- Open Access
Identification of novel Cyclooxygenase-2-dependent genes in Helicobacter pylori infection in vivo
- Anna K Walduck†1, 2,
- Matthias Weber†1,
- Christian Wunder†1,
- Stefan Juettner1, 3,
- Manfred Stolte4,
- Michael Vieth4,
- Bertram Wiedenmann3,
- Thomas F Meyer1Email author,
- Michael Naumann†5 and
- Michael Hoecker†3
© Walduck et al; licensee BioMed Central Ltd. 2009
- Received: 08 October 2008
- Accepted: 24 March 2009
- Published: 24 March 2009
Helicobacter pylori is a crucial determining factor in the pathogenesis of benign and neoplastic gastric diseases. Cyclooxygenase-2 (Cox-2) is the inducible key enzyme of arachidonic acid metabolism and is a central mediator in inflammation and cancer. Expression of the Cox-2 gene is up-regulated in the gastric mucosa during H. pylori infection but the pathobiological consequences of this enhanced Cox-2 expression are not yet characterized. The aim of this study was to identify novel genes down-stream of Cox-2 in an in vivo model, thereby identifying potential targets for the study of the role of Cox- 2 in H. pylori pathogenesis and the initiation of pre- cancerous changes.
Gene expression profiles in the gastric mucosa of mice treated with a specific Cox-2 inhibitor (NS398) or vehicle were analysed at different time points (6, 13 and 19 wk) after H. pylori infection. H. pylori infection affected the expression of 385 genes over the experimental period, including regulators of gastric physiology, proliferation, apoptosis and mucosal defence. Under conditions of Cox-2 inhibition, 160 target genes were regulated as a result of H. pylori infection. The Cox-2 dependent subset included those influencing gastric physiology (Gastrin, Galr1), epithelial barrier function (Tjp1, connexin45, Aqp5), inflammation (Icam1), apoptosis (Clu) and proliferation (Gdf3, Igf2). Treatment with NS398 alone caused differential expression of 140 genes, 97 of which were unique, indicating that these genes are regulated under conditions of basal Cox-2 expression.
This study has identified a panel of novel Cox-2 dependent genes influenced under both normal and the inflammatory conditions induced by H. pylori infection. These data provide important new links between Cox-2 and inflammatory processes, epithelial repair and integrity.
- Gastric Mucosa
- Pylorus Infection
- Infected Mouse
- Gastric Epithelial Cell
Helicobacter pylori infection is associated with a variety of gastric disorders including chronic gastritis, peptic ulcer disease, mucosa associated lymphatic tissue (MALT) lymphoma, and gastric adenocarcinoma [1, 2]. The pathogenicity of the bacterium is determined by epidemiological influences as well as bacterial and host factors [1, 3]. Bacterial colonization of the gastric mucosa leads to development of a chronic inflammatory infiltrate, which is accompanied by enhanced release of inflammatory mediators, growth factors and reactive oxygen metabolites [2, 4].
The inducible Cox-2 enzyme and its constitutively expressed isoform Cox-1 are the key regulators of human prostaglandin metabolism [5–7]. The end products of their enzymatic activity comprise a panel of prostaglandins and thromboxanes, which have been identified as critical regulators of fundamental physiological and pathological processes including platelet aggregation, parturition, T-cell development, inflammation and cancer [5–7]. Cox-2 enzymatic activity is largely regulated through de novo synthesis of Cox-2 protein [5, 6].
