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Su1826 Pseudokinase Tribbles 3 Enhances SHP2-Dependent ERK Signaling in Helicobacter pylori-Induced Gastric Carcinogenesis
AGA Abstracts
Su1826
Su1828
Pseudokinase Tribbles 3 Enhances SHP2-Dependent ERK Signaling in Helicobacter pylori-Induced Gastric Carcinogenesis Yutaka Kondo, Naoki Asano, Akira Imatani, Xiaoyi Jin, Katsunori Iijima, Tomoyuki Koike, Tooru Shimosegawa
MiR-155 Expression in Helicobacter pylori Infection, Inflammatory and Precancerous Gastric Conditions Wiebke Schirrmeister, Arne Kandulski, Cosima Langner, Thomas Wex, Alexander Link, Peter Malfertheiner
Background & Aim: Helicobacter pylori (H. pylori) infection is known to induce gastric inflammation, gastric intestinal metaplasia and gastric carcinogenesis, but the details are still ambiguous. In DDW2012, we reported that H. pylori infection induced the expression of pseudokinase Tribbles 3 (Trib3) both in vitro and in vivo, and overexpression of this pesudokinase led to augmented phosphorylation of tyrosine phosphatase SHP2. In this study, we aimed to identify the downstream signaling pathway of SHP2 affected by Trib3, which contributes to gastric carcinogenesis. Methods & Results: First, we generated Trib3 overexpressing cell line Trib3GSM06 from normal murine gastric epithelial cell line GSM06. We then infected these cells with H. pylori and investigated the signaling pathway that is affected by the overexpression of Trib3. Western blot analysis of these cells showed that although neither β-catenin nor STAT3 was affected by over-expression of Trib3, the expression of phosphorylated ERK1/2 (p44/42 MAPK) was augmented up to 2.4 folds in Trib3GSM06 compared to GSM06 cells. To verify if the phosphorylation of ERK1/2 was the result of SHP2 activation, we assessed the effect of SHP2 inhibitor (II-B08) on ERK1/2 phosphorylation by western blot. As a result, the addition of SHP2 inhibitor diminished the expression of phosphorylated ERK1/2. In addition, MTS assay of Trib3GSM06 and GSM06 cells showed that over-expression of Trib3 enhanced cell proliferation, and this enhancement was suppressed by the addition of SHP2 inhibitor. Moreover, expression of c-Myc was enhanced up to 2.0 folds by overexpression of Trib3, and this enhancement was abolished by the addition of SHP2 inhibitor. Finally, we investigated the physical interaction between Trib3 and SHP2 in H. pylori-infected GSM06 cells by immunoprecipitation and western blotting, which clearly showed the binding of these two molecules. These findings suggested that over-expression of Trib3 activates ERK1/2 by directly binding to and activating SHP2. Conclusion: Our study demonstrated that H. pylori-induced Trib3 directly interacts with SHP2 and activates ERK1/2 signaling, leading to gastric carcinogenesis. This finding suggests Trib3 as a possible candidate of therapeutic target for gastric cancers.
Introduction: H. pylori-triggered inflammation is a dynamic process that induces the cascade of gastric carcinogenesis. Understanding of this process in detail is crucial for the development of preventive strategies, in particular once potentially irreversible alterations, such as intestinal metaplasia (IM), have occurred. Recently, microRNA (miRNA) miR-155 has been linked to H. pylori infection of gastric mucosa, but systematic analyses are not available yet. Here, we extensively analyzed miR-155 expression in the stomach with regard to H. pylori infection/ genotype and inflammatory/precancerous condition. Methods: In this prospective study (ERANET), 186 patients (with normal mucosa (N), chronic gastritis (CG), atrophic gastritis (AG±IM) underwent upper GI endoscopy. Histological mucosa characterization was assessed and genotyping of H. pylori was performed following bacterial culturing. Expression of miR155 was analyzed using TaqMan assay in biopsies from antrum. Mucosal inflammation, atrophic or malignant changes were evaluated histologically according to Sydney classification. Results: MiRNA-155 expression was higher in antrum mucosa of CG and AG compared to N (p<0.001). While H. pylori-negative CG was associated with increased miR-155 expression, the highest expression was observed in H. pylori-related CG in antrum (p<0.001). Active H. pylori-infection, defined by direct H. pylori detection (microbiology, histology), was associated with increased miR-155 expression compared to patients with no detectable H. pylori despite H. pylori-positive serology (p=0.015). Expression of miR-155 was dependent on H. pylori cagA and vacA with highest expression in cagA-positive and vacA s1i1m1 genotype strains (p<0.001). Histological analyses revealed that miR-155 expression strongly correlated with inflammation score of the mucosa in a H. pylori-dependent manner. While chronic lymphocytic inflammation had a strong impact on miR-155 expression, only modest or no correlation was found for grade of active inflammation and IM. Discussion: miR-155 is a biomarker of chronic H. pylori-triggered inflammation in gastric mucosa. The extent of miR155 expression is dependent on H. pylori genotype and may be predictive for progression of H. pylori-triggered inflammation in gastric mucosa. Further studies are needed to evaluate the biomarker potential in clinical settings.
