H Pylori
Copyright ©2007 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Sep 7, 2007; 13(33): 4445-4451
Published online Sep 7, 2007. doi: 10.3748/wjg.v13.i33.4445
H pylori seropositivity and cytokine gene polymorphisms
Yasuaki Saijo, Eiji Yoshioka, Tomonori Fukui, Mariko Kawaharada, Fumihiro Sata, Hirokazu Sato, Reiko Kishi
Yasuaki Saijo, Department of Health Science, Asahikawa Medical College, Midorigaoka, E2-1-1-1, Asahikawa, Hokkaido 078-8510, Japan
Eiji Yoshioka, Tomonori Fukui, Mariko Kawaharada, Fumihiro Sata, Reiko Kishi, Department of Public Health, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo 060-8638, Japan
Hirokazu Sato, Health Administration Department, Sapporo Railway Hospital, Kita 3, Higashi 1, Cyuo-ku, Sapporo 060-0033, Japan
Author contributions: All authors contributed equally to the work.
Supported by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant-in-Aid for Scientific Research from the Ministry of Health, Labour and Welfare of Japan
Correspondence to: Dr. Yasuaki Saijo, Department of Health Science, Asahikawa Medical College, Midorigaoka, E2-1-1-1, Asahikawa, Hokkaido 078-8510, Japan. y-saijo@asahikawa-med.ac.jp
Telephone: +81-166-682402 Fax: +81-166-682409
Received: February 27, 2007
Revised: March 15, 2007
Accepted: March 21, 2007
Published online: September 7, 2007

Abstract

AIM: To investigate whether the pro- and anti-inflammatory cytokine gene polymorphisms, IL1B-511C/T, IL1B-31C/T, IL6-634C/G, TNF-1031T/C, TNF-857C/T, and IL10-1082A/G, interact with smoking and drinking habits to influence infection with H pylori.

METHODS: The subjects were 410 Japanese transit company employees. C-reactive protein and conventional cardiovascular risk factors were evaluated. Serum anti-H pylori antibodies were measured. The genotypes of IL1B-511C/T, IL1B-31C/T, IL6-634C/G, TNF-1031T/C, TNF-857C/T, and IL10-1082A/G polymorphisms were determined by allelic discrimination using fluorogenic probes and a 5´nuclease assay.

RESULTS: In gender- and age-adjusted logistic analyses, the subjects with TNF-857T/T had a significantly lower odds ratio (OR) for H pylori seropositivity (reference -857C/C; OR = 0.15, 95%CI: 0.03-0.59, P = 0.007). After stratification according to smoking and drinking status, among never-smokers, the subjects with IL1B-511C/T had a significantly lower OR (reference -511C/C; OR = 0.30, 95%CI: 0.10-0.90, P = 0.032). Among drinkers in the 1-5 times/wk category, the subjects with IL1B-511T/T had a significantly lower OR (reference C/C; OR = 0.38, 95%CI: 0.16-0.95, P = 0.039), and the subjects with IL1B-31C/T and T/T had a significantly higher OR (reference C/C; C/T: OR = 2.59, 95%CI, P = 0.042: 1.04-6.47; C/C: OR = 3.17, 95%CI: 1.23-8.14, P = 0.017). Among current smokers, the subjects with IL6-634C/G had a significantly higher OR (reference C/C; OR = 2.28, 95%CI: 1.13-4.58, P = 0.021). However, the interactions terms between the aforementioned genotypes and lifestyles were not statistically significant.

CONCLUSION: Contrary to previous findings, the results herein suggest that the TNF-857T/T genotype may be protective against chronic infection with H pylori. Drinking and smoking habits may influence the effect of cytokine gene polymorphisms. Further studies are required to clarify the effects of the pro- and anti-inflammatory cytokine polymorphisms and gene-environmental interactions on H pylori infection.

Key Words: H pylori seropositivity, Cytokines, Poly-morphisms



INTRODUCTION

The prevalence of H pylori infection is generally higher in developing countries than in developed countries[1]; however, the Japanese population has a high prevalence of H pylori seropositivity[2]. Infection with H pylori represents a key factor in the etiology of various gastrointestinal diseases, including asymptomatic chronic active gastritis, peptic ulceration, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma[3]. H pylori has also been implicated in a number of extra-gastrointestinal disorders, such as atherosclerosis[4], cerebral vascular disease[5], idiopathic thrombocytopenic purpura[6], and rosacea[7]. Because of the greater prevalence and various pathogenic activities of H pylori in Japanese, it is important to understand the basis for genetic susceptibility and identify the environmental factors that maintain chronic infection.