In the stomach, enhanced Cox-2 expression has been found during H. pylori-triggered gastritis as well as in mucosal stress lesions, gastroduodenal ulcers and after ischemia/reperfusion damage [8–10]. Cox-2 and its related prostanoids also appear to contribute to the pathogenesis of gastric cancer. Gastric adenocarcinoma and premalignant mucosal lesions frequently over-express the Cox-2 gene [11–14], and elevated intratumoral Cox-2 levels seem to be associated with deeper tumor invasion  and an increased frequency of lymphatic metastasis . In addition, Cox-2 inhibitors have been demonstrated to potently suppress proliferation of human gastric cancer cells in vitro [1, 17, 18] as well as experimental gastric adenocarcinomas in nude mice . Recently however, a number of reports have challenged the notion that this anti- tumour activity is due to inhibition of Cox-2 itself . Individuals taking Cox-inhibitors have been reported to display a reduced risk for development of gastric carcinoma , however the reported cardiovascular side effects associated with chronic coxib administration mean that clinical use of Cox- inhibitors for anti-carcinogenic treatment is controversial (Reviewed in).
Expression of the Cox-2 gene therefore appears to be an important step in the pathogenesis of benign and malignant gastric diseases and therefore, clarification not only of its contribution to H. pylori-dependent pathogenesis, but also the downstream effects of Cox inhibiting drugs is of special clinical significance.
We have previously demonstrated that H. pylori can directly influence expression of Cox-2 in gastric epithelial cells through transcriptional mechanisms, and identified MAPK-ERK-dependent activation of a proximal cis-regulatory CRE-Ebox element as a key step in the H. pylori-response of the Cox-2 gene . While these results further confirmed the pathophysiological link between the bacterium and Cox-2, molecular effectors located downstream of Cox-2 during gastric H. pylori infection remained unidentified.
Here we analysed gene expression in the gastric epithelium of mice treated with the Cox-2 specific inhibitor NS398, at different time points after H. pylori infection using DNA microarrays and were able to define gene expression profiles regulated by H. pylori through Cox-2-dependent and independent mechanisms.
Determination of Cox-2 inhibitor concentration
To determine the appropriate concentration of inhibitor in our H. pylori infection model, PGE2 levels were measured in the gastric mucosa after H. pylori infection in the presence or absence of Cox-2 inhibition with NS398. Infected mice showed a 50% increase in PGE2 level in the gastric mucosa. Treatment of infected mice with NS398 (10 mg/kg) led to a reduction in the PGE2 such that it did not differ from the control group (data not shown). We therefore concluded that a dose of 10 mg/kg was sufficient to suppress Cox 2 activity in the presence of a H. pylori infection.
Long term administration of the specific Cox-2 inhibitor NS398 does not significantly affect bacterial colonization or inflammatory scores
Global gene expression in the gastric mucosa of H. pylori infected mice
The RNA used in this study was extracted from the gastric mucosa only, and histological analyses of stomachs prepared in this way have confirmed that muscle or other connective tissue underlying the mucosa were not included in our preparations (not shown). The changes in gene expression seen in this study are therefore highly likely to reflect a transcriptional response restricted to that of gastric epithelial cells and the developing lymphocytic and granulocytic infiltrates which characterise chronic H. pylori infection. Cut-offs for significant changes in gene expression were set at p < 0.05 and three fold change .
In H. pylori infected mice, 385 genes passed the cut- off criteria at at least one time during the study (Figure 2A). H. pylori infected mice that were treated with NS398 had 160 differentially expressed genes. In mice treated with NS398 alone, 140 genes were differentially expressed (Figure 2A). Using a subtractive approach, genes were divided into subgroups reflecting the major experimental effects: H. pylori infection, Cox-2 suppression, and Cox-2 suppression during infection.
-4 & -3
NS398 (10 mg/kg)
Vehicle plus H. pylori (V+ Hp)
NS398 (10 mg/kg) plus H. pylori
Effect of NS398 on gastric gene expression profiles
Previous studies on the Cox-2 inhibitor NS398 in a variety of in vitro and in vivo models have demonstrated it is a specific inhibitor of Cox-2 activity with little or no influence on the closely related, constitutively expressed Cox-1 [26, 27]. In our study, long-term administration appeared to have effects on gene expression that may have been resolved or compensated for after several months, as a large proportion of the genes which were up-regulated after week 6 of administration were not differently expressed after week 13 (Figure 2B and Table 1). Only 33 of the genes that were influenced by NS398 were also regulated in response to infection. A complete list of the genes regulated as a result of NS398 treatment is shown (Additional File 1, Table S3.
