Pattern of Transcription Factor Activation in Helicobacter pylori–Infected Mongolian Gerbils
2007; Elsevier BV; Volume: 132; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2007.01.009
ISSN1528-0012
AutoresTakahiko Kudo, Hong Lü, Jeng–Yih Wu, Tomoyuki Ohno, Michael Wu, Robert M. Genta, David Y. Graham, Yoshio Yamaoka,
Tópico(s)Phytochemistry and Bioactive Compounds
ResumoBackground & Aims: Helicobacter pylori interact with epithelial cells resulting in activation of cellular signaling pathways leading to an inflammatory response. The pattern and timing of transcription factor activation in H pylori-infected gastric mucosa remain unclear. We investigated the roles of transcription factors in the gastric mucosa of H pylori-infected gerbils over the course of the infection. Methods: Six-week-old male Mongolian gerbils were inoculated orally with H pylori TN2GF4 or isogenic cagE mutants and examined at 1, 3, 9, and 18 months. We examined the expression of 54 transcription factors using DNA/protein arrays and electrophoretic mobility shift assays. Phosphorylation status of mitogen-activated protein kinases and IκB were evaluated by immunoblot and immunohistochemistry. Results: Ten transcription factors were up-regulated by H pylori infection. Six of these factors, including activator protein-1 (AP-1) and cAMP responsive element binding protein (CREB), reached maximal levels at 3 months and were strongly correlated with cellular inflammation and ulceration. Phosphorylation of extracellular signal-regulated kinase correlated with activation of AP-1 and CREB. Levels of nuclear factor-κB and interferon-stimulated responsive element (ISRE) peaked at 18 months and correlated with the presence of severe atrophy and with phosphorylation of Jun-N-terminal kinase (JNK), p38, and IκB. Conclusions: The gastric mucosal transcription factors induced by H pylori infection differed according to the phase and outcome of infection; AP-1 and CREB levels were early responders related to inflammation and ulceration, whereas NF-κB and ISRE were late responders related to atrophy. Background & Aims: Helicobacter pylori interact with epithelial cells resulting in activation of cellular signaling pathways leading to an inflammatory response. The pattern and timing of transcription factor activation in H pylori-infected gastric mucosa remain unclear. We investigated the roles of transcription factors in the gastric mucosa of H pylori-infected gerbils over the course of the infection. Methods: Six-week-old male Mongolian gerbils were inoculated orally with H pylori TN2GF4 or isogenic cagE mutants and examined at 1, 3, 9, and 18 months. We examined the expression of 54 transcription factors using DNA/protein arrays and electrophoretic mobility shift assays. Phosphorylation status of mitogen-activated protein kinases and IκB were evaluated by immunoblot and immunohistochemistry. Results: Ten transcription factors were up-regulated by H pylori infection. Six of these factors, including activator protein-1 (AP-1) and cAMP responsive element binding protein (CREB), reached maximal levels at 3 months and were strongly correlated with cellular inflammation and ulceration. Phosphorylation of extracellular signal-regulated kinase correlated with activation of AP-1 and CREB. Levels of nuclear factor-κB and interferon-stimulated responsive element (ISRE) peaked at 18 months and correlated with the presence of severe atrophy and with phosphorylation of Jun-N-terminal kinase (JNK), p38, and IκB. Conclusions: The gastric mucosal transcription factors induced by H pylori infection differed according to the phase and outcome of infection; AP-1 and CREB levels were early responders related to inflammation and ulceration, whereas NF-κB and ISRE were late responders related to atrophy. Helicobacter pylori causes chronic gastric inflammation in virtually all infected persons. The cellular inflammatory response initially consists of infiltration by neutrophils followed by T and B lymphocytes, plasma cells, and macrophages. Infiltration of the mucosa with inflammatory cells is associated with epithelial cell damage (reviewed in Suerbaum and Michetti1Suerbaum S. Michetti P. Helicobacter pylori infection.N Engl J Med. 2002; 347: 1175-1186Crossref PubMed Scopus (2236) Google Scholar) and with a marked increase in the expression of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, and IL-8,2Yamaoka Y. Kita M. Kodama T. Sawai N. Imanishi J. Helicobacter pylori cagA gene and expression of cytokine messenger RNA in gastric mucosa.Gastroenterology. 