H pylori NCTC11639 strain was stored at -70°C in our department. Bacteria were routinely cultured on Columbia agar plates supplemented with 10% defibrinated sheep blood, 0.004% triphenyltetrazolium chloride, and Dent selective supplement (Oxoid, Basingstoke, UK) at 37°C for 3 d under a microaerophilic atmosphere containing 50 mL/L O₂, 150 mL/L CO₂ and 800 mL/L N₂. Several colonies were then picked up and inoculated into 20 mL of Brucella broth (Becton Dickinson, Cockeysville, MS) containing 0.1% β-cyclodextrin supplemented with 5% (v/v) fetal calf serum. After 24 h, 2 mL of culture was transferred to 40 mL of fresh medium, and the same process was repeated twice. Finally, 1 mL of the incubated medium containing the bacterial cells, most of which were spiral rather than coccoid, was plated on Brucella agar (Becton Dickinson) containing 10% (v/v) defibrinated sheep blood and cultured at 37°C for an additional 3 d in a microaerophilic atmosphere containing 50 mL/L O₂, 150 mL/L CO₂ and 800 mL/L N₂. Bacterial cells were harvested, washed twice with cold phosphate-buffered saline (PBS, 25 mmol/L sodium phosphate, pH 7.2, 0.9% NaCl), and then sedimented by centrifugation at 5000 ×g for 10 min at 4°C. The cell pellet was stored at -80°C.

Human gastric epithelial cells (SGC7901) were cultured at 37°C in RPMI-1640 (Gibco, USA) containing 10% FBS (Gibco) in a humidified atmosphere containing 50 mL/L CO₂, and plated at 10⁶ cells/well in 24-well plates. The medium was changed every 3 d and replaced with RPMI-1640 without serum before experiment.

H pylori infection was diagnosed by histological examination of endoscopic biopsy specimens and CLO testing. Forty-three serum samples were collected from patients with gastric cancer at Southern Hospital, Guangzhou, China. The age of patients ranged 27-83 years. Twenty-eight serum samples were also collected from patients with peptic ulcer or chronic gastritis at Southern Hospital. The age of patients ranged 28-67 years. Finally, 89 serum samples were collected from healthy blood donors at the age of 18-70 years.

Genomic DNA of H pylori was prepared using a Takara kit (Takara, Japan) according to its manufacturer’s instructions. The extracted genomic DNA was then used as a template for amplification of the NAP coding region using a Taq DNA polymerase PCR kit (Takara, Japan)[2223]. Two primer sequences corresponding to the 5′ and 3′ ends of the coding gene, including EcoRI and XhoI restriction sites, were used: P1: 5'CCGGAATTCATGAAAACATTTGAA-3′, P2: 5'CCGCTCGAGTTAAGCCAAATGGGC-3′.

The PCR product was cloned into the expression vector pGEX-4T-1 (Amersham Biosciences). The plasmid was then transformed into E. coli strain TOP10 (Invitrogen BV, Leek, The Netherlands), and NAP expression was induced with 1 mmol/L isopropyl-β-d-1-thiogalactopyranoside when the cells were grown to the log phase at room temperature. After 4 h, the cells were harvested by centrifugation and washed with ice-cold PBS containing 5 mmol/L EDTA and 2 mmol/L PMSF. All subsequent procedures were performed at 4°C. The NAP-GST fusion protein was purified by glutathione-sepharose 4B column chromatography.

Anti-H pylori IgG antibodies in serum samples were assayed by indirect ELISA using a diagnostic kit (BIOCUP, Shenzhen, China). According to the instructions, serum (l00 &mgr;L, diluted 1/100 in PBS) was added to ELISA plates pre-coated with purified H pylori antigens in duplicate and incubated for 1 h. Controls consisted of wells with PBS alone, H pylori negative and positive serum, which were considered blank, negative and positive controls, respectively. Peroxidase-conjugated anti-human IgG (1/5000) was added and incubated for 30 min, after which the plates were washed with PBS, and color reaction was initiated by the addition of TMB (100 &mgr;L). After 10 min, the reaction was terminated by the addition of 1 mol/L H₂SO₄ (100 &mgr;L). The plate reader was calibrated to the blank well and the absorbance at 450 nm was read. Cut-off value (C.O.) = 2.1 × Nc (Nc = the mean absorbance value for three negative controls). Samples with absorbance > 0.21 were considered positive. The anti-H pylori IgG seropositive individuals were considered to be H pylori-infected healthy individuals.

