Gastrin Is an Essential Cofactor for Helicobacter-Associated Gastric Corpus Carcinogenesis in C57BL/6 Mice
2009; Elsevier BV; Volume: 175; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2009.081165
ISSN1525-2191
AutoresShigeo Takaishi, Shuiping Tu, Zinaida A. Dubeykovskaya, Mark T. Whary, Sureshkumar Muthupalani, Barry Rickman, Arlin B. Rogers, Nantaporn Lertkowit, Andrea Varró, James G. Fox, Timothy C. Wang,
Tópico(s)Cancer-related gene regulation
ResumoWe have previously described a synergistic interaction between hypergastrinemia and Helicobacter felis infection on gastric corpus carcinogenesis in FVB/N mice housed under specific-pathogen-free conditions. However, gastrin-deficient (GAS-KO) mice on a mixed C57BL/6/129Sv genetic background maintained in conventional housing were reported to develop spontaneous gastric antral tumors. Therefore, we investigated the role of gastrin in Helicobacter-associated gastric carcinogenesis in H. felis-infected mice on a uniform C57BL/6 background housed in specific-pathogen-free conditions. Hypergastrinemic transgenic (INS-GAS) mice, GAS-KO mice, and C57BL/6 wild-type mice were infected with H. felis for either 12 or 18 months. At 12 months postinfection, INS-GAS mice had mild corpus dysplasia, while B6 wild-type mice had either severe gastritis or metaplasia, and GAS-KO mice had only mild to moderate gastritis. At 18 months postinfection, both INS-GAS and B6 wild-type mice had both severe atrophic gastritis and corpus dysplasia, while GAS-KO mice had severe gastritis with mild gastric atrophy, but no corpus dysplasia. In contrast, both GAS-KO and B6 wild-type mice had mild to moderate antral dysplasia, while INS-GAS mice did not. H. felis antral colonization remained stable over time among the three groups of mice. These results point to a distinct effect of gastrin on carcinogenesis of both the gastric corpus and antrum, suggesting that gastrin is an essential cofactor for gastric corpus carcinogenesis in C57BL/6 mice. We have previously described a synergistic interaction between hypergastrinemia and Helicobacter felis infection on gastric corpus carcinogenesis in FVB/N mice housed under specific-pathogen-free conditions. However, gastrin-deficient (GAS-KO) mice on a mixed C57BL/6/129Sv genetic background maintained in conventional housing were reported to develop spontaneous gastric antral tumors. Therefore, we investigated the role of gastrin in Helicobacter-associated gastric carcinogenesis in H. felis-infected mice on a uniform C57BL/6 background housed in specific-pathogen-free conditions. Hypergastrinemic transgenic (INS-GAS) mice, GAS-KO mice, and C57BL/6 wild-type mice were infected with H. felis for either 12 or 18 months. At 12 months postinfection, INS-GAS mice had mild corpus dysplasia, while B6 wild-type mice had either severe gastritis or metaplasia, and GAS-KO mice had only mild to moderate gastritis. At 18 months postinfection, both INS-GAS and B6 wild-type mice had both severe atrophic gastritis and corpus dysplasia, while GAS-KO mice had severe gastritis with mild gastric atrophy, but no corpus dysplasia. In contrast, both GAS-KO and B6 wild-type mice had mild to moderate antral dysplasia, while INS-GAS mice did not. H. felis antral colonization remained stable over time among the three groups of mice. These results point to a distinct effect of gastrin on carcinogenesis of both the gastric corpus and antrum, suggesting that gastrin is an essential cofactor for gastric corpus carcinogenesis in C57BL/6 mice. Gastric cancer remains the second leading cause of cancer-related mortality in the world, although its incidence and mortality rates have been decreasing in the United States over the past 70 years.1Wang TC Gastrin transgenes and gastric cancer.in: Merchant JL Buchan AMJ Wang TC CURE foundation, Los Angeles2004: 235-252Google Scholar, 2Fox JG Wang TC Inflammation, atrophy, and gastric cancer.J Clin Invest. 