Fold change in gene expression of selected genes regulated as a result of infection with H. pylori and/or treatment with the specific Cox-2 inhibitor NS398
V vs V + Hp
V+Hp vs NS398+Hp
V vs Ns398
thymoma viral proto-oncogene 3
protein amino acid phosphorylation
chemokine (C-C motif) ligand 5
deleted in malignant brain tumors 1/crp ductin, muclin
scavenger receptor activity.
heat shock protein 1A
interferon consensus seq. binding protein 1
immune response; transcription regulation
interferon gamma inducible protein
ATP binding activity
lymphocyte antigen 75
major urinary protein 5
pheromone binding activity; transport; transporter activity
prostaglandin-endoperoxide synthase 1
prostaglandin-endoperoxide synthase 2
carboxy- lyase activity
Slc7a11. cd98 light
solute carrier family 7 member 11. CD98 light
cationic amino acid transporter
trefoil factor 1
response to wounding
T-cell specific GTPase
GTP binding activity
Confirmation of expression of selected Cox-2 dependent genes
The mean change in expression level of Cox1 (Ptgs1) and Cox2 (Ptgs2), and a selection of Cox2-dependent genes involved in inflammation (intracellular adhesion molecule 1, Icam1; Transforming growth factor b1,Tgfb1), gastric function (Gastrin, Gast), barrier function (Aquaporin 5, Aqp5; Tight junction protein 1, Tjp1) and proliferation/carcinogenesis (Ornithine decarboxylase, Odc1) was determined for individual mice at all time points by real-time PCR (Additional File 1, Table S4). In 94% of cases the change in expression could be confirmed (up- or down- regulated over the three-fold cut off criteria).
The aim of this study was to enhance our knowledge about the role of cyclooxygenase-2 in H. pylori-triggered mucosal inflammation by identifying new downstream molecular effectors. The in vivo approach taken here has also provided insight at the transcriptome level, into the complex changes that occur as a result of the chronic inflammatory response to H. pylori infection and to Cox-2 inhibition.
Both H. pylori infection, and NS398 treatment elicited unique transcriptional signatures in the gastric mucosa of mice, despite the similarity in pathology scores and colonization density between the different groups (Figure 1). This is of interest because in another report employing a model of mucosa-associated lymphoid tissue lymphoma (MALT lymphoma), transcriptional profiles in BALB/c mice infected with H. heilmannii were determined after 12 to 24 months, and the major changes in gene regulation occurred in the earlier stages of the disease (<12 months, mild to moderate pathology) . After this time, clustering of the 300 most differentially expressed genes allowed segregation of infected mice into groups corresponding almost exactly with their pathological characterization. In view of our data, we would conclude that during the early development phase of chronic active gastritis, standard pathology analysis is not able to detect subtle, but important changes in the mucosa.
NS398 specifically inhibits the activity of the Cox-2 protein, and alterations in Cox-2 gene expression might be expected when infected mice were treated with a specific Cox-2 inhibitor, either as a result of a possible feedback mechanism involving PGE2 , or as a compensatory mechanism to overcome the enzymatic inhibition. No significant change in Cox-2 expression was observed in vivo however, supporting the notion that the expression of the Cox-2 gene in the stomach is controlled by a variety of factors. Cox-2 is expressed by both inflammatory and gastric epithelial cells  and its expression may be controlled by different mechanisms in different cells types.
We were able to identify a subset of genes that were differentially expressed as a result of Cox-2 suppression, highlighting the scope of influence of Cox-inhibitors in gastric inflammation. Cox-2 dependent genes fell into numerous functional categories, chiefly those involved in gastric physiology (acid secretion, motility), epithelial repair and proliferation, and inflammatory mediators.
