Volume 124, Issue 5, Pages 1408-1419 (May 2003) Chronic Helicobacter pylori infections induce gastric mutations in mice1   Eliette Touati, Valérie Michel, Jean-Michel Thiberge, Nicole Wuscher, Michel Huerre, Agnès Labigne  Gastroenterology  Volume 124, Issue 5, Pages 1408-1419 (May 2003) DOI: 10.1016/S0016-5085(03)00266-X

Figure 1 Anti-H. pylori and anti-H. felis antibodies in the sera of C57BL/6 Big Blue mice infected with H. pylori SS1 (gray circles) and H. felis CS1 (closed circles), respectively, or noninfected (open circles) detected by enzyme-linked immunosorbent assay. The results are presented as A405–492 readings for diluted serum samples (1:100). Each point corresponds to one mouse. Horizontal bars represent the mean value in each condition for mice 1, 3, 6, 9, and 12 months after infection. Gastroenterology 2003 124, 1408-1419DOI: (10.1016/S0016-5085(03)00266-X)

Figure 2 Lesions of the antral mucosa of (C-H2) H. pylori- and (I-K2) H. felis-infected mice compared with (A and B) uninfected mice. In contrast to the normal glandular architecture of uninfected tissues (A and B; original magnification 100×), the gastric glands infected by H. pylori for (C and D) 6 and (F and G) 12 months and H. felis for (I) 6 and (J) 12 months are hyperplastic. Inflammatory infiltrates are seen by H&E staining in both (C and F) H. pylori- and (I and J) H. felis-infected samples, in the lamina propria and within the submucosa, as indicated by the arrows (bar = 150 μm). Polymorphonuclear cells are visible (E2) around and (H2 and I [inset]) inside the glands, as indicated by the arrowheads (bar = 37.5 μm). Note the large aggregates of lymphocytes (arrows in J and inset) and polymorphonuclear cells (arrowhead in J and inset) seen after 12 months of H. felis infection. These changes are accompanied by (D and G) an increase in the number of mucus-producing cells (periodic acid-Schiff stain indicated by the arrows) compared with (B) control (bar = 150 μm). The Warthin-Starry stains of (E1 and H1) H. pylori- and (K1 and K2) H. felis-infected samples show the presence of bacteria inside the glands at (E1 and K1) 6 and (H1 and K2) 12 months after infection as indicated by the arrows (bar =15 μm). Gastroenterology 2003 124, 1408-1419DOI: (10.1016/S0016-5085(03)00266-X)

Figure 3 Inflammation grading in long-term H. pylori- (gray symbols) and H. felis- (hatched symbols) infected and uninfected mice (open symbols) after (A) 6 months and (B) 12 months. The intensity of the lesions was evaluated semiquantitatively.36 Infiltrates of polymorphonuclear cells (PMN) and plasmocytes were graded as follows: 0, no infiltrates; 1, mild, multifocal infiltration; 2, mild, widespread infiltration; 3, mild, widespread, and moderate multifocal infiltration; 4, moderate, widespread infiltration; 5, moderate, widespread, and severe multifocal infiltration. Lymphoid aggregates were graded as 1 (mild, 1–10 glands), 2 (moderate, 10–20 glands), or 3 (severe, more than 20 glands). Each symbol corresponds to one mouse. The horizontal bars represent the mean scores, of which the values are reported in parentheses. No significant differences for PMN infiltrates, plasmocyte infiltrates, and lymphocyte aggregates were observed between H. pylori and H. felis time-matched infected mice (P > 0.05). Circles, antrum; squares, corpus. Gastroenterology 2003 124, 1408-1419DOI: (10.1016/S0016-5085(03)00266-X)

Figure 4 iNOS immunohistochemistry in gastric tissue sections of (A) noninfected, (B) H. felis CS1-, and (C) H. pylori SS1-infected samples at 6 months. Strong immunoreactivity for iNOS can be observed limited to the inflammatory cell infiltrate in the mucosa and deep mucosa in all infected samples. Note that there is weak iNOS immunoreactivity in epithelial cells in the case of H. felis infection (B). Uninfected samples were either negative or, when positive, only a very small number of iNOS-producing cells were detected in the lamina propria. Gastroenterology 2003 124, 1408-1419DOI: (10.1016/S0016-5085(03)00266-X)

Figure 5 RT-PCR analysis of iNOS mRNA in stomach mucosa from Big Blue mice. (A) Representative gel of iNOS and β-actin expression in samples from uninfected, H. pylori SS1-, and H. felis CS1-infected mice after 6 and 12 months. (B) Histograms corresponding to the quantification of the relative expression of iNOS vs. β-actin after 6 and 12 months. P values are indicated for significant differences between infected and uninfected samples. Each column represents the mean ± SD for one group of mice (n = 6 or 7) for each tested condition. Each experiment was performed 3 times. Gastroenterology 2003 124, 1408-1419DOI: (10.1016/S0016-5085(03)00266-X)

Figure 6 Gastric mutant frequencies measured using the Big Blue assay protocol29,31 in the stomach of age-matched uninfected and H. pylori- or H. felis-infected mice on the standard diet (open circles) and on the high-salt diet (closed circles) after 6 and 12 months. Each point corresponds to one mouse. Reported values are the mean gastric mutant frequency (× 10−5) for each group of mice. The positive control for the mutagenesis assay was Big Blue mice treated with the reference mutagen, R7000 (see Materials and Methods); the gastric mutant frequency 3 weeks after treatment was 30 ± 11 × 10−5, and that for the corresponding untreated mice was 6.7 ± 2 × 10−5. Gastroenterology 2003 124, 1408-1419DOI: (10.1016/S0016-5085(03)00266-X)

Figure 7 Distribution of the base pair substitution events in the cII gene as assessed using mutants isolated from the genomic gastric DNA from (A and C) uninfected and (B and D) H. pylori-infected mice after (A and B) 6 months and (C and D) 12 months. The number of each type of base pair substitution is expressed as a percentage of all substitution events observed. Gastroenterology 2003 124, 1408-1419DOI: (10.1016/S0016-5085(03)00266-X)