1996; 110: 1744-1752Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 3Yamaoka Y. Kita M. Kodama T. Sawai N. Kashima K. Imanishi J. Induction of various cytokines and development of severe mucosal inflammation by cagA gene-positive Helicobacter pylori strains.Gut. 1997; 41: 442-451Crossref PubMed Scopus (330) Google Scholar which are regulated at the level of transcription. The signal transduction pathways involve transcription factors that bind DNA at specific promoter or enhancer regions. Activated transcription factors regulate gene expression by modulating the frequency of transcription initiation by interacting with specific DNA-binding elements present in promoters. The details and sequence of transcription factor activation in H pylori infection are not well understood (reviewed in Naumann and Crabtree4Naumann M. Crabtree J.E. Helicobacter pylori-induced epithelial cell signaling in gastric carcinogenesis.Trends Microbiol. 2004; 12: 29-36Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Much of the available data comes from in vitro studies in which H pylori was cocultured with gastric cancer cells. In these experiments, transcription factor activation typically occurs within 1 hour, and the relationship between these in vitro observations and the phenomenon that occurs in H pylori-infected gastric mucosa remains largely unexplored. The Mongolian gerbil (Meriones unguiculatus) has proven to be a convenient animal model for studies of H pylori infection because the gross and histologic findings mimic the lesions induced by H pylori infection in the human gastric mucosa.5Hirayama F. Takagi S. Yokoyama Y. Iwao E. Ikeda Y. Establishment of gastric Helicobacter pylori infection in Mongolian gerbils.J Gastroenterol. 1996; 31: 24-28PubMed Google Scholar, 6Watanabe T. Tada M. Nagai H. Sasaki S. Nakao M. Helicobacter pylori infection induces gastric cancer in Mongolian gerbils.Gastroenterology. 1998; 115: 642-648Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar, 7Ikeno T. Ota H. Sugiyama A. Ishida K. Katsuyama T. Genta R.M. Kawasaki S. Helicobacter pylori-induced chronic active gastritis, intestinal metaplasia, and gastric ulcer in Mongolian gerbils.Am J Pathol. 1999; 154: 951-960Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 8Franco A.T. Israel D.A. Washington M.K. Krishna U. Fox J.G. Rogers A.B. Neish A.S. Collier-Hyams L. Perez-Perez G.I. Hatakeyama M. Whitehead R. Gaus K. O'Brien D.P. Romero-Gallo J. Peek Jr, R.M. Activation of β-catenin by carcinogenic Helicobacter pylori.Proc Natl Acad Sci U S A. 2005; 102: 10646-10651Crossref PubMed Scopus (413) Google Scholar, 9Yamaoka Y. Yamauchi K. Ota H. Sugiyama A. Ishizone S. Graham D.Y. Maruta F. Murakami M. Katsuyama T. Natural history of gastric mucosal cytokines expression in Helicobacter pylori gastritis in Mongolian gerbils.Infect Immun. 2005; 73: 2205-2212Crossref PubMed Scopus (45) Google Scholar In the present study, we investigated the roles of transcription factors in the gastric mucosa of H pylori-infected gerbils over the course of the infection in vivo. Six-week-old male Mongolian gerbils (MGS/Sea; Harlan Sprague Dawley, Inc., Indianapolis, IN) were housed in an air-conditioned biohazard room designed for infectious animals with a 12-hour light/12-hour dark cycle. They were provided rodent diet and water ad libitum. The animal facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. The experiment protocol was approved by the Animal Care Committee of Baylor College of Medicine and Michael E. DeBakey Veterans Affairs Medical Center, Houston, TX. H pylori strain TN2GF4 was isolated from a Japanese gastric ulcer patient and has been shown to colonize gerbils consistently for at least 1 year and to cause reproducible mucosal damage.6Watanabe T. Tada M. Nagai H. Sasaki S. Nakao M. Helicobacter pylori infection induces gastric cancer in Mongolian gerbils.Gastroenterology. 