Helicobacter pylori cag-Type IV Secretion System Facilitates Corpus Colonization to Induce Precancerous Conditions in Mongolian Gerbils  Gabriele Rieder, Juanita L. Merchant, Rainer Haas  Gastroenterology  Volume 128, Issue 5, Pages 1229-1242 (May 2005) DOI: 10.1053/j.gastro.2005.02.064 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Colonization density of antral (open symbols) and corpus (filled symbols) mucosa in Mongolian gerbils orally challenged with H pylori B128 WT (circle), B128ΔcagY (triangle), and B128ΔcagA (square) isogenic mutant strains for 32 weeks. The detection limit was <5 × 102 colony-forming units (CFU) per gram of stomach (dotted line). Gastric tissue specimens without H pylori reisolation are shown as null (*P < .05; #not significant). Gastroenterology 2005 128, 1229-1242DOI: (10.1053/j.gastro.2005.02.064) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Macroscopic findings. (A) Stomach size of noninfected, H pylori B128 WT, B128ΔcagY, and B128ΔcagA mutant infected Mongolian gerbils after 32 weeks (bar = 10 mm). (B) The total diameter of the stomach at the greater curvature is shown (line in box represents median) in gerbils challenged with H pylori B128 WT, B128ΔcagY, and B128ΔcagA mutant strains for 32 weeks compared with age-matched noninfected controls. All box plots show 25th to 75th percentiles (box) and 5th to 95th percentiles (whiskers). The line in the box represents the median (*P < .05; #not significant). Gastroenterology 2005 128, 1229-1242DOI: (10.1053/j.gastro.2005.02.064) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 (A and C) H&E staining and (B and D) histological grading of antral (A and B) and corpus (C and D) mucosa from gerbils challenged with H pylori B128 WT (circles), B128ΔcagY (triangles), and B128ΔcagA (squares) mutant strains for 32 weeks compared with age-matched noninfected controls. Shown are remaining parietal cells in the atrophic corpus of WT-infected gerbils (arrows) (C). Periodic acid-Schiff/alcian blue stains are shown of antral (E) and corpus (F) mucosa from noninfected and WT-infected gerbils. Mucous gland metaplasia in the corpus of WT-infected gerbils is shown (arrows) (F). Bar = 100 μm (**P < .001; *P < .05; #not significant). Gastroenterology 2005 128, 1229-1242DOI: (10.1053/j.gastro.2005.02.064) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 (A and C) H&E staining and (B and D) histological grading of antral (A and B) and corpus (C and D) mucosa from gerbils challenged with H pylori B128 WT (circles), B128ΔcagY (triangles), and B128ΔcagA (squares) mutant strains for 32 weeks compared with age-matched noninfected controls. Shown are remaining parietal cells in the atrophic corpus of WT-infected gerbils (arrows) (C). Periodic acid-Schiff/alcian blue stains are shown of antral (E) and corpus (F) mucosa from noninfected and WT-infected gerbils. Mucous gland metaplasia in the corpus of WT-infected gerbils is shown (arrows) (F). Bar = 100 μm (**P < .001; *P < .05; #not significant). Gastroenterology 2005 128, 1229-1242DOI: (10.1053/j.gastro.2005.02.064) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Immunohistochemical staining is shown with anti-Ki-67 antibody to detect proliferating cells in the corpus of noninfected (A) and WT-infected (B) gerbils (original magnification, 200×). (C) Box plot of the number of Ki-67–producing cells per antral (white box) and corpus (gray box) mucosa (original magnification, 100×) of gerbils challenged with H pylori B128 WT, B128ΔcagY, and B128ΔcagA mutant strains for 32 weeks compared with age-matched noninfected controls. All box plots show the 25th to 75th percentiles (box) and 5th to 95th percentiles (whiskers). The line in the box represents the median (**P < .001; *P < .05; #not significant). Gastroenterology 2005 128, 1229-1242DOI: (10.1053/j.gastro.2005.02.064) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Atrophy in corpus mucosa is associated with reduced H+K+-ATPase. (A) H&E stain of the corpus of an H pylori B128 WT-infected gerbil shows atrophy. The transition zone is marked with arrows (original magnification, 100×). (B) Real-time RT-PCR of H+K+-ATPase is shown normalized to the amount of 18S ribosomal RNA of antral (white box) and corpus (gray box) tissue of gerbils challenged with H pylori B128 WT, B128ΔcagY, and B128ΔcagA mutant strains for 32 weeks compared with age-matched noninfected controls. All box plots show the 25th to 75th percentiles (box) and 5th to 95th percentiles (whiskers). Line in box represents the median (**P < .001; #not significant). Gastroenterology 2005 128, 1229-1242DOI: (10.1053/j.gastro.2005.02.064) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Relative cytokine mRNA levels in the infected gerbil stomach. (A) IL-1β, (B) IFN-γ, (C) KC, and (D) IL-12p40 real-time RT-PCR normalized to 18S ribosomal RNA of antral (white box) and corpus (gray box) mucosa of gerbils challenged with H pylori B128 WT, B128ΔcagY, and B128ΔcagA mutant strains for 32 weeks compared with age-matched noninfected controls. All box plots show the 25th to 75th percentiles (box) and 5th to 95th percentiles (whiskers). Line in box represents the median (**P < .001; *P < .05; #not significant; antrum and corpus of WT-infected compared with mutant strains, respectively: ++P < .001; +P < .05; †not significant). Gastroenterology 2005 128, 1229-1242DOI: (10.1053/j.gastro.2005.02.064) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Hypochlorhydria and hypergastrinemia in the corpus of H pylori B128 WT-infected gerbils. (A) The pH of the corpus mucous surface measured with indicator test sticks (comparison of noninfected vs mutants was nonsignificant). (B) Plasma gastrin levels (detection limit <1 pmol/L) and relative antral (white box) and corpus (gray box) (C) somatostatin mRNA transcripts are shown for gerbils infected with H pylori B128 WT (circles), B128ΔcagY (triangles), and B128ΔcagA (squares) mutant strains for 32 weeks compared with age-matched noninfected controls. All box plots show the 25th to 75th percentiles (box) and 5th to 95th percentiles (whiskers). Line in box represents the median (**P < .001; *P < .05; #not significant; antrum of noninfected and WT-infected, compared with mutant strains, respectively: †not significant). Gastroenterology 2005 128, 1229-1242DOI: (10.1053/j.gastro.2005.02.064) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 In vitro stimulation of gastrin promoter. (A) AM0 and AM4 cells stably transfected with the WT and mutant gastrin promoter luciferase reporter construct, respectively, were stimulated with different multiplicities of infection (MOI) of the H pylori P12 WT strain: MOI of 100:1, 10:1, and 1:1 for 5 hours, as indicated. Relative induction is shown of (B) AM0 and AM4 cells stimulated with 50 nmol/L EGF, H pylori WT P12 and B128, B128ΔcagA and B128ΔcagY isogenic mutants, Tx30a, Campylobacter jejuni, and Escherichia coli DH5α strains or (C and D) AM0 cells stimulated with 50 nmol/L EGF, H pylori HP 26695, 26695ΔPAI, 26695ΔcagA, and cag-PAI isogenic mutants (hp0520-hp0546)14 at an MOI of 10:1 for 5 hours. Luciferase activities in relation to H pylori P12 MOI 100:1 (A) and B128 (represents 100%), MOI 10:1 (B and C), and IL-8 (percentage as compared to 26695) (D) were plotted as the mean and SE. Gastroenterology 2005 128, 1229-1242DOI: (10.1053/j.gastro.2005.02.064) Copyright © 2005 American Gastroenterological Association Terms and Conditions