Helicobacter pylori infection usually is acquired in childhood and may persist for a lifetime. There is greater prevalence of infection in developing countries (1,2). Infected subjects have chronic gastritis and, in about 10% of cases, other pathologic conditions such as duodenal ulcer (DU), gastric carcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma may develop (1,3–5). The factors associated with the development of these complications are not fully understood (3–7). However, attachment of the bacterium to gastric epithelium has been related to persistence of infection and disease outcome (8–10).
H. pylori colonization depends on both bacterial and host factors (11–18). Surface adhesins that recognize proteins or glycoproteins on the epithelial cell surface mediate bacterial attachment. A number of molecules have been implicated as H. pylori adhesin receptors (11,12,16,18), among them the fucosylated blood group antigen Lewis b (Leb) (11). BabA protein was identified as the adhesin responsible for H. pylori binding to Leb (14). Despite in vitro evidence showing the role of epithelial Leb on H. pylori colonization (11,14), data obtained from gastric biopsy specimens in adults and children are conflicting (19–22).
It has been suggested that Leb expression is less common in children than in adults, possibly because of epithelial immaturity and lower fucosyltransferase activity (21). Previously, we observed that increased cag A positivity was associated with increased age (6). Bacterial BabA phenotype is common among cag A-positive microorganisms (7,8,14), and it has been shown that the presence of cag A is associated with bacterial binding to the Leb antigen (14). Thus, a low frequency of infection by cag A-positive strains in young children may be related to low Leb expression. In this study, we aimed to determine in children whether the expression of Lewis antigens in gastric mucosa was associated with H. pylori infection, cag A status, patient age, or DU.
MATERIALS AND METHODS
Endoscopic biopsy specimens from 70 children were selected from a series of 631 biopsy specimens obtained from 529 consecutive patients undergoing upper intestinal endoscopy for investigation of upper digestive symptoms. Informed consent was obtained from all patients or their parents. The Ethical Committee of the University Hospital, Federal University of Minas Gerais, Brazil, approved the study. Biopsy specimens were taken from antral (lesser curvature, 2 cm above the pylorus) and oxyntic (greater curvature, middle portion) mucosa and processed routinely for histologic evaluation in 5-μm thick hematoxylin-and-eosin–stained sections. The presence of H. pylori was determined by urease test and inspection of carbolfuchsin stained sections; culture, serology, and polymerase chain reaction (PCR) for ure A and cag A detection were performed in several patients who were included in another study (6). The same pathologist (AMN) examined all of the histologic samples.
Seventy patients who had paraffin blocks and well preserved tissue from the antrum and corpus were selected for the study. Age, gender, H. pylori status, and associated DU were recorded. Patients were divided in four age groups: younger than 4 years, 4 to 8 years, 9 to 12 years, and 13 to 18 years. Thirty-one (44.3%) patients were male, and 39 were female (55.7%); 47 patients had H. pylori, 49 (70%) had chronic gastritis, 17 (24.3%) had DU, and 4 (5.7%) had normal histologic findings (Table 1).
Histologic slides were retrieved from files, and the diagnoses were revised if necessary. Inflammation and activity were scored according to the updated Sydney System (23). In 34 patients with H. pylori, cag A status was previously determined by PCR validated for our pediatric population (6). All patients without H. pylori included in the study had negative results of urease, culture, and PCR tests (6).
Epithelial expression of Lewis antigens was determined by immunohistochemistry in additional 5-μm thick histologic sections from the antrum and corpus. The streptavidin-biotin-peroxidase method for monoclonal antibodies was used, as previously described (24). The antibodies used were: BG6 (anti-Leb, diluted 1:40, Signet Laboratories, Dedham, MA, USA), CA3F4 (25) (anti-Lewis a [anti-Lea], diluted 1:2), H1 (26) (anti-Lewis x [Lex], diluted 1:2), and AH6 (27) (anti-Lewis y [Ley], diluted 1:2). Incubation was performed overnight at 4°C in a humidified chamber. All the series included positive controls. Negative controls were performed by the substitution of the primary antibodies with immunoglobulins of the same class and concentration.
Lewis expression was analyzed in the superficial/foveolar epithelium and in the glands. Expression of Lea and Leb was graded as: 0, negative or less than 5% of stained cells; 1, 5% to 25% of stained cells; 2, 25% to 50%; and 3, more than 50% of stained cells.
Two-tailed χ2, χ2 for trend and Mann-Whitney U tests were used in the analysis of the association between Lewis expression and H. pylori status, cag A status, patient's age, and DU. The significance level was set at P < 0.05.
Epithelial Expression of Lewis Antigens
Lea and Leb antigens were equally expressed in the antrum and the corpus on the apical surface of the superficial/foveolar epithelium (Fig. 1, A and B), with some variation in extent from case to case. Forty-five (64%) patients had expression of Lea, and 31 (44%) had Leb expression. Lea expression was more intense than Leb. Positivity in more than 50% of the cells was observed in 34 (49%) patients for Lea and in only 19 (27%) for Leb. The distribution of Lewis phenotypes is shown in Table 2. Expression of Lex and Ley was observed in all the patients and in all the glands of the antrum and corpus (Fig. 1, C and D). Expression of these antigens was also observed on the apical surface of the superficial/foveolar epithelium: Lex in 30 (43%) cases and Ley in 59 (84%) cases.
