- Email: [email protected]
904 Partner Burden in Irritable Bowel Syndrome (IBS)
AGA Abstracts
expression occurs with the induction of BE, and this expression drives the intestinalization of the Barrett's cells. Our previous study demonstrated that Cdx2 expression alone is not sufficient to induce the BE phenotype in keratinocytes. In the present study, we determined if other signaling pathways (COX2 and Wnt) could synergize with Cdx2 to induce greater intestinalization than Cdx2 alone. Materials and Methods: Human EPC2-hTERT cells were infected with a retrovirus to induce COX2 expression. A fusion protein called CatCLef was expressed using a lentiviral vector in order to drive Wnt target gene expression. CatCLef consists of the transcription activation domain from beta-catenin fused to the DNA binding domain of Lef1. Quantitative RT-PCR, Western, ELISA and TOPFLASH Luciferase assay were performed to check the expression level and protein activity. BrdU incorporation assay and WST-1 analysis were used to detect effect on cell proliferation. The expression of intestine- and Barrett's-specific genes was tested by qRT-PCR analysis. Cell migration analysis was performed with transwell assay. Results: EPC2-hTERT were engineered to overexpress COX2 and CatCLef1, confirmed by qPCR and Western blot. Moreover PGE2 levels were significantly increased in the cell culture media with COX2 overexpression. CatCLef protein expression increased luciferase activity seven-fold fold compared to controls. The overexpression of COX2 or CatCLef both increased EPC2-hTERT cell proliferation and increased Cyclin D1 protein levels. However, neither was able to drive proliferation enough to rescue Cdx2 expression. COX2 overexpression increased cell migration but did not alter the expression level of intestine- or Barrett's- specific genes. CatCLef1 overexpression did modestly induce Villin mRNA levels (10-fold), alkaline phosphatase (4-fold) and Muc2 (3.5-fold). The effects of COX2 and CatCLef expression on 3-d organotypic culture morphology will be determined by ongoing studies. Conclusion: In summary, COX2 and CatCLef1 increased cell proliferation and Cyclin D1 expression. However, this was not enough to rescue Cdx2 expression in esophageal keratinocytes. CatCLef1 could modestly induce intestine-specific gene expression at RNA level. COX2 increased cell migration dramatically. We conclude that COX2 and Wnt signaling can modestly support intestinalization of esophageal keratinocytes by Cdx2.
Results EMRs were rarely genetically homogeneous. Typically, dysplastic crypts within individual lesions were clonal for a founding p53 or p16 point-mutated clone, whereas the adjacent metaplastic (non-dysplastic) crypts analysed did not contain the point mutation. Mosaic subclones with distinct patterns of LOH (often 18q and/or 3p) were present. In one EMR, the same p53 and p16 point mutations and 5q and 17p LOH were detected across the entire EMR. Conclusions Clonal expansion of mutated cells is frequently restricted to small numbers of crypts. Interactions between genetically distinct clones appears to be predominantly competitive: A crypt acquiring an additional pro-tumorigenic mutation can outcompete, and so clonally expand to the detriment of, the original crypts. Further investigation will reveal whether this progression is driven by mutation and selection or by additional mutations altering the behaviour of neighboring pre-existing clones. 903 Impact of High-Level Sport Practice on the Prevalence of Anal Incontinence in a Young Healthy Female Population Veronique Vitton, Karine Baumstarck-Barrau, Sarah Brardjanian, Michel Bouvier, JeanCharles Grimaud Background and Aim: Regular physical activity usually confers long-term health benefits. However, high-level sport may also induce some harmful outcomes as pelvic floor dysfunction. Urinary incontinence was previously documented, but few data are available about anal incontinence in athlete female. The aim of this study was to determine the part of the high-level sport practice on fecal and urinary incontinences in a young healthy female population. Material and methods: This study incorporated a cross-sectional design and took place in a public university in the South of France (Luminy, Marseille, France). All women aged from 18 to 45 years were eligible. Anonymous self-administered questionnaires were consecutively delivered to each voluntary female member. Two groups of women were defined in accordance to the literature: 1. Intensive sport practice group: women practicing high-level sport and/or more than 8 hours weekly; 2. Non-intensive sport practice group: all other cases. Results: A total of 393 women were enrolled, 169 in the “intensive sport” group and 224 in the “non-intensive sport” group. Women of the “intensive sport” group were significantly younger than the others (21.74±4.28 vs 24.87±5.61, p<0.001), and have more deliveries (0.07±0.31 vs 0.20±0.62, p=0.005). The prevalence of anal incontinence was statistically higher in the “intensive sport” group than the “non-intensive sport” group (14.8% vs 4.9%, p=0.001), as urinary incontinence (33.1% vs 18.3%, p=0.001) and dyspareuny (20.1% vs 9.4%, p=0.002). To determine variables potentially linked to anal incontinence, multivariate analysis (logistic regression model) was performed showing that intensive sport practice was the only parameter linked to anal incontinence (OR=3.05, CI=[1.337.01], p=0.009) taking account for major confounding factors. In the “intensive sport” group, anal incontinence was mainly represented by loss of flatus in 91% with a need for protection use in 87%. Weekly leakage was observed in 39% and monthly leakage in 35% of the “intensive sport” group. Three sports were significantly associated with a higher prevalence of anal incontinence: ball games, climbing and alpine skiing. Conclusion: High-level sport appears to be a significant independent risk factor for anal incontinence in healthy young female. These results suggest that preventive measures such as pelvic floor muscle training may be proposed in this young population.
901 Barrett's Esophagus Correlates With Increased Putative Gastrointestinal Stem Cell Markers DCLK1 and CCK2R in a IL-1β Mouse Model and in Humans Michael Quante, Julian A. Abrams, Frederic Marrache, Timothy C. Wang Background: Barrett's esophagus (BE) is a premalignant, inflammation-induced condition of the esophagus, characterized by a metaplastic change from squamous to columnar intestinaltype epithelium. Il-1β is a proinflammatory cytokine involved in esophagitis. With a critical need for a good animal model to study BE, we aimed to generate a mouse model to analyze the underlying genetic changes associated with the development of BE. Barrett's mucosa expresses the cholecystokenin 2 receptor (CCK2R), and amidated gastrin induces proliferation in BE. Our recent findings suggest a role for CCK2R in the proliferation and survival of colonic stem cells (JCI 2008). Doublecortin-like kinase-1 (Dclk1) is a putative marker for quiescent stem cells in the gastrointestinal tract and might therefore be upregulated in intestinal metaplasia in BE. Methods: A mouse model of BE was generated through specific overexpression of IL-1β in mouse esophageal mucosa, using the Epstein Barr Virus L2 promoter. Transgenic mice were treated with bile acids (0.2% deoxycholate (DCA)) for up to 6 months, and histology was evaluated. Gene expression was analyzed with qRT-PCR and IHC at the squammo-columnar-junction (SCJ). 133 biopsies from 25 human patients with GERD, Barrett's Esophagus and Dysplasia were analyzed. Results: Mice with IL-1β overexpression developed spontaneous esophagitis and forestomach inflammation, resulting in intestinal metaplasia at the SCJ within 12 months, with characteristic expression of TFF2 and Cdx2. DCA exposure for four months resulted in accelerated development of intestinal metaplasia, tumor formation, and dysplasia in 6-8 month old mice. Putative Dclk1 stem cells were not observed in squamous epithelium but were increased at the SCJ in Barrett's metaplasia, suggesting an origin of metaplastic cells from the gastric epithelium. Similar, CCK2R was upregulated in mouse intestinal metaplasia but not in squamous epithelium. Both markers decreased with dysplasia. Identical findings could be obtained in human tissue, with upregulation of Dclk1 (4-fold) and CCK2R (30-fold) in BE compared to normal esophageal tissue, followed by decreasing expression in dysplasia. Conclusion: Overexpression of IL-1β in the mouse esophagus induces early esophagitis and late intestinal metaplasia. Additional application of bile acids results in Barrett's-like metaplasia and dysplasia, emphasizing the role of bile acids in the pathogenesis of BE. Human and mouse BE revealed expansion of putative gastrointestinal Dclk1+ and CCKBR+ stem cells, suggesting the potential origin of BE from the stomach and not the esophagus.
