Volume 126, Issue 3, Pages 674-682 (March 2004) Induction and regulation of Smad7 in the gastric mucosa of patients with Helicobacter pylori infection  Giovanni Monteleone, Giovanna Del Vecchio Blanco, Giampiero Palmieri, Piero Vavassori, Ivan Monteleone, Alfredo Colantoni, Serena Battista, Luigi Giusto Spagnoli, Marco Romano, Melissa Borrelli, Thomas T. MacDonald, Francesco Pallone  Gastroenterology  Volume 126, Issue 3, Pages 674-682 (March 2004) DOI: 10.1053/j.gastro.2003.11.048

Figure1 Active TGF-β1 in Hp-colonized gastric mucosa. The TGF-β1 ELISA was performed using total proteins extracted from gastric biopsy specimens of 20 Hp-positive patients and 19 Hp-negative controls who had no evidence of gastritis (normal controls). Each point represents the value of active TGF-β1 in mucosal samples taken from a single subject. Horizontal bars indicate the median value. Gastroenterology 2004 126, 674-682DOI: (10.1053/j.gastro.2003.11.048)

Figure2 Reduced phosphorylation of Smad3 in gastric mucosa from Hp-infected individuals. (A) Representative Western blot showing both phosphorylated (p)-Smad3 and total Smad3 protein in biopsy specimens from 3 Hp-positive patients and 3 normal controls. The example is representative of 4 separate experiments analyzing, in total, biopsy specimens from 13 Hp-positive patients and 13 negative controls who had no evidence of gastritis (normal controls). (B) Quantitative analysis of active/inactive Smad3 ratio in gastric biopsy specimens from 13 Hp-positive patients and 13 normal controls, as measured by densitometry scanning of Western blots. Values are expressed in arbitrary units (a.u.). Each point represents the value (a.u.) of active/inactive Smad3 ratio in biopsy specimens taken from a single subject. Horizontal bars indicate the median. A significant decrease in active/inactive Smad3 ratio is seen in patients with Hp infection in comparison with controls (P < 0.001, Mann-Whitney U test). (C) Western blot showing (active) phosphorylated (p)-Smad3 and total Smad3 protein in gastric lamina propria mononuclear cells (LPMC) isolated from 5 Hp-positive patients and 5 negative controls who had no evidence of gastritis (normal controls). Both p-Smad3 and total Smad3 were analyzed as indicated above. Immunoreactivity for p-Smad3 is consistently decreased in gastric LPMC isolated from patients with Hp infection in comparison with controls. Gastroenterology 2004 126, 674-682DOI: (10.1053/j.gastro.2003.11.048)

Figure3 Enhanced Smad7 in biopsy specimens from Hp-infected patients. (A) Representative expression of Smad7 and β-actin protein in gastric biopsy specimens taken from 3 Hp-positive patients and 3 negative controls who had no evidence of gastritis (normal controls). The example is representative of 4 separate experiments analyzing, in total, biopsy specimens from 13 Hp-positive patients and 13 normal controls. Inset: Quantitative data of Smad7 as measured by densitometry scanning of Western blots. Values are expressed in arbitrary units (a.u.). Each point represents the value (a.u.) of Smad7 in mucosal samples taken from a single subject. Horizontal bars indicate the median value. A significant increase in Smad7 is seen in patients with Hp infection in comparison with controls (P < 0.001, Mann-Whitney U test). (B) Western blot showing Smad7 and β-actin protein in gastric lamina propria mononuclear cells (LPMC) isolated from 5 Hp-positive patients and 5 negative controls who had no evidence of gastritis (normal controls). Immunoreactivity for Smad7 is consistently increased in gastric LPMC isolated from patients with Hp infection in comparison with controls. (C) Eradication of Hp leads to a down-regulation in Smad7 expression. Expression of Smad7 and β-actin protein in Hp-infected patients before (B) and after (A) a successful eradication therapy. Gastric mucosal biopsy specimens were taken from the antrum of 3 patients with Hp infection. The example is representative of 2 separate experiments using, in total, samples from 5 patients. (D) No Smad7 induction occurs in Hp-negative gastritis. Representative expression of Smad7 and β-actin protein in biopsy specimens taken from 5 patients with Hp-negative gastritis and 5 Hp-negative controls who had no evidence of gastritis (normal controls). The example is representative of 3 separate experiments analyzing, in total, biopsy specimens from 8 patients with Hp-negative gastritis and 13 normal controls. Gastroenterology 2004 126, 674-682DOI: (10.1053/j.gastro.2003.11.048)