Recombinant HP-NAP was prepared and purified, and ELISA was carried out as previously described[24–27]. Briefly, immunoplates (Nunc, Denmark) were coated with rNAP (5&mgr;g/well) and incubated overnight at 4°C. After washed three times with a washing buffer containing PBS (pH 7.2) and 0.1% Tween 20, the plates were blocked with 200 &mgr;L of 10% bovine serum in PBS and incubated in a moist chamber for 1 h at 37°C, then washed three times with a washing buffer containing PBS (pH 7.2) and 0.1% Tween 20. One hundred microlitre of serum samples (1:100) from patients or healthy individuals was then added to the microtiter wells, and the plates were incubated at 37°C for 60 min. After washed with PBS, 100 &mgr;L of secondary antibody (goat anti-human IgG-HRP, 1:10 000) was added to each well and incubated for 60 min at 37°C. After washed with PBS, 100 &mgr;L of TMB/H₂O₂ substrate was added to the wells and incubated at room temperature for about 10 min. The reaction was terminated by adding 100 &mgr;L of 2 mol/L H₂SO₄. The absorbance of each well was read at 450 nm. Samples with absorbance > 0.78 were considered positive. Each sample was tested in duplicate.

A receiver operating curve (ROC) was plotted to calculate the cut-off values for antibodies against HP-NAP at a 95% accuracy level[26]. In addition, the cut-off values were determined by mean plus 2 × SD and mean plus 3 × SD, derived from P values in healthy individuals.

Genomic DNA of 20 H pylori clinical isolates was prepared and used as a template for amplification of the NAP coding region with two primer sequences as preciously described[28]. PCR was carried out in a final volume of 60 &mgr;L. A preliminary denaturation step at 95°C for 5 min was followed by 30 amplification cycles, each consisting of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 45 s, and a final extension at 72°C for 7 min. Then, 10 &mgr;L of amplicon was transferred onto a 1.5% (w/v) agarose gel in 1 × TBE buffer, and electrophoresis was performed at 80 V for 30 min in a DNA submarine plate. The gel was then stained with ethidium bromide.

Before use, human gastric epithelial cells (SGC7901) growing in 24-well plates were washed and replaced with RPMI-1640 without serum, and then stimulated with 10 &mgr;g rHP-NAP for 12 h and 36 h, respectively. The supernatant was centrifuged to remove particulate debris and stored in aliquots at -70°C. IL-8 and GRO_α concentrations in the culture supernatant were measured by ELISA using a Quantikine immunoassay kit (R&D, USA) according to its manufacturer’s instructions. Before detection, the culture supernatant was filtered through a sterile membrane filter (0.22 &mgr;m. pore size).

Statistical analyses were conducted using the SPSS software package. One-way ANOVA was performed to assess differences among groups. Post hoc multiple comparisons between different infectious groups were done by LSD analysis. Comparisons between the seropositivity of anti-HP-NAP antibodies in patients with gastric cancer and other gastroduodenal diseases were done by chi-square test.

The gene encoding for HP-NAP was obtained by PCR amplification using genomic DNA extracted from H pylori as a template. Agarose gel electrophoresis analysis of the PCR product is shown in Figure 1.

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Figure 1 Agarose gel electrophoresis of napA gene amplified by PCR. Lane 1: DL2000 DNA marker; lane 2: H pylori napA gene (435 bp).

