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Monitoring gastric epithelial gene expression changes induced by Helicobacter pylori
AGAA739
April 2000
4001
4003
HIGH OXYGEN FREE RADICALS GENERATION IN GASTRIC MUCOSA INFECTED BY H. PYLORI CYTOTOXIC STRAINS. Silvio Danese, Marcello Candelli, Filippo Cremonini, Alessandro Armuzzi, Veronica Ojetti, Alfredo Pastorelli, Giancarlo Zannoni, Giovanni Camardese, Cristiano Padalino, Giovanni B. Gasbarrini, Pasquale De Sole, Antonio Gasbarrini, Catholic Univ, Rome, Italy. BACKGROUND: Reactive oxygen metabolites (ROMs) production by phagocytes is important for host-defence against microrganisms. Helicobacterpylori (HP) is the main aetiological agent of chronic active gastritis. HP-CagA positive strains induce more extensive gastric mucosal inflammation. AIMS: to assess whether HP infection and CagA status could correlate to neutrophil count and to gastric mucosa ROMs generation. METHODS: 60 dyspeptic patients (pts) (mean age 47::':16 yrs), consecutively submitted to gastroscopy with multiple biopsies, were enrolled. Histology was used to assess HP infection. CagA status was assessed through serology. Histological assessment of inflammation was graded from 0 (absence) to 3 (severe) and neutrophils were individually counted. Gastric biopsies were taken for ROMs assay by luminol enhanced-chemiluminescence (CLS). Each specimen was transferred with I ml of 100 Mmol luminol to a luminometer apparatus. Sample photons emission was continuosly measured up to 45 min. RESULTS: 40 out of 60 pts were infected by HP. 24 out of 40 infected pts were CagA positive. As regards histological grading, a statistically significant difference was found in HP-CagA positive compared to HP-CagA negative and HP negative (2,5::':0,5vs 1,8::':0,2vs 0,5::':0,5; p<0.05, respectively). Neutrophils count was also higher in HP CagA positive when compared to other groups (6,5::':0,5 vs 4,5::':0,5 vs 1,2::':0,2; p<0.05, respectively) Finally, CLS emission was significantly greater in HP-CagApositive pts when compared to other groups (51x104 vs 18xlO4 vs IOxlO ; p<0.05,respectively). A significant correlation between neutrophils count and ROMs production was found only in biopsies from HP-CagA positive pts (r=0.56, p=O.004). CONCLUSIONS: HP-CagA positive strains are associated with greater neutrophils counts and higher ROMs generation in gastric mucosa. Since ROMs production is associated to DNA oxydative damage, a long term stimulation by CagA positive strains might be relevant in the pathogenesis of gastric malignancies.
PERSISTENCE OF H. PYLORI: ROLE OF THE IMMUNOSUPPRESSIVE CYTOKINE IL·IO IN RHESUS MONKEYS. Andre Dubois, Anthony Welch, Thomas Wigginton, Leslie Jones, Robert Kampen, Allan Kirk, Usuhs, Bethesda, MD; Diagnon, Inc, Rockville, MD; Nrnrc, Bethesda, MD. Helieobaeterpylori is extraordinary among bacteria in its ability to persist in the stomach of over 50% of humans. Similarly, H. pylori persistently colonizes the stomach of up to 90% of socially-housed rhesus monkeys. Only a fraction of humans and rhesus monkeys appear to resist natural H. pylori infection, developing only transient infection following accidental or experimental inoculation (N Engl J Med 341:456-458; Infect Immun 1996;64:2885-91). To test the hypothesis that this resistance is due to specific differences in T cell- and/or macrophage-mediated local immune responses, we determined the effect of H. pylori inoculation on cytokine mRNA expression in gastric mucosal biopsies. 109 CFU of virulent H. pylori strains isolated from humans were injected into the stomach of 6 rhesus monkeys, and pinch biopsies were obtained at endoscopies performed before, and at 6, 14, 64 and 310 days after inoculation. H. pylori status was determined by culture, histology and PCR. mRNA expression for IL-lf3, IL-4, IL-8, IL-IO, TNF-a, and IFN--y was determined and normalized to f3-actin. By day 310, only 2 animals still were persistently infected (PER) whereas the 4 other ones were H. pylori-negative. Initially, gastritis was observed in all animals, but it persisted only in PER animals. mRNA expression of the immunosuppressive cytokine IL-IO increased at day 6 only in PER animals, decreased at day 14 and 60, and increased again at day 310 (Figure). In addition, IL-If3, IL-8, and IFN--yincreased earlier in PER animals, and expression of IL-4 was never detected in any animal. This observation suggests that persistence of H. pylori depends on IL-IO expression and on the subsequent suppression of yet undefined local immune responses.