Gastrin (Gast) is an important mediator in the stomach [30–34] and expression in the mucosa was strongly influenced, not only by infection with H. pylori, but also by suppression of Cox-2 activity (Figure 3, Additional File 1, Table S4). In addition to its role in regulating gastric acid secretion, gastrin has trophic effects and regulates proliferation and repair in the mucosa. Indeed INS-GAS transgenic mice which suffer from hypergastrinemia develop carcinoma after infection with H. pylori . The development of carcinoma is however restricted to males in this model . Other workers have also observed that Cox-2 inhibition influenced gastrin expression in an in vitro colorectal cancer model  and also in H. pylori positive gastric cancer patients .
Whereas expression of the apoptosis mediating growth differentiation factor 3 (Gdf3) and c-Myc (Mycs) genes peaked at week 13, the apoptosis inhibiting gene clusterin (Clu) strongly decreased at this time point. Taken together, the gene expression pattern is suggestive of a shift in the rate of proliferation/apoptosis of the epithelium after 13 weeks of infection as a result of NS398 treatment. Longer-term studies would be required to determine whether this effect continues or recurs. A number of genes which have been previously observed to be over-expressed in tumours (Mycs, Clu), tumor suppressors (Patched, Ptch), or otherwise involved in metastasis or DNA repair: bikunin (Ambp), ornithine decarboxylase gene (Odc), Trefoil factor 1 (Tff1), insulin like growth factor (Igf2)  and DNA repair protein 1 (Ddb1), were also differentially expressed in NS398 treated infected mice (Figure 3). The expression pattern was complex however, and some mediators tended to be enhanced by Cox-2 suppression, while the majority were down-regulated (Figure 3). This probably reflects a regulatory role for Cox-2 as part of a network of control mechanisms for epithelial maintenance. The Odc gene, for example, encodes a key regulatory enzyme in the production of polyamines which are essential for cell proliferation  and has been shown to play a role alongside Cox-2 in the development of atrophic gastritis [42, 43]. It has been proposed that Cox-2 inhibitors may inhibit Odc, and in this way be responsible for the observed anti-proliferative effects of Cox-2 inhibitors . Our observation of decreases in Odc expression as a result of NS398 treated H. pylori infection in both in vivo and in vitro studies (Table 1 and Figure 4C) is in keeping with this notion.
Helicobacter infection is strongly associated with the induction of a strong Th1-type inflammatory response, with high levels of Ifnγ, which induces expression of other inflammatory mediators such as iNOS and Cox-2, and also circulating growth factors such as gastrin . Supporting the notion that Ifnγ plays the key role in attracting and activating lymphocytes [46, 47], we observed that expression of T-cell surface markers Icam1 [48, 49] and Cd86 and ligands (Sell)  peaked at week 13 in infected mice. The interferon dependent GTPases (Igtp, Iigp-pending) regulate the anti-microbial activities of Ifnγ in a STAT1 dependent manner [51, 52], and their expression was dramatically reduced in NS398 treated mice by the end of the study. Overall, inhibition of Cox-2 activity led to a reduced expression of inflammatory mediators between weeks 13 and 19 (Figure 3); interestingly this change was not reflected in the pathology scores. Cox-2 has been shown to modulate the Th1/Th2 balance in inflammatory responses and inhibition of Cox-2 using NS398 led to a polarization of the response of in vitro stimulated human PBMCs toward Th1. The authors postulated that chronic expression of Cox-2 and production of PGE2 results in an inhibition of the effectiveness of the mucosal immune response by enhancing a state of tolerance. The gene expression pattern we observed here is indeed consistent with an effect of Cox-2 inhibition on the inflammatory response (See Figure 3 and Table 1) although the changes in expression of classical Th1/Th2 mediators such as IL-12, I-10 and IL-4 did not differ significantly in our study.