1998; 115: 642-648Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar This strain is cag pathogenicity island (PAI) positive, vacA s1 (production of the vacuolating cytotoxin) and has functional blood group antigen binding adhesin (BabA) and outer inflammatory protein (OipA). Long-term studies (eg, 18 months) used wild-type (WT) strain TN2GF4, whereas the cag PAI mutant (isogenic cagE mutant) was used for short-term studies (ie, 1 and 3 months). The isogenic cagE mutant was constructed previously10Yamaoka Y. Kwon D.H. Graham D.Y. A Mr 34,000 proinflammatory outer membrane protein (oipA) of Helicobacter pylori.Proc Natl Acad Sci U S A. 2000; 97: 7533-7538Crossref PubMed Scopus (362) Google Scholar and was chloramphenicol resistant. H pylori were grown in brain heart infusion (BHI) broth supplemented with 15% fetal bovine serum (FBS) for 20–30 hours at 37°C under microaerobic conditions and saturated humidity, with shaking at 200 rpm. After fasting for 12 hours, each animal was orogastrically inoculated 3 times (days 0, 1, and 2) with 1.0 mL inoculum of H pylori (108Franco A.T. Israel D.A. Washington M.K. Krishna U. Fox J.G. Rogers A.B. Neish A.S. Collier-Hyams L. Perez-Perez G.I. Hatakeyama M. Whitehead R. Gaus K. O'Brien D.P. Romero-Gallo J. Peek Jr, R.M. Activation of β-catenin by carcinogenic Helicobacter pylori.Proc Natl Acad Sci U S A. 2005; 102: 10646-10651Crossref PubMed Scopus (413) Google Scholar colony forming unit [CFU]/mL) or sterile BHI broth (as a control) using gastric intubation needles. No specific pretreatments were given before orogastric H pylori inoculation. Infected gerbils were killed and necropsied 1, 3, 9, or 18 months after H pylori inoculation. Five or 6 H pylori-inoculated gerbils were used for the 1-, 3-, and 9-month time points, and 18 inoculated gerbils were used for the 18-month time point. Three to 5 age- and sex-matched uninfected gerbils were used as controls at each time point. At necropsy, stomachs were opened along the greater curvature and were divided longitudinally into 2 parts. One half was fixed in 10% phosphate-buffered formalin for histologic examination. The other half was divided further into the pyloric gland mucosa (antrum) and the fundic gland mucosa (corpus) and stored at −80°C. The gastric mucosa was separated as much as possible from the underlying muscle by sharp dissection. In addition, one mm2Yamaoka Y. Kita M. Kodama T. Sawai N. Imanishi J. Helicobacter pylori cagA gene and expression of cytokine messenger RNA in gastric mucosa.Gastroenterology. 1996; 110: 1744-1752Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar piece of gastric mucosa from the antrum was taken to culture H pylori. The culture of H pylori from the gastric mucosa of gerbils was performed as previously described.9Yamaoka Y. Yamauchi K. Ota H. Sugiyama A. Ishizone S. Graham D.Y. Maruta F. Murakami M. Katsuyama T. Natural history of gastric mucosal cytokines expression in Helicobacter pylori gastritis in Mongolian gerbils.Infect Immun. 2005; 73: 2205-2212Crossref PubMed Scopus (45) Google Scholar, 11Sakai T. Fukui H. Franceschi F. Penland R. Sepulveda A.R. Fujimori T. Terano A. Genta R.M. Graham D.Y. Yamaoka Y. Cyclooxygenase expression during Helicobacter pylori Infection in Mongolian gerbils.Dig Dis Sci. 2003; 48: 2139-2146Crossref PubMed Scopus (16) Google Scholar The recovered strains were tested for chloramphenicol resistance to confirm the stability of the mutation for the cagE mutant-infected cases. Polymerase chain reaction (PCR) analysis was also performed for cagE mutants to confirm the preservation of chloramphenicol cassettes inside the target genes as previously described.12Yamaoka Y. Kita M. Kodama T. Imamura S. Ohno T. Sawai N. Ishimaru A. Imanishi J. Graham D.Y. Helicobacter pylori infection in mice: role of outer membrane proteins in colonization and inflammation.Gastroenterology. 2002; 123: 1992-2004Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Tissues were fixed in 10% neutral-buffered formalin and sliced along the longitudinal axis into 4 to 7 slips of equal width, embedded in paraffin, and cut into 4-μm sections. The sections were stained with H&E for morphologic observation and with Genta stain to detect H pylori and mucin-containing cells. Because the inflammatory component of the mucosa was virtually identical to that found in human H pylori gastritis,7Ikeno T. Ota H. Sugiyama A. Ishida K. Katsuyama T. Genta R.M. Kawasaki S. Helicobacter pylori-induced chronic active gastritis, intestinal metaplasia, and gastric ulcer in Mongolian gerbils.Am J Pathol. 