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Cag PAI-dependent gene expression in human cells induced by Helicobacter pylori strains in Japan
parameters. METHODS, Twenty-live H.pylori-inl~ective patients were enrolled. At an initial endoscopic examination, two each biotic tissue samples were obtained from the antrum and great curvature of the corpus. One of them was used tbr the ELISAq'neasurement of tissue contents of HGF. The other tissue samples were processed for histological assessment of IM and immunohistochemistry of Ki67. All patients were treated by onc-week course of triple therapy, One year after the snccessful therapy, biochemical and histological assessments were repeated. RESULTS. In all patients enrolled, no IM was detected in the corpus mucosa while IM was observed in 10 cases (40%) in the antrum. Thia prevalence ratio was not changed after the eradication, indicating that 1M did not vanish in spite of successful therapy. Slight reduction of mucosal HGF contents (pg/mg) was demonstrated (antrum: 102 +_ 15.3 to 70.6 -+ 20.4, corpus: 77,6 +- 28.2 to 60.1 _+ 13.8). However, the level of antra[ HGF significantly decreased in patients with 1M (115 +- 35.3 to 37.2 +- 12.8, pC0.05), not in ilmse without 1M (92.3 _+ 12.3 to 92.9 _+ 32.3). in patients without 1M, Ki67 labeling index in the antrum decreased one year after H.pylon-eradication, while it was unchanged in patients with IM. CONCLUSIONS. Cure of H.pylori-infection affords an influence on mucosal HGF content and its decline is significant in the gastric mncosa with IM compared with that without 1M. However, the proliferation of epithelial cell decreased in the mucosa without IM more than with IM after H.pylofi-eradication. These evidences suggest that the efficacy of H.pylori-emdication therapy for the prevention of gastric cancer may he iniluenced by the presence of intestinal metaplasia.
pylon on TGF-c~ release, we next examined CM from MKN-28 cells cocuhured with H. pylorL In cot~,tnlst, TGF-r was hmnd in the CM from cocuhure cells in the absence of PMA. Furthermore, TGFqa was also found in CM obtained from gastnc biopsy speciniens without PMA treatment. TGF-~* levels in the CM were correlated with scm~s {or activity and inflammation ot gastrius and the presence ot H. pylori. Conclusion: H pylon, but not IU113, might induce ectodomain shedding of TGF-~x in gastric epithelial cells.
W880 Long-standing Gastric Mucosal Barrier Dysfunction in Mongolian Gerbils with Helicobacter pylori-induccd Gastritis Yi-Qian Sun, Johan D. Soderholnl, Fredrik Petersson, Kurt Botch Background 6;r aims: kielicobacter Ey/ori infection causes chronic gastritis and may lead to peptic ulcer and gastric adenocarcinoma. A long-standing impaired function of the gastric mncosal barrier could be one pathogeneuc factor behind some of these associations. Our aim was to investigate the gastric mucosal barrier fnnction in Mongolian gerbils with ki. pylori-indnced gastritis. Methods: Stripped mucosal tissues from tire antrum and corpus of the stomach in H. pyloti-infected Mongolian gerbils [four weeks (short-term gastritis n = 10) and 70 weeks (long-standing gastritis n = 6) postmfection, respectively] and shamioocnlated controls (short-term controls n = 10, long-term controls n = 4) were mounted in Ussing chambers. The viability of the mounted tissues was monitored by transepithelial potential (PD,). The apparent t>ermeability coefficients (Papp) of 5~Cr-labeled ethylenediaminetetraacetic acid (5~Cr-EDTA) and horseradish pemxidase (kiRP) flux were used as indicators of gastric mucosal barrier thnction. Results: In the antrum, both short-term [2.34 (194~ 2.87) E-6 vs 160 (1.18-1.88) E-6 cm/s, p < 0.001] and long-standing [2.23 (1.86-2.64) E-6 vs 115 (0,78-130) E-6 