Infection with H. pylori has been reported to damage epithelial integrity and several potential mechanisms for this have been reported (reviewed in ). CagA, a major H. pylori pathogenicity factor, is translocated into epithelial cells via the type IV secretion apparatus [55, 56]. Studies in a canine kidney cell model (MDCK) showed that CagA associates with the tight junction adaptor protein zona occludens 1 (zo-1, mouse homologue -tight junction protein 1, Tjp1) and the junctional adhesion molecule (Jcam or Jam), leading to a long term disruption of epithelial barrier function in vitro . Whilst we observed increased expression of Tjp1 at the transcriptional level in NS398 treated infected mice, Jcam expression was not affected. In addition, an EST with homology to connexin 45 (AV148957, Gja7) and Aqp5 were influenced by H. pylori infection regardless of NS398 treatment (Figure 3). As both connexin 45 and Aqp5 are know to play a role in intercellular transport of water and small molecules, and there is experimental evidence that connexin 45 interacts directly with Tjp1 [58, 59], it would appear that Cox-2 also has a role in the maintenance of water balance in the gastric epithelium. This idea is supported by our in vitro observations of a cox-2 dependent increase in Zo-1 protein expression in MKN28 cells. In contrast to the reports by Amieva et al.  this effect was not related to the CagA status of H. pylori (Figure 4). Barrier function effects in the mouse model are in any case unlikely to be due to the actions of CagA, as although we found that H. pylori SS1 expressed CagA protein, we were not able to detect translocation in either in vitro or in vivo experiments (data not shown). Therefore, it would appear that H. pylori infection has additional mechanisms to influence epithelial integrity. It is also of note that another H. pylori pathogenicity factor, vacuolating toxin (VacA) causes formation of fluid-filled vacuoles in epithelial cells and furthermore, this activity can be inhibited in vitro by NS398 treatment . As the gastric aquaporin Aqp5 is expressed on the lateral and intercellular membranes in the gastric crypts , we speculate that this pore is influenced by changes to tight junction proteins, and that it plays a role in the development of oedema in the epithelium during infection.
A number of published reports have attempted to shed light on gene regulation in H. pylori infection using the microarray approach to study global gene expression in gastric epithelial cells in vitro [61–63] (reviewed in ), reporting a rapid up-regulation of inflammatory mediators and a variety of transcription factors to be the hallmarks of the expression pattern. In our study, none of the proliferation related genes (c-Fos, b-Fos, c-Jun and cyclinD1 (Pcna)) reported by Sepulveda et al., nor those reported by Cox et al. (amphiregulin, Adam10)  were differentially expressed in any group of mice. This probably reflects the differences between the in vitro and in vivo models of infection. The data reported here, and that of other reports [25, 65] strongly suggest that cells of the immune system influence not only this effect but also epithelial integrity, limiting the conclusions that can be drawn from in vitro studies using cell lines in isolation.
Some of the Cox-2 dependent genes from this study have been previously identified as being influenced by Cox-2 or Cox-2 inhibition (Gast, Icam1, Odc), but the remainder are novel and point to new links between Cox-2 and inflammatory processes, epithelial repair and integrity. This study has provided insights into not only the effects of prostaglandin inhibitors on inflammation, but also enabled us to identify novel Cox-2 dependent processes such as a role in epithelial integrity and as a result further targets for its role in transformation. Further investigation of these targets will help us to explain the protective effects of Cox-2 inhibitors and NSAIDs in gastritis patients and aid the development of new treatment strategies.
H. pylori strains
H. pylori strain P12, ΔPAI and ΔVacA were cultured on GC Agar plates as previously described [66, 67]. The mouse adapted H. pylori strain (SSI) (kind gift from A. Lee, University of Sydney, Australia) were cultured on GC Agar plates as previously described . For infection, bacteria were scraped off plates and grown overnight in liquid culture in Brain Heart Infusion (BHI) broth .