1999; 154: 951-960Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar the degree of inflammation and existence of intestinal metaplasia could be graded blindly by one pathologist (R.M.G) according to the Updated Sydney System.13Dixon M.F. Genta R.M. Yardley J.H. Correa P. Classification and grading of gastritis: the updated Sydney system.Am J Surg Pathol. 1996; 20: 1161-1181Crossref PubMed Scopus (4545) Google ScholarH pylori density was also evaluated by histology because only one mm2Yamaoka Y. Kita M. Kodama T. Sawai N. Imanishi J. Helicobacter pylori cagA gene and expression of cytokine messenger RNA in gastric mucosa.Gastroenterology. 1996; 110: 1744-1752Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar piece of gastric mucosa from the antrum was taken to culture H pylori, and the number of H pylori by culture should not be more reliable than scoring the H pylori density by histology. Nuclear extracts from gastric mucosal specimens were prepared using hypotonic/nonionic detergent lysis.14Brasier A.R. Jamaluddin M. Casola A. Duan W. Shen Q. Garofalo R.P. A promoter recruitment mechanism for tumor necrosis factor-α-induced interleukin-8 transcription in type II pulmonary epithelial cells Dependence on nuclear abundance of RelA, NF-κB1, and c-Rel transcription factors.J Biol Chem. 1998; 273: 3551-3561Crossref PubMed Scopus (147) Google Scholar After extraction, nuclear proteins were normalized by protein assay (Bio-Rad, Hercules, CA). Equal amounts (25 μg per sample) were used for the protein/DNA array (TranSignal Arrays, Panomics, Inc., Redwood City, CA). The protein/DNA array provides a profile of DNA binding activity for multiple transcription factors in a single array experiment and allows 54 transcription factors to be identified simultaneously. For quantitation, pooled nuclear extracts were prepared from a mixture of 3 gastric mucosal specimens obtained 9 months after inoculation of WT strains (standard sample). Six array membranes were set up simultaneously, with 1 membrane being used as a control. The density of dots for each transcription factor and the standard sample were scanned using Scion Image beta 4.02 software (Scion Corp. Frederick, MA). In the standard sample, the density of all dots from 54 transcription factors was summed (= density for all). The density of each transcription factor in the test samples was divided by the "density for all" and multiplied by 100. Electrophoretic mobility shift assay (EMSA) was performed using the same nuclear extracts used in the protein/DNA array. Equal amounts of nuclear proteins (10 μg per sample) were used to bind to duplex oligonucleotides corresponding to nuclear factor (NF)-κB, activator protein-1 (AP-1), cAMP responsive element binding protein (CREB), and interferon-stimulated responsive element (ISRE) binding sites. We used commercial consensus probes for these binding sites (Promega Corp, Madison, WI). EMSA was performed using standard methods as previously described.15Yamaoka Y. Kudo T. Lu H. Casola A. Brasier A.R. Graham D.Y. Role of interferon-stimulated responsive element-like element in interleukin-8 promoter in Helicobacter pylori infection.Gastroenterology. 2004; 126: 1030-1043Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar For semiquantitation, we used pooled nuclear extracts from the mixture of 3 WT H pylori-infected gastric mucosal specimens from the gastritis group at 18 months (standard sample). Density was measured by scanning, using the software Image J 1.36 software from the National Institutes of Health (http://rsbweb.nih.gov/ij/), and the density of samples was presented as a percentage of the standard sample. For competition assays, 5 pmol of unlabeled competitor was added at the time of probe addition. In the gel mobility supershift assays, commercial antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) against specific transcription factors were added to the binding reactions and incubated on ice prior to fractionation by 6% PAGE. Immunoblot analysis was performed using standard methods. We used