Imerio Angriman Background The inconsistency between dysplasia rate and incidence of cancer in ulcerative colitis (UC)suggests the presence of an immunosurveillance mechanism. The aim of our study was to analyse the expression of costimulatory molecules CD80 and CD86, the lamina propria mononuclear cells populations and the cytokines network at the different stages of carcinogenesis in UC compared to the non inflammatory omologues. Patients and methods Five groups of patients affected by UC (19 pts), UC with colonic dysplasia (10 pts), UC and cancer (7 pts), colonic adenoma (11 pts), sporadic colonic cancer (15 pts) and a group of healthy control (11 pts) were enrolled. Mucosal CD80 and CD86 mRNA levels were quantified with Real Time PCR. Mucosal expression of CD80, CD86, CD3, CD20, CD68 and p53 was analysed by immunohistochemistry. Mucosal levels of IL-1β, IL-2 and TNFα were measured with immunometric assays. Kruskal-Wallis ANOVA, Mann-Whitney U test and Kendall's tau correlation test were used. Results In UC, CD80 expression was higher in patients with dysplasia (p=0.017). This difference was not evident in the non inflammatory carcinogenesis (fig 1). CD80 expression directly correlated with IL-2 expression and with lamina propria T and B lymphocytes and monocytes populations. In UC, CD86 expression resulted significantly higher than in non inflammatory carcinogenesis without any differences between the steps of the carcinogenesis. CD86 expression correlated with inflammation severity and with IFNgamma expression. Conclusion In UC, CD80 is significantly more expressed in the dysplastic colonic tissue and it seems to be down regulated in cancer. CD80 mucosal levels correlate with T cell population and with IL-2 expression suggesting its involvement in the immunosurveillance mechanism in UC that prevent that all the dysplasia progress to cancer.
M1951 Gene Expression of the Truncated Neurokinin-1 Receptor as Well as Two NFkB Subunits is Altered in the Transition of Colonic Epithelial Cells From Quiescent Colitis to High Grade Dysplasia to Carcinoma in Colitis Associated Colorectal Cancer Earl Gillespie, Jeremy T. Hetzel, Jennifer Coukos, Francis A. Farraye, Sandra R. Cerda, Michael J. O'Brien, Susan E. Leeman, Arthur F. Stucchi, James M. Becker INTRODUCTION Patients with long-standing ulcerative colitis (UC) have an increased risk of developing colorectal cancer (CRC). Although chronic inflammation is a key risk factor, the mechanisms that initiate the transition of normal epithelia to dysplasia to cancer remain unclear. It is known that the neurokinin-1 receptor (NK-1R) is pro-inflammatory and is overexpressed in many cancers. The NK-1R exists in two forms: A full-length and truncated form, but the differences in their expression in colitis-associated CRC have not been previously studied. The aim of this study was to examine changes in gene expression in the progression of colitis-associated CRC. METHODS Archived formalin-fixed, paraffin-embedded (FFPE) samples were obtained from 5 colectomy specimens of CRC patients with pre-existing UC. From each colon, samples were identified and graded by pathologists (MO, SC) as carcinoma (CA), high-grade dysplasia (HGD), or quiescent colitis with no evidence of dysplasia or cancer (QC). Colonic crypt epithelial cells were microdissected using laser capture microscopy, and RNA was extracted and purified using MDS Analytical Technologies' Paradise Plus kit. Reverse transcription and pre-amplification of 88 target genes related to cancer and inflammation was performed using RT2 FFPE Pre-Amp kit; and gene expression was assayed by RT2 Profiler PCR Array (SABiosciences). The data was analyzed by paired t-test using R (HTqPCR Bioconductor package). RESULTS The expression of several genes downstream of the NK1R and NF-kB is significantly changed in the progression from QC to HGD to CA in colitisassociated CRC. Of particular interest are the increased expression of the truncated NK-1R gene, TACR1S, the lack of change in the full-length NK-1R gene, and the increased expression of the NF-kB subunits REL and NFKB2 (see table). CONCLUSION The genes identified in this study, especially the truncated NK-1R, further our understanding of the mechanisms by which chronic inflammation promotes the progression to CRC.
AGA Abstracts
M1953 JC Virus T-Antigen Expression in Gastrointestinal Mucosa of Immunosuppressed Patients - A Prospective, Controlled Study Alex Vilkin, Doron Boltin, Zohar Levi, Ori Elkayam OBJECTIVES: JC virus (JCV), a polyoma virus, is the etiological agent of progressive multifocal leukoencephalopathy in immunosuppressed patients. JCV T-Ag has proven oncogenic potential and is expressed in colonic polyps and carcinomas. We proposed that the prevalence of JCV T-Ag is higher in the normal gastrointestinal (GI) mucosa of immunosuppressed patients compared to their immune competent counterparts. METHODS: Morphologically normal samples of upper and lower GI mucosa were obtained from 38 immunosuppressed patients. A control group included samples from 19 immune competent inflammatory bowel disease (IBD) and 29 non-IBD cases. DNA was extracted and PCR was performed using primers specific for T-Ag. RESULTS: JCV T-Ag was found in 9 of the immunosuppressed patients (23.7%) and in 3 of the controls (6.3%), P=0.02. Transplant recipients had a particularly high prevalence of JCV T-Ag (35.3%). Patients with IBD receiving immunosuppressive drugs had a higher prevalence of JCV T-Ag in comparison with IBD patients who did not receive immunosuppression, 22.2% versus 10.5% respectively, but this difference was not statistically significant (P=0.574). CONCLUSION: JCV T-Ag is more prevalent in
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