cm/s, p < 00011 gastritis were associated with increased permeability to ' Cr-EDTA compared with controls. [n long-standing gastritis of' the antrum there was also an increased kiRP flux compared with controls [49.37 (2.30-68.54) vs 0 6 3 (0 55-1.05) pmobql/cm 2, p < 0.051 In the corpus, permeabihty to "~Cr-EDTA was only increased in long-standing gastritis as compared with controls [2.84 (1.86-3.42) E-6 vs 1.39 (1.26-193) E-6 cm/s, p = 0 0 1 ] The gastric mncosal permeability" to 5~Cr-EDTA was correlated to the degree of mncosal inflammation (lnononuclear cells infiltration) (Rho = 0.57, p < 0.0001) and inllammatory activity (nentrophils intihration) (P,ho = 0.55, p < 0.0001), Conclusions: H. Eylor/-induced gastritis in Mongolian gerbils was associated with a long-standing gastric mucosal barner dysfuntion to both small molecules and macromolecues. 1"he barrier defect extended from the antrnm into the corpus over time. This may contribute to perpetuation of chromc inflammation as well as to H. pylori-accociated carcinogenesis,
W883 ENHANCED PLASMA GHRELIN LEVEL REFLECTS GHRELIN SECRETION FROM THE INFLAMED STOMACH OF H.PYLORI-INFECTEDMONGOLIAN GERBILS Hidekazu Suzuki, Tatsuhiro Masaoka, Hiroshi Hosoda, Takayuki Ota, Yuriko Minegishi, Kenji Kangawa, Hiromasa Ishii Ghrelin, a novel growth-hormone-rekasing peptide, was isolated from rat and human stomach and demonsu~ted to localize mainly in gastric endncrine cells. Although ghrelin accelerates food intake and gastrointestinal motility', the regulation of gastric ghrelin secretion in H.pylori inl;ection remains unknown. The present study was designed to investigate plasma and gastric ghrelin levels in H.pylori-coloinzed Mongohan gerbils, a suitable model for H.pylo*{ infection. Methods, Male Mongolian gerbils (MGS/Sea, 5 weeks) were orally" inoculated with ki.pylo~(ATCC43504, 10sXCFUs/animal) (klp group) and other cohorts were inoculated with cuhure media (Cont.). Seventeen and 23 wee~ later, gerbils were examined after overnight fasting. To examine mRNA expression of preproghrelin in gerbils, cDNA encoding the gerbil counterpart of prepmghrelin and GAPDki was isolated and sequenced (460 bp, 117 amino acids; 83% identity with human, GenBank:AF442491) and the real-time RTPCR system for preproghrelin mRNA was established. Ghrelin levels in plasma and in the stomach were measured by radioimmnnoassay. Immunohistochemistry of ghrelin in the stomach was also performed. Gastric mucosal inflamnration was evaluated by myeloperoxidase (MPO) activity. Results. In the Hp group, although plasma ghrelin level (hnol/ml) was siguificantly increased as compared to Cont. (17 weeks; Cont. 535_+ 59, Hp 886-+ 61, 23 weeks; Cont. 600+_65, Hp 799+_81, pC0.01), the mRNA expression of preproghrelin (/ GAPDH mRNA, pC0.05), total ghrehn levels in the stomach (fmol/mg; 23 weeks; Cont. 3735 +_293 vs Hp 1741 +_212, pC0.001) and the number of ghrelimimmunoreactive cells (%) were signitlcanily decreased. In ihe kip group, however, since stomach weight was siguificantly inereased, the ghrdin level per stomach increased, showing a positive correlation vvlth the plasma ghrelin level (r=0.63, pC0.05). Gastric MPO acti~qty was correlated well with the plasma ghrelin level (pC0.01). Conclusion. In H.pyIori-infected gerbils, while preproghrelin mRNA expression and ghrelin-immunoreactive cell number in the stomach decleased significantly as inflanImatoD' ceils and hyperplastic epithelial cells increased, the level of ghrelin secretion from one stomach was rather increased due to a significant increase in stomach weight. Since enhanced plasma ghrelin level could reflect total ghrelin secretion from the inflamed stomach, it is a possible candidate for a clinical marker of ki.pyloriassociated gastritis.
