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Long Type dupA but Not Short Type dupA Associated With Peptic UlcerDisease in JapanAyaka Takahashi, Seiji Shiota, Miyuki Matsuda, Saori Nakachi, Osamu Matsunari,Masahide Watada, Rumiko Suzuki, Katsuhiro Hanada, Yoshio Yamaoka

Background and aims: Helicobacter pylori dupA (duodenal ulcer promoting) gene was reportedas a virulence factor that induced duodenal ulcer and had a suppressive action on gastriccancer. However, the association of the dupA gene on clinical outcomes is different indifferent populations. Full-sequenced data of H. pylori showed that the length of the dupAgene is depends on the strain; Shi470 and G27 have approximately 600bp longer than thatof other strains (e.g., strain J99) in the C-terminal region of the dupA gene. This suggeststhat the dupA gene has two genotypes: long and short type. In addition, previous studiesindicated that some strains have a presence of the mutation (insertion or mutation) in thedupA gene, which created a premature stop codon . In this study, we aimed to examine thedupA genotypes (long type or short type), presence of the mutation, and examined theassociation with clinical outcomes in Japanese population. Methods: A total of 388 patients(97 with gastritis, 137 with duodenal ulcer [DU], 121 with gastric ulcer [GU], and 24 withgastric cancer [GC]) were included in this study. The status of the dupA genes was examinedby polymerase chain reaction. The oligonucleotide primers for the dupA typing were designedaccording to the dupA gene sequence deposited in Genbank. First primers were designedto include the additional region of C-terminal of the dupA gene (long type dupA). Secondprimers were designed to include the conventional dupA gene (short type dupA). We alsosequenced full-length dupA gene and evaluated the presence of mutation. Results: Theprevalence of short type dupA gene alone was not significantly different among four groups(gastritis 14.4%, DU 8.0%, GU 6.5%, and GC 4.2%, respectively). Interestingly, the preval-ence of the long type dupA gene in strains from DU was significantly higher than that fromgastritis (19.7 v.s. 9.3%, p = 0.02). Those of GU, GC were also significantly higher thanthat of gastritis (25.8, 25.0 and 9.3%, p = 0.0001, p = 0.03, respectively). Full-lengthsequence of the dupA gene was completed in 33 strains. Among them, point mutation leadto stop codon was found in only one strain. Conclusions: The long type but not the shorttype dupA gene can be used as discriminating factor of peptic ulcer and gastric cancer fromgastritis in Japan. These observations suggest that only strains that are intact dupA-positivemight be involved in gastroduodenal diseases.

Su1665

Higher Frequency of CagA EPIYA-C Phosphorylation Sites in H. pylori StrainsFrom Relatives of Gastric Cancer PatientsGifone A. Rocha, Dulciene M. Queiroz, Andreia Maria C. Rocha, Sérgio A. Batista, CíceroM. Silva, Manuel B. Braga Neto, André M. Fialho, Andrea B. Fialho, Lúcia Libanês B.Braga

Infection with CagA-positive H. pylori strains has been associated with an increased risk forgastric cancer. The protein is translocated into the gastric epithelial cell by the type foursecretion system encoded by the cagPAI. Once translocated, CagA localizes to the innersurface of the plasma membrane where it is phosphorylated by a multiple members of theSrc kinase family within a repeated specific five aminoacid EPIYAmotif. Once phosphorylatedCagA forms a physical complex with SHP-2 phosphatase and triggers abnormal cellularsignals which enhance the risk of damaged cells to acquire pre cancerous genetic changes.It has also been shown that infection with CagA strains with higher number of EPIYA-Csegments is a risk for gastric cnacer. Since H. pylori infection is predominantly acquired inchildhood in the familiar environment and the infection persists lifelong and it has beenreported that relatives of patients with gastric cancer are at increased risk of gastric cancer,we aimed to evaluate the frequency of CagA EPIYA-C motifs in cagA-positive H. pylori strainsisolated from relatives of gastric cancer patients (n=45) and from age and gender matchedpatients with no family history of gastric cancer (n=30, control group), selected among thosewho underwent upper gastrointestinal endoscopy for investigation of dyspeptic symptomsin Ceará, Northeastern Brazil. The number of EPIYA-C segments was determined accordingto the methods proposed by Yamaoka's group and Atherton's group. The results wereconfirmed by sequencing. Statistical analysis was performed by using SPSS version 17.0.Associations were evaluated by chi-square test with Yates correction. Odds ratios and 95%confidence intervals were calculated. Colonization by H. pylori CagA-positive strains pos-sessing two or three EPIYA-C motifs was more frequently observed (OR=3.65, 95%CI=1.1- 13.3, p = 0.04) in the group of relatives of patients with gastric cancer (19/45, 42.0%)than in the control group (5/30, 16.7%). Higher number of EPIYA-C segments was alsoassociated with gastric corpus inflammation (p = 0.04), gastric corpus foveolar hyperplasia(p = 0.05) and gastric corpus atrophy (p = 0.05). In conclusion, we found that infectionby H. pylori CagA-positive strains harboring multiple EPIYA-C repeats is more frequentlyobserved in relatives of patients with gastric cancer. These results, suggested that in additionto familial predisposing factors, relatives of patients with gastric cancer are infected withmore virulent H. pylori strains.

