Volume 131, Issue 1, Pages 246-258 (July 2006) Gastric Inflammation, Metaplasia, and Tumor Development in Gastrin–Deficient Mice  Lennart Friis–Hansen, Klaus Rieneck, Hans–Olof Nilsson, Torkel Wadström, Jens F. Rehfeld  Gastroenterology  Volume 131, Issue 1, Pages 246-258 (July 2006) DOI: 10.1053/j.gastro.2006.04.031 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Semiquantitative analysis of the load of gastric enterococci and lactobacilli. The type and number of gastric bacteria were determined in wild-type, gastrin KO, omeprazole-treated wild-type, and gastrin KO mice after 1 week of gastrin infusion. All mice were 12–16 weeks old. Half of the stomach was smeared on bile-esculin agar and the other half on MRS agar. After 48 hours of aerobic incubation at 37°C, the number of colonies was counted. The data are given as 109 colonies per stomach. Single colonies were identified using the api-test (BioMéreux). The mice were housed in a barrier mouse facility with 12-hour light/dark cycles. The mouse colony was monitored according to the FELASA guidelines,67 and during the experiments the colony tested negative for all monitored viruses and bacteria except Helicobacter bilis. n = 6–8 in each group. *P < .05. Gastroenterology 2006 131, 246-258DOI: (10.1053/j.gastro.2006.04.031) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Hierarchical cluster analysis of gastric gene expression profiles from wild-type, gastrin KO, gastrin KO + gastrin, and omeprazole-treated wild-type mice. The 2 groups of wild-type mice were more closely related to each other than they were to the 2 groups of gastrin KO mice. Exogenous gastrin infusion restores acid secretion but does not normalize gene expression. Similarly, even though omeprazole-treated mice are achlorhydric, their gastric gene expression is still closer to wild-type mice than to the achlorhydric gastrin KO mice. (Gastric RNAs from 5 mice aged 12–16 weeks were pooled and subsequently analyzed using the Affymetrix u74 GeneChips.) Gastroenterology 2006 131, 246-258DOI: (10.1053/j.gastro.2006.04.031) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Intestinal metaplasia in gastrin-deficient mice. (A and B) Periodic acid–Schiff/Alcian staining of the stomach showed that there are no major differences between young (12–16 weeks old) gastrin KO and wild-type mice. However, at 1 year of age, there are increasing signs of metaplasia with the accumulation of neutral and acid mucins. (D and I) Furthermore, lymphocytes infiltrated both the fundic and the antral mucosa (arrow). At 1½–2 years of age, there is increasing accumulation of mucins within the gastric glands, (H and I) development of cyst changes, and (J) ultimately the development of resulting polyp/tumor development. Immunohistochemical identification of lymphocytes is shown in Supplementary Figure 1 (see supplemental material online at www.gastrojournal.org). Bar = 50 μm. Gastroenterology 2006 131, 246-258DOI: (10.1053/j.gastro.2006.04.031) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 Gastric mucosal expression of inflammatory response genes in gastrin KO mice. Expression of Best5 is not detectable in (A) fundic mucosal cells in wild-type mice; expression is activated in (B) fundic cells but not (C) antral cells. (D) However, in antral polyps in the gastrin KO mice, there is an abundant expression (the arrows indicate clusters of cells with abundant Best5 expression). Schlafen4 is not detectable in (E) wild-type but present in (F) the nucleus of mucosal cells in the gastrin KO mice (the arrows point to typical cells). ISG15 is not detectable in (G) wild-type but present in (H) the nucleus of mucosal cells in gastrin KO mice (some of these are indicated by the arrows). The mice in A–C and E–H were 12–16 months old except in D, where they were 1½ years old. Gastroenterology 2006 131, 246-258DOI: (10.1053/j.gastro.2006.04.031) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Cdx2 expression in wild-type and KO gastric mucosa. (A and B) Cdx2 expression was absent in wild-type fundic and antral mucosa. However, in the gastrin KO mice, Cdx2 expression was found in (C and D) metaplastic fundic mucosa as well as (E) some parts of the antral foveolar polyps that developed in 1½-year-old gastrin KO mice. (F) The abundant Cdx2 expression in the wild-type duodenum. The tissues in A–D and F were from 1-year-old mice, whereas the tissue in E is from 1/12-year-old mice. Bar = 100 μm. Gastroenterology 2006 131, 246-258DOI: (10.1053/j.gastro.2006.04.031) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Immunocytochemical localization of IGF-BP2, syndecan 4, and NOV in wild-type gastric mucosa. Immunocytochemistry was used to localize 3 of the genes whose expression was down-regulated in the gastrin KO mice. IGF-BP2 was expressed in few cells (arrow) in the base of the glands. Syndecan 4 was detected in the extracellular space at low levels throughout the mucosa; areas with stronger staining are indicated by the arrows. NOV was expressed by few cells in each gland (arrows). All mice were 12–16 weeks old. Gastroenterology 2006 131, 246-258DOI: (10.1053/j.gastro.2006.04.031) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions