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653 Case-Finding Is Insufficient to Detect Most Persons With Celiac Disease: A Population-Based Study
of PHD1 in microvascular colonic endothelial cells is sufficient to restore endothelial function and to dampen experimental colitis.
653
597 Transcription Factor ZBP-89 Protects the Colon From AOM/DSS-Induced Tumorigenesis by Interacting With β-Catenin Bryan E. Essien, Alexander Yang, Juanita L. Merchant
Background and Aims: The natural history of undiagnosed celiac disease (CD) is incompletely understood. We evaluated the prevalence of undiagnosed CD, rate of subsequent clinical diagnosis, and survival in a Midwestern community with both high awareness for CD and an active case-finding effort - our community has one of the highest incidence rates of CD reported so far, 17.4 per 100 000 person-years (Ludvigsson J, et al. Am J Gastroenterol 2013; 108: 818)-. Methods: Blood samples from 35,299 unique Olmsted County adults residents without a previous diagnosis of CD were tested for immunoglobulin A tissue transglutaminase antibodies (tTGA). A tTGA test <2.0 U/ml was considered negative. Subjects with tTGA test ≥2.0 U/ml were further analyzed for endomysial antibodies (EMA). Undiagnosed CD was established if tTGA test ≥2.0 U/ml and subsequent EMA positive. Medical records were reviewed for demographics, laboratory, and survival. The medical records linkage system available at Olmsted County (part of the Rochester Epidemiology Project) was used to assure complete incident case ascertainment (clinical diagnosis). The 5-year Kaplan-Meier (K-M) rate of subsequent clinical detection (incident cases) after the date of blood draw was determined for both undiagnosed CD and seronegative individuals. Survival was summarized by K-M curves. Cause of death was obtained from the medical record (or death certificate). Results: We identified 327 (prevalence of 0.93%; 95% confidence interval: 0.83%-1.03%) subjects with undiagnosed CD (mean age 46.6 years, 56% female). Table 1 The 5-year K-M rate of subsequent clinical detection after blood draw was just 5% among undiagnosed CD and less than 0.1% among seronegative individuals (p<0.001) (Figure). Overall, 22 subjects with undiagnosed CD had a subsequent clinical diagnosis of CD. The 5year K-M survival rate was 94% among persons with undiagnosed CD similar to seronegative individuals (94% vs. 96%). The cause of death in subjects with undiagnosed CD (n=9) was cancer (n=4), lymphoma (n=2), ischemic heart disease (n=2), and pneumonia (n=1). Conclusions: This study represents the largest general population screening experience for CD in a single community. The prevalence of undiagnosed CD was 0.93%, confirming the substantial burden of unrecognized CD. Case-finding is insufficient to detect most persons with undiagnosed CD. Summary of results of two-steps serology protocol
Background: Transcription factor Zinc-finger Binding Protein-89 (ZBP-89, ZNF148) expression is regulated by butyrate and forms protein-protein complexes with tumor suppressor factors, e.g. p53, p300, ataxia-telangiectasia mutated (ATM). To study the role of ZBP-89 in vivo, we generated a conditional knockout in the intestine and colon (ZBP-89ΔInt). Mice exhibited increased morbidity and mortality when challenged with Salmonella typhimurium, in part due to reduced tryptophan hydroxylase 1(Tph1) expression and reduced colonic antimicrobial peptide secretion (defensins). We found that β-catenin cooperates with ZBP89 to induce tryptophan hydroxylase 1(Tph1) expression in enterochromaffin cells. Aim: To determine whether ZBP-89 interacts directly with β-catenin to prevent colonic transformation. Methods: ZBP-89ΔInt and WT littermates were treated with 7.4mg/kg azoxymethane (AOM) and water containing 2% dextran sulfate sodium (DSS). After 3 cycles, mice were sacrificed 5 weeks later for tumor evaluation, mRNA and histological analysis. Expression of β-cateninTCF gene targets was determined by rt-qPCR on colonic lysates. The ZBP-89 expression vector was co-transfected with or without β-catenin into HEK293 or SW480, a human colorectal cell line with the TCF reporter plasmid TOPFLASH to assess direct regulation of Wnt-β-catenin-TCF transcriptional activity. To demonstrate the association of ZBP-89 with β-catenin, cell lysates of SW480 were used to perform co-immunoprecipitation. Results: We observed 