904 Partner Burden in Irritable Bowel Syndrome (IBS) Reuben K. Wong, Douglas A. Drossman, Carolyn B. Morris, Stephan R. Weinland, Jane Leserman, Yuming J. Hu, Shrikant I. Bangdiwala Background and Aims: Studies have described the burden experienced by caregivers and next-of-kin of organic diseases such as cancer. The concept of partner burden has never been studied in functional GI disorders. Our study had three aims: (1) to quantify the degree of burden in IBS, (2) to describe the factors affecting the burden perceived, and (3) to identify the areas of relationship that are impacted. Methods: We surveyed 67 Rome III diagnosed IBS patients and their partners from a tertiary GI clinic. The non-IBS partners completed questionnaires including the Zarit Burden Interview (ZBI), Relationship Satisfaction Scale (RSS) and questions on sexual relationships. IBS patients were rated on disease severity using the Functional Bowel Disease Severity Index (FBDSI). Results: Of the participating couples, almost all were spouses (97%) who had been together for 24.1 ± 12.6 years. Non-IBS partners were mainly males (71%) with a mean age of 49.5 years. Relationship burden was high with an overall mean ZBI score of 22.6 (greater than caregivers of cancer patients 18.51). Those rating their relationship as more burdensome (ZBI) had IBS partners with worse disease severity (FBDSI; p=0.003). Partners' rating of burden (ZBI) was also significantly negatively correlated with both quality of the relationship (RSS; R=-0.60, p<0.001) and sexual satisfaction (R=-0.56, p<0.0001). Demographics had no effect on burden. Conclusions: IBS poses a significant degree of partner burden, at least comparable to or greater than cancer. Perceived burden is increased with worsening IBS severity, poorer sexual and relationship satisfaction. [Supported by R24 DK067674 and Takeda Pharmaceuticals] 1Caregiver assessment of patients with advanced cancer: concordance with patients, effects of burden and positivity. Higginson IJ, Gao W. Health and Quality Outcomes 2008; 6:42
902 Investigating Clonal Competition in Barrett's Associated Tumorigenesis Using Spatial Maps of Genetic Heterogeneity Trevor A. Graham, Shabuddin Khan, Dahmane Oukrif, Alicia E. Green, Laura J. Gay, Hugh Barr, Marco Novelli, Janusz A. Jankowski, Neil A. Shepherd, Simon Leedham, Stuart A. McDonald, Nicholas A. Wright Hypothesis Clonal competition promotes tumorigenesis of Barrett's associated adenocarcinoma. Introduction Genetic heterogeneity within Barrett's mucosa is significantly associated with the development of oesophageal adenocarcinoma (OA). Why genetic diversity promotes tumorigenesis is unclear. Pro-tumorigenic interaction between clones is a possible explanation. To look for evidence of clonal interaction within Barrett's lesions, we have undertaken a high resolution clonal-ordering analysis to produce phylogenetic trees and spatial maps that illustrate the spread of genetically distinct clones in endoscopic mucosal resection specimens (EMRs). Methods Entire EMR specimens were serially sectioned. Each crypt was classified according to histological grade. Aneuploidy was assessed using image cytometry. More than 50 individual crypts were then laser-capture microdissected from each EMR. The genetic mutation burden of individual crypts was assessed (K-ras, p53 and p16 point mutations and microsatellite loss of heterozygosity (LOH) data for up to16 loci on chromosomes 3p (FHIT), 5q (APC), 9p (p16), 17p (p53), 17q and 18q (SMAD4)). In each EMR, a point mutation in at least one gene was identified. The relative location of each analysed crypt was recorded and used to reconstruct a genetic and histological map of each EMR.
