Differences in Surface-Exposed Antigen Expression between Helicobacter pylori Strains Isolated from Duodenal Ulcer Patients and from Asymptomatic Subjects

Patients and AS.Twenty-one patients suffering from active or inactive DU disease (15 males; mean age 54 years, range 26 to 84 years) and 20 AS (14 men; mean age 50 years, range 29 to 60 years) participated in this study. The DU patients were recruited from patients seeking care for gastroduodenal disease at the Sahlgrenska University Hospital in Göteborg, Sweden, and the AS were recruited from healthy, age-matched blood donors with no symptoms of dyspepsia at the blood bank of the Sahlgrenska University Hospital as previously described (20). Informed consent was obtained from all patients and AS who participated in the study, and the protocol was approved by the Ethical Committee of the Medical Faculty, Göteborg University, Sweden. All subjects were screened for the presence of H. pylori-specific antibodies in serum as described (19), and AS were recruited based on positiveH. pylori serology. Infection was confirmed by culturing of biopsy specimens provided by gastroscopy (20).

The ABO blood group status of each subject was analyzed by a serological test in which equal volumes of serum and erythrocytes of known ABO blood groups were mixed for 10 min. Serum antibodies agglutinating specific blood groups were recorded, and the relationship between the ABO blood group status and the expression of BabA on the best-growing H. pylori strain in the antrum and the duodenum was analyzed for each subject.

Design of study.Initial screening for the presence of specific H. pylori antigens was determined by enzyme-linked immunosorbent assay (ELISA) on H. pylori isolated from one of the two antral biopsy specimens (except for the BabA, which was analyzed on H. pylori isolated from each of the two antral biopsy specimens) and on all H. pylori isolates that were recovered from four to five different duodenal biopsy specimens collected from each subject. Some of the antigens positive in the qualitative analyses were also tested for quantitative analyses, i.e., the concentrations of the HpaA, flagellins, urease, and the 26-kDa protein were determined on H. pylori strains isolated from one of the two antral strains isolated from each subject by using inhibition ELISA. Possible relationships between the presence of specific antigens on H. pylori and active or chronic inflammatory score in the duodenum were based on analyses of the best-growing strain from either type of the four to five duodenal biopsy specimens from each subject.

Culture conditions for H. pylori and reference strains.Each biopsy specimen was stored in 1 ml of saline for approximately 1 h after gastroscopy and was thereafter analyzed for growth of H. pylori by disintegrating the tissue with a homogenizer (Ultra-Turrax T25; IKA-Laboratechnik) at a rate of 8,000 to 9,500 rpm for 20 to 40 s. H. pylori strains were cultured on 8.5% horse blood Columbia agar plates and selective agar plates supplemented with 3 μg of vancomycin per ml, 5 μg of trimethoprim per ml, 10 μg of nalidixic acid per ml, and 10 μg of nystatin per ml and were incubated under microaerobic conditions at 37°C for 3 to 6 days and stored in a specific freeze-drying medium at −80°C.

The following reference strains positive and, in some instances, negative for the different antigens were included in the study:H. pylori CCUG (Culture Collection, Göteborg University) 17874, a spontaneous H. pylori urease-negative mutant (provided by G. I. Perez-Perez, Nashville, Tenn.), H. pylori strains G21 and G39 (provided by N. Figura, Siena, Italy),H. pylori strains P466 and MO19 (provided by J. Çelik, Karolinska Institute, Stockholm, Sweden), H. pylori E32,Helicobacter felis CCUG 28540, and Helicobacter nemestrinae CCUG 32350. Two Escherichia coli reference strains, E1392 and WM73494, were cultured on 5% horse blood agar plates and incubated at 37°C for 24 h under aerobic conditions.

Microbiological examination of H. pyloristrains.The typical rod-shaped morphology of H. pyloristrains was identified by phase-contrast microscopy. The activities of urease (10), catalase (Merck, Darmstadt, Germany), and oxidase (Sigma-Aldrich Co., Tyresö, Sweden) were measured on allH. pylori strains. Growth of H. pylori was confirmed by using a specific monoclonal antibody (MAb) against HpaA in a dot blot method, as previously described (5).

H. pylori antigen preparations.Whole membrane proteins were prepared from selected H. pylori strains, CCUG 17874 and Hel 305, by sonication followed by differential centrifugation as described previously (1). These strains were selected since they had previously been shown to be positive for most of the antigens analyzed by ELISA and in other immunological analyses. NAP (15, 17), HpaA (5, 14), and the 26-kDa proteins (28) were recombinantly produced in E. coli. E. coli cells were transformed with an expression vector containing the H. pylori napA gene under the control of the T7 promoter. Cells were disrupted, and the soluble proteins were applied to a Q-Sepharose column (Pharmacia BioTech, Uppsala, Sweden) at pH 8.0 in a buffer containing 25 mM Tris-HCl and 50 mM NaCl. The recombinant NAP (rNAP) was found in the nonbinding fraction. The fractions containing the rNAP were concentrated and purified by gel filtration on a Superdex 200 column (Pharmacia). The rNAP eluted as a single peak. The recombinant HpaA (rHpaA) protein was purified fromE. coli cells transformed with an expression vector containing the H. pylori hpaA gene under the control of the T7 promoter. Cells were dissolved with 1.5% Triton X-114 (TX-114; Sigma) and were subjected to two-phase partitioning (6). The detergent phase was diluted to approximately 1% TX-114 and was applied to a Q-Sepharose column (Pharmacia) at pH 8.0 in a buffer containing 50 mM Tris, 2 mM EDTA, 50 mM NaCl, and 0.1% Triton X-100. The rHpaA was found in the nonbinding fraction. The fractions containing the rHpaA were pooled and applied to a new Q-Sepharose column (Pharmacia) at pH 8.6, and the rHpaA was collected by isocratic elution with a buffer containing 10 mM Tris, 2 mM EDTA, 1 M NaCl, and 0.1% Triton X-100, pH 8.6.