2007; 117: 60-69Crossref PubMed Scopus (662) Google Scholar, 3Jemal A Siegel R Ward E Hao Y Xu J Murray T Thun MJ Cancer statistics, 2008.CA Cancer J Clin. 2008; 58: 71-96Crossref PubMed Scopus (10258) Google Scholar The risk of developing gastric adenocarcinoma is strongly associated with Helicobacter pylori infection, which is gradually disappearing from western societies. Despite the overall decline in gastric cancer prevalence, the treatment of stomach cancer remains a challenging clinical problem, since most patients who undergo surgical resection develop regional or distant recurrences and the overall 5-year survival rate for gastric cancer patients remains around 20% in western countries.3Jemal A Siegel R Ward E Hao Y Xu J Murray T Thun MJ Cancer statistics, 2008.CA Cancer J Clin. 2008; 58: 71-96Crossref PubMed Scopus (10258) Google Scholar H. pylori, first identified in the gastric antrum of patients with active chronic gastritis and peptic ulcers,4Marshall BJ Warren JR Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration.Lancet. 1984; 1: 1311-1315Abstract PubMed Scopus (4308) Google Scholar is now recognized as the major cause of gastric cancer, and has been classified as a group I carcinogen by World Health Organization.5Schistosomes, liver flukes and Helicobacter pylori. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, Lyon, 7–14 June 1994.IARC Monogr Eval Carcinog Risks Hum. 1994; 61: 1-241PubMed Google Scholar, 6Fox JG Wang TC Helicobacter pylori–not a good bug after all!.N Engl J Med. 2001; 345: 829-832Crossref PubMed Scopus (87) Google ScholarH. pylori infection causes persistent chronic gastritis, which in susceptible individuals may progress to atrophy, intestinal metaplasia, dysplasia, and finally, intestinal-type gastric cancer. This sequence, commonly referred to as Correa's cascade, is considered the primary histological pathway for the development of intestinal type of gastric cancer,7Correa P Houghton J Carcinogenesis of Helicobacter pylori.Gastroenterology. 2007; 133: 659-672Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar and is both initiated and promoted by H. pylori infection. It has generally been recognized that H. pylori infection results in a mild (1.5- to 2-fold elevation) hypergastrinemia that occurs early on in the course of the infection in many individuals. Given the known properties of gastrin as a mucosal growth factor, hypergastrinemia was postulated to be a factor promoting the development of gastric cancer. Indeed, previous studies have suggested a possible association between hypergastrinemia, Helicobacter infection, and gastric cancer.8Fox JG Wang TC Rogers AB Poutahidis T Ge Z Taylor N Dangler CA Israel DA Krishna U Gaus K Peek Jr, RM Host and microbial constituents influence Helicobacter pylori-induced cancer in a murine model of hypergastrinemia.Gastroenterology. 2003; 124: 1879-1890Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 9Wang TC Dangler CA Chen D Goldenring JR Koh T Raychowdhury R Coffey RJ Ito S Varro A Dockray GJ Fox JG Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.Gastroenterology. 2000; 118: 36-47Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, 10Fox JG Rogers AB Ihrig M Taylor NS Whary MT Dockray G Varro A Wang TC Helicobacter pylori-associated gastric cancer in INS-GAS mice is gender specific.Cancer Res. 2003; 63: 942-950PubMed Google Scholar, 11Wang TC Koh TJ Varro A Cahill RJ Dangler CA Fox JG Dockray GJ Processing and proliferative effects of human progastrin in transgenic mice.J Clin Invest. 1996; 98: 1918-1929Crossref PubMed Scopus (261) Google Scholar, 12Ohtani M Garcia A Rogers AB Ge Z Taylor NS Xu S Watanabe K Marini RP Whary MT Wang TC Fox JG Protective role of 17 beta -estradiol against the development of Helicobacter pylori-induced gastric cancer in INS-GAS mice.Carcinogenesis. 2007; 28: 2597-2604Crossref PubMed Scopus (63) Google Scholar Therefore, to study the role of gastrin and the potential mechanisms involved in gastric carcinogenesis, we developed a mouse model of gastric cancer through the generation of insulin-gastrin (INS-GAS) transgenic mice that overexpressed human amidated gastrin. In the absence of Helicobacter infection, INS-GAS mice on an FVB/N genetic background exhibited mild hypergastrinemia in association with elevated gastric acid secretion and an increased parietal cell number at 1 to 3 months of age. With increasing age, the INS-GAS mice showed progressive loss of parietal cells and significant changes in the corpus, including hypochlorhydria, gastric atrophy, metaplasia, and dysplasia. At 20 months of age, INS-GAS mice developed invasive gastric cancer.9Wang TC Dangler CA Chen D Goldenring JR Koh T Raychowdhury R Coffey RJ Ito S Varro A Dockray GJ Fox JG Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.Gastroenterology. 