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95 Exome Sequencing Analysis of Candidate Genes in Very Early Onset IBD
101
Looking Beyond Deep Remission: Effect of Biologic Therapy on Transmural Healing Evaluated by Ultrasound in Pediatric Ileal Crohn's Disease Fortunata Civitelli, Federica Nuti, Manuel Murciano, Giovanni Di Nardo, Lorena Messina, Salvatore Oliva, Marina Aloi, Silvia Bloise, Giuseppe La Torre, Franca Viola, Salvatore Cucchiara
Indoleamine-2,3-Dioxygenase 1 (IDO1) Mediates Gastric Metaplasia via the Induction of Caspase 1 and the Production of Bioactive IL-1β Mohamad El-Zaatari, Yu-Ming Chang, Min Zhang, Andrew Shreiner, John Y. Kao BACKGROUND: Longstanding chronic inflammation is associated with cancer transformation. What is not known is whether an on-switch of pro-tumor immunoediting occurs when inflammation progresses from early to late stage. Our preliminary work using a model of Helicobacter-induced gastric metaplasia showed that metaplasia was detected at 6 months, but not at 2 months, post-infection with significant induction of IDO1. Thus, the aim of this study is to determine the role of IDO1 in the development of gastric metaplasia during Helicobacter felis (H. felis) infection of the stomach. METHODS: The immunopathology of H. felis was compared between wild type (WT) and IDO1-/- mice (age-matched males). The activity of IDO1 in converting tryptophan to kynurenine in the stomach was assayed by measuring tissue kynurenine levels using mass spectrometry. The host's immunopathology was measured by a combination of immunohistochemistry, RT-qPCR, pathological scoring (including TFF2/IF co-localization), and flow cytometry. Genome-wide screening of IDO1target pathways in the stomach was performed by microarray. RESULTS: H. felis infection induced IDO1 mRNA expression at 6 months but not 2 month postinfection (6-months = 41.5-fold vs 2-months = 0.4-fold, p < 0.01). This induction was restricted to myeloid and few epithelial cells. Kynurenine levels corroborated with IDO1 induction and were significantly increased in the stomach of 6 month-infected mice (6.3-fold, p < 0.05). H. felis infection did not induce downstream metabolites of kynurenine (kynurenic acid, 3-hydroxy-L-kynurenine, and anthranilic acid) indicating that the H. felis effect was specific to tryptophan-kynurenine conversion. IDO1 deletion prevented the H. felis-mediated breakdown of tryptophan into kynurenine. This breakdown was necessary for H. felis immunopathology as gastric metaplasia was significantly reduced in IDO1-/- mice. To examine the mechanism of IDO1 function and its role in mediating metaplasia, we assayed myeloid cell composition and cytokine production in infected WT versus IDO1-/- stomachs. Interestingly, IDO1-/- gastric mucosa did not exhibit any changes in myeloid cell composition as measured by FACS, or in cytokine production of IL-1β, TNF-α or IFN-γ. Therefore, we performed microarray analysis to identify the pathways regulated by IDO1, which identified caspase 1 as a major target (WT = 10-fold vs IDO-/- = 2-fold, p < 0.05). Consistent with this finding, the production of cleaved / bioactive IL-1β was induced in infected WT but not IDO1-/- gastric mucosa. In vitro, kynurenine was able to stimulate bioactive IL-1β secretion from myeloid cells during H. felis infection. CONCLUSIONS: Chronic Helicobacter infection induces myeloid expression of IDO1 and increases kynurenine production, which promotes caspase 1 induction and cleaved IL-1β production known to trigger gastric metaplasia.