phospho-specific antibodies to detect phospho-p38, phospho-extracellular signal-regulated kinase (ERK), phospho-Jun-N-terminal kinase (JNK), phospho-inhibitor of IκB α, and control antibodies against total p38, ERK, and JNK as well as β-actin (1:1500 dilution) (antibodies for mitogen-activated protein kinase [MAPKs] were from Promega, and antibodies for phospho-IκBα and β-actin were from Cell Signaling Technology, Beverly, MA). These antibodies have been reported to cross-react with Mongolian gerbils.16Walton K.M. DiRocco R. Bartlett B.A. Koury E. Marcy V.R. Jarvis B. Schaefer E.M. Bhat R.V. Activation of p38 MAPK in microglia after ischemia.J Neurochem. 1998; 70: 1764-1767Crossref PubMed Scopus (150) Google Scholar, 17Sugino T. Nozaki K. Takagi Y. Hattori I. Hashimoto N. Moriguchi T. Nishida E. Activation of mitogen-activated protein kinases after transient forebrain ischemia in gerbil hippocampus.J Neurosci. 2000; 20: 4506-4514Crossref PubMed Google Scholar, 18Kawano T. Fukunaga K. Takeuchi Y. Morioka M. Yano S. Hamada J. Ushio Y. Miyamoto E. Neuroprotective effect of sodium orthovanadate on delayed neuronal death after transient forebrain ischemia in gerbil hippocampus.J Cereb Blood Flow Metab. 2001; 21: 1268-1280Crossref PubMed Scopus (86) Google Scholar, 19Koyama M. Spicer S.S. Schulte B.A. Distribution of IκB proteins in gastric mucosa and other organs of mouse and gerbil.J Histochem Cytochem. 2000; 48: 191-200Crossref PubMed Scopus (6) Google Scholar, 20Shibata W. Hirata Y. Maeda S. Ogura K. Ohmae T. Yanai A. Mitsuno Y. Yamaji Y. Okamoto M. Yoshida H. Kawabe T. Omata M. CagA protein secreted by the intact type IV secretion system leads to gastric epithelial inflammation in the Mongolian gerbil model.J Pathol. 2006; 210: 306-314Crossref PubMed Scopus (56) Google Scholar Cytoplasmic extract (10 μg per sample) obtained during the preparation of nuclear extracts was fractionated by SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane. Detection was performed using ECL reagents (Amersham Life Science, Arlington Heights, IL). For semiquantitation, pooled cytoplasmic extracts from the mixture of 3 WT H pylori-infected gastric mucosal specimens with gastritis at 18 months were used as the standard sample. Density was measured by scanning, using Image J 1.36 software (NIH), and the density of samples was calculated as a percentage of the standard sample. Primary gastric epithelial cells were isolated enzymatically from gerbil stomachs using previously established methods.21Tanahashi T. Kita M. Kodama T. Yamaoka Y. Sawai N. Ohno T. Mitsufuji S. Wei Y.P. Kashima K. Imanishi J. Cytokine expression and production by purified Helicobacter pylori urease in human gastric epithelial cells.Infect Immun. 2000; 68: 664-671Crossref PubMed Scopus (79) Google Scholar, 22Lu H. Wu J.Y. Kudo T. Ohno T. Graham D.Y. Yamaoka Y. Regulation of interleukin-6 promoter activation in gastric epithelial cells infected with Helicobacter pylori.Mol Biol Cell. 2005; 16: 4954-4966Crossref PubMed Scopus (80) Google Scholar Briefly, the surface mucosal layer (combined antrum and corpus) was removed with a razor blade, immediately minced, and then incubated in Ham's F-12 culture medium containing collagenase type I (0.2 mg/mL) (Invitrogen) for 10 minutes. Cells from the final incubation were washed and cultured in Ham's F-12 medium supplemented with 10% FBS and streptomycin (300 μg/mL) at 37°C in a humidified 5% CO2 atmosphere. Cultured cells (1 × 106Watanabe T. Tada M. Nagai H. Sasaki S. Nakao M. Helicobacter pylori infection induces gastric cancer in Mongolian gerbils.Gastroenterology. 1998; 115: 642-648Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar cells/mL) formed subconfluent monolayers within 24 hours of inoculation in a 24-well collagen-coated dish. Approximately 93% of cultured cells in the monolayers had periodic acid-Schiff-positive material in the cytoplasm, confirming that the population consisted of mucus-producing epithelial cells with minimal contamination by other cells. Epithelial cells could be maintained up to 1 week in culture. H pylori TN2GF4 were suspended in cell culture medium, and the subconfluent epithelial cells (1 × 106Watanabe T. Tada M. Nagai H. Sasaki S. Nakao M. Helicobacter pylori infection induces gastric cancer in Mongolian gerbils.Gastroenterology. 