The mucosal production of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL6, and tumor necrosis factor (TNF)-α, appears to be enhanced by infection with H pylori[8,11]. Interleukin-1β and TNF-α inhibit gastric acid secretion, providing a favorable condition for H pylori to survive in the stomach[12]. Although one study failed to show inhibition of gastric acid secretion by IL-6[13], several studies have shown that gastric colonization with H pylori leads to elevated IL-6 levels in the gastric mucosa[14,15]. Thus, IL-6 may be one of the factors that maintain chronic infection with H pylori. Furthermore, IL-10, an anti-inflammatory cytokine, may reduce the inflammation associated with H pylori infection[16].

The host’s ability to regulate cytokine production has been shown to be influenced by the presence of cytokine gene polymorphisms. Therefore, H pylori-susceptible cytokine gene backgrounds have recently been investigated. Regarding the IL1B gene, Japanese subjects with the -31T/T genotype have a significantly higher odds ratio (OR) for H pylori seropositivity as compared to subjects with the -31C/C or C/T genotypes[17]. A strong relationship involving ILIB -31T/T has been demonstrated in Japanese Brazilians[18]; however, such an association has not been shown to exist in Italians[19] or Jamaicans[20]. Regarding the TNF gene, Japanese subjects with the -1031C/C genotype have a significantly lower OR compared to those with the -1031T/T genotype[21]; however, an association was not found in Italians[19], Jamaicans[20], or Japanese Brazilians[22]. Thus, the effect IL1B and TNF polymorphisms on infection with H pylori remains controversial. Furthermore, among Jamaicans, the IL6-634C/G polymorphism (denoted -572G/C) was not associated with H pylori seropositivity[20], and the IL10-1082C/G polymorphism was not associated with H pylori infection among Jamaicans[20] or Italians[19]. Little is known regarding the effects of the IL6 and IL10 promoter polymorphisms on infection with H pylori.

Smoking cigarettes and drinking alcohol may have an effect on chronic infection with H pylori[23,26]. Therefore, interactions between the genome and lifestyle factors should be elucidated. An interaction between the IL1B genotype and one’s cigarette smoking status on the eradication of H pylori has been reported[27]. It has also been reported that the effect of the IL1B-31T/T genotype on H pylori infection is modified by smoking cigarettes and drinking alcohol[18,28], but the interactions between other cytokine gene polymorphisms and lifestyle factors on H pylori infection have not been fully investigated.

The aim of this study was to investigate whether the pro- and anti-inflammatory cytokine gene polymorphisms, IL1B-511C/T, IL1B-31C/T, IL6-634C/G, TNF-1031T/C, TNF-857C/T, and IL10-1082A/G, interact with smoking cigarettes and drinking alcohol to influence infection with H pylori in Japanese.

MATERIALS AND METHODS
Subjects

The subjects were transit company employees (1255 men and 94 women, 35-60 years of age), who had their annual health checkup between April 2003 and March 2004. We used a self-administered questionnaire that included items regarding clinical history, smoking cigarettes, and consumption of alcohol. The questionnaire was distributed to the subjects prior to their annual health checkup, and was collected at the time of the checkup. Answers to the questionnaire and written informed consent to view pertinent health checkup data were obtained from 413 men and 5 women, for a response rate of 32.9% and 5.3%, respectively. Eight subjects were excluded due to inadequate blood samples. Ultimately, we analyzed a total of 410 employees (405 men and 5 women). No subject had a history of an internal malignancy or gastric surgery.

This study was conducted with written informed consent from all the subjects and approved by the institutional ethical board for epidemiological studies and human gene and genome studies of the Hokkaido University Graduate School of Medicine.

Data collection

Subjects were classified as current, never- or ex-smokers. Alcohol consumption habits were categorized as never/rarely, 1-5 times/wk, or 6-7 times/wk.