BACKGROUND AND AIM: Therapeutic goals for Crohn's disease (CD) have evolved from a mere control of symptoms to the concept of deep remission (DR), including clinical and biomarker remission and mucosal healing (MH). Biologic therapy with anti-TNFα is effective in achieving MH. Yet, CD is a transmural disease, characterized by a progressive bowel damage leading to complications. This is the first pediatric study prospectively evaluating the efficacy of anti-TNFα therapy in inducing clinical remission, MH and transmural healing (TH) in ileal CD. METHODS: Pediatric patients (pts) with ileal CD starting biological therapy with Infliximab or Adalimumab were prospectively enrolled. All pts were Naive to biologics. Clinical activity (Pediatric Crohn's Disease Activity Index, PCDAI), laboratory tests (CRP, ESR), endoscopic activity (simple endoscopic score, SES-CD) and transmural disease assessed by small intestine contrast ultrasonography (SICUS) were evaluated before starting (T0) and after 9-12 months of therapy (T1). Complete MH was defined as a SES-CD of 0-1, partial MH as 50% decrease vs T0. At US the evaluated parameters were: extension of disease (cm), bowel wall thickness >3 mm (BWT), bowel wall vascularity, stratification of the BW (BWS), presence of stricture, fistulae and abscess. Wilcoxon signed rank test was used for pair comparison (T1-T0). RESULTS: 26 pts (mean age 13.3 ± 4, 16 males) were included. The mean PCDAI, ileal SES-CD, CRP, ESR, BWT and disease extension values significantly decreased at T1 (table). Increased bowel wall vascularity was present in 80% of pts at T0 and in 24% at T1 (p<0.0001). In pts with complete and partial MH the extension of disease and the mean BWT at US were significantly reduced at T1 (p<0.02); in pts without endoscopic response the US parameters didn't change significantly, despite clinical response. Presence of strictures and BWS didn't modify during therapy in any group. CONCLUSIONS: Biologics are effective in inducing clinical and laboratory remission and in achieving MH in pediatric CD. Transmural inflammation significantly improves during therapy, however when a substantial bowel damage (stricture) is present, the effect on TH might be poorer. Further studies are needed to evaluate the impact of TH on the long term outcome of CD. CLINICAL, ENDOSCOPIC AND US DATA AT T0 AND T1
102 Resumption of Low-Dose Aspirin and the Risk of Recurrent Lower GI Bleeding and CV Outcomes: A Long-Term Cohort Study Francis K. L. Chan, En-Ling Leung Ki, Yee Kit Tse, Kim WL Au, Jessica Ching, Grace Wong, Justin C. Wu Table: Mean ± SD values
BACKGROUND: Among low-dose aspirin (ASA) users who developed lower gastrointestinal (GI) bleeding, the long-term risk of recurrent lower GI bleeding with ASA is unknown. METHODS: This was a single-center, retrospective cohort study. We identified a cohort of ASA users with lower GI bleeding between January 2000 and December 2007 according to prespecified criteria. Patients were included if they had used ASA within one week of onset of bleeding, had documented melena or hematochezia, and without a source of upper GI bleeding confirmed by gastroscopy. The exclusion were hemorrhoidal bleeding, colorectal cancer, and concomitant NSAID use. After being discharged from the hospital, eligible patients were reviewed for recurrent lower GI bleeding, major adverse CV events, and death for up to 5 years. They were divided into 2 groups according to their cumulative exposure to ASA, namely, ASA group (defined as ASA use in at least 50% of the follow-up period) and non-user group (ASA use in less than 20% of the follow-up period). The primary endpoint was recurrent lower GI bleeding. Secondary endpoints included major adverse CV events as defined by the APTC definition and death from other causes. An independent, blinded adjudication committee evaluated these endpoints according to predefined criteria. RESULTS: A total of 295 patients were eligible for the study (174 in the ASA group and 121 in the non-user group). The median follow-up was 2.7 years (range, 0.6-5.0) in the ASA group and 1.7 years (range, 0.2-4.7) in the non-user group. In the ASA group, 84% of patients received ASA prescriptions in ≥75% of the follow-up period whereas 87% patients in the non-user group received ASA in ≤10% of the study period. Comparing to the ASA group, the non-user group was older, had fewer smokers and received more transfusion at the index bleed (Table 1). The cumulative incidence of recurrent lower GI bleeding in 5 years was 18.9% (95% CI, 13.3%-25.3%) in the ASA group compared to 6.9% (95% CI, 3.2%-12.5%) in the non-user group (p=0.007). Major adverse CV events at 5 years was 22.8% (16.6%-29.6%) in the ASA group compared to 36.5% (27.4%-45.6%) in the nonuser group (p=0.017). Other causes of death at 5 years was 8.2% (4.6% to 13.2%) in ASA group compared to 26.7% (18.7% to 35.4%) in the non-user group (p<0.001). CONCLUSIONS: Among ASA users with a history of lower GI bleeding, continuation of ASA increased the risk of recurrent lower GI bleeding by 2.7 folds but reduced the risk of major adverse CV events by 1.6 folds and other causes of death by 3.2 folds as compared to non-users.
95 Exome Sequencing Analysis of Candidate Genes in Very Early Onset IBD Judith R. Kelsen, Christopher J. Moran, Ariella Sasson, Mahdi Sarmady, Kernika Gupta, Helen Pauly-Hubbard, Eric Rappaport, Catherine Stolle, Petar Mamula, Andrew B. Grossman, David Piccoli, Harland Winter, Robert Baldassano, Marcella Devoto Background: Very early onset IBD (VEO-IBD) frequently presents with a different and more severe phenotype than older onset IBD, suggesting a more pronounced genetic susceptibility and dysregulated immune response. We hypothesized that rare or novel variants, including mutations in genes associated with primary immunodeficiencies (PI), were enriched in patients with severe VEO-IBD. Methods: IBD patients 5 years and younger, and parents were recruited. DNA was isolated from whole blood. Exome capture was performed by Agilent SureSelect V4, and sequencing was done using the Illumina HiSeq platform at an average coverage depth of 100X. Alignment to human genome GRCh37 was performed followed by post processing and variant calling. Following functional annotation, only variants likely to alter protein function, such as missense and loss of function mutations, were kept for subsequent analysis. Further filtering included only those with minor allele frequency less than 5% in controls from the 1000 Genomes Project and The Children's Hospital of Philadelphia's internal whole exome cohort (>400 samples). Primary focus of the analysis was on known IBD risk loci and genes associated with PI and related pathways (> 870 genes). Results: 44 samples were included, comprising 28 patients with VEO-IBD and 16 parents. Patients' age ranged from 8 weeks to 4 years, and 21 were under 2 years. The IBD risk loci and genes associated with PI contain 14,060 coding exons totaling more than 2.5 Mbp. In these regions, 87.7% of coding exons were fully covered at more than 20x. Analysis revealed novel and rare putative causative variants within these genes in multiple families that are currently being evaluated. Examples of non-synonymous variants detected in heterozygosity include rare missense variants in LRBA (rs72719663; c.21720A>G, p.T1724V), and in TNFRSF18 (rs114666761; c.128C>G, p.T43R) in a male with severe colonic IBD diagnosed at age 3. Additionally, a novel MSH5 variant (c.1661G>C p.S554T) was detected in a 3 year old male with severe ileocolonic VEO-IBD and fistulizing perianal disease. Laboratory evaluation demonstrated low IgG and IgM, and low end of normal IgA and IgE. Defects in these genes are associated with common variable immunodeficiency (CVID). CVID is a heterogeneous disorder characterized by hypogammaglobulinemia, recurrent infections and GI manifestations. Conclusions: Candidate causative mutations in VEOIBD can be identified by exome sequencing, and it is likely that some patients harbor mutations in several genes in the same or different pathways. Thus, the heterozygous variants detected may have contributed to the phenotype of VEO-IBD. Ongoing analysis is being performed, including evaluation of parental transmission; however, we anticipate that additional causative variants may occur in genes not previously associated with IBD.