4002 ROLE OF REACTIVE OXYGEN SPECIES IN ENHANCED APOPTOSIS OF GASTRIC EPITHELIAL CELLS ASSOCIATED WITH HEUCOBACTER PYLORI INFECTION. Song-Ze Ding, Yutaka Minohara, Bernadette Dirden-Kramer, Istvan Boldogh, Xue-Jun Fan, Jide Wang, Victor E. Reyes, Peter B. Ernst, Sheila E. Crowe, Univ of Texas Med Branch, Galveston, TX. Background: Helieobaeterpylori infection is associated with altered gastric epithelial cell growth that may contribute to the development of peptic ulcer disease or carcinoma. Since recent studies suggest that generation of reactive oxygen species (ROS) is increased in H. pylori infected gastric epithelial cells and as ROS have been shown to be involved in apoptosis mediated by multiple mechanisms in other cell types, we evaluated the role oxidative stress plays in H. pylori-induced apoptosis of gastric epithelial cells. Methods: N87, Kato III and AGS gastric epithelial cells were exposed to various strains of H. pylori, inflarnrnatory cytokines (lFN--y, TNF-a, IL-I (3) and hydrogen peroxide (H2 0 2 ) in the absence or presence of antioxidant agents. Increased intracellular ROS were detected using a redox-sensitive fluorescent dye, a cytochrome c reduction assay and measurements of glutathione. Apoptosis was evaluated by detecting DNA fragmentation and caspase activation. Epithelial cells isolated from human gastric mucosal biopsy specimens were used in some experiments. Results: Infection with H. pylori or exposure of cultured gastric epithelial cells to H2 0 2 resulted in increased DNA degradation, caspase activation and a dose-dependent increase in ROS generation that was enhanced by pretreatment with inflammatory cytokines. Different methods of ROS detection and inhibition of ROS generation by various pharmacologic agents indicated accumulation of superoxide, hydrogen peroxide, hydroxyl radical, and peroxynitrite after H. pylori infection. Basal levels of ROS were greater in epithelial cells isolated from H. pylori infected human subjects compared to cells from uninfected individuals. H. pylori strains bearing the eag pathogenicity island induced a greater and earlier accumulation of intracellular oxygen metabolites than their isogenic mutants. Antioxidants inhibited both ROS generation and apoptosis by H. pylori. The pattern of activation of caspase 8 was similar after stimulation with bacteria or H2 0 2 • Conclusion: Together, these results indicate that both bacterial factors and the host inflammatory response serve as sources of oxidative stress to the gastric epithelium during H. pylori infection. Our data suggest that ROS generation may playa role in the enhanced programmed cell death that is associated with H. pylori infection.
z
91 00 Oil 200 ~ ~
-+- PERSISTENT
=r:::Q,J
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- 0-
RESISTANT
~1100 ~Ili-I zo ~~
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o J.-~~ . o
100
--- ------ I 200
300
TIME (days from inoculation) 4004 MONITORING GASTRIC EPITHELIAL GENE EXPRESSION CHANGES INDUCED BY HEUCOBACTER PYWRI. F. Falciani, J. M. Cox, C. L. Clayton, P. A. Robinson, S. Blakemore, D. M. Wallace, M. K. Trower, P. Sanseau, J. E. Crabtree, Glaxo Wellcome, Stevenage, United Kingdom; St James Hosp, Leeds, United Kingdom. To investigate the molecular response of gastric epithelial cells to H. pylori we have used high density cDNA array technology to identify differentially regulated genes. The aim of this study was to use statistical analysis to examine temporal changes in gene expression patterns induced by H. pylori. mRNA was extracted from Kato-3 cells 45 rnins, 3 and 24 hours following infection. Radiolabelled first strand cDNA was hybridised to cDNA arrays derived from a collection of 46,302 non-redundant LM.A.G.E. clones. Hybridisation signals were acquired using Glaxo Wellcome developed software. The large gene expression matrix generated was studied using cluster and principal component analysis. 4,500 genes mapped into discrete clusters suggesting that they belong to defined pathways of gene expression. Infected epithelial cells respond with two coordinated waves of transient early gene activation, a wave of down regulated genes and a small cluster of upregulated genes at 24 hours. Using cluster analysis and Pearson correlation coefficient analysis, two ESTs with very high correlation coefficients with a down regulated elastase inhibitor were identified, suggesting they may be involved in functionally correlated pathways. These results demonstrate the use of statistical analysis monitoring differential gene expression. This approach has applications for identifying bacterial induced epithelial response genes of relevance to acute and chronic enteric infections. This research was funded by Yorkshire Cancer Research and Glaxo Wellcome.