AGA Abstracts
905 A Novel Gastrointestinal Prokinetic, ATI-7505, Increased Spontaneous Bowel Movements (Sbms) in a Phase II, Randomized, Placebo-Controlled Study of Patients With Chronic Idiopathic Constipation (CIC) Monica Palme, Peter G. Milner, Dave J. Ellis, Tonya Marmon, Daniel M. Canafax ATI-7505 is a potent, selective agonist of human serotonin 5-HT4 receptors. ATI-7505 has minimal activity at the hERG cardiac ion channel and is not metabolized by CYP450 enzymes. In a thorough QT study (ICH E14), ATI-7505 did not affect cardiac repolarization at doses up to 800 mg per day. ATI-7505 is being developed for treatment of both upper and lower
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expression occurs with the induction of BE, and this expression drives the intestinalization of the Barrett's cells. Our previous study demonstrated that Cdx2 expression alone is not sufficient to induce the BE phenotype in keratinocytes. In the present study, we determined if other signaling pathways (COX2 and Wnt) could synergize with Cdx2 to induce greater intestinalization than Cdx2 alone. Materials and Methods: Human EPC2-hTERT cells were infected with a retrovirus to induce COX2 expression. A fusion protein called CatCLef was expressed using a lentiviral vector in order to drive Wnt target gene expression. CatCLef consists of the transcription activation domain from beta-catenin fused to the DNA binding domain of Lef1. Quantitative RT-PCR, Western, ELISA and TOPFLASH Luciferase assay were performed to check the expression level and protein activity. BrdU incorporation assay and WST-1 analysis were used to detect effect on cell proliferation. The expression of intestine- and Barrett's-specific genes was tested by qRT-PCR analysis. Cell migration analysis was performed with transwell assay. Results: EPC2-hTERT were engineered to overexpress COX2 and CatCLef1, confirmed by qPCR and Western blot. Moreover PGE2 levels were significantly increased in the cell culture media with COX2 overexpression. CatCLef protein expression increased luciferase activity seven-fold fold compared to controls. The overexpression of COX2 or CatCLef both increased EPC2-hTERT cell proliferation and increased Cyclin D1 protein levels. However, neither was able to drive proliferation enough to rescue Cdx2 expression. COX2 overexpression increased cell migration but did not alter the expression level of intestine- or Barrett's- specific genes. CatCLef1 overexpression did modestly induce Villin mRNA levels (10-fold), alkaline phosphatase (4-fold) and Muc2 (3.5-fold). The effects of COX2 and CatCLef expression on 3-d organotypic culture morphology will be determined by ongoing studies. Conclusion: In summary, COX2 and CatCLef1 increased cell proliferation and Cyclin D1 expression. However, this was not enough to rescue Cdx2 expression in esophageal keratinocytes. CatCLef1 could modestly induce intestine-specific gene expression at RNA level. COX2 increased cell migration dramatically. We conclude that COX2 and Wnt signaling can modestly support intestinalization of esophageal keratinocytes by Cdx2.
Results EMRs were rarely genetically homogeneous. Typically, dysplastic crypts within individual lesions were clonal for a founding p53 or p16 point-mutated clone, whereas the adjacent metaplastic (non-dysplastic) crypts analysed did not contain the point mutation. Mosaic subclones with distinct patterns of LOH (often 18q and/or 3p) were present. In one EMR, the same p53 and p16 point mutations and 5q and 17p LOH were detected across the entire EMR. Conclusions Clonal expansion of mutated cells is frequently restricted to small numbers of crypts. Interactions between genetically distinct clones appears to be predominantly competitive: A crypt acquiring an additional pro-tumorigenic mutation can outcompete, and so clonally expand to the detriment of, the original crypts. Further investigation will reveal whether this progression is driven by mutation and selection or by additional mutations altering the behaviour of neighboring pre-existing clones. 903 Impact of High-Level Sport Practice on the Prevalence of Anal Incontinence in a Young Healthy Female Population Veronique Vitton, Karine Baumstarck-Barrau, Sarah Brardjanian, Michel Bouvier, JeanCharles Grimaud Background and Aim: Regular physical activity usually confers long-term health benefits. However, high-level sport may also induce some harmful outcomes as pelvic floor dysfunction. Urinary incontinence was previously documented, but few data are available about anal incontinence in athlete female. The aim of this study was to determine the part of the high-level sport practice on fecal and urinary incontinences in a young healthy female population. Material and methods: This study incorporated a cross-sectional design and took place in a public university in the South of France (Luminy, Marseille, France). All women aged from 18 to 45 years were eligible. Anonymous self-administered questionnaires were consecutively delivered to each voluntary female member. Two groups of women were defined in accordance to the literature: 1. Intensive sport practice group: women practicing high-level sport and/or more than 8 hours weekly; 2. Non-intensive sport practice group: all other cases. Results: A total of 393 women were enrolled, 169 in the “intensive sport” group and 224 in the “non-intensive sport” group. Women of the “intensive sport” group were significantly younger than the others (21.74±4.28 vs 24.87±5.61, p<0.001), and have more deliveries (0.07±0.31 vs 0.20±0.62, p=0.005). The prevalence of anal incontinence was statistically higher in the “intensive sport” group than the “non-intensive sport” group (14.8% vs 4.9%, p=0.001), as urinary incontinence (33.1% vs 18.3%, p=0.001) and dyspareuny (20.1% vs 9.4%, p=0.002). To determine variables potentially linked to anal incontinence, multivariate analysis (logistic regression model) was performed showing that intensive sport practice was the only parameter linked to anal incontinence (OR=3.05, CI=[1.337.01], p=0.009) taking account for major confounding factors. In the “intensive sport” group, anal incontinence was mainly represented by loss of flatus in 91% with a need for protection use in 87%. Weekly leakage was observed in 39% and monthly leakage in 35% of the “intensive sport” group. Three sports were significantly associated with a higher prevalence of anal incontinence: ball games, climbing and alpine skiing. Conclusion: High-level sport appears to be a significant independent risk factor for anal incontinence in healthy young female. These results suggest that preventive measures such as pelvic floor muscle training may be proposed in this young population.