Su1827 Su1829 High Prevalence of Atrophic Autoimmune Gastritis in the Rio Grande Valley Kevin Turner, Carmela P. Morales, Elsa S. Canales, Monte D. Allen, Robert M. Genta
BIoinformatic Approach to Mapping Global H. pylori cagA Sequences for Targeted Vaccine Development Songhua Zhang, Tu Pham, Christopher A. Lopes, Frances Terry, Leonard Moise, William Martin, Anne S. De Groot, Steven F. Moss
Background: Corpus-restricted atrophic gastritis without H. pylori infection is essentially diagnostic of autoimmune atrophic gastritis (AIG), a rare entity believed to affect mostly elderly women of European descent. While the female proclivity has been confirmed, several studies have reported AIG in Hispanic patients. This study was designed to determine prevalence of AIG in a population of mostly Hispanic ancestry residing in a South Texas region. Methods: Three groups were extracted from a nationwide computerized database of patients who had EGD with Sydney System-compliant gastric biopsies (1.2008 to 10.2014): 1) all subjects seen at gastroenterology practices located along the Rio Grande Valley (study group); 2) patients from US regions with a prevalence of H. pylori infection similar or higher than the study group (high-prevalence area controls); and 3) patients from US regions with low H. pylori prevalence (low-prevalence area controls). We then used strict histopathologic criteria to establish a presumptive diagnosis of AIG: 1) atrophy, metaplasia (intestinal, pseudopyloric, or pancreatic), and chronic inflammation in the oxyntic mucosa; 2) linear or micronodular ECL-cell hyperplasia; and 3) normal or reactive antrum with no inflammation, atrophy, or metaplasia. The prevalence of AIG in the groups was compared using unadjusted odds ratios (OR). Results: There were 6,283 study group patients (median age 58 years, 72% female); 27,810 high-prevalence area controls (median age 55 years, 56% female), and 21,872 low-prevalence area controls (median age 69 years, 62% female). The prevalence of histologic AIG in each group is depicted in Figure 1. Patients from the Rio Grande Valley were more likely to have AIG than subjects from both low H. pylori prevalence areas (OR 6.27 95%CI 5.01-7.84) and high H. pylori prevalence areas (OR 3.97 95%CI 3.30-4.79). The median age of patients with AIG was similar in all groups (66 to 69). In low H. pylori prevalence areas AIG was equally prevalent in males and females, but there was significant females predominance in both high-prevalence area controls (OR 1.41 95%CI 1.08-1.82) and the Rio Grande population (OR 2.06 95%CI 1.44-2.94). Conclusions: Corpus-restricted atrophic gastritis with features typical of AIG had an unusually high prevalence in a South Texas community predominantly of Mexican ancestry. The high prevalence of H. pylori infection in this area is unlikely to be etiologically relevant since a control group with a slightly higher prevalence of the infection (21.0% versus 19.4%) showed a rate of AIG four times lower than in the Rio Grande population and only slightly higher than a control group with low (5.8%) H. pylori prevalence. These data reemphasize the need to consider AIG in patients who do not fit the traditional - and inaccurate - stereotype of the "elderly woman of European descent."