W88 l Cag PAbDependent Gene Expression in Human Ceils Induced by Helicobacter pylori Strains in Japan Wataru Sbibata Yoshilriro Hirata, Yuzo Mitsuno, Ayako Yanai, Shin Maeda, Motoynki Otsuka, Yujin Hoshida, Hamhiko Yoshida, Takao Kawabe, Masao Omaha
Background & Aims: Helic&act*'r pyIori induces various responses in host cells related to intlamma~tion, apoptosis, and cell proliferation although their interaction is not well kno'am. We sequemially analyzed rug-dependent and independent gene expression induced by H, Dqor/by using high-density cDNA microarray. Methods: Gastric cancer cells (AGS) were cocuhmvd with type I H. pyEri (TN2) or its isogenic mutant TN2-AcagE mRNA expression was analyzed with cDNA microarray containing 4600 genes Changes in expression with a tactor of 5 or greater in either direction were considered significant. Result: On co
W884 Cross Talk Between EGFR and NF-KB Altered by H. Pylori in Human Gastric Epithelial Cells , Hassan Ashktorab, Alfred Johnson, Mohammad Daremipouran, Awana Ferguson, Bimam Kifle, Duane T Smoot Helicobacter pylori (HP) mf~:ction of gastric mucosa is strongly associated with gastritis and peptic ulcer disease. However, the mechanisms of the ulcemgenic action of HP and/or the interference of liP with uker healing are unknown. Through binding to its receptor, epidermal growth tactor accelerates cells migration and triggers epithelial cell s~gnahng which are both important for the healing of gastroduodenal ulcers. In addition, both NF-kB and epidermal growth factor receptor (EGFR) are modulator oI reactive oxygen species (ROS) in response to HP.In tfus study, we investigated the efl~ects of H. pylori on EGFR-, NF-kB-mediated signal transdnction pathways in a gastric carcinoma cell lines (AGS, AGS-E6, and AGS-Neo), Cells wei~ or were not transiently transfected with and without, EGFR-Lnciferase (a reporter vector containing the entire promoter region of EGFR), IK-BSR3236, backbone vector pCDNA The number of cell death was examined with TUNEL assay. Exposure of gastnc cells to HP resulted in increased activity and phosphorylation of the EGFR, increased ERK1/ 2 phosphorylation, and increased ROS levels, AGS cells transferred with EGFR showed an increase in EGFR promoter acUvity (~ 2 tbld pC0.05) through 3 hours after HP treatment. Inhibition of NF-kB in the AGS calls by forced expression of IKB-SR3236 increased EGFR activity and ROS through 3 h; indicating role of NF-kB protection, The cell death was increased in AGS cells in presence of NF&B inhibitor (IK-B4R32-36), In contrast, AGS-E6 cells (p53 null) showed less activity for EGFR confirming the role of p53 in the cross talk in ROS compare to AGS~Neo ceils (functional p53), in summary, exposure to HP induce EGFR activity that alters NF-kB signal transduction pathways However, EGFR activity is also increased in gastric epithelial cell lines with NF&B inhibited. This indicates that cells deficient in p53 or lack of NF-kB activation may respond differently to the cell death process
Numberof genes tnduced by TN2 More than 54old Less than 0.2-fold Total
At 1,5 hrs (%,~__ N 3 hrs (%) 64(1,8) 1t4(3,2) 25(0,7) 36(1,0) 89{Z5) 150(4,2)
C~-depandent ~mes at 3 hrs(%) 91/114(79,8) 35136(97.2) 126/150(84)