Flagellins (subunits FlaA and FlaB), kindly provided by I. Bölin, were purified from strain E32 as previously described (23). The flagellin fraction was further purified by flow pressure low chromatography fractionation on a Resource Q column (Pharmacia). Urease was purified from strain E32 by using a modified version of the method described by Dunn et al. (13) and Evans et al. (16). The positive fractions were pooled, dialyzed against phosphate-buffered saline (PBS), filtered through a 0.45-μm-pore-size membrane, and purified by flow pressure low chromatography.

The purity of the proteins was confirmed by Coomassie staining of material that had undergone sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoblotting, using rabbit polyclonal antisera against whole H. pylori bacteria as well as MAbs reacting specifically with the respective protein (24).

Production of MAbs.The properties of the manufactured and in-house MAbs used in the study are shown in Table1. MAbs against Lewis antigens were purchased (Signet Laboratories, Inc., Amsterdam, The Netherlands) and also provided as a gift from B. Appelmelk, Amsterdam, The Netherlands. The MAbs specific to the 26-kDa protein, the HpaA, and the flagellin FlaA and FlaB subunits of H. pylori have previously been described (5, 24).

Qualitative determination of H. pylori antigens.The presence of NAP, a 26-kDa protein, HpaA, urease, flagellins, and Lewis blood group antigens (Le^x, Le^y, Le^a, Le^b, and sialyl-Le^x) on the different H. pylori isolates was determined by optimized whole-cell ELISAs as previously described (29). Briefly,H. pylori isolates were grown on 8.5% horse blood Columbia agar plates, resuspended in PBS at a final concentration of ∼2 × 10⁹ bacteria/ml (the lower limit for detection of the antigens adjusted according to standard curves for the individual antigen). Thereafter, 100 μl of the bacterial suspensions was applied to each well of a 96-well polystyrene plate as a solid-phase antigen and was incubated at 4°C overnight. After blocking with 1% bovine serum albumin (BSA) in PBS at 37°C for 30 min and washing two times with PBS, the different MAbs described in Table 1 were diluted in 1% BSA–PBS–0.05% Tween to the lowest concentration giving clear cutoff absorbance values for positive and negative antigen expression, were added to the wells, and were incubated at 37°C for 90 min. After washing, the plates were developed with horseradish peroxidase-conjugated anti-mouse immunoglobulin M (IgM) and IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.), followed by addition of H₂O₂ ando-phenylenediamine in sodium citrate buffer (pH 4.5), and were read at 450 nm. All H. pylori samples were run in duplicate on two different occasions. The same number of bacteria and concentration of MAb were used when testing samples and the negative and positive controls. Titers were determined as the reciprocal dilution giving an absorbance of 0.5 above background (i.e., slightly above the mean absorbance value plus three times the standard deviations of negative control samples).

Analyses of H. pylori BabA using Le^boligosaccharide probe.Agar-grown H. pylori strains from the antrum and duodenum (H. pylori isolated from two antral biopsy specimens and one to three duodenal biopsy specimens) were directly transferred to nitrocellulose filters probed with biotinylated Le^b oligosaccharides and incubated with streptavidin-alkaline phosphatases as previously described (9). The filters were developed by using the 5-bromo-4-chlor-3-indolylphosphate substrate and the nitroblue tetrazolium color reagent (BCIP/NBT tablets; Sigma). The intensity of the color reaction was observed by eye, and results were compared with those positive and negative H. pylori control strains, as well as E. coli controls.

Quantitative determination of H. pylori antigens by inhibition ELISA.The concentrations of HpaA, flagellins, urease, and the 26-kDa protein in different H. pylori isolates were measured by an optimized inhibition ELISA (24). The lower limit for detection of H. pylori antigens studied varied between 2 and 25 μg for individual antigens. Plates were coated with one of the following antigen preparations: whole membrane proteins of CCUG strain 17874 (25 μg/ml), 26-kDa protein (10 μg/ml), urease (2 μg/ml), or a crude flagellin preparation (2 μg/ml) and incubated at room temperature (RT) overnight. Suspensions of frozen H. pylori bacteria (∼2 × 10¹⁰ bacteria/ml) were serially diluted threefold in 0.1% BSA-PBS in noncoated plates. Thereafter, an equal volume of each of the specific MAbs, diluted in BSA-PBS to a concentration corresponding to 10 to 20 times the ELISA titer against the respective antigen, was added to each of the bacterial dilutions and incubated at RT for 1 h with moderate shaking. The mixtures were then transferred to the antigen-coated plates and were incubated at RT for 90 min. Plates were washed and developed as described for ELISA. The bacterial concentrations causing 50% inhibition of the binding of the respective MAb to the solid-phase antigen were determined (24). All samples and reference strains were tested in duplicate on two different occasions.

Histological examination of duodenal biopsy specimens.In each biopsy specimen, the severity and activity of chronic inflammation (infiltration of lymphocytes and plasma cells) and active inflammation (infiltration of neutrophils) were graded on a scale of 0 to 3, which correspond to no inflammation, 0; mild inflammation, 1; moderate inflammation, 2; and severe mucosal inflammation (duodenitis), 3. The severity of active inflammation was expressed as the mean score of the total number of biopsies, whereas the activity of active inflammation was expressed as the highest score observed among the different duodenal biopsies from each subject.