2000; 118: 36-47Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar The gastric cancer phenotype was accelerated by gastric Helicobacter spp. infection, and lesion severity was more profound in male INS-GAS mice.10Fox JG Rogers AB Ihrig M Taylor NS Whary MT Dockray G Varro A Wang TC Helicobacter pylori-associated gastric cancer in INS-GAS mice is gender specific.Cancer Res. 2003; 63: 942-950PubMed Google Scholar The cause of this gender-specific incidence was due in part to ovarian-dependent estrogen production, since H. pylori infected ovariectomized female INS-GAS mice also developed severe gastric neoplasia, and 17beta-estradiol treatment significantly suppressed this phenotype.12Ohtani M Garcia A Rogers AB Ge Z Taylor NS Xu S Watanabe K Marini RP Whary MT Wang TC Fox JG Protective role of 17 beta -estradiol against the development of Helicobacter pylori-induced gastric cancer in INS-GAS mice.Carcinogenesis. 2007; 28: 2597-2604Crossref PubMed Scopus (63) Google Scholar However, determining the role of gastrin in predisposing individuals to gastric cancer has not been straightforward. Some H. pylori-infected patients have lower levels of gastrin and acid secretion relative to non-infected healthy persons, and hypochlorhydria probably plays an important role in the carcinogenic process through altered bacterial colonization along with changes in nitrite levels.13McColl KE el-Omar EM Gillen D Alterations in gastric physiology in Helicobacter pylori infection: causes of different diseases or all epiphenomena?.Ital J Gastroenterol Hepatol. 1997; 29: 459-464PubMed Google Scholar Gastrin-deficient mice on a mixed C57BL6/129Sv background developed spontaneous gastric antral tumors when maintained under conventional housing conditions at 12 months of age, while C57BL/6 wild-type and somatostatin-deficient mice did not develop tumors.14Zavros Y Eaton KA Kang W Rathinavelu S Katukuri V Kao JY Samuelson LC Merchant JL Chronic gastritis in the hypochlorhydric gastrin-deficient mouse progresses to adenocarcinoma.Oncogene. 2005; 24: 2354-2366Crossref PubMed Scopus (123) Google Scholar The authors concluded that neoplastic transformation of the antrum does not require gastrin, and that gastrin may actually suppress the development of gastric antral tumors. In addition, there have been other genetic models reported, such as the gp130757F/F mouse, which do not appear to be dependent on gastrin for tumor development.15Judd LM Alderman BM Howlett M Shulkes A Dow C Moverley J Grail D Jenkins BJ Ernst M Giraud AS Gastric cancer development in mice lacking the SHP2 binding site on the IL-6 family co-receptor gp130.Gastroenterology. 2004; 126: 196-207Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar It has been difficult to reconcile these observations regarding the influence of gastrin on gastric cancer, given the different genetic backgrounds, housing conditions, and Helicobacter infection status. Thus, the purpose of this study is to examine the effect of gastrin in Helicobacter-associated gastric carcinogenesis using hypergastrinemic (INS-GAS) mice and gastrin deficient (GAS-KO) mice on a uniform C57BL/6 background and housed under SPF conditions. The animal protocol was reviewed and approved by the Columbia University Medical Center Institutional Animal Care and Use Committee. Eight- to twelve-week-old, male and female hypergastrinemic transgenic (INS-GAS) mice, gastrin-deficient (GAS-KO) mice, both backcrossed with C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) more than 10 generations, and C57BL/6J wild-type mice were used in this study.9Wang TC Dangler CA Chen D Goldenring JR Koh T Raychowdhury R Coffey RJ Ito S Varro A Dockray GJ Fox JG Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.Gastroenterology. 2000; 118: 36-47Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, 11Wang TC Koh TJ Varro A Cahill RJ Dangler CA Fox JG Dockray GJ Processing and proliferative effects of human progastrin in transgenic mice.J Clin Invest. 