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Mo1243 An Analysis of the High Eradication Rate for Vonoprazan-Based Triple Therapy Against Helicobacter pylori Resistant to Clarithromycin
for free in collaborating hospitals near their high schools, and the eradication ratio and side effect will be presented. Conclusion Kyoto started the test and treat project of HP eradication for high school students, aiming to spread this project into whole prefecture in three years.
Mo1245 Identification, Molecular Basis, and Function of Diverse Helicobacter pylori ITAM-Like GroEL Chaperonins Associated With CagA Momoyo Asahi Abstract Diverse ITAM-like CagA proteins are important Helicobacter pylori virulence factors. We found that Hp-GroEL, Hp-urease beta subunit and Hp-OipA also have ITAMlike motifs. The details of moleculer interactions between diverse Hp-CagAs and Hp-GroEL, Hp-urease B subunit, Hp-OipA and T4SS-CagII components remain unclear. Methods: We used Western blot and confocal fluorescence microscopy (CFM) to analyze the molecular interactions and translocations in Hp-infected AGS cells and virus binding Viro-Adembeads (VA-beads) and PCR to detect VA-bead-bound Hp-periplsmic cDNAs. We analyzed VAbeads-bound 34 kDa OipA-like proteins by LC/MS/MS. IL-8 and IL-6 productions were measured by ELISA. Results: We found the presence of diverse 34 kDa functional OipA homologues in Hp-periplasm, Hp-cytoplasm and Hp-membrane fractions. In oipA-knockout mutants of strains NCTC11637 and of TN2GF4, our PCR analysis using oipA-primers indicated that diverse hp-periplasmic groEL (hsp60) genes including hp-N-terminal groEL (828 bp) have complementary homology with oipA in both an oipA-dependent and oipA loss-dependent manner. We also found two types of gene-loss stress-inducible hp-groEL cDNAs (1641 bp and/or 828 bp) induced by oipA-gene-loss stress. LC/MS/MS analysis of a VA-beads-bound 34 kDa OipA-like protein that cross-reacted with OipA-antisera, identified H. pylori GroEL chaperonin, which dominantly-posseses N-terminal GroEL-peptide fragments, together with Hp-urease subunits and diverse CagAs. PCR showed that expression of diverse stress-inducible groEL (hsp60) genes correlated with Hp-induced IL-8 and IL-6 production with or without serum in an oipA-gene-dependent and oipA-gene loss-dependent and in an IgG-dependent and IgG-independent manner. Our rabbit pY- and non-pY-ITAMlike-peptide pAbs and the ITAM-like peptides were effective against neutralizing of diverse Hp-ITAM-like virulent antigens in IgG-dependent manner. Conclusion: Diverse stressinducible GroELs including VA-beads-bound (Hp-bacteriophage-encoded) 34 kDa N-terminal GroEL are secreted with diverse CagAs and urease subunits via T4SS-CagII components associated with Hp-periplasmic OipA-homologue and OipA-related OMPs, and are involved in the production of IL-8 and IL-6 in tyrosine-phosphorylated (pY), CagA-dependent and non-pY-CagA-dependent ways and in IgG-dependent and IgG-independent ways. Rabbit ITAM-like-peptide monoclonal antibody and the ITAM-like peptides may provide therapeutic benefit to diverse Hp-VA-beads-bound (bacteriophage-encoded) ITAM-like virulent antigens including ITAM-like GroEL.