100% increase in tumor incidence and size in the distal colon and rectum of ZBP-89ΔInt compared to WT mice after the administration of AOM/DSS, N=16 mice/group (P<0.05). mRNA for cyclinD1, c-myc and Axin2 was significantly increased 3-fold in the ZBP89ΔInt mice after AOM/DSS treatment. Ectopic expression of ZBP-89 mitigated induction of the TOPFLASH reporter by β-catenin by 60% in both HEK293 and SW480 cell lines. Coimmunoprecipitation/western blotting showed that ZBP-89 and β-catenin formed a complex in cytoplasmic and nuclear extracts. Confocal microscopy demonstrated that ZBP-89 colocalized with β-catenin in the nucleus. In addition to protein-protein interactions, we found that knockdown of ZBP-89 by siRNA reduced β-catenin protein levels, suggesting that ZBP89 regulates β-catenin expression. Indeed, in silico analysis revealed a putative ZBP-89 binding site in the human and mouse β-catenin promoter at -83 to -78. Conclusion: Collectively, the data suggests that ZBP-98 modulates β-catenin activity and subsequently the induction of Wnt-β-catenin-TCF pathway in vivo and in vitro. Pull-down assays demonstrated that ZBP-89 modulates β-catenin activity through both protein-protein interactions and possibly transcriptional regulation of β-catenin expression, providing a possible mechanism by which this transcription factor inhibits colonic tumor formation. 598
*Prevalence 0.93% (95% confidence interval, 0.83-1.03)
Transforming Growth Factor-Beta Signaling on Dendritic Cells Regulates Bacterial Communities and Gut Homeostasis Sozaburo Ihara, Yoshihiro Hirata, Takako Serizawa, Nobumi Suzuki, Hiroto Kinoshita, Hayato Nakagawa, Hideaki Ijichi, Kazuhiko Koike BACKGROUND: Dendritic cells (DCs) are essential mediators of the host immune response to microbes, and serve a critical role in gut homeostasis. However, the molecular mechanisms how DCs regulate the gut microbes are not fully elucidated. Recent studies show the deletion of TGFB signaling on DCs leads to spontaneous colitis with autoimmunity. Here we investigated the involvement of TGFB signaling on DCs in the gut microbiota and the contribution to the pathogenesis of colitis. METHODS: We used DC specific TGFBRII knockout mice (KO; CD11c-cre Tgfbr2 fl/fl, HT; CD11c-cre Tgfbr2 fl/+), and wild type mice (WT). WT and HT mice received 2.0% dextran sulfate sodium (DSS) with or without Antibiotics (ABX) in the drinking water for 5 days. The mice were killed 10 days after initiation of DSS treatment. The colon and feces were harvested for histological and bacterial assessment. DNA was extracted from feces and quantitative PCR of bacterial 16s rRNA gene was analyzed. We next performed fecal microbiota transplantation (FMT) to evaluate whether microbiota harvested from KO mice could develop colitis. Antibiotic-pretreated WT mice were orogastrically gavaged with frozen stocks of intestinal contents from WT or KO mice. Other groups of the mice were gavaged with mixed cultures grown on MacConkey or Blood agar derived from intestinal contents of KO mice. All mice received DSS treatment followed by gavage. RESULTS: KO mice died early weeks of age (5-14w) with colitis. HT mice were more susceptible to DSS-induced colitis than WT mice. In contrast, WT and HT mice with DSS + ABX treatment did not exhibit significant loss of body weight and colitis. In the analysis of 16s rRNA gene, Enterobacteriaceae in feces were 100-fold enriched in KO mice compared to that in WT mice without DSS treatment, and were 10-fold enriched in HT mice compared to that in WT mice with DSS treatment. In contrast, the abundance of Enterobacteriaceae did not significantly differ between WT and HT mice without DSS and with DSS + ABX treatment. In FMT, mice gavaged with intestinal contents from KO mice were more susceptible to DSS-induced colitis than that from WT mice. Moreover, mice gavaged with mixed cultures of intestinal contents from KO mice grown on MacConkey agar were more susceptible to DSS-induced colitis than that on Blood agar. CONCLUSIONS: DC specific TGFBRII-deficient mice develop colitis, and Enterobacteriaceae are enriched. ABx prevent the colitis and the increase in Enterobacteriaceae. FMT shows Enterobacteriaceae selectively grown on MacConkey derived from KO mice are associated with the development of DSS-induced colitis. These findings reveal TGFB signaling on DCs is important for regulating microbiota and gut homeostasis.