Background: Helicobacter pylori (H. pylori) strains carrying the cytotoxin-associated gene pathogenicity island (cag PAI) tend to be associated with more severe clinical outcomes compared to cagA-negative H. pylori infections, including increased risk for non-cardia gastric adenocarcinoma. In the era of progressive antibiotic resistance, a T cell-directed anti-H. pylori CagA vaccine could be a cost-effective strategy to prevent H. pylori-associated disease. Aim: To investigate and identify potentially immunogenic T cell epitopes as H. pylori CagA vaccine candidates through the use of immunoinformatic tools and human in vitro assays. Methods: Forty one publicly available and geographically and genetically diverse H. pylori cagA sequences were computationally mapped to identify T-cell epitopes with promising antigenic properties. An epitope mapping algorithm for a panel of eight archetypal HLA alleles was used to select strong class II human leukocyte antigen (HLA) binders among conserved 9-mer cagA sequences. We then utilized EpiAssembler software to assemble putative immunogenic 9-mers to maximize epitope density over a 15-25 amino acids stretch and construct immunogenic consensus sequences (ICS). Subsequently, JanusMatrix analysis was employed to exclude cross-reactivity of the ICS with the human genome and microbiome at T cell receptor-facing positions. Selected ICS were used for peptide synthesis for HLA binding assays and for evaluation of immune responses in human peripheral mononuclear cells (PBMC) extracted from H. pylori- infected and -uninfected subjects. Results: After the multistep computational selection process, we excluded three sequences with cross-reactivity with human genome and human microbiome. We then ranked H. pylori cagA conserved ICS among 41 H. pylori cagA sequences according to EpiMatrix scores. The top 32 ICS with predicted binding to at least four HLA-DR alleles were selected for peptide synthesis and immunogenicity validation. Thirty one (97%) synthesized peptides showed moderate to strong HLA DR alleles binding affinity in vitro. We performed human IFN-gamma and IL10 ELISpot as well as ELISA assays to further evaluate and identify immunogenic H. pylori CagA vaccine candidates, and exclude peptides with immunoregulatory function for DNA vaccine construction. Peptides co-cultured with PBMC in vitro for 24 hours stimulated interferon gamma secretion. We are currently recruiting H. pylori- infected and -uninfected subjects to complete this validation. Conclusion: This bioinformatic approach rapidly facilitates T cell epitopes selection among geographically and genetically diverse H. pylori cagA sequences, showing promising properties for T cell-based CagA vaccine development. (Supported by NIH U19 AI082642) Su1830 Immunization Combined With Inhibiting the Anti-Inflammatory Effects of Adenosine Enhances Gastritis and Attenuates Bacterial Load Associated With Persistent Infection by H. pylori Sujay Singh, Victoria Derr, Ioannis Drygiannakis, Sheila E. Crowe, Salony Roongta, Steven F. Josephs, Joel Linden, Soumita Das, Peter B. Ernst Background/Aims: Over half of the world's population is infected with Helicobacter pylori which typically occurs in childhood and usually persists for life. According to the WHO, gastric cancer caused by H. pylori is the third leading cause of cancer mortality in the world. One explanation for the persistence of H. pylori is the presence of Treg. Two mediators produced by Treg have been implicated in the persistence of H. pylori: IL-10 and adenosine.
AGA Abstracts
S-528
Su1826
Su1828
Pseudokinase Tribbles 3 Enhances SHP2-Dependent ERK Signaling in Helicobacter pylori-Induced Gastric Carcinogenesis Yutaka Kondo, Naoki Asano, Akira Imatani, Xiaoyi Jin, Katsunori Iijima, Tomoyuki Koike, Tooru Shimosegawa
MiR-155 Expression in Helicobacter pylori Infection, Inflammatory and Precancerous Gastric Conditions Wiebke Schirrmeister, Arne Kandulski, Cosima Langner, Thomas Wex, Alexander Link, Peter Malfertheiner
Background & Aim: Helicobacter pylori (H. pylori) infection is known to induce gastric inflammation, gastric intestinal metaplasia and gastric carcinogenesis, but the details are still ambiguous. In DDW2012, we reported that H. pylori infection induced the expression of pseudokinase Tribbles 3 (Trib3) both in vitro and in vivo, and overexpression of this pesudokinase led to augmented phosphorylation of tyrosine phosphatase SHP2. In this study, we aimed to identify the downstream signaling pathway of SHP2 affected by Trib3, which contributes to gastric carcinogenesis. Methods & Results: First, we generated Trib3 overexpressing cell line Trib3GSM06 from normal murine gastric epithelial cell line GSM06. We then infected these cells with H. pylori and investigated the signaling pathway that is affected by the overexpression of Trib3. Western blot analysis of these cells showed that although neither β-catenin nor STAT3 was affected by over-expression of Trib3, the expression of phosphorylated ERK1/2 (p44/42 MAPK) was augmented up to 2.4 folds in Trib3GSM06 compared to GSM06 cells. To verify if the phosphorylation of ERK1/2 was the result of SHP2 activation, we assessed the effect of SHP2 inhibitor (II-B08) on ERK1/2 phosphorylation by western blot. As a result, the addition of SHP2 inhibitor diminished the expression of phosphorylated ERK1/2. In addition, MTS assay of Trib3GSM06 and GSM06 cells showed that over-expression of Trib3 enhanced cell proliferation, and this enhancement was suppressed by the addition of SHP2 inhibitor. Moreover, expression of c-Myc was enhanced up to 2.0 folds by overexpression of Trib3, and this enhancement was abolished by the addition of SHP2 inhibitor. Finally, we investigated the physical interaction between Trib3 and SHP2 in H. pylori-infected GSM06 cells by immunoprecipitation and western blotting, which clearly showed the binding of these two molecules. These findings suggested that over-expression of Trib3 activates ERK1/2 by directly binding to and activating SHP2. Conclusion: Our study demonstrated that H. pylori-induced Trib3 directly interacts with SHP2 and activates ERK1/2 signaling, leading to gastric carcinogenesis. This finding suggests Trib3 as a possible candidate of therapeutic target for gastric cancers.
