Journal: Gut

Article Title: Helicobacter pylori potentiates epithelial:mesenchymal transition in gastric cancer: links to soluble HB-EGF, gastrin and matrix metalloproteinase-7

doi: 10.1136/gut.2009.199794

Figure Lengend Snippet: Epithelial:mesenchymal transition gene expression following co-culture of gastric epithelial cells with Helicobacter pylori pathogenic (60190) and non-pathogenic (Tx30a) strains. (a) Snail gene expression: *significantly higher gene expression level compared with untreated control cells (strain 60190 treated: AGS p<0.001, MGLVA1 p<0.05, ST16 p<0.005; strain Tx30a treated: AGS p<0.005, MGLVA1 p<0.05, ST16 p<0.02); #significantly higher gene expression level compared with Tx30a treated cells (AGS p<0.05, ST16 p<0.05). (b) Slug gene expression: *significantly higher gene expression level compared with untreated control cells (strain 60190 treated: p<0.001 in all cell lines; strain Tx30a treated: AGS p<0.001, MGLVA1 p<0.005, ST16 p<0.005); #significantly higher gene expression level compared with Tx30a treated cells (AGS p<0.04, MGLVA1 p<0.05, ST16 p<0.002). (c) Vimentin gene expression: *significantly higher gene expression level compared with untreated control cells (strain 60190 treated: AGS p<0.05, MGLVA1 p<0.001, ST16 p<0.005; strain Tx30a treated: MGLVA1 p<0.05); #significantly higher gene expression level compared with Tx30a treated cells (MGLVA1 p<0.04). (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence).

Article Snippet: H pylori strains 60190 (American Type Culture Collection (ATCC) 49503) and Tx30a (ATCC 51932) were used in this study.

Techniques: Gene Expression, Co-Culture Assay, Control

Journal: Gut

Article Title: Helicobacter pylori potentiates epithelial:mesenchymal transition in gastric cancer: links to soluble HB-EGF, gastrin and matrix metalloproteinase-7

doi: 10.1136/gut.2009.199794

Figure Lengend Snippet: Effect of co-culture of gastric epithelial cells with Helicobacter pylori strain 60190 on heparin-binding epidermal growth factor (HB-EGF) expression. (a) HB-EGF gene expression following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains; *p<0.001 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (b) Cell-associated HB-EGF expression following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains. Cells were stained for HB-EGF (red fluorescence) and nuclei were counterstained with Hoechst (blue fluorescence); (i) AGS, (ii) AGS/ H pylori 60190, (iii) AGS/ H pylori Tx30a. Magnification ×20. (c) HB-EGF protein expression level quantified by image analysis following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains; *significantly higher HB-EGF protein expression level compared with untreated control cells (p<0.03). (d) HB-EGF shedding level following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains; *significantly higher HB-EGF shedding level compared with untreated control cells (strain 60190 treated: p<0.01 in all cell lines; strain Tx30a treated: p<0.05 in AGS and ST16); #significantly higher HB-EGF shedding level compared with Tx30a treated (p<0.05 in AGS and ST16). (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence).

Article Snippet: H pylori strains 60190 (American Type Culture Collection (ATCC) 49503) and Tx30a (ATCC 51932) were used in this study.

Techniques: Co-Culture Assay, Binding Assay, Expressing, Gene Expression, Staining, Fluorescence, Control

Journal: Gut

Article Title: Helicobacter pylori potentiates epithelial:mesenchymal transition in gastric cancer: links to soluble HB-EGF, gastrin and matrix metalloproteinase-7

doi: 10.1136/gut.2009.199794

Figure Lengend Snippet: Effect of co-culture of gastric epithelial cells with Helicobacter pylori strain 60190 on matrix metalloproteinase 7 (MMP-7) expression. (a) MMP-7 gene expression following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains. *p<0.001 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (b) MMP-7 protein expression following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains. Cells were stained for MMP-7 (green fluorescence) and nuclei were counterstained with Hoechst (blue fluorescence). (i) AGS, (ii) AGS/ H pylori 60190, (iii) AGS H pylori Tx30a. Magnification ×20. (c) MMP-7 protein expression level quantified by image analysis following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains; *significantly higher MMP-7 protein expression level compared with untreated control cells (p<0.001). (d) MMP-7 and heparin-binding epidermal growth factor (HB-EGF) co-localisation: MMP-7 expression was stained by green fluorescence (i), HB-EGF by red fluorescence (ii), nuclei were stained with Hoechst blue fluorescence (iii). Merged image shows co-localisation of MMP-7 and HB-EGF (iv). Magnification ×20. (e) MMP-7 gene expression in MMP-7 siRNA treated gastric cell lines. MMP-7 siRNA significantly reduced MMP-7 mRNA expression in all three gastric cell lines tested. *p<0.001 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (f) HB-EGF gene expression in MMP-7 siRNA treated gastric cell lines exposed to H pylori 60190. MMP-7 siRNA significantly reduced HB-EGF shedding in all three gastric cell lines tested. *p<0.02, **p<0.03 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (g) HB-EGF gene expression in MMP-7 neutralising antibody treated gastric cell lines exposed to H pylori 60190. *p<0.03 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (h) HB-EGF shedding levels in gastric cell lines exposed to H pylori 60190 treated with an MMP-7 neutralising antibody. *p<0.04, **p<0.03 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (i) EMT gene expression in MMP-7 siRNA treated gastric cell lines exposed to H pylori strain 60190: Snail, p<0.01; Slug, p<0.005; vimentin, p<0.001 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence).