5-year cumulative incidence of clinical diagnosis among persons with undiagnosed CD and seronegative persons 654 Celiac Disease Screening is Suboptimal in a Tertiary Gastroenterology Setting Heba Iskandar, Darrell M. Gray, Hongha T. Vu, Faiz Mirza, Mary K. Rude, Kara Regan, Adil Abdalla, Srinivas Gaddam, Sami A. Almaskeen, Michael Mello, Evelyn O. Marquez, Claire Meyer, Ahmed Bolkhir, Navya D. Kanuri, Gregory S. Sayuk, C. Prakash Gyawali BACKGROUND: Celiac disease (CD) is widely prevalent in North America, with estimated incidence as high as 17.4 cases per 100,000 person-years. There are no population-based screening programs, and case-finding techniques currently utilized may not be adequate. We aimed to determine the adequacy of CD screening in an academic gastroenterology (GI) practice. METHODS: Consecutive initial visits to a tertiary academic GI practice were surveyed over a 3-month period as part of a fellow-initiated quality improvement project. Eligibility for CD testing was determined as per published guidelines (AGA 2006, ACG 2013). Each patient's electronic record was scrutinized to evaluate indications for CD screening, if screening had been performed prior to or after referral, and the screening method. Data analysis assessed CD screening practices across GI subspecialty clinics. RESULTS: 619 consecutive patients (49±0.6 yr, range 16-87 yr, 58.5%F, 94% Caucasian) fulfilled inclusion criteria. CD testing was indicated in 336 (54.5%); breakdown by clinic consisted of 63.1% of 252 luminal GI patients, 89.8% of 91 inflammatory bowel disease (IBD) patients, 31.2% of 202 hepatology patients, and 47.3% of 74 biliary patients. Indications for testing included: chronic GI symptoms (49.4%), IBD (18.5%), irritable bowel syndrome (12%), abnormal transaminases (9.2%), iron deficiency anemia (4.8%), other conditions (osteopenia, type I
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AGA Abstracts
AGA Abstracts
Case-Finding Is Insufficient to Detect Most Persons With Celiac Disease: A Population-Based Study Alberto Rubio-Tapia, Tricia L. Brantner, Carol T. Van Dyke, Deanna L. Brogan, Vincent S. Rajkumar, Joseph A. Murray
653
597 Transcription Factor ZBP-89 Protects the Colon From AOM/DSS-Induced Tumorigenesis by Interacting With β-Catenin Bryan E. Essien, Alexander Yang, Juanita L. Merchant
Background and Aims: The natural history of undiagnosed celiac disease (CD) is incompletely understood. We evaluated the prevalence of undiagnosed CD, rate of subsequent clinical diagnosis, and survival in a Midwestern community with both high awareness for CD and an active case-finding effort - our community has one of the highest incidence rates of CD reported so far, 17.4 per 100 000 person-years (Ludvigsson J, et al. Am J Gastroenterol 2013; 108: 818)-. Methods: Blood samples from 35,299 unique Olmsted County adults residents without a previous diagnosis of CD were tested for immunoglobulin A tissue transglutaminase antibodies (tTGA). A tTGA test <2.0 U/ml was considered negative. Subjects with tTGA test ≥2.0 U/ml were further analyzed for endomysial antibodies (EMA). Undiagnosed CD was established if tTGA test ≥2.0 U/ml and subsequent EMA positive. Medical records were reviewed for demographics, laboratory, and survival. The medical records linkage system available at Olmsted County (part of the Rochester Epidemiology Project) was used to assure complete incident case ascertainment (clinical diagnosis). The 5-year Kaplan-Meier (K-M) rate of subsequent clinical detection (incident cases) after the date of blood draw was determined for both undiagnosed CD and seronegative individuals. Survival was summarized by K-M curves. Cause of death was obtained from the medical record (or death certificate). Results: We identified 327 (prevalence of 0.93%; 95% confidence interval: 0.83%-1.03%) subjects with undiagnosed CD (mean age 46.6 years, 56% female). Table 1 The 5-year K-M rate of subsequent clinical detection after blood draw was just 5% among undiagnosed CD and less than 0.1% among seronegative individuals (p<0.001) (Figure). Overall, 22 subjects with undiagnosed CD had a subsequent clinical diagnosis of CD. The 5year K-M survival rate was 94% among persons with undiagnosed CD similar to seronegative individuals (94% vs. 96%). The cause of death in subjects with undiagnosed CD (n=9) was cancer (n=4), lymphoma (n=2), ischemic heart disease (n=2), and pneumonia (n=1). Conclusions: This study represents the largest general population screening experience for CD in a single community. The prevalence of undiagnosed CD was 0.93%, confirming the substantial burden of unrecognized CD. Case-finding is insufficient to detect most persons with undiagnosed CD. Summary of results of two-steps serology protocol