An Analysis of the High Eradication Rate for Vonoprazan-Based Triple Therapy Against Helicobacter pylori Resistant to Clarithromycin Hidetoshi Ohta, Tamotsu Sagawa Introduction: The newly developed drug Vonoprazan (VP; potassium channel competitive acid blocker) has an extremely high level of acid control in the stomach due to its rapid and stable action against acid secretion. In Japan, the eradication rate for treating H. pylori (HP) using a PPI-based triple therapy with Clarithromycin (CAM) has gradually fallen from 80% to 70% due to HP's resistance to CAM. In comparison, standard Vonoprazan-based triple therapy (sVP: Vonoprazan 20mg + AMPC 750mg + CAM 200mg, bid 7 days) currently achieves a 92.6% eradication rate in Japan. Aims: The aim was to analyze why Vonoprazan could achieve a high eradication rate and allude to its eradication rate in the future. Materials & Methods: 210 patients whose endoscopic specimens tested positive for urease were enrolled in this study. 122 patients (as a control arm) from September 2013 to February 2015 underwent an esomeprazole (EO) -based therapy (EAC: EO 20mg + AMPC 750mg + CAM 200mg bid 7 days). In comparison, 88 patients from March to November 2015 underwent a modified VP-based triple therapy (mVP: VP 20mg day 0 + sVP bid day1-7). Furthermore, four factors related to the eradication rate of the mVP were evaluated by, 1)continuous monitoring of the pH level during the treatment of two volunteers using a newly developed ingestible wireless sensor anchored in the stomach, 2) searching the literature for data on CAM concentrations in the stomach and pH-dependent Minimum Inhibitory Concentrations (MIC) , 3) clarifying the MIC of the CAM-resistant HP, 4) checking for mutations at positions 2142 and 2143 on the HP 23S rRNA gene (GENECUBE) in the failed cases, and 5) evaluating the rate of adverse reactions. Results: Eradication was achieved in 85/88 of the patients (96.6%) in the mVP group vs. 96/122 (78.6%) in the EAC group (p=0.0002, c2 test). 1) pH>6.0 holding time during the treatment was 86% compared to 46% in PPI reported studies. 2) Concentrations for CAM in the sera and mucus at 2 hours after ingestion were 2.0µg/mL and 300µg/mL respectively. MIC was minimal around pH7.4. 3) The MICs of the EAC-resistant HPs were classified into 2 groups i) the Intermediate MIC (1~10µg/ml): 11 patients and ii) the higher MIC (>10µg/ml): 4 patients. 4) mVP-resistant HP had a much higher MIC (‡32µg/ml) and a gene mutation, A2142G. 5) bearable soft stools or diarrhea were 15% higher in the mVP group compared to the EAC group. Conclusion: The strict pH control during the mVP made it possible to eradicate the H. pylori with the intermediate MIC even though it was partially resistant to CAM. It resulted in the high eradication rate. However, if Japanese practitioners continue to overuse CAM for common infectious diseases and 23S rRNA gene mutations continue to increase, it could result in the eradication rate for VP-based triple therapy following a similar course to current PPI therapies.
Mo1244 Role of Helicobacter pylori sabA and Other Host-Interactive Genes in Iron Deficiency Anemia, As Evaluated by Genome-Wide cDNA Analysis Seiichi Kato, Takako Osaki, Shigeru Kamiya, Xue-Song Zhang, Martin J. Blaser Background & Aim: Gastric Helicobacter pylori colonization leads to iron deficiency anemia (IDA), especially in children and adolescents. The pathogenesis of H. pylori-associated IDA remains to be unclear and few studies have focused upon iron-associated H. pylori genes in the context of IDA pathogenesis. Knowledge of the metabolism of iron uptake in H. pylori is limited. Using a whole-genome DNA microarray, we sought to identify specific H. pylori IDA-associated genes by comparing bacterial gene expression profiling in patients with or without IDA. Methods: We isolated H. pylori strains from four children (2 males and 2 females; median, 14.5 years) with IDA and from four age- and sex-matched controls without IDA. Based on these isolates, we performed cDNA microarrays under iron-replete or depleted conditions to systematically compare gene expression profiles at the whole genome level. We used real-time reverse-transcriptase (RT-) PCR to confirm the profile differentiation of the identified H. pylori IDA-associated genes. Moreover, in each H. pylori isolate, the vacA genotype and the synthesis of the VacA protein using both cytotoxic assay and immunoblot analysis were studied. Results: Compared to the control (non-IDA) strains, in the IDA strains 29 and 11 genes showed significantly higher or lower expression, respectively. Especially notable were sialic acid-binding adhesin gene sabA (4-fold), sabB (11-fold), and coaX (HP0682, 84-fold). Real-time RT-PCR study confirmed that sabA expression was consistently higher in the IDA than in the control isolates ( p= 0.029), whereas sabB and coaX varied substantially among eight isolates. Iron-depletion in vitro led to up-regulation of fecA1 and frpB1 and down-regulation of pfr, as predicted; known iron-regulated genes such as fur, pfr, fecA, and feoB did not significantly differ between the IDA and control strains. The IDA isolates had significantly higher vacA expression than in controls, consistent with the results of VacA protein assays: all four IDA and one control isolate secreted detectable amounts of the VacA protein. The vacA genotype analysis showed that three IDA and one control strains carried the s1 and m1 alleles. There were no significant differences in bacterial growth after 24-h incubation between IDA (0.18±0.11) and control groups (0.25±0.13) ( p= 0.17). Conclusions: The IDA strains had significantly altered expression of 41 genes, with the greatest consistency for sabA. It is possible that host-interactive genes including vacA, sabA and sabB, may play synergistic roles in H. pylori-associated IDA development.
Mo1246 Virulence Factors of Helicobactor pylori Among Patients With Functional Dyspepsia and Peptic Ulcer in the Community: A Study by CagA and vacA Genotyping Mohammed M. Rahman, Uday C. Ghoshal, Shamsun Nahar, Mian Mashhud Ahmad, Md.Golam Kibria, Faruque Ahmed, Ahm Rowshon, Nigar Sultana, Mohammad Abdullah Yusuf, Mahmud Hasan Background: Though the role of Helicobactor pylori (H. pylori) in peptic ulcer (PU) is wellestablished, its role in functional dyspepsia (FD) is controversial. Hence, we undertook a study in a Bangladeshi rural community to evaluate the virulence-associated genes of H. pylori (CagA, vacA and specifically the vacA allelic variants) among patients with FD as compared to PU with a hypothesis that H. pylori may be as virulent among these patients as compared to those with PU. Methods: In a door-to-door survey, adult subjects with dyspepsia (>18-years) living in four villages in Bangladesh (Charcharia, Chatia and Churain of Dhaka District; Kharrah of Munshiganj district) were identified by trained interviewers using Bengali translated and validated Enhanced Asian Rome III Questionnaire. All the patients with dyspepsia defined by Rome III criteria were offered and those agreed underwent upper gastrointestinal endoscopy. The findings at endoscopy were recorded in predefined criteria. Absence of organic lesion at endoscopy was defined as FD. H. pylori was identified and genotyped using multiplex PCR on antral biopsies among dyspeptic subjects. Results: Of 268 dyspeptic subjects (mean age 41.72 ± 14.52; female 161), 190 (71%; mean age 40.12 ± 13.59, female 126) had FD and 78 (29%; mean age 45.63±15.99; female 35) had PU (active PU and ulcer in remission). H. pylori was detected as commonly among patients with FD as those with PU (170/190 [89.5%] vs. 74/78 [95%]; p=0.238). H. pylori infected patients with PU had higher frequency of CagA positivity than those with FD. The frequency of vacA genotype s1m1 was higher among patients with PU compared to FD. The frequency of CagA and vacA genotypes s1m1, s1m2, s2m1 and s2m2 among patients with FD and PU are shown in the table. Conclusion: These data suggest that patients with PU in the community had more virulent H. pylori compared to those with FD. These data contradict some of the earlier studies that suggested that in hyper-endemic areas such as India and Bangladesh frequency of virulent strains of H. pylori is comparable among patients with FD and PU in hospital-based studies.
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Mo1245 Identification, Molecular Basis, and Function of Diverse Helicobacter pylori ITAM-Like GroEL Chaperonins Associated With CagA Momoyo Asahi Abstract Diverse ITAM-like CagA proteins are important Helicobacter pylori virulence factors. We found that Hp-GroEL, Hp-urease beta subunit and Hp-OipA also have ITAMlike motifs. The details of moleculer interactions between diverse Hp-CagAs and Hp-GroEL, Hp-urease B subunit, Hp-OipA and T4SS-CagII components remain unclear. Methods: We used Western blot and confocal fluorescence microscopy (CFM) to analyze the molecular interactions and translocations in Hp-infected AGS cells and virus binding Viro-Adembeads (VA-beads) and PCR to detect VA-bead-bound Hp-periplsmic cDNAs. We analyzed VAbeads-bound 34 kDa OipA-like proteins by LC/MS/MS. IL-8 and IL-6 productions were measured by ELISA. Results: We found the presence of diverse 34 kDa functional OipA homologues in Hp-periplasm, Hp-cytoplasm and Hp-membrane fractions. In oipA-knockout mutants of strains NCTC11637 and of TN2GF4, our PCR analysis using oipA-primers indicated that diverse hp-periplasmic groEL (hsp60) genes including hp-N-terminal groEL (828 bp) have complementary homology with oipA in both an oipA-dependent and oipA loss-dependent manner. We also found two types of gene-loss stress-inducible hp-groEL cDNAs (1641 bp and/or 828 bp) induced by oipA-gene-loss stress. LC/MS/MS analysis of a VA-beads-bound 34 kDa OipA-like protein that cross-reacted with OipA-antisera, identified H. pylori GroEL chaperonin, which dominantly-posseses N-terminal GroEL-peptide fragments, together with Hp-urease subunits and diverse CagAs. PCR showed that expression of diverse stress-inducible groEL (hsp60) genes correlated with Hp-induced IL-8 and IL-6 production with or without serum in an oipA-gene-dependent and oipA-gene loss-dependent and in an IgG-dependent and IgG-independent manner. Our rabbit pY- and non-pY-ITAMlike-peptide pAbs and the ITAM-like peptides were effective against neutralizing of diverse Hp-ITAM-like virulent antigens in IgG-dependent manner. Conclusion: Diverse stressinducible GroELs including VA-beads-bound (Hp-bacteriophage-encoded) 34 kDa N-terminal GroEL are secreted with diverse CagAs and urease subunits via T4SS-CagII components associated with Hp-periplasmic OipA-homologue and OipA-related OMPs, and are involved in the production of IL-8 and IL-6 in tyrosine-phosphorylated (pY), CagA-dependent and non-pY-CagA-dependent ways and in IgG-dependent and IgG-independent ways. Rabbit ITAM-like-peptide monoclonal antibody and the ITAM-like peptides may provide therapeutic benefit to diverse Hp-VA-beads-bound (bacteriophage-encoded) ITAM-like virulent antigens including ITAM-like GroEL.