Article Snippet: H pylori strains 60190 (American Type Culture Collection (ATCC) 49503) and Tx30a (ATCC 51932) were used in this study.

Techniques: Co-Culture Assay, Expressing, Gene Expression, Staining, Fluorescence, Control, Binding Assay

Journal: Gut

Article Title: Helicobacter pylori potentiates epithelial:mesenchymal transition in gastric cancer: links to soluble HB-EGF, gastrin and matrix metalloproteinase-7

doi: 10.1136/gut.2009.199794

Figure Lengend Snippet: Effect of co-culture of gastric epithelial cells with Helicobacter pylori strain 60190 on gastrin expression and the effect of gastrin siRNA on matrix metalloproteinase 7 (MMP-7) and epithelial:mesenchymal transition (EMT). (a) Gastrin gene expression following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains. *p<0.001 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (b) Gastrin gene expression in gastrin siRNA treated gastric cell lines. Gastrin siRNA significantly reduced gastrin mRNA expression in all three gastric cell lines tested. *p<0.001 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (c) MMP-7 expression in gastric cells exposed to H pylori and gastrin siRNA.*p<0.02 and **p<0.05 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (d) MMP-7 protein expression in gastrin siRNA transfected gastric cell lines exposed to H pylori . Cells were stained for MMP-7 (green fluorescence) and nuclei were counterstained with Hoescht (blue fluorescence). Magnification ×20. (e) MMP-7 protein expression level quantified by image analysis in gastrin siRNA transfected gastric cell lines exposed to H pylori ; *significantly higher MMP-7 protein expression level compared with untreated control cells (p<0.001). (f) Gastrin siRNA reverses H pylori induced EMT in gastric cell lines.*p<0.01 (n=3 replicates per condition and a representative graph is shown. Error bars indicate 95% confidence).

Article Snippet: H pylori strains 60190 (American Type Culture Collection (ATCC) 49503) and Tx30a (ATCC 51932) were used in this study.

Techniques: Co-Culture Assay, Expressing, Gene Expression, Transfection, Staining, Fluorescence, Control

Journal: Infection and Immunity

Article Title: The Intermediate Region of Helicobacter pylori VacA Is a Determinant of Toxin Potency in a Jurkat T Cell Assay

doi: 10.1128/IAI.00052-12

Figure Lengend Snippet: Amino acid sequences of the VacA i-region in different H. pylori strains. The secreted p88 VacA protein (comprising 821 amino acids in H. pylori strain 60190) contains two domains, designated p33 and p55. Amino acid sequence variation in VacA proteins from different H. pylori strains is especially prominent in three regions, which are designated s-, i-, and m-regions. The i-region and a portion of the s-region are localized within the p33 domain, and the m-region is localized within the p55 domain. The lower portion of this figure shows the sequences of VacA i-regions from strain 60190 (GenBank no. U05676), Tx30a (GenBank no. U29401), and X47. The figure illustrates the sequence of the secreted VacA protein of H. pylori strain 60190 (amino acids G120 to L236) and corresponding regions of VacA in two other wild-type strains (Tx30a and X47). Clusters of amino acid polymorphisms defined as clusters A, B, and C are indicated. Asterisks (*) indicate amino acids that are identical in all three sequences. The sequence of VacA from strain 60190 is designated a prototype for the i1 region, and the sequence of VacA from strain Tx30a is designated a prototype for the i2 region. VacA from strain X47 contains a type i2 sequence in cluster C, and cluster B is chimeric.