103 Gaviscon Double Action (Antacid + Alginate) Versus Equivalent Antacid for Postprandial Acid Reflux: A Double-Blind Crossover Study in GERD Patients Annemijn de Ruigh, Joan Chen, John E. Pandolfino, Peter J. Kahrilas Background: Most individuals treat GERD symptoms with over-the-counter medications such as antacids and alginates. Antacids neutralize gastric acid while the proposed working mechanism of alginates (such as Gaviscon) is formation of a gel-antacid matrix floating on top of the chyme co-localizing with and neutralizing the postprandial acid pocket. This study aimed to compare the effectiveness of Gaviscon to an equivalent strength non-alginate
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AGA Abstracts
AGA Abstracts
94
Looking Beyond Deep Remission: Effect of Biologic Therapy on Transmural Healing Evaluated by Ultrasound in Pediatric Ileal Crohn's Disease Fortunata Civitelli, Federica Nuti, Manuel Murciano, Giovanni Di Nardo, Lorena Messina, Salvatore Oliva, Marina Aloi, Silvia Bloise, Giuseppe La Torre, Franca Viola, Salvatore Cucchiara
Indoleamine-2,3-Dioxygenase 1 (IDO1) Mediates Gastric Metaplasia via the Induction of Caspase 1 and the Production of Bioactive IL-1β Mohamad El-Zaatari, Yu-Ming Chang, Min Zhang, Andrew Shreiner, John Y. Kao BACKGROUND: Longstanding chronic inflammation is associated with cancer transformation. What is not known is whether an on-switch of pro-tumor immunoediting occurs when inflammation progresses from early to late stage. Our preliminary work using a model of Helicobacter-induced gastric metaplasia showed that metaplasia was detected at 6 months, but not at 2 months, post-infection with significant induction of IDO1. Thus, the aim of this study is to determine the role of IDO1 in the development of gastric metaplasia during Helicobacter felis (H. felis) infection of the stomach. METHODS: The immunopathology of H. felis was compared between wild type (WT) and IDO1-/- mice (age-matched males). The activity of IDO1 in converting tryptophan to kynurenine in the stomach was assayed by measuring tissue kynurenine levels using mass spectrometry. The host's immunopathology was measured by a combination of immunohistochemistry, RT-qPCR, pathological scoring (including TFF2/IF co-localization), and flow cytometry. Genome-wide screening of IDO1target pathways in the stomach was performed by microarray. RESULTS: H. felis infection induced IDO1 mRNA expression at 6 months but not 2 month postinfection (6-months = 41.5-fold vs 2-months = 0.4-fold, p < 0.01). This induction was restricted to myeloid and few epithelial cells. Kynurenine levels corroborated with IDO1 induction and were significantly increased in the stomach of 6 month-infected mice (6.3-fold, p < 0.05). H. felis infection did not induce downstream metabolites of kynurenine (kynurenic acid, 3-hydroxy-L-kynurenine, and anthranilic acid) indicating that the H. felis effect was specific to tryptophan-kynurenine conversion. IDO1 deletion prevented the H. felis-mediated breakdown of tryptophan into kynurenine. This breakdown was necessary for H. felis immunopathology as gastric metaplasia was significantly reduced in IDO1-/- mice. To examine the mechanism of IDO1 function and its role in mediating metaplasia, we assayed myeloid cell composition and cytokine production in infected WT versus IDO1-/- stomachs. Interestingly, IDO1-/- gastric mucosa did not exhibit any changes in myeloid cell composition as measured by FACS, or in cytokine production of IL-1β, TNF-α or IFN-γ. Therefore, we performed microarray analysis to identify the pathways regulated by IDO1, which identified caspase 1 as a major target (WT = 10-fold vs IDO-/- = 2-fold, p < 0.05). Consistent with this finding, the production of cleaved / bioactive IL-1β was induced in infected WT but not IDO1-/- gastric mucosa. In vitro, kynurenine was able to stimulate bioactive IL-1β secretion from myeloid cells during H. felis infection. CONCLUSIONS: Chronic Helicobacter infection induces myeloid expression of IDO1 and increases kynurenine production, which promotes caspase 1 induction and cleaved IL-1β production known to trigger gastric metaplasia.