1996; 98: 1918-1929Crossref PubMed Scopus (261) Google Scholar, 16Koh TJ Goldenring JR Ito S Mashimo H Kopin AS Varro A Dockray GJ Wang TC Gastrin deficiency results in altered gastric differentiation and decreased colonic proliferation in mice.Gastroenterology. 1997; 113: 1015-1025Abstract Full Text PDF PubMed Scopus (217) Google Scholar Male hypergastrinemic transgenic (INS-GAS) mice in a FVB/N background with or without H. felis infection for 9 to 10 months, and FVB/N wild-type mice (Jackson Laboratory, ME), with or without H. felis infection for 12 months, were also included in the study for comparison as previously described.9Wang TC Dangler CA Chen D Goldenring JR Koh T Raychowdhury R Coffey RJ Ito S Varro A Dockray GJ Fox JG Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.Gastroenterology. 2000; 118: 36-47Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar All mice were bred under SPF conditions and thus free from murine-specific pathogens such as Lymphocytic choriomeningitis virus, Sendai virus, Mouse hepatitis virus, Ectromelia virus, Mycoplasma pulmonis, Clostridium piliforme, Salmonella spp., Pasteurella pneumotropica, Corynebacterium kutscheri, Citrobactor rodentium, Giardia muris, and Spironucleus muris. Mice were maintained in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and housed on hard wood bedding in micro-isolator, solid-bottomed polycarbonate cages, and given a commercial rodent diet and water ad libitum. Mice were infected by oral gavage with H. felis in 0.2 ml trypticase broth three times per week on every other day for a total dose of 100 million colony-forming units per mouse as previously described.9Wang TC Dangler CA Chen D Goldenring JR Koh T Raychowdhury R Coffey RJ Ito S Varro A Dockray GJ Fox JG Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.Gastroenterology. 2000; 118: 36-47Abstract Full Text Full Text PDF PubMed Scopus (519) Google ScholarH. felis-uninfected mice were sham dosed with 0.2 ml of broth. Mice were euthanized and analyzed at 12 and 18 months post-infection (MPI). Following isoflurane inhalation, blood was immediately collected into serum collection vials (BD Biosciences, San Jose, CA) by incision of brachial artery or vein. The mice were then sacrificed by cervical dislocation. The stomach and proximal duodenum were removed and the stomach was incised along the greater curvature. Linear gastric strips from the lesser curvature were fixed overnight in 10% neutral-buffered formalin, embedded, cut to 5-μm thickness, and stained with H&E. Tissue sections were scored for gastric lesions using previously published criteria by board certified veterinary pathologists (S.M., B.H. R., and A.B.R.) blinded to sample identity.17Takaishi S Cui G Frederick DM Carlson JE Houghton J Varro A Dockray GJ Ge Z Whary MT Rogers AB Fox JG Wang TC Synergistic inhibitory effects of gastrin and histamine receptor antagonists on Helicobacter-induced gastric cancer.Gastroenterology. 2005; 128: 1965-1983Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 18Lee CW Rickman B Rogers AB Ge Z Wang TC Fox JG Helicobacter pylori eradication prevents progression of gastric cancer in hypergastrinemic INS-GAS mice.Cancer Res. 2008; 68: 3540-3548Crossref PubMed Scopus (99) Google Scholar A dysplasia score of 3.0 was considered carcinoma in situ or low-grade gastrointestinal intraepithelial neoplasia. Ki-67 immunostaining (Abcam, see immunostaining section) was used to measure epithelial proliferation of gastric mucosa. The ratio of Ki-67-positive to total epithelial nuclei in glands occupying the full length of the proximal corpus was quantified manually for the Ki-67 labeling index, and the results were averaged from two to three mice in each group. The remainder of the gastric tissue was snap-frozen in dry ice and stored at −80°C for mRNA analysis. Small pieces of gastric tissue, 1 to 2 mm square in size, from the corpus and antrum respectively, were digested with proteinase K at 55°C for 24 to 48 hours, followed by genomic DNA extraction using DNA isolation kit (Lamda Biotech, St. Louis, MO) based on the manufacturer's instruction. H. felis colonization levels in gastric tissue were quantified by real-time PCR assay with H. felis flagellar filament B (flaB) primers using QuantiTect SYBR Green PCR kit (QIAGEN, Valencia, CA) and 7300 Real Time PCR System (Applied Biosystems, Foster City, CA) as previously described.17Takaishi S Cui G Frederick DM Carlson JE Houghton J Varro A Dockray GJ Ge Z Whary MT Rogers AB Fox JG Wang TC Synergistic inhibitory effects of gastrin and histamine receptor antagonists on Helicobacter-induced gastric cancer.Gastroenterology. 