W882 H,PylorbEradication Reduces Tissue Content of Hepatocyte Growth Factor (HGF) in The Antral Mucosa with Intestinal Metaplasia Masa~aki Suznki, Hidekazu Suzuki, Tatsuhiro Masaoka Shin Tanaka, Kouichi Suzuki, kiiromasa Ishfi Hehcobacter pylori-associated mucosal inflammation has been thought to provoke intestinal metaplasia (IM) which is a pre-malignant lesion of human stomach, Hepatocyte growth factor (HGF) is mainly produced from interstitial cells, and plays a cntical role in the development ot gastric cancer by accelerating cell proliferation of surface epithelium. However, the intluence of H.pylori-infection status on the prevalence of IM, epithelial cell prolilerauon and mncosal HGF content remains obscure. The aim of this clinical study is to determine the long-term effect of kiD, ion-eradication therapy on these carcinogenetic
A-587
AGA Abstracts
pylon on TGF-c~ release, we next examined CM from MKN-28 cells cocuhured with H. pylorL In cot~,tnlst, TGF-r was hmnd in the CM from cocuhure cells in the absence of PMA. Furthermore, TGFqa was also found in CM obtained from gastnc biopsy speciniens without PMA treatment. TGF-~* levels in the CM were correlated with scm~s {or activity and inflammation ot gastrius and the presence ot H. pylori. Conclusion: H pylon, but not IU113, might induce ectodomain shedding of TGF-~x in gastric epithelial cells.
W880 Long-standing Gastric Mucosal Barrier Dysfunction in Mongolian Gerbils with Helicobacter pylori-induccd Gastritis Yi-Qian Sun, Johan D. Soderholnl, Fredrik Petersson, Kurt Botch Background 6;r aims: kielicobacter Ey/ori infection causes chronic gastritis and may lead to peptic ulcer and gastric adenocarcinoma. A long-standing impaired function of the gastric mncosal barrier could be one pathogeneuc factor behind some of these associations. Our aim was to investigate the gastric mucosal barrier fnnction in Mongolian gerbils with ki. pylori-indnced gastritis. Methods: Stripped mucosal tissues from tire antrum and corpus of the stomach in H. pyloti-infected Mongolian gerbils [four weeks (short-term gastritis n = 10) and 70 weeks (long-standing gastritis n = 6) postmfection, respectively] and shamioocnlated controls (short-term controls n = 10, long-term controls n = 4) were mounted in Ussing chambers. The viability of the mounted tissues was monitored by transepithelial potential (PD,). The apparent t>ermeability coefficients (Papp) of 5~Cr-labeled ethylenediaminetetraacetic acid (5~Cr-EDTA) and horseradish pemxidase (kiRP) flux were used as indicators of gastric mucosal barrier thnction. Results: In the antrum, both short-term [2.34 (194~ 2.87) E-6 vs 160 (1.18-1.88) E-6 cm/s, p < 0.001] and long-standing [2.23 (1.86-2.64) E-6 vs 115 (0,78-130) E-6 cm/s, p < 00011 gastritis were associated with increased permeability to ' Cr-EDTA compared with controls. [n long-standing gastritis of' the antrum there was also an increased kiRP flux compared with controls [49.37 (2.30-68.54) vs 0 6 3 (0 55-1.05) pmobql/cm 2, p < 0.051 In the corpus, permeabihty to "~Cr-EDTA was only increased in long-standing gastritis as compared with controls [2.84 (1.86-3.42) E-6 vs 1.39 (1.26-193) E-6 cm/s, p = 0 0 1 ] The gastric mncosal permeability" to 5~Cr-EDTA was correlated to the degree of mncosal inflammation (lnononuclear cells infiltration) (Rho = 0.57, p < 0.0001) and inllammatory activity (nentrophils intihration) (P,ho = 0.55, p < 0.0001), Conclusions: H. Eylor/-induced gastritis in Mongolian gerbils was associated with a long-standing gastric mucosal barner dysfuntion to both small molecules and macromolecues. 