Su1666

Novel Definition of Helicobacter pylori as a “Gastrophile” Based on ItsRequirement for Acid to Maintain Urease Activity, RNA Stability and BacillaryMorphologyDavid R. Scott, Elizabeth A. Marcus, Yi Wen, Jing Feng, George Sachs

Background: The In Vitro bioenergetic profile of H. pylori is that of a neutralophile. In theabsence of urea, H. pylori grows maximally at pH 7.4. However, in the presence of physiologicurea concentrations in unbuffered medium, this gastric pathogen requires a pH of 3.5 orless for growth and survival, to avoid elevation of medium pH to lethal levels (Clyne, M.etal, Infect Immun 63, 1669-73, 1995, Meyer-Rosberg, K et al, Gastroenterology 111, 886-900,1996). Although controversial, the pH at the gastric surface, the site of H. pylori colonization,is most likely ~pH 3.0 for most of the 24 hr period, as determined by pH electrodes,fluorescent dye probes and In Vivo transcriptomics. Traditionally, studies of the H. pylori

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acid stress response have been performed by growing the organism at neutral pH followedby exposure to low pH. However, this is the reverse of the In Vivo condition, where theorganism is exposed to low pH with excursions to higher pHs during the digestive phaseafter food intake. Aims: To determine the effect of mimicking the In Vivo pH excursions ofhigh to low acidity on urease activity, RNA stability, and H. pylori morphology. Methods:Urease activity and RNA stability of ureA and ureB transcripts were determined in H. pylorigrown for 18 hours at pH 4.5, then exposed to pH 7.4. Urease activity was determinedby release of 14CO2. RNA stability was determined by Northern analysis and capillaryelectrophoresis. The effect of the pH change on H. pylori morphology was determined bylight microscopy. Results: After growth at pH 4.5 and exposure to neutral pH, ureaseactivity progressively declined over 180 minutes. H. pylori remaining at pH 4.5 for the sametime course maintained urease activity at stable levels. Likewise, the stability of the ureA/ureB RNA transcript and 16s and 23s RNA rapidly declined after exposure to neutral pH.H. pylori rapidly converted from a bacillary to a coccoid morphology with exposure toneutral pH after growth at acidic pH. Conclusion: In its natural environment, the gastricepithelial surface, H. pylori is exposed to high acidity and urea. Under these conditions theorganism thrives. Excursions of intragastric pH from high acidity to relatively more neutralpH leads to loss of urease activity and RNA stability and likely death and/or coccoidconversion. Although H. pylori is bioenergetically classified as a neutralophile, given itsrequirement for an acidic niche in the stomach, it more uniquely should be thought of asa “Gastrophile”. These results also suggest that if gastric pH is maintained at neutrality,eradication of this pathogen should occur, barring coccoid to bacillary conversion In Vivo.

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Gastrokine Gene Expression During H. pylori Infection in MiceNoura Choudhury, Sicheng Wen, Victoria E. Ruiz, Songhua Zhang, Jason T. Machan,Steven F. Moss

Background: Gastric cancer is strongly associated with chronic Helicobacter pylori infectionbut the mechanisms linking infection to carcinogenesis in the stomach are poorly defined.The gastrokines comprise a family of largely homologous proteins expressed exclusively inmucous-secreting gastric epithelial cells. Evidence exists for functional and physical interac-tions between gastrokine family members and some trefoil family peptides, though theirfunction remains lagely unknown. Gastrokine 1 and 2 are down-regulated in patients withH. pylori infection and in gastric cancer, indicating potential tumor suppressive functions.Gastrokine 3 is not expressed in humans, but in mice it has been reported to be over-expressed during gastric atrophy. Aim: To use a mouse model to investigate the effects ofH. pylori infection over time on gastrokine gene family expression. Methods: Wild-typeC57/B6 mice were gavaged with 109 CFUs of the mouse-adapted H. pylori SS1 strain andgroups of five to ten mice were sacrificed at 2, 4, or 8 months post-infection. Control miceof similar ages and genotypes were included, for a total of 64 mice in the study. H. pyloriinfection status was determined by serology and confirmed by histology. GKN1,GKN2 andGKN3 expression in stomach was detected using real-time RT-PCR, with β-actin as thehousekeeping gene. Results: The ratio of the expression of GKN1 and GKN2 in infectedto uninfected mice decreased as length of infection increased, while the ratio of GKN3expression in infected to uninfected mice reached a maximum at 4 months before declining.None of these trends reached statistical significance. Infection for 2 months resulted inincreased expression of GKN1 and GKN2 compared to control mice (p< 0.05), but by 8months the infected mice had decreased expression of both GKN1 and GKN2 compared tocontrols (p< 0.05). At 4 months after infection, levels of GKN1 and GKN2 expressionwere not different between infected and uninfected mice. GKN3 levels were slightly (non-significantly) higher in infected mice for all time points (see figure). Conclusion: Comparedto the data reported in patients with established chronic H. pylori infection, mRNA expressionof the gastrokine gene family in mice is little changed over the course of 6 months infectionwith H. pylori SS1 strain. Since SS1 lacks a functional cag pathogenicity island, it remainspossible that changes in gastrokine gene expression are cag-dependent and/or that evenmore prolonged infection is necessary to mimic the changes in GKN1 and GKN2 observedin humans.

Gastrokine expression in mice; * = p<0.05

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