An Analysis of the High Eradication Rate for Vonoprazan-Based Triple Therapy Against Helicobacter pylori Resistant to Clarithromycin Hidetoshi Ohta, Tamotsu Sagawa Introduction: The newly developed drug Vonoprazan (VP; potassium channel competitive acid blocker) has an extremely high level of acid control in the stomach due to its rapid and stable action against acid secretion. In Japan, the eradication rate for treating H. pylori (HP) using a PPI-based triple therapy with Clarithromycin (CAM) has gradually fallen from 80% to 70% due to HP's resistance to CAM. In comparison, standard Vonoprazan-based triple therapy (sVP: Vonoprazan 20mg + AMPC 750mg + CAM 200mg, bid 7 days) currently achieves a 92.6% eradication rate in Japan. Aims: The aim was to analyze why Vonoprazan could achieve a high eradication rate and allude to its eradication rate in the future. Materials & Methods: 210 patients whose endoscopic specimens tested positive for urease were enrolled in this study. 122 patients (as a control arm) from September 2013 to February 2015 underwent an esomeprazole (EO) -based therapy (EAC: EO 20mg + AMPC 750mg + CAM 200mg bid 7 days). In comparison, 88 patients from March to November 2015 underwent a modified VP-based triple therapy (mVP: VP 20mg day 0 + sVP bid day1-7). Furthermore, four factors related to the eradication rate of the mVP were evaluated by, 1)continuous monitoring of the pH level during the treatment of two volunteers using a newly developed ingestible wireless sensor anchored in the stomach, 2) searching the literature for data on CAM concentrations in the stomach and pH-dependent Minimum Inhibitory Concentrations (MIC) , 3) clarifying the MIC of the CAM-resistant HP, 4) checking for mutations at positions 2142 and 2143 on the HP 23S rRNA gene (GENECUBE) in the failed cases, and 5) evaluating the rate of adverse reactions. Results: Eradication was achieved in 85/88 of the patients (96.6%) in the mVP group vs. 96/122 (78.6%) in the EAC group (p=0.0002, c2 test). 1) pH>6.0 holding time during the treatment was 86% compared to 46% in PPI reported studies. 2) Concentrations for CAM in the sera and mucus at 2 hours after ingestion were 2.0µg/mL and 300µg/mL respectively. MIC was minimal around pH7.4. 3) The MICs of the EAC-resistant HPs were classified into 2 groups i) the Intermediate MIC (1~10µg/ml): 11 patients and ii) the higher MIC (>10µg/ml): 4 patients. 4) mVP-resistant HP had a much higher MIC (‡32µg/ml) and a gene mutation, A2142G. 5) bearable soft stools or diarrhea were 15% higher in the mVP group compared to the EAC group. Conclusion: The strict pH control during the mVP made it possible to eradicate the H. pylori with the intermediate MIC even though it was partially resistant to CAM. It resulted in the high eradication rate. However, if Japanese practitioners continue to overuse CAM for common infectious diseases and 23S rRNA gene mutations continue to increase, it could result in the eradication rate for VP-based triple therapy following a similar course to current PPI therapies.