1998; 115: 642-648Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar cells/mL) were cocultured with H pylori (multiplicity of infection [MOI] of 100) or kept uninfected in a 24-well collagen-coated dish for 3 hours. Cell viability was approximately 90% during the coculturing experiments (data not shown). Nuclear extracts were prepared using hypotonic/nonionic detergent lysis, and equal amounts (10 μg per sample) were used for protein/DNA arrays. Semiquantitation was performed as described previously. In some experiments, we performed luciferase reporter gene assays using primary gastric epithelial cells isolated from gerbil stomachs as described above. Commonly used nonviral transfection methods (eg, Lipofectamine 2000 [Invitrogen]) did not succeed using primary gastric epithelial cells. Therefore, we used a novel nonviral transfection technology specially designed for primary cells and difficult to transfect cell lines, which allows transfected DNA to enter directly the nucleus of nondividing cells within a few hours (Nucleofector; Amaxa Biosystems, Allemagne, Germany). The PathDetect cis-reporting plasmids pNF-κBluc, pAP-1luc, and pCREluc, which contain the luciferase reporter gene driven by the TATA box plus multiple repeats of consensus NF-κB, AP-1, and CRE binding sequences, respectively, were purchased from Stratagene (La Jolla, CA). Luciferase assays were performed using the Dual-Luciferase reporter assay system according to the manufacturer's instructions (Promega). Epithelial cells (1 × 106Watanabe T. Tada M. Nagai H. Sasaki S. Nakao M. Helicobacter pylori infection induces gastric cancer in Mongolian gerbils.Gastroenterology. 1998; 115: 642-648Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar cells/mL) were cultured in a 24-well collagen-coated dish for 24 hours before transfection, and, 6 hours after transfection, H pylori (MOI of 100) were added or kept uninfected. Nine hours after stimulation with H pylori, cells were harvested and lysed using passive lysis buffer (Promega), and the lysates were assayed for luciferase activity. Cell viability was approximately 85% to 90% during the coculturing experiments (data not shown). Normalized luciferase activity was calculated as firefly luciferase activity/Renilla luciferase activity. Luciferase activity is reported as fold increase of luciferase activity in treated cells relative to uninfected or mock-treated controls. Immunohistochemistry was performed for p38, ERK, JNK, and IκBα. We used phospho-specific antibodies to detect phospho-p38, phospho-ERK, phospho-JNK, and phospho-IκBα (Promega and Cell Signaling Technology). Tissues were placed in a microwave oven at high power in 0.1 mol/L citrate buffer, pH 6.5, and treated 4 times for 5 minutes each. This was followed by a 2-hour incubation at room temperature with each antibody diluted 1:50. The immunoperoxidase reaction was developed using the biotin-streptavidin immunoperoxidase method (DAKO, Santa Barbara, CA). Areas of the surface/foveolar epithelium, glands, and submucosal lymphocytes were estimated, and the results were scored 0 to 3: 3, high level of staining (approximately 80%–100%); 2, medium level of staining (25%–80%); 1, low level of staining (1%–25%); and 0, virtually no staining (<1%). Total RNA was extracted from the gastric mucosa using an RNA extraction kit (Qiagen). After DNase treatment, 5 μg total RNA was subjected to reverse transcription (RT) using 200 U Moloney murine leukemia-virus reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD) and 1 μmol/L of oligo(dT)16 primers. We used gerbil-specific IL-6 complementary DNA (cDNA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequences (GenBank accession number AB164706 and Yamaoka,10Yamaoka Y. Kwon D.H. Graham D.Y. A Mr 34,000 proinflammatory outer membrane protein (oipA) of Helicobacter pylori.Proc Natl Acad Sci U S A. 2000; 97: 7533-7538Crossref PubMed Scopus (362) Google Scholar respectively) and gerbil-specific keratinocyte-derived chemokine (KC) and interferon (IFN)-γ cDNA sequences (GenBank accession number: AJ877921 and L37782, respectively). Specific primers and TaqMan probes for KC, IL-6, IFN-γ, and GAPDH were described previously.23Rieder G. Merchant J.L. Haas R. Helicobacter pylori cag-type IV secretion system facilitates corpus colonization to induce precancerous conditions in Mongolian gerbils.Gastroenterology. 