Detection of prostaglandin E2 in stomach after daily administration of NS398
After infection with a single oral dose of 1 × 109 of a mouse adapted strain of H. pylori (SS1) suspended in sterile PBS, female C57 BL/6 mice (n = 18) then received daily subcutaneous injections of either vehicle (1% Tween 80) or 10 mg/kg of NS398 (Merck) in 1% Tween 80 for 14 days. On day 14, mice were euthanized and the stomach was removed, opened, washed in ice cold PBS, and frozen in liquid nitrogen. The gastric mucosa was scraped off and pulverized in liquid nitrogen. The material was weighed, suspended in 0.1 M phosphate buffer, vortexed for 1 min, and then clarified by centrifugation at 10 000 g for 15 min at 4°C. The supernatant was purified using a Prostaglandin E2 Affinity Column (Cayman) according to the manufacturer's instructions. The amount of prostaglandin E2 was determined by enzyme immunoassay (PGE2 EIA kit; Cayman).
Effect of administration of NS398 on inflammation in chronic H. pylori infection
Function of selected Cox-2 dependent genes shown in Figure 3
Receptor for galanin, pain perception, inhibits basal and gastrin-induced acid secretion
Tight junction protein, epithelial integrity. H. pylori interaction
Regulator of cell growth and differentiation
Inhibits plasminogen activator. important role in cell matrix degradation. gastrin dependent
Cell fate. tumor suppressor
Oncogene. mediates apoptosis
Protects mucosa from insults. stabilizes mucus layer. epithelial healing. suppresses proliferation
Regulates anti-microbial activities of IFNγ in a STAT 1 dependent manner
Tissue scavenger of leukocyte elastase
Regulation of gastric acid secretion. Growth factor activity in gastric mucosa. Selective inhibition of Cox-2 reverses trophic activity.
Heat shock protein, chaperone.
Mediates lymphocyte migration.
Mediates lymphocyte migration
Inhibits apoptosis, levels elevated in both mouse and human tumours
In concert with TLR4, controls B cell recognition and signalling of lipopolysaccharide (LPS)
Suppresses urokinase expression and tumour metastasis
DNA repair protein
Anti microbial peptide
Preparation of stomach tissue
Mice were killed and the stomach was removed and cut along the greater curvature into two tissue fragments encompassing antral and oxyntic mucosa, as well as part of the non-secretory epithelium. The stomach was cut longitudinally into 3 parts. One third of the tissue was frozen immediately in liquid N2. One third was weighed, homogenized and then serially diluted and plated out on Blood Agar plates (blood agar base No.2, 10% defibrinated horse blood, Vancomycin, Amphotericin B, Polymyxin B, Bacitracin, Nalidixic acid) to determine the number of colony forming units (cfu) of H. pylori. The remaining 1/3 of tissue was fixed in 4% buffered paraformaldehyde and processed for histological analysis. Tissue was embedded in paraffin and 2–4 μm sections were stained with hematoxylin and eosin, plus Warthin Starry stain and assessed by 2 independent pathologists (M.S & M.V) blinded to the experimental design. Pathology and colonization were scored according to the modified Sydney System .
RNA isolation and quality control
RNA was isolated from 1/3 the gastric mucosa by scraping tissue over liquid nitrogen as previously described . RNA from 3 mice with similar colonization levels were selected and pooled for microarray analysis.
In situ Oligonucleotide Arrays
A custom oligonucleotide glass array of specific 60 mer oligonucleotides representing 8187 mouse genes was designed based on Unigene Clusters (Unigene build#148), and produced by Agilent Technologies (Palo Alto, USA). Oligonucleotide probes were synthesized and deposited by Agilent using in situ ink-jet printing technology as proposed by Blanchard et al. .