Blood samples were drawn from the antecubital vein of the subject after a 12 h fast while in a seated position and with minimal tourniquet use. The anti-H pylori antibody titer was measured using an enzyme immunoassay (E plate; Eiken Chemical, Tokyo, Japan)[29]; an assay value < 10 U/mL was considered negative and a value > 10 U/mL was considered positive.

Genomic DNA was extracted from each subject’s peripheral blood lymphocytes using an EZ1 DNA blood kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. We genotyped the IL1B-511C/T (dbSNP: rs16944), IL1B-31C/T (dbSNP: rs1143627), IL6-634C/G (rs1800796), TNF-1031T/C (dbSNP: rs1799964), TNF-857C/T (dbSNP: rs1799724), and IL10-1082A/G (dbSNP: rs1800896) polymorphisms by allelic discrimination using fluorogenic probes and the 5´nuclease (TaqMan) assay, as previously described[30,31]. To detect a polymorphism in IL-6-634C/G, the following MGB probes were prepared: a C allele-specific probe, 5'-FAM-CAACAGCCCCTCACAG-MGB-3', and a G allele-specific probe, 5'-VIC-CAACAGCCGCTCACAG-MGB-3'. Each of the reporters was quenched with MGB, which was typically located at the 3' end. The primers for the PCR involving the promoter region, including the -634C/G polymorphism of IL-6, were as follows: forward, 5'-GGATGGCCAGGCAGTTCTA-3', and reverse, 5'-CCAGTCATCTGAGTTCTTCTGTGTT-3'. The reaction mixture contained approximately 40 ng of template DNA, 5.0 μL of TaqMan Universal PCR master mixture, and 0.3 μL of 40 × assay mixture, in a volume of 10 μL. The IL1B-511C/T, IL1B-31C/T, TNF-1031T/C, TNF-857C/T, and IL10-1082A/G polymorphisms were similarly genotyped using the TaqMan® SNP genotyping products: C_1839943_10, C_1839944_10, C_7514871_10, C_11918223_10, and C_1747360_10, respectively (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed on a 7500 Real-time PCR System (Applied Biosystems) using a protocol consisting of incubation at 50°C for 2 min and 95°C for 10 min, followed by 50 cycles for IL6 or 40 cycles for the other genotypes, denaturation at 92°C for 15 s, and annealing/extension at 60°C for 1 min. The FAM and VIC fluorescence levels of the PCR products were measured at 60°C for 1 min, resulting in the clear identification of all six genotypes of IL1B, IL6, TNF, or IL10 on a two-dimensional graph.

Statistical analysis

The differences in the frequency of each characteristic between the H pylori-seropositive and -seronegative groups were examined by the chi-square test. Hardy-Weinberg equilibrium analyses were performed to compare the observed and expected genotype frequencies using the chi-square test. A logistic regression analysis was used to evaluate the associations between each cytokine genotype and H pylori seropositivity, with adjustment for age and gender, to obtain the OR and 95% confidence intervals (CI). After stratification according to cigarette smoking and alcohol consumption status, the adjusted OR for each genotype of H pylori seropositivity was calculated. The interaction term for the genotype/lifestyle factors was included in the logistic model with the main effect.

The haplotype was analyzed using Haploview, version 3.32[32], and linkage disequilibrium between loci was measured using Lewontin’s D’[33]. The adjusted OR for the IL1B and TNF haplotypes was analyzed by logistic regression models. Statistical analyses were conducted with SPSS software for Windows, version 14.0 (SPSS; Chicago, IL, USA).

RESULTS

The characteristics of the groups according to H pylori seropositivity are shown in Table 1. Two hundred thirty-seven subjects (57.8%) were H pylori-seropositive. The H pylori-seropositive group was older and drank alcohol more frequently than the H pylori-seronegative group.

Table 1 Characteristics of H pylori-seropositive and -seronegative subjects.
H pylori-seropositive(n = 237)
H pylori-seronegative(n = 173)
P-value
n (%)n (%)
Gender0.645
Male23498.717198.8
Female31.321.2
Age (yr)< 0.001
< 453012.76336.4
45-496226.25330.6
50-548535.94324.9
≥ 556025.3148.1
Smoking0.281
Never5924.93218.5
Former7632.15732.9
Current10243.08448.6
Drinking0.023
Never or rarely3514.84324.9
1-5 times/wk13155.39152.6
6-7 times/wk7130.03922.5

H pylori seropositivity, according to the genotypes of IL1B, IL-6, TNF, and IL-10, are shown in Table 2. The distribution of genotypes in each group was in the Hardy-Weinberg equilibrium. TNF-857C/T genotypes were significantly different between the H pylori-seropositive and -seronegative subjects.