April 2000
4001
4003
HIGH OXYGEN FREE RADICALS GENERATION IN GASTRIC MUCOSA INFECTED BY H. PYLORI CYTOTOXIC STRAINS. Silvio Danese, Marcello Candelli, Filippo Cremonini, Alessandro Armuzzi, Veronica Ojetti, Alfredo Pastorelli, Giancarlo Zannoni, Giovanni Camardese, Cristiano Padalino, Giovanni B. Gasbarrini, Pasquale De Sole, Antonio Gasbarrini, Catholic Univ, Rome, Italy. BACKGROUND: Reactive oxygen metabolites (ROMs) production by phagocytes is important for host-defence against microrganisms. Helicobacterpylori (HP) is the main aetiological agent of chronic active gastritis. HP-CagA positive strains induce more extensive gastric mucosal inflammation. AIMS: to assess whether HP infection and CagA status could correlate to neutrophil count and to gastric mucosa ROMs generation. METHODS: 60 dyspeptic patients (pts) (mean age 47::':16 yrs), consecutively submitted to gastroscopy with multiple biopsies, were enrolled. Histology was used to assess HP infection. CagA status was assessed through serology. Histological assessment of inflammation was graded from 0 (absence) to 3 (severe) and neutrophils were individually counted. Gastric biopsies were taken for ROMs assay by luminol enhanced-chemiluminescence (CLS). Each specimen was transferred with I ml of 100 Mmol luminol to a luminometer apparatus. Sample photons emission was continuosly measured up to 45 min. RESULTS: 40 out of 60 pts were infected by HP. 24 out of 40 infected pts were CagA positive. As regards histological grading, a statistically significant difference was found in HP-CagA positive compared to HP-CagA negative and HP negative (2,5::':0,5vs 1,8::':0,2vs 0,5::':0,5; p<0.05, respectively). Neutrophils count was also higher in HP CagA positive when compared to other groups (6,5::':0,5 vs 4,5::':0,5 vs 1,2::':0,2; p<0.05, respectively) Finally, CLS emission was significantly greater in HP-CagApositive pts when compared to other groups (51x104 vs 18xlO4 vs IOxlO ; p<0.05,respectively). A significant correlation between neutrophils count and ROMs production was found only in biopsies from HP-CagA positive pts (r=0.56, p=O.004). CONCLUSIONS: HP-CagA positive strains are associated with greater neutrophils counts and higher ROMs generation in gastric mucosa. Since ROMs production is associated to DNA oxydative damage, a long term stimulation by CagA positive strains might be relevant in the pathogenesis of gastric malignancies.
PERSISTENCE OF H. PYLORI: ROLE OF THE IMMUNOSUPPRESSIVE CYTOKINE IL·IO IN RHESUS MONKEYS. Andre Dubois, Anthony Welch, Thomas Wigginton, Leslie Jones, Robert Kampen, Allan Kirk, Usuhs, Bethesda, MD; Diagnon, Inc, Rockville, MD; Nrnrc, Bethesda, MD. Helieobaeterpylori is extraordinary among bacteria in its ability to persist in the stomach of over 50% of humans. Similarly, H. pylori persistently colonizes the stomach of up to 90% of socially-housed rhesus monkeys. Only a fraction of humans and rhesus monkeys appear to resist natural H. pylori infection, developing only transient infection following accidental or experimental inoculation (N Engl J Med 341:456-458; Infect Immun 1996;64:2885-91). To test the hypothesis that this resistance is due to specific differences in T cell- and/or macrophage-mediated local immune responses, we determined the effect of H. pylori inoculation on cytokine mRNA expression in gastric mucosal biopsies. 109 CFU of virulent H. pylori strains isolated from humans were injected into the stomach of 6 rhesus monkeys, and pinch biopsies were obtained at endoscopies performed before, and at 6, 14, 64 and 310 days after inoculation. H. pylori status was determined by culture, histology and PCR. mRNA expression for IL-lf3, IL-4, IL-8, IL-IO, TNF-a, and IFN--y was determined and normalized to f3-actin. By day 310, only 2 animals still were persistently infected (PER) whereas the 4 other ones were H. pylori-negative. Initially, gastritis was observed in all animals, but it persisted only in PER animals. mRNA expression of the immunosuppressive cytokine IL-IO increased at day 6 only in PER animals, decreased at day 14 and 60, and increased again at day 310 (Figure). In addition, IL-If3, IL-8, and IFN--yincreased earlier in PER animals, and expression of IL-4 was never detected in any animal. This observation suggests that persistence of H. pylori depends on IL-IO expression and on the subsequent suppression of yet undefined local immune responses.