BACKGROUND AND AIM: Therapeutic goals for Crohn's disease (CD) have evolved from a mere control of symptoms to the concept of deep remission (DR), including clinical and biomarker remission and mucosal healing (MH). Biologic therapy with anti-TNFα is effective in achieving MH. Yet, CD is a transmural disease, characterized by a progressive bowel damage leading to complications. This is the first pediatric study prospectively evaluating the efficacy of anti-TNFα therapy in inducing clinical remission, MH and transmural healing (TH) in ileal CD. METHODS: Pediatric patients (pts) with ileal CD starting biological therapy with Infliximab or Adalimumab were prospectively enrolled. All pts were Naive to biologics. Clinical activity (Pediatric Crohn's Disease Activity Index, PCDAI), laboratory tests (CRP, ESR), endoscopic activity (simple endoscopic score, SES-CD) and transmural disease assessed by small intestine contrast ultrasonography (SICUS) were evaluated before starting (T0) and after 9-12 months of therapy (T1). Complete MH was defined as a SES-CD of 0-1, partial MH as 50% decrease vs T0. At US the evaluated parameters were: extension of disease (cm), bowel wall thickness >3 mm (BWT), bowel wall vascularity, stratification of the BW (BWS), presence of stricture, fistulae and abscess. Wilcoxon signed rank test was used for pair comparison (T1-T0). RESULTS: 26 pts (mean age 13.3 ± 4, 16 males) were included. The mean PCDAI, ileal SES-CD, CRP, ESR, BWT and disease extension values significantly decreased at T1 (table). Increased bowel wall vascularity was present in 80% of pts at T0 and in 24% at T1 (p<0.0001). In pts with complete and partial MH the extension of disease and the mean BWT at US were significantly reduced at T1 (p<0.02); in pts without endoscopic response the US parameters didn't change significantly, despite clinical response. Presence of strictures and BWS didn't modify during therapy in any group. CONCLUSIONS: Biologics are effective in inducing clinical and laboratory remission and in achieving MH in pediatric CD. Transmural inflammation significantly improves during therapy, however when a substantial bowel damage (stricture) is present, the effect on TH might be poorer. Further studies are needed to evaluate the impact of TH on the long term outcome of CD. CLINICAL, ENDOSCOPIC AND US DATA AT T0 AND T1
102 Resumption of Low-Dose Aspirin and the Risk of Recurrent Lower GI Bleeding and CV Outcomes: A Long-Term Cohort Study Francis K. L. Chan, En-Ling Leung Ki, Yee Kit Tse, Kim WL Au, Jessica Ching, Grace Wong, Justin C. Wu Table: Mean ± SD values
BACKGROUND: Among low-dose aspirin (ASA) users who developed lower gastrointestinal (GI) bleeding, the long-term risk of recurrent lower GI bleeding with ASA is unknown. METHODS: This was a single-center, retrospective cohort study. We identified a cohort of ASA users with lower GI bleeding between January 2000 and December 2007 according to prespecified criteria. Patients were included if they had used ASA within one week of onset of bleeding, had documented melena or hematochezia, and without a source of upper GI bleeding confirmed by gastroscopy. The exclusion were hemorrhoidal bleeding, colorectal cancer, and concomitant NSAID use. After being discharged from the hospital, eligible patients were reviewed for recurrent lower GI bleeding, major adverse CV events, and death for up to 5 years. They were divided into 2 groups according to their cumulative exposure to ASA, namely, ASA group (defined as ASA use in at least 50% of the follow-up period) and non-user group (ASA use in less than 20% of the follow-up period). The primary endpoint was recurrent lower GI bleeding. Secondary endpoints included major adverse CV events as defined by the APTC definition and death from other causes. An independent, blinded adjudication committee evaluated these endpoints according to predefined criteria. RESULTS: A total of 295 patients were eligible for the study (174 in the ASA group and 121 in the non-user group). The median follow-up was 2.7 years (range, 0.6-5.0) in the ASA group and 1.7 years (range, 0.2-4.7) in the non-user group. In the ASA group, 84% of patients received ASA prescriptions in ≥75% of the follow-up period whereas 87% patients in the non-user group received ASA in ≤10% of the study period. Comparing to the ASA group, the non-user group was older, had fewer smokers and received more transfusion at the index bleed (Table 1). The cumulative incidence of recurrent lower GI bleeding in 5 years was 18.9% (95% CI, 13.3%-25.3%) in the ASA group compared to 6.9% (95% CI, 3.2%-12.5%) in the non-user group (p=0.007). Major adverse CV events at 5 years was 22.8% (16.6%-29.6%) in the ASA group compared to 36.5% (27.4%-45.6%) in the nonuser group (p=0.017). Other causes of death at 5 years was 8.2% (4.6% to 13.2%) in ASA group compared to 26.7% (18.7% to 35.4%) in the non-user group (p<0.001). CONCLUSIONS: Among ASA users with a history of lower GI bleeding, continuation of ASA increased the risk of recurrent lower GI bleeding by 2.7 folds but reduced the risk of major adverse CV events by 1.6 folds and other causes of death by 3.2 folds as compared to non-users.