Table 2 H pylori seropositivity according to cytokine genotypes.
H pylori-seropositive(n = 237)
H pylori-seronegative(n = 173)
P-value
n(%)n(%)
IL1B-511C/T0.243
CC9339.25431.2
CT10946.08951.4
TT3514.83017.3
IL1B-31C/T0.434
CC3313.92916.8
CT11247.38750.3
TT9238.85732.9
IL6-634C/G0.753
CC13858.210460.1
CG8837.15934.1
GG114.6105.8
TNF-1031T/C0.00.170
TT15264.111566.5
CT8033.85129.5
CC52.174.0
TNF-857C/T0.018
CC17071.711566.5
CT6427.04727.2
TT31.3116.4
IL10-1082A/G0.852
AA21189.015388.4
AG/GG12611.02011.6

The age- and gender-adjusted ORs of the genotypes for H pylori seropositivity are shown in Table 3. The subjects with TNF-857T/T had a significantly lower OR for H pylori seropositivity (reference -857C/C; OR = 0.15, 95%CI 0.03-0.59).

Table 3 Age, gender-adjusted ORs for H pylori seropositivity according to cytokine genotypes.
nHp (+)%1Adjusted OR (95%CI)P-value
IL1B-511C/T
CC14763.31.00
CT19855.10.69 (0.43-1.10)0.121
TT6553.80.70 (0.37-1.32)0.270
IL1B-31C/T
CC6253.21.00
CT19956.31.11 (0.61-2.05)0.726
TT14961.71.47 (0.78-2.78)0.234
IL6-634C/G
CC24257.01.00
CG14759.91.06 (0.68-1.66)0.785
GG2152.40.63 (0.24-1.62)0.335
TNF-1031T/C
TT26756.91.00
CT13161.11.24 (0.78-1.95)0.361
CC1241.70.48 (0.13-1.70)0.253
TNF-857C/T
CC28559.61.00
CT11157.70.93 (0.58-1.49)0.760
TT1421.40.15 (0.03-0.59)0.007
IL10-1082A/G
AA36458.01.00
AG/GG24656.51.08 (0.56-2.09)0.811

After stratification according to cigarette smoking and alcohol consumption status, the age- and gender-adjusted ORs of IL1B and IL-6 genotypes for H pylori seropositivity are shown in Table 4. Among never-smokers, subjects with IL1B-511C/T had a significantly lower OR for H pylori seropositivity (reference -511C/C; OR = 0.30, 95%CI: 0.10-0.90). Among the 1-5 times/wk drinkers, IL1B -511T/T had a significantly lower OR (reference C/C; OR = 0.38, 95%CI: 0.16-0.95), and IL1B-31C/T and -T/T had significantly higher ORs (reference C/C; C/T: OR = 2.59, 95%CI: 1.04-6.47; C/C: OR = 3.17, 95%CI: 1.23-8.14). Among current smokers, IL6-634C/G had a significantly higher OR (reference C/C; OR = 2.28, 95%CI: 1.13-4.58); however, the interaction terms between the aforementioned genotypes and lifestyles were not statistically significant. The remaining genotypes revealed no statistically significant ORs after stratification (data not shown).