Labeling and hybridization to arrays
RNA (5 μg) from each pool was amplified and labelled as described previously . Pairs of target RNAs were mixed and hybridized to arrays to perform 3 experimental comparisons i.e. infected versus non infected, infected only versus NS398 treated and infected and non infected versus non infected and NS398 treated (Figure 2A). After data extraction and normalization  data from duplicate hybridizations were combined and expressed as log ratios or fold changes. Statistical and bioinformatic analysis was performed using the Rosetta Resolver™ software package (V 3.2 (build 184.108.40.206.40)), Rosetta Biosoftware, Kirkland, USA).
Culture of gastric cell lines, and in vitro infection experiments
MKN28 cells (ATCC, Rockville, USA) were cultured in RPMI 1640 (Gibco BRL, Eggenstein, Germany) medium containing 10% foetal calf serum (FCS, Invitrogen, Carlsbad, Germany) using standard methods. H. pylori strains P12, P12-PAI and P12-VacA were added to epithelial cells at a multiplicity of infection (MOI) of 100. Western blots were performed as described previously . Proteins were transferred to a PVDF membrane (Perkin Elmer, Jügesheim, Germany) and detected using specific antibodies (Cox-2, Cayman, MI, USA; Zo1; Odc and Aquaporin 5, Santa Cruz, CA, USA,). An HRP-labelled secondary goat anti-rabbit antibody was used (diluted 1:3000) and the Renaissance Western Blot system (ECL, Eschwege, Germany) according to the manufacturer's recommendations.
Real-Time PCR assay
Total RNA was isolated from tissue as described above and subsequently used for cDNA synthesis. Real-Time quantitative RT-PCR analyses for Cox-2 sense 5'-AGA AGG AAA TGG CTG CAG AA-3, antisense 5'AGG TGC TCG GCT TCC AG TAT); Aqp5 sense 5'GGC CCT CTT AAT AGG CAA CC-3, antisense 5'TTG CCT GGT GTT GTG TTG TT-3; Gast sense 5' ACC AAT GAG GAC CTG GAA CA-3, antisense 5'CAT CCA TCC GTA TGC TTC CT-3; Tjp1 sense 5'-TCC ACC TCT GTC CAG CTC TT-3', antisense 5'-CAC CGG AGT GAT GGT TTT CT-3'; Icam1 sense, 5'-TTC ACA CTG AAT GCC AGC TC-3' antisense 5'-GCC ACA GTT CTC AAA GCA CA-3'; Odc sense 5'-TTG CCA CTG ATG ATT CCA AA-3', antisense 5'-AGC CAC CAC CAA TAT CAA GC-3' and TGFβ sense 5'-ACC TTC TGA TCC ATC GGT TG-3' TGFβ antisense 5'-TTC CTG TTG GCT GAG TTG TG-3' cDNA were performed using the ABM PRISM 7700 Sequence Detection System instrument and software (PE Applied Biosystems, Inc., Foster City, CA) and SYBR Green PCR Master mix (Applied Biosystems) with thermocycler conditions recommended by the manufacturer. PCRs were performed in duplicate in a total volume of 30 μl containing 10 μM primers. Data were normalized to HPRT (hypoxanthine phosphoribosyl transferase) expression to perform relative quantifications (Primers: sense 5'-GTT GGA TAC AGG CCA GCA TTT GT-3', antisense 5'-CAC AGG ACT ACT AGA ACA CCT GC-3'). Relative gene expression was calculated using the method described by Pfaffl  and expressed as fold change.
The authors wish to thank Marina Drabkina, Jörg Angermann, Frauke Schreiber, Dagmar Frahm, Kirsten Hoffmann and Annette Dietrich for excellent technical assistance. We are indebted to Yevhen Vainshtein and Hans Mollenkopf for their contributions to the design of the mouse oligonucleotide array and Claudia Scheppers for maintenance of the Resolver database. The support and co-operation of Götz Frommer and Andreas Rhülman of Agilent Technologies, and proofreading of the manuscript by Lesley Ogilvie are also gratefully acknowledged. This work was funded by the Max-Planck Society Tandem Project 'Significance of Helicobacter pylori induced COX-2 gene expression'.
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