Mo1244 Role of Helicobacter pylori sabA and Other Host-Interactive Genes in Iron Deficiency Anemia, As Evaluated by Genome-Wide cDNA Analysis Seiichi Kato, Takako Osaki, Shigeru Kamiya, Xue-Song Zhang, Martin J. Blaser Background & Aim: Gastric Helicobacter pylori colonization leads to iron deficiency anemia (IDA), especially in children and adolescents. The pathogenesis of H. pylori-associated IDA remains to be unclear and few studies have focused upon iron-associated H. pylori genes in the context of IDA pathogenesis. Knowledge of the metabolism of iron uptake in H. pylori is limited. Using a whole-genome DNA microarray, we sought to identify specific H. pylori IDA-associated genes by comparing bacterial gene expression profiling in patients with or without IDA. Methods: We isolated H. pylori strains from four children (2 males and 2 females; median, 14.5 years) with IDA and from four age- and sex-matched controls without IDA. Based on these isolates, we performed cDNA microarrays under iron-replete or depleted conditions to systematically compare gene expression profiles at the whole genome level. We used real-time reverse-transcriptase (RT-) PCR to confirm the profile differentiation of the identified H. pylori IDA-associated genes. Moreover, in each H. pylori isolate, the vacA genotype and the synthesis of the VacA protein using both cytotoxic assay and immunoblot analysis were studied. Results: Compared to the control (non-IDA) strains, in the IDA strains 29 and 11 genes showed significantly higher or lower expression, respectively. Especially notable were sialic acid-binding adhesin gene sabA (4-fold), sabB (11-fold), and coaX (HP0682, 84-fold). Real-time RT-PCR study confirmed that sabA expression was consistently higher in the IDA than in the control isolates ( p= 0.029), whereas sabB and coaX varied substantially among eight isolates. Iron-depletion in vitro led to up-regulation of fecA1 and frpB1 and down-regulation of pfr, as predicted; known iron-regulated genes such as fur, pfr, fecA, and feoB did not significantly differ between the IDA and control strains. The IDA isolates had significantly higher vacA expression than in controls, consistent with the results of VacA protein assays: all four IDA and one control isolate secreted detectable amounts of the VacA protein. The vacA genotype analysis showed that three IDA and one control strains carried the s1 and m1 alleles. There were no significant differences in bacterial growth after 24-h incubation between IDA (0.18±0.11) and control groups (0.25±0.13) ( p= 0.17). Conclusions: The IDA strains had significantly altered expression of 41 genes, with the greatest consistency for sabA. It is possible that host-interactive genes including vacA, sabA and sabB, may play synergistic roles in H. pylori-associated IDA development.
Mo1246 Virulence Factors of Helicobactor pylori Among Patients With Functional Dyspepsia and Peptic Ulcer in the Community: A Study by CagA and vacA Genotyping Mohammed M. Rahman, Uday C. Ghoshal, Shamsun Nahar, Mian Mashhud Ahmad, Md.Golam Kibria, Faruque Ahmed, Ahm Rowshon, Nigar Sultana, Mohammad Abdullah Yusuf, Mahmud Hasan Background: Though the role of Helicobactor pylori (H. pylori) in peptic ulcer (PU) is wellestablished, its role in functional dyspepsia (FD) is controversial. Hence, we undertook a study in a Bangladeshi rural community to evaluate the virulence-associated genes of H. pylori (CagA, vacA and specifically the vacA allelic variants) among patients with FD as compared to PU with a hypothesis that H. pylori may be as virulent among these patients as compared to those with PU. Methods: In a door-to-door survey, adult subjects with dyspepsia (>18-years) living in four villages in Bangladesh (Charcharia, Chatia and Churain of Dhaka District; Kharrah of Munshiganj district) were identified by trained interviewers using Bengali translated and validated Enhanced Asian Rome III Questionnaire. All the patients with dyspepsia defined by Rome III criteria were offered and those agreed underwent upper gastrointestinal endoscopy. The findings at endoscopy were recorded in predefined criteria. Absence of organic lesion at endoscopy was defined as FD. H. pylori was identified and genotyped using multiplex PCR on antral biopsies among dyspeptic subjects. Results: Of 268 dyspeptic subjects (mean age 41.72 ± 14.52; female 161), 190 (71%; mean age 40.12 ± 13.59, female 126) had FD and 78 (29%; mean age 45.63±15.99; female 35) had PU (active PU and ulcer in remission). H. pylori was detected as commonly among patients with FD as those with PU (170/190 [89.5%] vs. 74/78 [95%]; p=0.238). H. pylori infected patients with PU had higher frequency of CagA positivity than those with FD. The frequency of vacA genotype s1m1 was higher among patients with PU compared to FD. The frequency of CagA and vacA genotypes s1m1, s1m2, s2m1 and s2m2 among patients with FD and PU are shown in the table. Conclusion: These data suggest that patients with PU in the community had more virulent H. pylori compared to those with FD. These data contradict some of the earlier studies that suggested that in hyper-endemic areas such as India and Bangladesh frequency of virulent strains of H. pylori is comparable among patients with FD and PU in hospital-based studies.
S-677
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Page 677
AGA Abstracts
AGA Abstracts
Mo1243