Table 4 Age, gender-adjusted ORs for H pylori seropositivity according to cytokine genotypes and lifestyle factors.
IL1B–511C/T
nHp (+)%1C/CC/TP-valueT/TP-value
All subjects41057.81.000.69 (0.43-1.10)0.1210.70 (0.37-1.32)0.270
Smoking
Never9164.81.000.30 (0.10-0.90)0.0320.40 (0.10-1.62)0.200
Former13357.11.000.90 (0.39-2.10)0.8190.74 (0.26-2.15)0.583
Current18654.81.000.83 (0.42-1.63)0.5820.80 (0.30-2.16)0.658
Drinking
Never or rarely7844.91.000.58 (0.20-1.66)0.3101.38 (0.31-6.23)0.676
1-5 times/wk22259.01.000.81 (0.43-1.55)0.5310.38 (0.16-0.95)0.039
6-7 times/wk11064.51.000.66 (0.25-1.69)0.3831.11 (0.33-3.77)0.862
IL1B–31C/T
nHp (+)%1C/CC/TP-valueT/TP-value
All subjects41057.81.001.11 (0.61-2.05)0.7261.47 (0.78-2.78)0.234
Smoking
Never9164.81.000.79 (0.22-2.29)0.7232.25 (0.56-9.08)0.257
Former13357.11.001.34 (0.50-3.62)0.5601.55 (0.55-4.42)0.409
Current18654.81.001.20 (0.44-3.24)0.7221.22 (0.44-3.40)0.705
Drinking
Never or rarely7844.91.000.44 (0.11-1.82)0.2580.68 (0.15-3.12)0.624
1-5 times/wk22259.01.002.59 (1.04-6.47)0.0423.17 (1.23-8.14)0.017
6-7 times/wk11064.51.000.72 (0.23-2.29)0.5790.81 (0.24-2.73)0.735
IL6–634C/G
nHp (+)%1C/CC/GP-valueG/GP-value
All subjects41057.81.001.06 (0.68-1.66)0.7850.63 (0.24-1.62)0.335
Smoking
Never9164.81.000.54 (0.22-1.38)0.2002.79 (0.28-27.73)0.382
Former13357.11.000.63 (0.28-1.41)0.2590.00 (0.00)0.999
Current18654.81.002.28 (1.13-4.58)0.0210.84 (0.22-3.15)0.796
Drinking
Never or rarely7844.91.001.07 (0.41-2.82)0.8860.00 (0.00)1.000
1-5 times/wk22259.01.001.02 (0.55-1.91)0.9400.40 (0.11-0.41)0.153
6-7 times/wk11064.51.001.61 (0.63-4.12)0.3251.60 (0.28-9.28)0.599

Complete linkage disequilibrium existed between the two IL1B promoter lesions (D’ = 1, r2 = 0.048) and strong linkage disequilibrium existed between the two TNF promoter lesions (D’ = 0.953). The estimated haplotype frequency of TNF (-1031T/C and -857C/T) was as follows: TC = 64.1%, CC = 18.9%, TT = 17.0%, and CT = 0%. The estimated haplotype frequency of IL1B (-511C/T and -31C/T) was as follows: CT = 58.9%, TC = 38.3%, TT = 1.7%, and CC = 1.1%.

The adjusted ORs of the combination of the two IL1B promoter genotypes and the TNF genotypes for H pylori seropositivity are shown in Table 5. The subjects with TNF-1031T/T and -857T/T had significantly lower ORs for H pylori seropositivity (reference, all the remaining combinations of genotypes; OR = 0.15, 95%CI: 0.04-0.60); however, subjects with TNF-1031T/T and -857T/T were similar to the subjects with -857T/T.

Table 5 Age, gender-adjusted ORs for H pylori seropositivity according to the combination of the two promoter genotypes of TNF and IL1B.
nHp (+)%1Adjusted OR (95%CI)P-value
TNF-1031/-857
TT/CC216958.61.07 (0.70-1.64)0.743
TC/CC210463.51.38 (0.85-2.25)0.195
CC/CC21241.70.44 (0.13-1.57)0.207
TT/CT28459.51.06 (0.63-1.78)0.830
TC/CT22751.90.89 (0.39-2.02)0.781
TT/TT21421.40.15 (0.04-0.60)0.007
IL1B-511/-313
CC/TT214161.71.29 (0.83-2.01)0.259
CT/CT219154.50.74 (0.48-1.13)0.159
TT/CC25952.50.77 (0.43-1.39)0.384
DISCUSSION

In the current study, the TNF-857T/T genotype had a significantly reduced OR for H pylori seropositivity. Because the subjects with both TNF-1031T/T and -857T/T genotypes were similar to the subject who was classified with the TNF -857T/T genotype only, the combination of genotypes also had a reduced OR.