Figure4 IFN-γ induces Smad7 in normal gastric mucosa. (A) Representative expression of Smad7 and β-actin protein in gastric mucosal samples taken from negative controls who had no evidence of gastritis (normal control) that were cultured in medium alone (UNS) or in the presence of Hp culture broth filtrate (Hp broth) or a Brucella culture broth filtrate (control broth) or rh-IFN-γ (100 ng/mL) for 2 hours. Smad7 was examined as indicated in the Patients and Methods section. The example is representative of 3 separate experiments analyzing, in total, mucosal samples from 5 normal controls. Identical results were seen in each experiment. (B) Representative RT-PCR gel showing IL-8 transcripts in AGS cells cultured in medium alone (UNS) or in the presence of Hp culture broth filtrate (Hp broth) or a control Brucella culture broth filtrate (control broth) or rh-IFN-γ (100 ng/mL) for 1 hour. (C) Neutralization of endogenous IFN-γ inhibits Smad7 in Hp-colonized gastric mucosa. Representative Western blot showing Smad7 protein in gastric mucosal samples taken from a Hp-positive patient and cultured in medium alone (UNS) or in the presence of a neutralizing IFN-γ or control antibody for 24 hours. The example is representative of 3 separate experiments analyzing, in total, mucosal samples from 5 Hp-positive patients. Identical results were seen in each experiment. Gastroenterology 2004 126, 674-682DOI: (10.1053/j.gastro.2003.11.048)

Figure5 (A) IFN-γ enhances STAT1 activation in normal gastric mucosa. Normal gastric biopsy specimens were preincubated with Tyrphostin B42 (TB42), a JAK2-STAT1 inhibitor, or DMSO for 30 minutes before the stimulation with IFN-γ (100 ng/mL) for a further 1 hour. UNS, biopsy specimens cultured with medium alone. STAT1 was analyzed as indicated in the Patients and Methods section. The example is representative of 3 separate experiments analyzing, in total, mucosal samples from 4 normal controls. Identical results were seen in each experiment. (B) Inhibition of STAT1 activation prevents the IFN-γ-mediated induction of Smad7. Normal gastric biopsy specimens were preincubated with Tyrphostin B42 (TB42) or DMSO for 30 minutes before the stimulation with IFN-γ (100 ng/mL) for a further 2 hours. Smad7 and β-actin were then examined by Western blotting as indicated in the Patients and Methods section. One of 3 experiments analyzing, in total, mucosal samples from 4 normal controls is shown. Identical results were seen in each experiment. Gastroenterology 2004 126, 674-682DOI: (10.1053/j.gastro.2003.11.048)

Figure6 Inhibition of Smad7 protein by a specific Smad7 antisense oligonucleotide (AS) in Hp-infected gastric mucosa results in increased p-Smad3 (A) and decreased tissue IFN-γ and T-bet expression (B). Gastric biopsy specimens taken from a Hp-positive patient were cultured in medium alone (UNS) or Smad7 sense (S) or antisense (AS) oligonucleotide for 24 hours. The example is representative of 4 separate experiments, in each of which gastric mucosal biopsy specimens from 1 Hp-positive patient were used. Identical results were seen in each patient. (C) Inhibition of IFN-γ secretion in the organ culture supernatants of Hp-infected biopsy specimens by Smad7 antisense (AS). After 24 hours in medium alone (UNS), the amount of IFN-γ in the supernatants was 345 ± 82 pg/100 μg total protein. With the sense oligonucleotide, there was no decrease (366 ± 98.2 pg/100 μg total protein). However, with antisense to Smad7, IFN-γ concentrations decreased in all samples to a mean of 208 ± 62 pg/100 μg total protein (P < 0.03). Gastroenterology 2004 126, 674-682DOI: (10.1053/j.gastro.2003.11.048)