A 435 bp DNA fragment was detected, which corresponded to the size of the HP-NAP gene. The DNA sequence of the hypothetical HP-NAP gene of H pylori was determined (data not shown) and submitted to GenBank (accession No. DQ341279). To facilitate purification, HP-NAP was expressed as a GST fusion protein in E. coli Top10 cells. Expression of the recombinant NAP-GST was examined by SDS-PAGE. A pronounced band with an approximate molecular weight of 44 kDa appeared in the supernatant of cell lysate after induction but not in control cells, suggesting that the fusion protein can be successfully expressed in bacterial cells (Figure 2).

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Figure 2 SDS-PAGE analysis of the expression of pGEX-4T-1/HP-NAP in E. coli. Lane 1: Protein marker; Lane 2: pGEX-4T-1 before induction; Lane 3: pGEX-4T-1 after induction; Lane 4: pGEX-4T-1/HP-NAP before induction; Lane 5: pGEX-4T-1/HP-NAP after induction; Lane 6: Supernatant of E. coli after induction; Lane 7: Lysate of E. coli after induction.

The purified NAP-GST fusion protein was further confirmed by Western blotting. Serum from H pylori-infected patients specifically recognized the recombinant NAP-GST fusion protein, while negative serum did not (Figure 3).

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Figure 3 Western blotting analysis of HP-NAP reactivity with the positive serum from H pylori-infected patients. Lane 1: Serum from a control patient not infected with H pylori; Lane 2: Serum from a patient infected with H pylori; M: Protein marker.

A cut-off value of 0.78 was determined for ELISA of HP-NAP antibodies using ROC analysis (Figure 4). The area under the ROC curve was 0.97. Overall, ELISA yielded a sensitivity of 95.5% (95% confidence interval) and a specificity of 91.5% (95% confidence interval) for the detection of antibodies against HP-NAP in serum.

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Figure 4 Receiver operating characteristic curve analysis showing the cut-off value for anti-HP-NAP antibodies in serum from patients with gastric cancer, peptic ulcer, chronic gastritis and healthy controls, respectively.

Anti-H pylori IgG seropositivity was detected in 47 of the 89 healthy subjects who were considered H pylori-infected healthy individuals. As shown in Table 1, the anti-H pylori IgG and anti-HP-NAP seroposivity in healthy individuals was 52.8% (47/89) and 14.6% (13/89), respectively. The anti-H pylori IgG seropositivity was much higher than the anti-HP-NAP antibody seropositivity (P < 0.005). The anti-HP-NAP antibody seropositivity, however, was 27.7% (13/47) in H pylori-infected healthy individuals.

The seropositivity for antibodies against HP-NAP in gastric cancer and peptic ulcer patients was 97.7% (42/43) and 92.8% (26/28), respectively, while the seropositivity for antibodies against HP-NAP in chronic gastritis patients was 85.7% (24/28). The seropositivity for antibodies against HP-NAP in infected healthy controls was 27.7%. The seropositivity for HP-NAP-specific antibodies in gastric cancer patients was higher than that in chronic gastritis patients (P < 0.05) and in infected healthy controls (P < 0.01). The difference in the mean absorbance value for antibodies against HP-NAP between groups was also confirmed by one-way ANOVA analysis (F = 4.014, P = 0.023). The mean absorbance value for anti-HP-NAP antibodies in gastric cancer patients was 1.01 ± 0.24 (range 0.748-1.269), significantly higher than that in chronic gastritis patients (0.89 ± 0.14, range 0.711-1.122, P < 0.005) and in infected healthy controls (0.65 ± 0.18, range 0.451-0.948, P < 0.001). There was no significant difference, however, in the mean absorbance value for anti-HP-NAP antibodies between patients with gastric cancer (1.01 ± 0.24, range 0.748-1.269) and peptic ulcer (0.98 ± 0.32, range 0.771-1.265) (Table 2).

Box plots of the antibodies against HP-NAP in sera from patients with gastric cancer, peptic ulcer, chronic gastritis, and healthy controls are shown in Figure 5, with the 90th percentile range and 75th and 25th percentiles.