95 Exome Sequencing Analysis of Candidate Genes in Very Early Onset IBD Judith R. Kelsen, Christopher J. Moran, Ariella Sasson, Mahdi Sarmady, Kernika Gupta, Helen Pauly-Hubbard, Eric Rappaport, Catherine Stolle, Petar Mamula, Andrew B. Grossman, David Piccoli, Harland Winter, Robert Baldassano, Marcella Devoto Background: Very early onset IBD (VEO-IBD) frequently presents with a different and more severe phenotype than older onset IBD, suggesting a more pronounced genetic susceptibility and dysregulated immune response. We hypothesized that rare or novel variants, including mutations in genes associated with primary immunodeficiencies (PI), were enriched in patients with severe VEO-IBD. Methods: IBD patients 5 years and younger, and parents were recruited. DNA was isolated from whole blood. Exome capture was performed by Agilent SureSelect V4, and sequencing was done using the Illumina HiSeq platform at an average coverage depth of 100X. Alignment to human genome GRCh37 was performed followed by post processing and variant calling. Following functional annotation, only variants likely to alter protein function, such as missense and loss of function mutations, were kept for subsequent analysis. Further filtering included only those with minor allele frequency less than 5% in controls from the 1000 Genomes Project and The Children's Hospital of Philadelphia's internal whole exome cohort (>400 samples). Primary focus of the analysis was on known IBD risk loci and genes associated with PI and related pathways (> 870 genes). Results: 44 samples were included, comprising 28 patients with VEO-IBD and 16 parents. Patients' age ranged from 8 weeks to 4 years, and 21 were under 2 years. The IBD risk loci and genes associated with PI contain 14,060 coding exons totaling more than 2.5 Mbp. In these regions, 87.7% of coding exons were fully covered at more than 20x. Analysis revealed novel and rare putative causative variants within these genes in multiple families that are currently being evaluated. Examples of non-synonymous variants detected in heterozygosity include rare missense variants in LRBA (rs72719663; c.21720A>G, p.T1724V), and in TNFRSF18 (rs114666761; c.128C>G, p.T43R) in a male with severe colonic IBD diagnosed at age 3. Additionally, a novel MSH5 variant (c.1661G>C p.S554T) was detected in a 3 year old male with severe ileocolonic VEO-IBD and fistulizing perianal disease. Laboratory evaluation demonstrated low IgG and IgM, and low end of normal IgA and IgE. Defects in these genes are associated with common variable immunodeficiency (CVID). CVID is a heterogeneous disorder characterized by hypogammaglobulinemia, recurrent infections and GI manifestations. Conclusions: Candidate causative mutations in VEOIBD can be identified by exome sequencing, and it is likely that some patients harbor mutations in several genes in the same or different pathways. Thus, the heterozygous variants detected may have contributed to the phenotype of VEO-IBD. Ongoing analysis is being performed, including evaluation of parental transmission; however, we anticipate that additional causative variants may occur in genes not previously associated with IBD.
103 Gaviscon Double Action (Antacid + Alginate) Versus Equivalent Antacid for Postprandial Acid Reflux: A Double-Blind Crossover Study in GERD Patients Annemijn de Ruigh, Joan Chen, John E. Pandolfino, Peter J. Kahrilas Background: Most individuals treat GERD symptoms with over-the-counter medications such as antacids and alginates. Antacids neutralize gastric acid while the proposed working mechanism of alginates (such as Gaviscon) is formation of a gel-antacid matrix floating on top of the chyme co-localizing with and neutralizing the postprandial acid pocket. This study aimed to compare the effectiveness of Gaviscon to an equivalent strength non-alginate
S-27
AGA Abstracts
AGA Abstracts
94