4002 ROLE OF REACTIVE OXYGEN SPECIES IN ENHANCED APOPTOSIS OF GASTRIC EPITHELIAL CELLS ASSOCIATED WITH HEUCOBACTER PYLORI INFECTION. Song-Ze Ding, Yutaka Minohara, Bernadette Dirden-Kramer, Istvan Boldogh, Xue-Jun Fan, Jide Wang, Victor E. Reyes, Peter B. Ernst, Sheila E. Crowe, Univ of Texas Med Branch, Galveston, TX. Background: Helieobaeterpylori infection is associated with altered gastric epithelial cell growth that may contribute to the development of peptic ulcer disease or carcinoma. Since recent studies suggest that generation of reactive oxygen species (ROS) is increased in H. pylori infected gastric epithelial cells and as ROS have been shown to be involved in apoptosis mediated by multiple mechanisms in other cell types, we evaluated the role oxidative stress plays in H. pylori-induced apoptosis of gastric epithelial cells. Methods: N87, Kato III and AGS gastric epithelial cells were exposed to various strains of H. pylori, inflarnrnatory cytokines (lFN--y, TNF-a, IL-I (3) and hydrogen peroxide (H2 0 2 ) in the absence or presence of antioxidant agents. Increased intracellular ROS were detected using a redox-sensitive fluorescent dye, a cytochrome c reduction assay and measurements of glutathione. Apoptosis was evaluated by detecting DNA fragmentation and caspase activation. Epithelial cells isolated from human gastric mucosal biopsy specimens were used in some experiments. Results: Infection with H. pylori or exposure of cultured gastric epithelial cells to H2 0 2 resulted in increased DNA degradation, caspase activation and a dose-dependent increase in ROS generation that was enhanced by pretreatment with inflammatory cytokines. Different methods of ROS detection and inhibition of ROS generation by various pharmacologic agents indicated accumulation of superoxide, hydrogen peroxide, hydroxyl radical, and peroxynitrite after H. pylori infection. Basal levels of ROS were greater in epithelial cells isolated from H. pylori infected human subjects compared to cells from uninfected individuals. H. pylori strains bearing the eag pathogenicity island induced a greater and earlier accumulation of intracellular oxygen metabolites than their isogenic mutants. Antioxidants inhibited both ROS generation and apoptosis by H. pylori. The pattern of activation of caspase 8 was similar after stimulation with bacteria or H2 0 2 • Conclusion: Together, these results indicate that both bacterial factors and the host inflammatory response serve as sources of oxidative stress to the gastric epithelium during H. pylori infection. Our data suggest that ROS generation may playa role in the enhanced programmed cell death that is associated with H. pylori infection.
z
91 00 Oil 200 ~ ~
-+- PERSISTENT
=r:::Q,J
~~
- 0-
RESISTANT
~1100 ~Ili-I zo ~~
~-
o J.-~~ . o
100
--- ------ I 200
300
TIME (days from inoculation) 4004 MONITORING GASTRIC EPITHELIAL GENE EXPRESSION CHANGES INDUCED BY HEUCOBACTER PYWRI. F. Falciani, J. M. Cox, C. L. Clayton, P. A. Robinson, S. Blakemore, D. M. Wallace, M. K. Trower, P. Sanseau, J. E. Crabtree, Glaxo Wellcome, Stevenage, United Kingdom; St James Hosp, Leeds, United Kingdom. To investigate the molecular response of gastric epithelial cells to H. pylori we have used high density cDNA array technology to identify differentially regulated genes. The aim of this study was to use statistical analysis to examine temporal changes in gene expression patterns induced by H. pylori. mRNA was extracted from Kato-3 cells 45 rnins, 3 and 24 hours following infection. Radiolabelled first strand cDNA was hybridised to cDNA arrays derived from a collection of 46,302 non-redundant LM.A.G.E. clones. Hybridisation signals were acquired using Glaxo Wellcome developed software. The large gene expression matrix generated was studied using cluster and principal component analysis. 4,500 genes mapped into discrete clusters suggesting that they belong to defined pathways of gene expression. Infected epithelial cells respond with two coordinated waves of transient early gene activation, a wave of down regulated genes and a small cluster of upregulated genes at 24 hours. Using cluster analysis and Pearson correlation coefficient analysis, two ESTs with very high correlation coefficients with a down regulated elastase inhibitor were identified, suggesting they may be involved in functionally correlated pathways. These results demonstrate the use of statistical analysis monitoring differential gene expression. This approach has applications for identifying bacterial induced epithelial response genes of relevance to acute and chronic enteric infections. This research was funded by Yorkshire Cancer Research and Glaxo Wellcome.