Background: Transcription factor Zinc-finger Binding Protein-89 (ZBP-89, ZNF148) expression is regulated by butyrate and forms protein-protein complexes with tumor suppressor factors, e.g. p53, p300, ataxia-telangiectasia mutated (ATM). To study the role of ZBP-89 in vivo, we generated a conditional knockout in the intestine and colon (ZBP-89ΔInt). Mice exhibited increased morbidity and mortality when challenged with Salmonella typhimurium, in part due to reduced tryptophan hydroxylase 1(Tph1) expression and reduced colonic antimicrobial peptide secretion (defensins). We found that β-catenin cooperates with ZBP89 to induce tryptophan hydroxylase 1(Tph1) expression in enterochromaffin cells. Aim: To determine whether ZBP-89 interacts directly with β-catenin to prevent colonic transformation. Methods: ZBP-89ΔInt and WT littermates were treated with 7.4mg/kg azoxymethane (AOM) and water containing 2% dextran sulfate sodium (DSS). After 3 cycles, mice were sacrificed 5 weeks later for tumor evaluation, mRNA and histological analysis. Expression of β-cateninTCF gene targets was determined by rt-qPCR on colonic lysates. The ZBP-89 expression vector was co-transfected with or without β-catenin into HEK293 or SW480, a human colorectal cell line with the TCF reporter plasmid TOPFLASH to assess direct regulation of Wnt-β-catenin-TCF transcriptional activity. To demonstrate the association of ZBP-89 with β-catenin, cell lysates of SW480 were used to perform co-immunoprecipitation. Results: We observed 100% increase in tumor incidence and size in the distal colon and rectum of ZBP-89ΔInt compared to WT mice after the administration of AOM/DSS, N=16 mice/group (P<0.05). mRNA for cyclinD1, c-myc and Axin2 was significantly increased 3-fold in the ZBP89ΔInt mice after AOM/DSS treatment. Ectopic expression of ZBP-89 mitigated induction of the TOPFLASH reporter by β-catenin by 60% in both HEK293 and SW480 cell lines. Coimmunoprecipitation/western blotting showed that ZBP-89 and β-catenin formed a complex in cytoplasmic and nuclear extracts. Confocal microscopy demonstrated that ZBP-89 colocalized with β-catenin in the nucleus. In addition to protein-protein interactions, we found that knockdown of ZBP-89 by siRNA reduced β-catenin protein levels, suggesting that ZBP89 regulates β-catenin expression. Indeed, in silico analysis revealed a putative ZBP-89 binding site in the human and mouse β-catenin promoter at -83 to -78. Conclusion: Collectively, the data suggests that ZBP-98 modulates β-catenin activity and subsequently the induction of Wnt-β-catenin-TCF pathway in vivo and in vitro. Pull-down assays demonstrated that ZBP-89 modulates β-catenin activity through both protein-protein interactions and possibly transcriptional regulation of β-catenin expression, providing a possible mechanism by which this transcription factor inhibits colonic tumor formation. 598
*Prevalence 0.93% (95% confidence interval, 0.83-1.03)
Transforming Growth Factor-Beta Signaling on Dendritic Cells Regulates Bacterial Communities and Gut Homeostasis Sozaburo Ihara, Yoshihiro Hirata, Takako Serizawa, Nobumi Suzuki, Hiroto Kinoshita, Hayato Nakagawa, Hideaki Ijichi, Kazuhiko Koike BACKGROUND: Dendritic cells (DCs) are essential mediators of the host immune response to microbes, and serve a critical role in gut homeostasis. However, the molecular mechanisms how DCs regulate the gut microbes are not fully elucidated. Recent studies show the deletion of TGFB signaling on DCs leads to spontaneous colitis with autoimmunity. Here we investigated the involvement of TGFB signaling on DCs in the gut microbiota and the contribution to the pathogenesis of colitis. METHODS: We used DC specific TGFBRII knockout mice (KO; CD11c-cre Tgfbr2 fl/fl, HT; CD11c-cre Tgfbr2 