Article Snippet: H. pylori liquid cultures were grown in brucella broth supplemented with either activated charcoal or 5% fetal bovine serum (FBS) ( 11 ). table ft1 table-wrap mode="anchored" t5 caption a7 Strain/plasmid Relevant characteristics Reference Strains 60190 Wild type (ATCC 49503); vacA s1/i1/m1 40 60190 Δ rdxA Same as 60190 except HP0954 ( rdxA ) gene deleted; metronidazole resistant This study 60190 vacA :: cat rdxA Same as 60190 Δ rdxA except cat cassette and rdxA inserted in vacA ; chloramphenicol resistant and metronidazole sensitive; expression of VacA is disrupted This study 60190 i2B Same as 60190 Δ rdxA except vacA cluster B changed to i2 This study 60190 i2C Same as 60190 Δ rdxA except vacA cluster C changed to i2 This study 60190 i2BC Same as 60190 Δ rdxA except vacA clusters B and C changed to i2 This study 60190 i1/i2C Same as 60190 Δ rdxA except vacA cluster C has 4 amino acids changed to i2 This study X47 Wild type; vacA s1/m2, chimeric i-region 32 X47 Δ rdxA Same as X47 except HP0954 ( rdxA ) gene deleted; metronidazole resistant This study X47 vacA :: cat rdxA Same as X47 Δ rdxA except cat cassette and rdxA inserted in vacA ; chloramphenicol resistant and metronidazole sensitive; expression of VacA is disrupted This study X47 i1C Same as X47 Δ rdxA except vacA cluster C changed to i1 This study Plasmids pMM672 Allows deletion of rdxA in H. pylori strains 41 pCGR1 Contains cat-rdxA cassette in StuI site; derived from pA178 plasmid This study pCGR2 Contains cat-rdxA cassette in EcoRV site from X47 vacA This study pCGR3 60910 cluster B changed from i1 to i2 by inverse PCR using primers B1F and B1R This study pCGR4 60190 cluster C changed from i1 to i2 by inverse PCR using primers C1F and C1R This study pCGR5 60190 clusters B and C changed from i1 to i2 by inverse PCR using primers C1F and C1R and pCGR3 as the template This study pCGR6 A portion of 60190 cluster C changed from i1 to i2 by inverse PCR using primers C2F and C2R This study pCGR7 X47 cluster C changed from i2 to i1 by inverse PCR using primers C3F and C3R This study p55 Expresses VacA p55 27 p33 Expresses VacA p33 31 p33 i2 Expresses p33 i2 This study Open in a separate window H. pylori strains and plasmids

Techniques: Sequencing

Journal: Infection and Immunity

Article Title: The Intermediate Region of Helicobacter pylori VacA Is a Determinant of Toxin Potency in a Jurkat T Cell Assay

doi: 10.1128/IAI.00052-12

Figure Lengend Snippet: H. pylori strains and plasmids

Article Snippet: H. pylori liquid cultures were grown in brucella broth supplemented with either activated charcoal or 5% fetal bovine serum (FBS) ( 11 ). table ft1 table-wrap mode="anchored" t5 caption a7 Strain/plasmid Relevant characteristics Reference Strains 60190 Wild type (ATCC 49503); vacA s1/i1/m1 40 60190 Δ rdxA Same as 60190 except HP0954 ( rdxA ) gene deleted; metronidazole resistant This study 60190 vacA :: cat rdxA Same as 60190 Δ rdxA except cat cassette and rdxA inserted in vacA ; chloramphenicol resistant and metronidazole sensitive; expression of VacA is disrupted This study 60190 i2B Same as 60190 Δ rdxA except vacA cluster B changed to i2 This study 60190 i2C Same as 60190 Δ rdxA except vacA cluster C changed to i2 This study 60190 i2BC Same as 60190 Δ rdxA except vacA clusters B and C changed to i2 This study 60190 i1/i2C Same as 60190 Δ rdxA except vacA cluster C has 4 amino acids changed to i2 This study X47 Wild type; vacA s1/m2, chimeric i-region 32 X47 Δ rdxA Same as X47 except HP0954 ( rdxA ) gene deleted; metronidazole resistant This study X47 vacA :: cat rdxA Same as X47 Δ rdxA except cat cassette and rdxA inserted in vacA ; chloramphenicol resistant and metronidazole sensitive; expression of VacA is disrupted This study X47 i1C Same as X47 Δ rdxA except vacA cluster C changed to i1 This study Plasmids pMM672 Allows deletion of rdxA in H. pylori strains 41 pCGR1 Contains cat-rdxA cassette in StuI site; derived from pA178 plasmid This study pCGR2 Contains cat-rdxA cassette in EcoRV site from X47 vacA This study pCGR3 60910 cluster B changed from i1 to i2 by inverse PCR using primers B1F and B1R This study pCGR4 60190 cluster C changed from i1 to i2 by inverse PCR using primers C1F and C1R This study pCGR5 60190 clusters B and C changed from i1 to i2 by inverse PCR using primers C1F and C1R and pCGR3 as the template This study pCGR6 A portion of 60190 cluster C changed from i1 to i2 by inverse PCR using primers C2F and C2R This study pCGR7 X47 cluster C changed from i2 to i1 by inverse PCR using primers C3F and C3R This study p55 Expresses VacA p55 27 p33 Expresses VacA p33 31 p33 i2 Expresses p33 i2 This study Open in a separate window H. pylori strains and plasmids

Techniques: Expressing, Derivative Assay, Plasmid Preparation, Inverse PCR