1"he barrier defect extended from the antrnm into the corpus over time. This may contribute to perpetuation of chromc inflammation as well as to H. pylori-accociated carcinogenesis,
W883 ENHANCED PLASMA GHRELIN LEVEL REFLECTS GHRELIN SECRETION FROM THE INFLAMED STOMACH OF H.PYLORI-INFECTEDMONGOLIAN GERBILS Hidekazu Suzuki, Tatsuhiro Masaoka, Hiroshi Hosoda, Takayuki Ota, Yuriko Minegishi, Kenji Kangawa, Hiromasa Ishii Ghrelin, a novel growth-hormone-rekasing peptide, was isolated from rat and human stomach and demonsu~ted to localize mainly in gastric endncrine cells. Although ghrelin accelerates food intake and gastrointestinal motility', the regulation of gastric ghrelin secretion in H.pylori inl;ection remains unknown. The present study was designed to investigate plasma and gastric ghrelin levels in H.pylori-coloinzed Mongohan gerbils, a suitable model for H.pylo*{ infection. Methods, Male Mongolian gerbils (MGS/Sea, 5 weeks) were orally" inoculated with ki.pylo~(ATCC43504, 10sXCFUs/animal) (klp group) and other cohorts were inoculated with cuhure media (Cont.). Seventeen and 23 wee~ later, gerbils were examined after overnight fasting. To examine mRNA expression of preproghrelin in gerbils, cDNA encoding the gerbil counterpart of prepmghrelin and GAPDki was isolated and sequenced (460 bp, 117 amino acids; 83% identity with human, GenBank:AF442491) and the real-time RTPCR system for preproghrelin mRNA was established. Ghrelin levels in plasma and in the stomach were measured by radioimmnnoassay. Immunohistochemistry of ghrelin in the stomach was also performed. Gastric mucosal inflamnration was evaluated by myeloperoxidase (MPO) activity. Results. In the Hp group, although plasma ghrelin level (hnol/ml) was siguificantly increased as compared to Cont. (17 weeks; Cont. 535_+ 59, Hp 886-+ 61, 23 weeks; Cont. 600+_65, Hp 799+_81, pC0.01), the mRNA expression of preproghrelin (/ GAPDH mRNA, pC0.05), total ghrehn levels in the stomach (fmol/mg; 23 weeks; Cont. 3735 +_293 vs Hp 1741 +_212, pC0.001) and the number of ghrelimimmunoreactive cells (%) were signitlcanily decreased. In ihe kip group, however, since stomach weight was siguificantly inereased, the ghrdin level per stomach increased, showing a positive correlation vvlth the plasma ghrelin level (r=0.63, pC0.05). Gastric MPO acti~qty was correlated well with the plasma ghrelin level (pC0.01). Conclusion. In H.pyIori-infected gerbils, while preproghrelin mRNA expression and ghrelin-immunoreactive cell number in the stomach decleased significantly as inflanImatoD' ceils and hyperplastic epithelial cells increased, the level of ghrelin secretion from one stomach was rather increased due to a significant increase in stomach weight. Since enhanced plasma ghrelin level could reflect total ghrelin secretion from the inflamed stomach, it is a possible candidate for a clinical marker of ki.pyloriassociated gastritis.
