Volume 116, Issue 6, Pages 1319-1329 (June 1999) Increased expression and cellular localization of inducible nitric oxide synthase and cyclooxygenase 2 in Helicobacter pylori gastritis  Sidong Fu, Kalathur S. Ramanujam, Annie Wong, George T. Fantry, Cinthia B. Drachenberg, Stephen P. James, Stephen J. Meltzer, Keith T. Wilson  Gastroenterology  Volume 116, Issue 6, Pages 1319-1329 (June 1999) DOI: 10.1016/S0016-5085(99)70496-8 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 RT-PCR analysis of iNOS and COX-2 mRNA expression in gastric tissues. RNA extracted from endoscopic biopsy specimens of the antrum was PCR-amplified with primers yielding the following size products: iNOS, 237 bp; COX-2, 756 bp; COX-1, 462 bp; β-actin, 436 bp. (A) Representative gel of samples from patients with normal mucosa (NL), histological gastritis in the absence of H. pylori, and H. pylori–positive gastritis. M, DNA markers. (B and C) Densitometry of all samples from normal (n = 10), gastritis (n = 13), and H. pylori–positive gastritis (n = 20) tissues for (B) iNOS and (C) COX-2, standardized to β-actin. *P < 0.05, **P < 0.01 vs. normal; and §§P < 0.01 vs. gastritis; Student–Newman–Keuls test. Gastroenterology 1999 116, 1319-1329DOI: (10.1016/S0016-5085(99)70496-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Northern blot analysis of iNOS mRNA expression in gastric tissues. N, normal mucosa; G, gastritis without H. pylori; HP, H. pylori–positive gastritis. Ten micrograms of total RNA per land was electrophoresed on a denaturing formaldehyde agarose gel, transferred to Hybond-N membrane, and hybridized with a specific probe for human iNOS (4.5-kb transcript) followed by a probe for GAPDH (1.8-kb transcript). The blot was washed and exposed to BioMax film at −80°C for 3 days (iNOS) or overnight (GAPDH). Gastroenterology 1999 116, 1319-1329DOI: (10.1016/S0016-5085(99)70496-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 COX-2 protein levels (70-kilodalton band) by immunoblotting in representative gastric tissues. N, normal mucosa; G, gastritis without H. pylori; HP, H. pylori–positive gastritis. In the last two lanes on the right, antrum (A) and body (B) tissues from a patient with H. pylori–positive gastritis are shown. Equal concentrations of protein (100 μg) were loaded in each lane. A monoclonal antibody to human COX-2 was used as described in Materials and Methods. Gastroenterology 1999 116, 1319-1329DOI: (10.1016/S0016-5085(99)70496-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 RT-PCR analysis of iNOS and COX-2 mRNA expression in gastric antrum vs. body in H. pylori–positive gastritis tissues. RNA extracted from endoscopic biopsy specimens was PCR-amplified as in Figure 1. (A) Representative gel of samples from patients with H. pylori–positive gastritis comparing (A) antrum and (B) body pairs. M, DNA markers. (B and C) Densitometry of all patient paired samples (n = 13) for (B) iNOS and (C) COX-2, standardized to β-actin. ***P < 0.0001, antrum vs. body; paired Student t test. Gastroenterology 1999 116, 1319-1329DOI: (10.1016/S0016-5085(99)70496-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Immunohistochemical detection of (A-F) iNOS and (G-L) COX-2 in tissues from gastric antrum. Tissues in A and G are normal mucosa; other tissues are H. pylori gastritis. Staining was performed on formalin-fixed tissues using specific monoclonal antibodies (see Materials and Methods) detected with peroxidase–3,3'-diaminobenzidine (brownish pigment). C, F, I, and L are high-power views (original magnification 600×) of tissues in B, E, H, and K (original magnification 200×). iNOS staining was absent in normal gastric mucosa (A). In H. pylori–positive gastritis, there was iNOS staining of endothelium (B and C, arrows), lamina propria immune cells (B, C, E, and F, *) and epithelium (E and F, arrowheads). D is a serial section from the tissue shown in E and H, which was incubated with mouse IgG rather than primary antibody; the absence of staining validates the specificity of the antibody staining. COX-2 protein is present at a low level in normal gastric mucosa (G) and strongly increased in the lamina propria of gastric mucosa from H. pylori gastritis (H, I, K, and L) with staining of the monocytic cells (open arrows) and myofibroblasts (open arrowheads). H is a serial section from the tissue in D and E, showing the markedly different staining pattern for COX-2 than for iNOS. J is an IgG control replacing primary antibody in a serial section from the tissue in K. Gastroenterology 1999 116, 1319-1329DOI: (10.1016/S0016-5085(99)70496-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions