APE1 regulates Nox1 expression.

<p>(A) Nox1 expression was examined in AGS pSIREN and shRNA cells infected with H. pylori for 1, 3 and 6h or left uninfected (0h). Corresponding densitometry results of three experiments are shown to the right of the western blot. (B) Immunofluorescence staining showing Rac1 and Nox1 after infection in AGS and shRNA cells. Scale bars indicate 10 μm. (C) Gene transcription levels of Nox1 (left panel) and APE1 (right panel) in gastric biopsies from H. pylori infected and uninfected patients. Each value was normalized to 18S and data were normalized to one of the uninfected samples. Levels of significance are indicated as follows: * p<0.05 and ** p<0.01.</p

H. pylori-induced ROS generation is mediated by Rac1.

<p>(A-C) ROS generation was measured by luminol oxidation in AGS cells (A), NCI-N87 cells (B) and primary gastric epithelial cells (C) after infection with H. pylori 26695 for 10, 30 or 60 min or left uninfected. (D) H. pylori-induced ROS generation (after 30 min) was measured in AGS cells after transfection with empty vector (pcDNA), constitutively active Rac1 (V12) or after treatment with Rac1 inhibitor (NSC23766). For A-C fold change of luminol oxidation compared to uninfected cells are shown as mean values (± SEM) of three independent experiments. (E-F) Rac1 activation was measured by a GTP pull down assay in AGS (E) and NCI-N87 (F) cells following infection with H. pylori at the indicated times. A representative western blot was selected from three independent experiments and densitometry results from multiple experiments are shown. Levels of significance are indicated as follows: * p<0.05, ** p<0.01 and *** p< 0.001.</p

APE1 inhibits H. pylori-induced ROS generation.

<p>ROS generation was measured by luminol oxidation (A) in WT AGS, control shRNA (pSIREN) or APE1 shRNA (shRNA) cells after infection with H. pylori for 30 min or left uninfected, (B) in infected cells that were either left untreated or treated with NSC23766 or DPI, (C) in shRNA cells either transfected with empty vector (pcDNA) or APE1; or treated with NAC or DPI before infection with H. pylori for 30 min or left uninfected and (D) in shRNA cells that were either transfected with pcDNA, APE1, V12 or both APE1 and V12 or treated with NSC23766 before infection with H. pylori or left uninfected. For A-D fold change of luminol oxidation compared to uninfected cells are shown as mean values (± SEM) of three independent experiments. (E) ROS generation was measured by confocal microscopy in AGS and shRNA cells after infection with H. pylori for 30 min or left uninfected. After infection ROS was detected using CM-H₂DCFDA and nuclei were stained with DAPI. Scale bar indicates 100 μm. The bar graph at the bottom shows quantification of multiple images from three independent experiments. Levels of significance are indicated as follows: * p<0.05, ** p<0.01 and *** p< 0.001.</p

H. pylori-induced ROS generation is regulated by NADPH oxidase.

<p>ROS generation was measured in (A) AGS cells infected with H. pylori for 30 min or left uninfected in the presence of 10 mM NAC or 10 μM DPI, (B) in Nox1 downregulated AGS cells following infection with H. pylori for 30 min or left uninfected and (C) in AGS cells either transfected with pcDNA or Rac1 V12 following infection with H. pylori for 30 min. In other experimental conditions, AGS cells were treated with NSC23766 (NSC), or DPI or both before infection with H. pylori for 30 min. For A-C fold change of luminol oxidation compared to uninfected cells are shown as mean values (± SEM) of three independent experiments. (D) Nox1 expression was measured in uninfected and H. pylori infected AGS cells transfected with either vector (pcDNA) or Rac1 V12 or were pre-treated with NSC23766 (NSC). The representative western blot with Nox1 expression was selected from three independent experiments and densitometry results are shown. Levels of significance are indicated as follows: ** p<0.01 and *** p< 0.001.</p

Multiple linear regression models including ferritin or haemoglobin as dependent variables and age, gender, country of birth and <i>H. pylori</i> infection as independent variables in children from United Kingdom, Chile and Brazil (n = 311).

<p>Multiple linear regression models including ferritin or haemoglobin as dependent variables and age, gender, country of birth and <i>H. pylori</i> infection as independent variables in children from United Kingdom, Chile and Brazil (n = 311).</p

Histological comparison of antral (n = 85) and corpus (n = 83) gastric mucosa of <i>H. pylori (HP)</i>-positive children and antral (n = 202) and corpus (n = 214) gastric mucosa of <i>H. pylori</i>-negative children.^*.

*<p>Twenty-four antral and 13 corpus gastric biopsy specimens were deemed to be inadequate for histology assessment. n, number.</p

Multiple linear regression models including ferritin or haemoglobin as dependent variables and age, gender, country of birth and <i>H. pylori</i> infection as independent variables in children from Chile (n = 105) and Brazil (n = 125).

<p>Multiple linear regression models including ferritin or haemoglobin as dependent variables and age, gender, country of birth and <i>H. pylori</i> infection as independent variables in children from Chile (n = 105) and Brazil (n = 125).</p

Multiple linear regression models including ferritin or haemoglobin as dependent variables and age, gender and <i>H. pylori</i> (HP) infection as independent variables in children from United Kingdom (n = 81), Chile (n = 105) and Brazil (n = 125).

<p>Multiple linear regression models including ferritin or haemoglobin as dependent variables and age, gender and <i>H. pylori</i> (HP) infection as independent variables in children from United Kingdom (n = 81), Chile (n = 105) and Brazil (n = 125).</p

Comparison of the iron deficiency/iron deficiency anaemia parameters between <i>H. pylori</i>-positive (n = 86) and negative (n = 225) children from Brazil, Chile and United Kingdom.

*<p>obtained by dividing the serum iron concentration by the total iron binding capacity;</p><p>SD, standard deviation; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin.</p