2005; 128: 1229-1242Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 24Saito H. Yamaoka Y. Ishizone S. Maruta F. Sugiyama A. Graham D.Y. Yamauchi K. Ota H. Miyagawa S. Roles of virD4 and cagG genes in the cag pathogenicity island of Helicobacter pylori using a Mongolian gerbil model.Gut. 2005; 54: 584-590Crossref PubMed Scopus (19) Google Scholar Real-time PCR was performed as previously described.24Saito H. Yamaoka Y. Ishizone S. Maruta F. Sugiyama A. Graham D.Y. Yamauchi K. Ota H. Miyagawa S. Roles of virD4 and cagG genes in the cag pathogenicity island of Helicobacter pylori using a Mongolian gerbil model.Gut. 2005; 54: 584-590Crossref PubMed Scopus (19) Google Scholar Cytokine messenger RNA (mRNA) levels were expressed as the ratio of cytokine mRNA to GAPDH mRNA (100,000 × cytokine mRNA [unit/μL]/GAPDH mRNA [unit/μL]). Each assay was performed in triplicate. In some experiments, we used RNA extracted from formalin-fixed, paraffin-embedded, tissue blocks. Serial 8-μm-thick sections of selected tissue blocks were placed onto glass slides, and the areas of interest (submucosal infiltrating cells) were microdissected visually after matching with an adjacent section stained with H&E. Microdissected samples were digested with proteinase K, and RNA was purified by phenol and chloroform extraction and reversed transcribed as published.25Specht K. Richter T. Muller U. Walch A. Werner M. Hofler H. Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue.Am J Pathol. 2001; 158: 419-429Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar Statistical analysis was performed using the Mann–Whitney rank sum test and the paired t test depending on the data set. The data are presented as mean ± standard error (SE). A P value of less than .05 was accepted as statistically significant. Mongolian gerbils were inoculated orally with H pylori TN2GF4 or isogenic cagE mutants. H pylori infection was confirmed by culture and/or histology in all cases. All cagE mutants recovered from gerbils were resistant to chloramphenicol, confirming that the chloramphenicol cassette remained functional. The disruption of cagE gene was also confirmed based on the size of the amplicons by PCR. One month after inoculation, infiltration of neutrophils and monocytes into the gastric mucosa, mainly located in the submucosa, was observed in WT-infected gerbils. Infiltration into the stomach appeared to spread from the antrum to the corpus over time, with focal lymphocyte aggregates observed in the submucosa at the glandular border of the pyloric mucosa (antrum) and the fundic mucosa (corpus). Three months after inoculation, inflammation and H pylori density reached maximal levels (Figure 1). Inflammation and H pylori density were more severe in the antrum than in the corpus in each time point (P < .01). Macroscopically, pyloric channel ulcers were present in 1 of 5 gerbils (20%) at 9 months and in 8 of 18 gerbils (44%) at 18 months. Microscopically, intestinal metaplasia was present in 10 of 18 (56%) infected gerbils at 18 months, including 5 gerbils with gastric ulcers. The intestinal metaplasia occurred frequently in the transitional zone between pyloric and fundic mucosa, and Paneth cells were rarely found, indicating that the metaplasia was mostly of the incomplete type. Eighteen months after inoculation, gerbils were divided into 3 groups based on the gross and histologic outcomes. Eight gerbils had gastric ulcers (ulcer group), 5 gerbils had severe atrophic gastritis with intestinal metaplasia, but without ulcers (atrophy group), and 5 gerbils had gastritis without intestinal metaplasia or ulcers (gastritis group). Cellular infiltration in the antrum was more severe in the ulcer group than in the atrophy group (Figure 1). In contrast to a previous study using strain6Watanabe T. Tada M. Nagai H. Sasaki S. Nakao M. Helicobacter pylori infection induces gastric cancer in Mongolian gerbils.Gastroenterology. 1998; 115: 642-648Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar TN2GF4, no infected gerbils developed gastric cancer. Infiltration of
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