W88 l Cag PAbDependent Gene Expression in Human Ceils Induced by Helicobacter pylori Strains in Japan Wataru Sbibata Yoshilriro Hirata, Yuzo Mitsuno, Ayako Yanai, Shin Maeda, Motoynki Otsuka, Yujin Hoshida, Hamhiko Yoshida, Takao Kawabe, Masao Omaha
Background & Aims: Helic&act*'r pyIori induces various responses in host cells related to intlamma~tion, apoptosis, and cell proliferation although their interaction is not well kno'am. We sequemially analyzed rug-dependent and independent gene expression induced by H, Dqor/by using high-density cDNA microarray. Methods: Gastric cancer cells (AGS) were cocuhmvd with type I H. pyEri (TN2) or its isogenic mutant TN2-AcagE mRNA expression was analyzed with cDNA microarray containing 4600 genes Changes in expression with a tactor of 5 or greater in either direction were considered significant. Result: On co
W884 Cross Talk Between EGFR and NF-KB Altered by H. Pylori in Human Gastric Epithelial Cells , Hassan Ashktorab, Alfred Johnson, Mohammad Daremipouran, Awana Ferguson, Bimam Kifle, Duane T Smoot Helicobacter pylori (HP) mf~:ction of gastric mucosa is strongly associated with gastritis and peptic ulcer disease. However, the mechanisms of the ulcemgenic action of HP and/or the interference of liP with uker healing are unknown. Through binding to its receptor, epidermal growth tactor accelerates cells migration and triggers epithelial cell s~gnahng which are both important for the healing of gastroduodenal ulcers. In addition, both NF-kB and epidermal growth factor receptor (EGFR) are modulator oI reactive oxygen species (ROS) in response to HP.In tfus study, we investigated the efl~ects of H. pylori on EGFR-, NF-kB-mediated signal transdnction pathways in a gastric carcinoma cell lines (AGS, AGS-E6, and AGS-Neo), Cells wei~ or were not transiently transfected with and without, EGFR-Lnciferase (a reporter vector containing the entire promoter region of EGFR), IK-BSR3236, backbone vector pCDNA The number of cell death was examined with TUNEL assay. Exposure of gastnc cells to HP resulted in increased activity and phosphorylation of the EGFR, increased ERK1/ 2 phosphorylation, and increased ROS levels, AGS cells transferred with EGFR showed an increase in EGFR promoter acUvity (~ 2 tbld pC0.05) through 3 hours after HP treatment. Inhibition of NF-kB in the AGS calls by forced expression of IKB-SR3236 increased EGFR activity and ROS through 3 h; indicating role of NF-kB protection, The cell death was increased in AGS cells in presence of NF&B inhibitor (IK-B4R32-36), In contrast, AGS-E6 cells (p53 null) showed less activity for EGFR confirming the role of p53 in the cross talk in ROS compare to AGS~Neo ceils (functional p53), in summary, exposure to HP induce EGFR activity that alters NF-kB signal transduction pathways However, EGFR activity is also increased in gastric epithelial cell lines with NF&B inhibited. This indicates that cells deficient in p53 or lack of NF-kB activation may respond differently to the cell death process
Numberof genes tnduced by TN2 More than 54old Less than 0.2-fold Total
At 1,5 hrs (%,~__ N 3 hrs (%) 64(1,8) 1t4(3,2) 25(0,7) 36(1,0) 89{Z5) 150(4,2)
C~-depandent ~mes at 3 hrs(%) 91/114(79,8) 35136(97.2) 126/150(84)
W882 H,PylorbEradication Reduces Tissue Content of Hepatocyte Growth Factor (HGF) in The Antral Mucosa with Intestinal Metaplasia Masa~aki Suznki, Hidekazu Suzuki, Tatsuhiro Masaoka Shin Tanaka, Kouichi Suzuki, kiiromasa Ishfi Hehcobacter pylori-associated mucosal inflammation has been thought to provoke intestinal metaplasia (IM) which is a pre-malignant lesion of human stomach, Hepatocyte growth factor (HGF) is mainly produced from interstitial cells, and plays a cntical role in the development ot gastric cancer by accelerating cell proliferation of surface epithelium. However, the intluence of H.pylori-infection status on the prevalence of IM, epithelial cell prolilerauon and mncosal HGF content remains obscure. The aim of this clinical study is to determine the long-term effect of kiD, ion-eradication therapy on these carcinogenetic
A-587
AGA Abstracts