901 Barrett's Esophagus Correlates With Increased Putative Gastrointestinal Stem Cell Markers DCLK1 and CCK2R in a IL-1β Mouse Model and in Humans Michael Quante, Julian A. Abrams, Frederic Marrache, Timothy C. Wang Background: Barrett's esophagus (BE) is a premalignant, inflammation-induced condition of the esophagus, characterized by a metaplastic change from squamous to columnar intestinaltype epithelium. Il-1β is a proinflammatory cytokine involved in esophagitis. With a critical need for a good animal model to study BE, we aimed to generate a mouse model to analyze the underlying genetic changes associated with the development of BE. Barrett's mucosa expresses the cholecystokenin 2 receptor (CCK2R), and amidated gastrin induces proliferation in BE. Our recent findings suggest a role for CCK2R in the proliferation and survival of colonic stem cells (JCI 2008). Doublecortin-like kinase-1 (Dclk1) is a putative marker for quiescent stem cells in the gastrointestinal tract and might therefore be upregulated in intestinal metaplasia in BE. Methods: A mouse model of BE was generated through specific overexpression of IL-1β in mouse esophageal mucosa, using the Epstein Barr Virus L2 promoter. Transgenic mice were treated with bile acids (0.2% deoxycholate (DCA)) for up to 6 months, and histology was evaluated. Gene expression was analyzed with qRT-PCR and IHC at the squammo-columnar-junction (SCJ). 133 biopsies from 25 human patients with GERD, Barrett's Esophagus and Dysplasia were analyzed. Results: Mice with IL-1β overexpression developed spontaneous esophagitis and forestomach inflammation, resulting in intestinal metaplasia at the SCJ within 12 months, with characteristic expression of TFF2 and Cdx2. DCA exposure for four months resulted in accelerated development of intestinal metaplasia, tumor formation, and dysplasia in 6-8 month old mice. Putative Dclk1 stem cells were not observed in squamous epithelium but were increased at the SCJ in Barrett's metaplasia, suggesting an origin of metaplastic cells from the gastric epithelium. Similar, CCK2R was upregulated in mouse intestinal metaplasia but not in squamous epithelium. Both markers decreased with dysplasia. Identical findings could be obtained in human tissue, with upregulation of Dclk1 (4-fold) and CCK2R (30-fold) in BE compared to normal esophageal tissue, followed by decreasing expression in dysplasia. Conclusion: Overexpression of IL-1β in the mouse esophagus induces early esophagitis and late intestinal metaplasia. Additional application of bile acids results in Barrett's-like metaplasia and dysplasia, emphasizing the role of bile acids in the pathogenesis of BE. Human and mouse BE revealed expansion of putative gastrointestinal Dclk1+ and CCKBR+ stem cells, suggesting the potential origin of BE from the stomach and not the esophagus.