Introduction: H. pylori-triggered inflammation is a dynamic process that induces the cascade of gastric carcinogenesis. Understanding of this process in detail is crucial for the development of preventive strategies, in particular once potentially irreversible alterations, such as intestinal metaplasia (IM), have occurred. Recently, microRNA (miRNA) miR-155 has been linked to H. pylori infection of gastric mucosa, but systematic analyses are not available yet. Here, we extensively analyzed miR-155 expression in the stomach with regard to H. pylori infection/ genotype and inflammatory/precancerous condition. Methods: In this prospective study (ERANET), 186 patients (with normal mucosa (N), chronic gastritis (CG), atrophic gastritis (AG±IM) underwent upper GI endoscopy. Histological mucosa characterization was assessed and genotyping of H. pylori was performed following bacterial culturing. Expression of miR155 was analyzed using TaqMan assay in biopsies from antrum. Mucosal inflammation, atrophic or malignant changes were evaluated histologically according to Sydney classification. Results: MiRNA-155 expression was higher in antrum mucosa of CG and AG compared to N (p<0.001). While H. pylori-negative CG was associated with increased miR-155 expression, the highest expression was observed in H. pylori-related CG in antrum (p<0.001). Active H. pylori-infection, defined by direct H. pylori detection (microbiology, histology), was associated with increased miR-155 expression compared to patients with no detectable H. pylori despite H. pylori-positive serology (p=0.015). Expression of miR-155 was dependent on H. pylori cagA and vacA with highest expression in cagA-positive and vacA s1i1m1 genotype strains (p<0.001). Histological analyses revealed that miR-155 expression strongly correlated with inflammation score of the mucosa in a H. pylori-dependent manner. While chronic lymphocytic inflammation had a strong impact on miR-155 expression, only modest or no correlation was found for grade of active inflammation and IM. Discussion: miR-155 is a biomarker of chronic H. pylori-triggered inflammation in gastric mucosa. The extent of miR155 expression is dependent on H. pylori genotype and may be predictive for progression of H. pylori-triggered inflammation in gastric mucosa. Further studies are needed to evaluate the biomarker potential in clinical settings.
Su1827 Su1829 High Prevalence of Atrophic Autoimmune Gastritis in the Rio Grande Valley Kevin Turner, Carmela P. Morales, Elsa S. Canales, Monte D. Allen, Robert M. Genta
BIoinformatic Approach to Mapping Global H. pylori cagA Sequences for Targeted Vaccine Development Songhua Zhang, Tu Pham, Christopher A. Lopes, Frances Terry, Leonard Moise, William Martin, Anne S. De Groot, Steven F. Moss
Background: Corpus-restricted atrophic gastritis without H. pylori infection is essentially diagnostic of autoimmune atrophic gastritis (AIG), a rare entity believed to affect mostly elderly women of European descent. While the female proclivity has been confirmed, several studies have reported AIG in Hispanic patients. This study was designed to determine prevalence of AIG in a population of mostly Hispanic ancestry residing in a South Texas region. Methods: Three groups were extracted from a nationwide computerized database of patients who had EGD with Sydney System-compliant gastric biopsies (1.2008 to 10.2014): 1) all subjects seen at gastroenterology practices located along the Rio Grande Valley (study group); 2) patients from US regions with a prevalence of H. pylori infection similar or higher than the study group (high-prevalence area controls); and 3) patients from US regions with low H. pylori prevalence (low-prevalence area controls). We then used strict histopathologic criteria to establish a presumptive diagnosis of AIG: 1) atrophy, metaplasia (intestinal, pseudopyloric, or pancreatic), and chronic inflammation in the oxyntic mucosa; 2) linear or micronodular ECL-cell hyperplasia; and 3) normal or reactive antrum with no inflammation, atrophy, or metaplasia. The prevalence of AIG in the groups was compared using unadjusted odds ratios (OR). Results: There were 6,283 study group patients (median age 58 years, 72% female); 27,810 high-prevalence area controls (median age 55 years, 56% female), and 21,872 low-prevalence area controls (median age 69 years, 62% female). The prevalence of histologic AIG in each group is depicted in Figure 1. Patients from the Rio Grande Valley were more likely to have AIG than subjects from both low H. pylori prevalence areas (OR 6.27 95%CI 5.01-7.84) and high H. pylori prevalence areas (OR 3.97 95%CI 3.30-4.79). The median age of patients with AIG was similar in all groups (66 to 69). In low H. pylori prevalence areas AIG was equally prevalent in males and females, but there was significant females predominance in both high-prevalence area controls (OR 1.41 95%CI 1.08-1.82) and the Rio Grande population (OR 2.06 95%CI 1.44-2.94). Conclusions: Corpus-restricted atrophic gastritis with features typical of AIG had an unusually high prevalence in a South Texas community predominantly of Mexican ancestry. The high prevalence of H. pylori infection in this area is unlikely to be etiologically relevant since a control group with a slightly higher prevalence of the infection (21.0% versus 19.4%) showed a rate of AIG four times lower than in the Rio Grande population and only slightly higher than a control group with low (5.8%) H. pylori prevalence. These data reemphasize the need to consider AIG in patients who do not fit the traditional - and inaccurate - stereotype of the "elderly woman of European descent."