Lewis Epithelial Expression and H. pylori Infection
Leb expression was more common among patients without H. pylori (15/23, 65%) than in those with H. pylori (16/47, 34%) (P = 0.03). The expression of Lea, Lex, and Ley was not associated with H. pylori infection. Lewis expression was not associated with cag A status (P = 0.7). Lea was detected in 14 of 23 (60.9%) patients with positive cag A status and in 8 of 11 (72.4%) with negative cag A status. In the same way, Leb was detected in 9 (39.1%) patients with positive cag A status and in 3 (27.3%) with negative status.
Lewis Epithelial Expression and Patient Age
In 23 patients without H. pylori, Leb and superficial Ley expression increased with the patient age (P = 0.02 and P = 0.04, respectively) (Fig. 2). In patients with H. pylori, superficial Lex expression was significantly more common in older children (P = 0.04) (Fig. 3).
Lewis Epithelial Expression and H. pylori-Associated Disease
Leb epithelial expression was more common and more intense among children with chronic gastritis than in those with DU (P = 0.03, P = 0.006, respectively) (Fig. 4). Lex superficial/foveolar expression was present in 11 of 17 (65%) children with DU and in 9 of 30 (30%) of those with H. pylori-associated gastritis. The expression of Lex on the superficial epithelium was significantly related to the presence of DU (OR = 4.28, 1.03 < 95% CI < 18.69, P = 0.04) (Fig. 5).
H. pylori colonization depends on several bacterial and host factors (11–18). The Leb blood group antigen expressed in the epithelium mediates H. pylori attachment to gastric mucosa (11) through the interaction with the BabA adhesin (14). Because H. pylori infection is acquired in childhood, the study of Lewis blood group antigen expression in the gastric mucosa of children can contribute to the understanding of the underlying adhesion and pathogenic mechanisms.
In our series, Lea and Leb expression were observed on the apical surface of superficial/foveolar epithelium. Lex and Ley were expressed in the glands of the antrum and corpus in all the patients and in superficial/foveolar epithelium of several patients. Except for the reports of Su et al. (15) and Çelik et al. (21), who observed Lea expression mainly in the deep glands, this pattern of expression is in accordance with previous results (22,28). As reported by others, immunoexpression of Lea and Leb antigens was not homogenous in terms of extent and staining intensity (20). It has been suggested that flow cytometry might be preferable because this method provides quantitation of antigen expression. In our study, we scored the immunoexpression of Lea and Leb antigens considering the percentage of immunoreactive cells in the gastric epithelium.
Lea and Leb immunoreactivity was detected in 64% and 44% of our pediatric patients, respectively, at variance to what has been reported previously in adults, in whom Leb expression ranged from 70% to 96% and was more common than Lea. Çelik et al. (21) observed Lea expression on surface mucous cells in children, but not in adults. The authors suggested that children might present lower fucosyltransferase activity and lower Leb expression than do adults.
Leb expression was less common in patients with H. pylori than in those without it. This result was unexpected because several studies have shown that Leb is expressed in the gastric mucosa of most of the subjects, regardless of H. pylori infection (12,15,20,22,28). One possible explanation for our result may be the low sensitivity of our method. It has been reported that conventional immunohistochemistry is not as sensitive as immunoelectron microscopic analysis (22). We speculate that an impaired immunoreactivity caused by antigenic site blocking by attached microorganisms might explain our observations. This negative result might be observed only in children because they probably express small amounts of Leb (21). Other studies on this subject using different methods, such as in situ adhesion assays and double immunohistochemistry, are needed.
We demonstrated that Leb and superficial Ley expression were associated with age and that Leb immunoreaction was weaker than that of Lea. These observations corroborate previous suggestions that fucosyltransferase enzymes may have low activity in children (21). Curiously, α1-2fucosyltransferase is involved in both Leb and Ley synthesis. The current data could also explain the low frequency of infection by cag A-positive strains among young children (6). It has been suggested that BabA phenotype is related to cag A status (14). Thus, if young children express few Leb receptors, they might have less chances of being infected by cag A-/ bab A2-positive strains. Additional studies focusing on H. pylori infection by bab A2-positive strains in children are needed to confirm this hypothesis. The pattern of Lewis expression changed in children with H. pylori. Lex superficial expression was significantly more common among older patients, and neither Leb nor Ley were associated with patient age. One must be cautious in the analysis of these results because the number of children was small in some subgroups.
Leb expression was significantly less common in children with DU than in those with gastritis only. We also observed that Lex superficial expression was significantly associated with DU in children with H. pylori. Our findings confirm published reports that DU is related to infection by cag A-positive strains (1,6,8). Despite some evidence showing that cag A-/ bab A-positive strains are related to DU in adults (8), other studies concerning the relationship between BabA-positive strains and DU do not support the association (29,30). Thus, other adhesins and receptors may be involved in H. pylori infection and in patients with DU, especially children. It had been suggested that molecular mimicry involving Lex expression by both the host and the bacterium might have a role in H. pylori adhesion (16). Recently, a new adhesin named “SabA” has been described (18). The authors pointed out that H. pylori adherence might involve two separated receptor-ligand systems, one mediated by Leb and the other by sialyl-Lex (sLex) (18). Thus, it is possible that an alternative adhesion system may be involved in children with low or absent Leb expression. Patients with superficial Lex expression may be at increased risk of DU, or alternatively, H. pylori infection may induce superficial Lex expression, mainly in patients with a more intense inflammatory reaction, as it was reported for sLex (18).
In conclusion, we have found that the expression of Leb and Ley in the superficial gastric epithelium of children seems to depend on the patient age and that other receptors, such as Lex, may have a role in H. pylori colonization, especially in patients with DU. Studies assessing the expression of Lewis antigens in children may contribute to the understanding of the mechanisms of acquisition of H. pylori infection.
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