904 Partner Burden in Irritable Bowel Syndrome (IBS) Reuben K. Wong, Douglas A. Drossman, Carolyn B. Morris, Stephan R. Weinland, Jane Leserman, Yuming J. Hu, Shrikant I. Bangdiwala Background and Aims: Studies have described the burden experienced by caregivers and next-of-kin of organic diseases such as cancer. The concept of partner burden has never been studied in functional GI disorders. Our study had three aims: (1) to quantify the degree of burden in IBS, (2) to describe the factors affecting the burden perceived, and (3) to identify the areas of relationship that are impacted. Methods: We surveyed 67 Rome III diagnosed IBS patients and their partners from a tertiary GI clinic. The non-IBS partners completed questionnaires including the Zarit Burden Interview (ZBI), Relationship Satisfaction Scale (RSS) and questions on sexual relationships. IBS patients were rated on disease severity using the Functional Bowel Disease Severity Index (FBDSI). Results: Of the participating couples, almost all were spouses (97%) who had been together for 24.1 ± 12.6 years. Non-IBS partners were mainly males (71%) with a mean age of 49.5 years. Relationship burden was high with an overall mean ZBI score of 22.6 (greater than caregivers of cancer patients 18.51). Those rating their relationship as more burdensome (ZBI) had IBS partners with worse disease severity (FBDSI; p=0.003). Partners' rating of burden (ZBI) was also significantly negatively correlated with both quality of the relationship (RSS; R=-0.60, p<0.001) and sexual satisfaction (R=-0.56, p<0.0001). Demographics had no effect on burden. Conclusions: IBS poses a significant degree of partner burden, at least comparable to or greater than cancer. Perceived burden is increased with worsening IBS severity, poorer sexual and relationship satisfaction. [Supported by R24 DK067674 and Takeda Pharmaceuticals] 1Caregiver assessment of patients with advanced cancer: concordance with patients, effects of burden and positivity. Higginson IJ, Gao W. Health and Quality Outcomes 2008; 6:42
902 Investigating Clonal Competition in Barrett's Associated Tumorigenesis Using Spatial Maps of Genetic Heterogeneity Trevor A. Graham, Shabuddin Khan, Dahmane Oukrif, Alicia E. Green, Laura J. Gay, Hugh Barr, Marco Novelli, Janusz A. Jankowski, Neil A. Shepherd, Simon Leedham, Stuart A. McDonald, Nicholas A. Wright Hypothesis Clonal competition promotes tumorigenesis of Barrett's associated adenocarcinoma. Introduction Genetic heterogeneity within Barrett's mucosa is significantly associated with the development of oesophageal adenocarcinoma (OA). Why genetic diversity promotes tumorigenesis is unclear. Pro-tumorigenic interaction between clones is a possible explanation. To look for evidence of clonal interaction within Barrett's lesions, we have undertaken a high resolution clonal-ordering analysis to produce phylogenetic trees and spatial maps that illustrate the spread of genetically distinct clones in endoscopic mucosal resection specimens (EMRs). Methods Entire EMR specimens were serially sectioned. Each crypt was classified according to histological grade. Aneuploidy was assessed using image cytometry. More than 50 individual crypts were then laser-capture microdissected from each EMR. The genetic mutation burden of individual crypts was assessed (K-ras, p53 and p16 point mutations and microsatellite loss of heterozygosity (LOH) data for up to16 loci on chromosomes 3p (FHIT), 5q (APC), 9p (p16), 17p (p53), 17q and 18q (SMAD4)). In each EMR, a point mutation in at least one gene was identified. The relative location of each analysed crypt was recorded and used to reconstruct a genetic and histological map of each EMR.
AGA Abstracts
905 A Novel Gastrointestinal Prokinetic, ATI-7505, Increased Spontaneous Bowel Movements (Sbms) in a Phase II, Randomized, Placebo-Controlled Study of Patients With Chronic Idiopathic Constipation (CIC) Monica Palme, Peter G. Milner, Dave J. Ellis, Tonya Marmon, Daniel M. Canafax ATI-7505 is a potent, selective agonist of human serotonin 5-HT4 receptors. ATI-7505 has minimal activity at the hERG cardiac ion channel and is not metabolized by CYP450 enzymes. In a thorough QT study (ICH E14), ATI-7505 did not affect cardiac repolarization at doses up to 800 mg per day. ATI-7505 is being developed for treatment of both upper and lower
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