Background: Helicobacter pylori (H. pylori) strains carrying the cytotoxin-associated gene pathogenicity island (cag PAI) tend to be associated with more severe clinical outcomes compared to cagA-negative H. pylori infections, including increased risk for non-cardia gastric adenocarcinoma. In the era of progressive antibiotic resistance, a T cell-directed anti-H. pylori CagA vaccine could be a cost-effective strategy to prevent H. pylori-associated disease. Aim: To investigate and identify potentially immunogenic T cell epitopes as H. pylori CagA vaccine candidates through the use of immunoinformatic tools and human in vitro assays. Methods: Forty one publicly available and geographically and genetically diverse H. pylori cagA sequences were computationally mapped to identify T-cell epitopes with promising antigenic properties. An epitope mapping algorithm for a panel of eight archetypal HLA alleles was used to select strong class II human leukocyte antigen (HLA) binders among conserved 9-mer cagA sequences. We then utilized EpiAssembler software to assemble putative immunogenic 9-mers to maximize epitope density over a 15-25 amino acids stretch and construct immunogenic consensus sequences (ICS). Subsequently, JanusMatrix analysis was employed to exclude cross-reactivity of the ICS with the human genome and microbiome at T cell receptor-facing positions. Selected ICS were used for peptide synthesis for HLA binding assays and for evaluation of immune responses in human peripheral mononuclear cells (PBMC) extracted from H. pylori- infected and -uninfected subjects. Results: After the multistep computational selection process, we excluded three sequences with cross-reactivity with human genome and human microbiome. We then ranked H. pylori cagA conserved ICS among 41 H. pylori cagA sequences according to EpiMatrix scores. The top 32 ICS with predicted binding to at least four HLA-DR alleles were selected for peptide synthesis and immunogenicity validation. Thirty one (97%) synthesized peptides showed moderate to strong HLA DR alleles binding affinity in vitro. We performed human IFN-gamma and IL10 ELISpot as well as ELISA assays to further evaluate and identify immunogenic H. pylori CagA vaccine candidates, and exclude peptides with immunoregulatory function for DNA vaccine construction. Peptides co-cultured with PBMC in vitro for 24 hours stimulated interferon gamma secretion. We are currently recruiting H. pylori- infected and -uninfected subjects to complete this validation. Conclusion: This bioinformatic approach rapidly facilitates T cell epitopes selection among geographically and genetically diverse H. pylori cagA sequences, showing promising properties for T cell-based CagA vaccine development. (Supported by NIH U19 AI082642) Su1830 Immunization Combined With Inhibiting the Anti-Inflammatory Effects of Adenosine Enhances Gastritis and Attenuates Bacterial Load Associated With Persistent Infection by H. pylori Sujay Singh, Victoria Derr, Ioannis Drygiannakis, Sheila E. Crowe, Salony Roongta, Steven F. Josephs, Joel Linden, Soumita Das, Peter B. Ernst Background/Aims: Over half of the world's population is infected with Helicobacter pylori which typically occurs in childhood and usually persists for life. According to the WHO, gastric cancer caused by H. pylori is the third leading cause of cancer mortality in the world. One explanation for the persistence of H. pylori is the presence of Treg. Two mediators produced by Treg have been implicated in the persistence of H. pylori: IL-10 and adenosine.
AGA Abstracts
S-528