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Figure 5 Box plot demonstrating the level of antibodies against HP-NAP in serum from patients with gastric cancer, peptic ulcer, chronic gastritis and healthy controls, respectively. The box plot showing the 5th and 95th percentiles (bars), the 75th and 25th percentiles (boxes), and the median (bars in boxes), respectively. n: Number of individuals in each group.

Most H pylori-associated diseases occurred in subjects at the ages of 40-70 years (53/63). The mean absorbance value for anti-HP-NAP antibodies was not significantly different in patients at different ages (Figure 6).

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Figure 6 Comparison of HP-NAP antibody levels in different age groups.

The napA gene was detected by PCR in 20 H pylori clinical isolates. All examined H pylori strains carried the napA gene. The electrophoresis results of PCR products of the napA gene from 8 representative H pylori strains are shown in Figure 7.

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Figure 7 Agarose gel electrophoresis analysis of PCR product of napA gene from H pylori clinic strains. Lane 1: DL2000 DNA marker; Lanes 2-9: napA gene (435 bp) from eight H pylori clinic strains.

The IL-8 and GRO_α levels in culture supernatant of human gastric epithelial cell line SGC7901 were measured by ELISA, showing that HP-NAP slightly up-regulated the IL-8 production (Table 3) but had no effect on GRO_α production by gastric epithelial cells (data not shown).

Helicobacter pylori (H pylori) infection increases the risk of developing peptic ulcer, gastric adenocarcinoma, and gastric B cell lymphoma. H pylori neutrophil-activating protein (HP-NAP), a virulence factor, promotes the adherence of neutrophils to gastric mucosa endothelial cells and stimulates high production of reactive oxygen species (ROS) in neutrophils. Since ROS can mediate DNA damage and enhance cell turnover, HP-NAP may be a risk factor for H pylori-associated gastric cancer. To prevent H pylori-related cancer, mass screening for and treatment of H pylori infection are cost-effective. Whether HP-NAP is related to the occurrence of gastric cancer is still uncertain.

HP-NAP is a virulence factor for H pylori infection and a vaccine candidate antigen. The majority of infected patients have antibodies against this antigen, and vaccination of HP-NAP in mice can protect against a subsequent challenge with H pylori. H pylori colonization is followed by infiltration of neutrophils, macrophages and lymphocytes in gastric mucosa. The degree of mucosal damage is closely correlated with the extent of neutrophil infiltration. It has been shown that HP-NAP can be involved in the extravasation of leukocytes, and ROS can play a role in the carcinogenic process of gastric mucosa during chronic H pylori infection.

An understanding of the relation between HP-NAP and gastric cancer should lead to improved approaches to the effective control of H pylori-associated gastric cancer. Seropositivity and mean absorbance value for HP-NAP-specific antibodies in gastric cancer patients were significantly higher than those in chronic gastritis patients and in infected healthy controls. There was no difference, however, in serum positivity or mean absorbance value for HP-NAP antibodies between patients with gastric cancer and peptic ulcer. These findings indicate that HP-NAP specific antibodies are correlated with severe gastroduodenal diseases and HP-NAP may contribute to the pathogenesis of H pylori-associated stomach cancer.

ELISA used for the detection of antibodies against HP-NAP in this study had a sensitivity of 95.5% and a specificity of 91.5%. Since the level of HP-NAP-specific antibodies may be correlated with the severity of H pylori infection, ELISA can be used to screen for a high-risk population with H pylori-associated gastric cancer.

H pylori, a gram-negative microaerophilic bacterium, causes a long-term mild inflammation of stomach lining and is strongly linked to the development of duodenal and gastric ulcers and stomach cancer. Over 50% of the world’s population harbor H pylori in their upper gastrointestinal tract, and infection is more prevalent in developing countries.

In this study, the authors detected and evaluated the level of antibodies against HP-NAP in patients with gastric cancer and other gastroduodenal diseases. The results suggest that HP-NAP-specific antibodies are correlated with severe gastroduodenal diseases and HP-NAP may contribute to the pathogenesis of H pylori-associated stomach cancer. ELISA can be used to screen for a high-risk population with H pylori-associated gastric cancer.