fl/+), and wild type mice (WT). WT and HT mice received 2.0% dextran sulfate sodium (DSS) with or without Antibiotics (ABX) in the drinking water for 5 days. The mice were killed 10 days after initiation of DSS treatment. The colon and feces were harvested for histological and bacterial assessment. DNA was extracted from feces and quantitative PCR of bacterial 16s rRNA gene was analyzed. We next performed fecal microbiota transplantation (FMT) to evaluate whether microbiota harvested from KO mice could develop colitis. Antibiotic-pretreated WT mice were orogastrically gavaged with frozen stocks of intestinal contents from WT or KO mice. Other groups of the mice were gavaged with mixed cultures grown on MacConkey or Blood agar derived from intestinal contents of KO mice. All mice received DSS treatment followed by gavage. RESULTS: KO mice died early weeks of age (5-14w) with colitis. HT mice were more susceptible to DSS-induced colitis than WT mice. In contrast, WT and HT mice with DSS + ABX treatment did not exhibit significant loss of body weight and colitis. In the analysis of 16s rRNA gene, Enterobacteriaceae in feces were 100-fold enriched in KO mice compared to that in WT mice without DSS treatment, and were 10-fold enriched in HT mice compared to that in WT mice with DSS treatment. In contrast, the abundance of Enterobacteriaceae did not significantly differ between WT and HT mice without DSS and with DSS + ABX treatment. In FMT, mice gavaged with intestinal contents from KO mice were more susceptible to DSS-induced colitis than that from WT mice. Moreover, mice gavaged with mixed cultures of intestinal contents from KO mice grown on MacConkey agar were more susceptible to DSS-induced colitis than that on Blood agar. CONCLUSIONS: DC specific TGFBRII-deficient mice develop colitis, and Enterobacteriaceae are enriched. ABx prevent the colitis and the increase in Enterobacteriaceae. FMT shows Enterobacteriaceae selectively grown on MacConkey derived from KO mice are associated with the development of DSS-induced colitis. These findings reveal TGFB signaling on DCs is important for regulating microbiota and gut homeostasis.
5-year cumulative incidence of clinical diagnosis among persons with undiagnosed CD and seronegative persons 654 Celiac Disease Screening is Suboptimal in a Tertiary Gastroenterology Setting Heba Iskandar, Darrell M. Gray, Hongha T. Vu, Faiz Mirza, Mary K. Rude, Kara Regan, Adil Abdalla, Srinivas Gaddam, Sami A. Almaskeen, Michael Mello, Evelyn O. Marquez, Claire Meyer, Ahmed Bolkhir, Navya D. Kanuri, Gregory S. Sayuk, C. Prakash Gyawali BACKGROUND: Celiac disease (CD) is widely prevalent in North America, with estimated incidence as high as 17.4 cases per 100,000 person-years. There are no population-based screening programs, and case-finding techniques currently utilized may not be adequate. We aimed to determine the adequacy of CD screening in an academic gastroenterology (GI) practice. METHODS: Consecutive initial visits to a tertiary academic GI practice were surveyed over a 3-month period as part of a fellow-initiated quality improvement project. Eligibility for CD testing was determined as per published guidelines (AGA 2006, ACG 2013). Each patient's electronic record was scrutinized to evaluate indications for CD screening, if screening had been performed prior to or after referral, and the screening method. Data analysis assessed CD screening practices across GI subspecialty clinics. RESULTS: 619 consecutive patients (49±0.6 yr, range 16-87 yr, 58.5%F, 94% Caucasian) fulfilled inclusion criteria. CD testing was indicated in 336 (54.5%); breakdown by clinic consisted of 63.1% of 252 luminal GI patients, 89.8% of 91 inflammatory bowel disease (IBD) patients, 31.2% of 202 hepatology patients, and 47.3% of 74 biliary patients. Indications for testing included: chronic GI symptoms (49.4%), IBD (18.5%), irritable bowel syndrome (12%), abnormal transaminases (9.2%), iron deficiency anemia (4.8%), other conditions (osteopenia, type I
S-113
AGA Abstracts
AGA Abstracts
Case-Finding Is Insufficient to Detect Most Persons With Celiac Disease: A Population-Based Study Alberto Rubio-Tapia, Tricia L. Brantner, Carol T. Van Dyke, Deanna L. Brogan, Vincent S. Rajkumar, Joseph A. Murray