2005; 128: 1965-1983Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar The genomic copies of H. felis colonies were normalized by comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level determined by quantitative PCR, which is assumed to represent endogenous stomach genomic DNA quantity. Primer sequences used in this experiment as follows: H. felis flaB: forward 5′-TTCGATTGGTCCTACAGGCTCAGA-3′, reverse 5′-TTCTTGTTGATGACATTGACCAACGCA-3′; mouse GAPDH: forward 5′-GACATCAAGAAGGTGGTGAAGCAG-3′, and reverse 5′-ATACCAGGAAATGAGCTTGACAAA-3′. PCR conditions are: 95°C for 15 minutes followed by 40 cycles of 94°C for 10 seconds, 55°C for 20 seconds, and 72°C for 30 seconds. Any sample detecting <10 copies of the H. felis genome was considered negative for H. felis colonization. Longitudinal strips of gastric tissue from the anterior wall and the posterior wall were harvested and snap-frozen in dry ice and kept in −80 freezer until processed for analysis. Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA) and cDNA was synthesized from 4 μg of total RNA with Superscript III First Strand cDNA synthesis kit (Invitrogen, CA). Expression levels of cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4), growth factors such as Reg I and Amphiregulin, matrix metalloproteinases (MMPs) such as MMP-9 and -13, were quantified by real-time PCR assays using PCR conditions: 95°C for 15 minutes followed by 40 cycles of 94°C for 10 seconds, 55°C for 20 seconds, and 72°C for 30 seconds, using QuantiTect SYBR Green PCR kit (Qiagen) and 7300 Real Time PCR System (Applied Biosystems, CA). Primer sequences used in this experiment are as follows: TNF-alpha: forward 5′-TGGCCCAGACCCTCACACTCAG-3′, reverse 5′-ACCCATCGGCTGGCACCACT-3′; IL-4, forward 5′-ATCGGCATTTTGAACGAGGTCA-3′, reverse 5′-CATCGAAAAGCCCGAAAGAGTCT-3′; Reg I, forward 5′-AAGGAGAGTGGCACTACAGACG-3′, reverse 5′-GTATTGGGCATCACAGTTGTCA-3′; Amphiregulin, forward 5′-GGCAAAAATGGAAAAGGCAGAA-3′, reverse 5′-CGAGGATGATGGCAGAGACAAA-3′; MMP-9, forward 5′-CGTCATTCGCGTGGATAAGGAG-3′, reverse 5′-CCTGGTTCACCTCATGGTCCAC-3′; MMP-13, forward 5′-AGATTATCCCCGCCTCATAGAAGA-3′, reverse 5′-GGAATTTGTTGGCATGACTCTCAC-3′; and GAPDH, forward 5′-GACATCAAGAAGGTGGTGAAGCAG-3′, reverse 5′-ATACCAGGAAATGAGCTTGACAAA-3′. mRNA level of each gene was normalized to the mRNA level of internal control GAPDH using the ΔΔCt method.17Takaishi S Cui G Frederick DM Carlson JE Houghton J Varro A Dockray GJ Ge Z Whary MT Rogers AB Fox JG Wang TC Synergistic inhibitory effects of gastrin and histamine receptor antagonists on Helicobacter-induced gastric cancer.Gastroenterology. 2005; 128: 1965-1983Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Mouse serum samples were assayed for total amidated gastrin concentrations by radioimmunoassay using antibody L2 (which reacts with G17 and G34, but not progastrin or Gly-gastrins), and L6 for human G17 (which reacts with human G17 only), as previously described.12Ohtani M Garcia A Rogers AB Ge Z Taylor NS Xu S Watanabe K Marini RP Whary MT Wang TC Fox JG Protective role of 17 beta -estradiol against the development of Helicobacter pylori-induced gastric cancer in INS-GAS mice.Carcinogenesis. 2007; 28: 2597-2604Crossref PubMed Scopus (63) Google Scholar, 17Takaishi S Cui G Frederick DM Carlson JE Houghton J Varro A Dockray GJ Ge Z Whary MT Rogers AB Fox JG Wang TC Synergistic inhibitory effects of gastrin and histamine receptor antagonists on Helicobacter-induced gastric cancer.Gastroenterology. 2005; 128: 1965-1983Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Mouse serum samples collected at necropsy were evaluated for H. felis–specific, total IgG, Th2-associated IgG1, and Th1-associated IgG2c, by enzyme-linked immunosorbent assay using an outer membrane protein preparation from H. felis, as previously described.19Fox JG Beck P Dangler CA Whary MT Wang TC Shi HN Nagler-Anderson C Concurrent enteric helminth infection modulates inflammation and gastric immune responses and reduces helicobacter-induced gastric atrophy.Nat Med. 2000; 6: 536-542Crossref PubMed Scopus (420) Google Scholar In brief, 96-well flat-bottomed plates were coated with 100 μl of antigen (10 μg/ml) overnight at 4°C; sera were diluted to a ratio of 1:100 and added to the wells. Biotinylated secondary goat anti-mouse antibodies, clones A85–1 and 5.7 (BD Biosciences, CA), were used for detecting IgG, IgG1, and IgG2c respectively. Incubation with extravidin peroxidase (Sigma-Aldrich, St. Louis, MO) was followed by treatment with 2,2′-azinobis 3-ethylbenzthiazolinesulfonic acid substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for color development. The absorbance was recorded at A405 and A595 with a plate reader per manufacturer's protocol (Power WaveX Select, Bio-Tek Instruments, Winooski, VT). Tissues were fixed in 10% formalin, embedded in paraffin, and processed by standard histological methods. From each selected paraffin block, 5-μm serial sections were cut. Immunohistochemical studies were performed with avidin-biotin-peroxidase complex kits (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. The following primary antibodies were used: anti-Ki-67 (1:100, rabbit polyclonal, Abcam, Cambridge, MA), anti-mouse-trefoil factor (TFF)2 antibody (1:100, rabbit polyclonal antibody, raised in our laboratory), and anti-human MMP-13 antibody (1:100, mouse monoclonal, Chemicon, Billerica, MA). Primary antibodies were incubated at room temperature for 1 hour or overnight, in a humidified chamber. Diaminobenzidine (Vector Laboratories) was used as the chromogen and slides were counterstained with Mayer's hematoxylin. Gastric lesion scores were compared by Student's t-test or Mann-Whitney U-test. Expression levels of cytokines and IgG titers were compared using Student's t-test. Statistical analysis was performed using Microsoft Excel or GraphPad Prism 4.0 (GraphPad Software Inc., La Jolla, CA) with significance at P < 0.01 or 0.05. We first analyzed the gastric histology of H. felis-infected INS-GAS, GAS-KO, and B6 wild-type mice at 12 MPI. The stomachs of H. felis-infected INS-GAS mice had grossly apparent thickened gastric corpus with pale-appearing mucosa, while GAS-KO and B6 wild-type mice did not exhibit any notable changes (Figure 1A). Histologically, H. felis-infected INS-GAS mice had moderate gastritis, foveolar hyperplasia with mild metaplasia, and dysplastic changes in the corpus. GAS-KO or B6 wild-type mice had similar levels of gastritis, but minimal changes were noted for metaplasia and dysplasia in the gastric corpus (Figure 1B). With respect to the gastric antrum, all three groups of mice had mild antral hyperplasia (Figure 1C). These features were confirmed by histological scoring for each parameter. The scores for epithelial defects, oxyntic atrophy, foveolar hyperplasia, intestinal metaplasia, and gastric dysplasia in the corpus were significantly higher in H. felis infected INS-GAS mice than those in GAS-KO and B6 wild-type mice (Figure 1D, P < 0.01 or 0.05), while the scores for inflammation were not significantly different among three groups. Next we analyzed the gastric histology of H. felis-infected INS-GAS, GAS-KO, and B6 wild-type mice at 18 MPI. Histologically, H. felis-infected INS-GAS and B6 wild-type mice both had remarkable foveolar hyperplasia with severe metaplasia, as well as dysplastic changes in the corpus. GAS-KO mice had similar levels of severe gastritis but minimal metaplastic or dysplastic changes in the gastric corpus (Figure 2A). In contrast, H. felis-infected GAS-KO and B6 wild-type mice had moderate antral hyperplasia with mild dysplastic changes, while infected INS-GAS mice had mild antral hyperplasia, which was similar with gastric lesions observed at 12 MPI (Figure 2B). The histological scoring for inflammation, foveolar hyperplasia, pseudo-pyloric metaplasia, and gastric dysplasia in the corpus were significantly higher in H. felis-infected INS-GAS and B6 wild-type mice than those in GAS-KO mice (Figure 2C, P < 0.01 or 0.05) but no significant difference was detected between infected INS-GAS and B6 wild-type mice. However, the histological scores for epithelial defects, antral hyperplasia, and gastric dysplasia in antrum were significantly higher in H. felis-infected GAS-KO and B6 wild-type mice compared with those noted in INS-GAS mice (Figure 2D, P < 0.05), while no significant difference was detected between infected GAS-KO and B6 wild-type mice. In addition, the scores for antral inflammation were nearly identical. Finally, there were no gender differences in the degree of gastric corpus and antral histopathology among the mice in all three groups (see supplemental Figure 1, see http://ajp.amjpathol.org). We have previously reported that the serum gastrin levels in INS-GAS mice in an FVB/N background gradually increased over time.9Wang TC Dangler CA Chen D Goldenring JR Koh T Raychowdhury R Coffey RJ Ito S Varro A Dockray GJ Fox JG Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.Gastroenterology. 2000; 118: 36-47Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, 17Takaishi S Cui G Frederick DM Carlson JE Houghton J Varro A Dockray GJ Ge Z Whary MT Rogers AB Fox JG Wang TC Synergistic inhibitory effects of gastrin and histamine receptor antagonists on Helicobacter-induced gastric cancer.Gastroenterology. 2005; 128: 1965-1983Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar In this study, we confirmed a similar result of progressive hypergastrinemia in H. felis-infected INS-GAS mice on a C57BL/6 background. As shown in Figure 3A and B, total amidated gastrin, as well as human-specific gastrin (G-17) in serum of infected INS-GAS mice on a C57BL/6 background gradually increased at 6, 12, and 18 MPI compared with uninfected INS-GAS/B6 mice. In contrast, serum amidated gastrin levels in H. felis infected B6 wild-type mice did not show significant changes during the course of H. felis infection (Figure 3C). Similar results were also obtained in H. felis-infected INS-GAS mice on an FVB/N background and H. felis-infected FVB/N wild-type mice, with progressive hypergastrinemia in the INS-GAS mice and little change in the wild-type mice (see supplemental Figure 2, http://ajp.amjpathol.org). No gastrin was detected in serum of GAS-KO mice with and without H. felis infection (data not shown). For further analysis of the gastric phenotypes observed in H. felis-infected INS-GAS, GAS-KO and B6 wild-type mice, we investigated H. felis colonization status using two distinct methods. First we analyzed H. felis colonization by quantitative real-time PCR using genomic DNAs obtained from the corpus and antrum of H. felis-infected stomachs among each group at 18 MPI. DNA copy numbers of H. felis relative to those of GAPDH in the corpus were similar among three groups, while in the antrum H. felis DNA copy numbers relative to those of GAPDH were the highest in infected GAS-KO and the lowest in infected INS-GAS mice (Figure 4A). Of note, H. felis DNA copy numbers in the antrum were always higher than those in the corpus for each group (Figure 4A). We also analyzed H. felis-specific antibody titers in serum of the mice, and no significant differences were detected among three groups at 6, 12, and 18 MPI (Figure 4B). We and other groups have previously reported that Th1–Th2 immunobalance is critical for the progression of gastric atrophy, as well as metaplastic/dysplastic changes and progression to gastric cancer.19Fox JG Beck P Dangler CA Whary MT Wang TC Shi HN Nagler-Anderson C Concurrent enteric helminth infection modulates inflammation and gastric immune responses and reduces helicobacter-induced gastric atrophy.Nat Med. 2000; 6: 536-542Crossref PubMed Scopus (420) Google Scholar, 20Stoicov C Whary M Rogers AB Lee FS Klucevsek K Li H Cai X Saffari R Ge Z Khan IA Combe C Luster A Fox JG Houghton J Coinfection modulates inflammatory responses and clinical outcome of Helicobacter felis and Toxoplasma gondii infections.J Immunol. 2004; 173: 3329-3336PubMed Google Scholar Therefore, we investigated whether gastrin overexpression as well as gastrin deficiency influenced the Th1–Th2 immunobalance. First, we analyzed the expression level of Th1 directed cytokine TNF-α, and Th2 directed cytoki
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