It has been reported that Japanese subjects with the -1031C/C genotype have a significantly lower OR for H pylori infection when compared to those with the -1031T/T genotype, and that subjects with -857T/T and -1013T/T have significantly higher ORs for H pylori infection when compared to those with -1031C/C and -857C/C[21]. However, neither -1031T/C nor -857C/T polymorphisms were associated with H pylori infection in Italians[19] or Jamaicans[20], and neither the genotypes nor the combination of genotypes were associated with Japanese Brazilians[22]. The genotype distributions of -1031T/C and -857C/T among the aforementioned Japanese subjects were quite similar to the distributions in the subjects enrolled in our study. However, the subjects between the studies differed as follows: (1) our subjects were younger than in the previously published study, (2) nearly all of our subjects were male, while approximately one-half of the previous study subjects were female, and (3) our study subjects were healthy workers, unlike the subjects in the previous study that included outpatients participating in a H pylori eradication program, outpatients with chronic diseases, as well as health checkup examinees; these differences may have been the basis for the discrepant results. Further studies are needed to elucidate age and sex specific effects of TNF-857C/T polymorphism on H pylori infection.

In the current study, the subjects with the TNF-857T/T genotype had the highest level of TNF-α secretion, resulting in low gastric acid secretion, and they were resistant to chronic H pylori infection. Higuchi et al[34] reported that the level of TNF-α and the transcription promoter activity produced by concanavalin A-activated peripheral blood mononuclear cells in subjects with -1031C or -857T alleles were higher than in those subjects with the -1031T or -857C alleles. Skoog et al[35] reported that subjects with -863A tightly linked with -1031C had a significantly lower serum TNF-α level. Moreover, in another study it was shown that ex vivo lipopolysaccharide-stimulated whole-blood TNF production was higher in healthy TNF-857C homozygotes[34]. Thus, further studies will be needed to clarify the effect of the TNF genotype on susceptibility to infection with H pylori and production of TNF-α.

The TNF gene has more than three relatively frequent bi-allelic single-nucleotide polymorphisms in the promoter region: -863C/A, -308G/A, and -238G/A[35]. It has been reported that the TNF-308A allele is highly associated with H pylori infection in Italy[19], but the -308A allele is rare in Japan, and the other major allele, -238A, is also rare in Japan (1.7% and 2.0%, respectively)[35]. Moreover, the -863C/A allele is tightly linked with -1031C/T[36]. Therefore, we investigated the two promoter region polymorphisms of TNF. However, since the TNF-857 T/T genotype is not frequent in the population, simple and easy methods for genotyping are required for practical use.

The two IL1B promoter genotypes were not associated with H pylori infection in our entire group of subjects. In like manner, no association was found in Italians[19] or Jamaicans[20]. However, a previous Japanese study showed that subjects with the -31T/T genotype had a significantly higher OR (1.74, 95%CI: 1.15-5.63) for H pylori infection as compared to those subjects with the -31C/T or -31CC genotypes[17]. Furthermore, a study of Japanese Brazilian subjects found an association (OR of T/T = 1.45, 95%CI: 1.02-2.07)[28]. The subjects in the two previous studies involved an adequate number of female subjects and the Japanese study subjects were older than our study subjects. These differences may have accounted for our inability to obtain statistically significant results. In addition, the sample size of the previous Japanese study was nearly the same as that of our study (n = 437), but the sample size of the Japanese Brazilian study was almost twice as large as that of our study (n = 963). If a real OR of the T/T genotype was approximately 1.5, a smaller sample size as in our study may have failed to reach statistical significance.

Smoking cigarettes and drinking alcohol augment the T/T genotype effect on H pylori infection[18,28]. In our study, 1-5 times/week drinkers with T/C and T/T genotypes had significant ORs. In previous studies, the subjects were divided into drinkers or non-drinkers[18,28] and the pattern of drinking enhanced the T/T genotype effect on chronic H pylori infection. The drinkers in the previous study involved moderate and heavy consumption of alcohol, but the difference in T/T genotype augmentation between moderate and heavy consumption of alcohol was not analyzed. In our study, the results suggested that moderate drinking enhanced the T/T genotype effect on chronic H pylori infection. Therefore, further studies are needed to elucidate the interactions betweens the volume of alcohol consumption and genotypes on H pylori infection.

In our study, 1-5times/wk drinkers with the -511T/T genotype had a significantly lower OR since -31C and -511T were tightly linked (-511T/C and -31C/T combinations: 59.8% for T-C, 1.7% for T-T, 38.3% for C-T, and 1.1% for C-C). Non-smokers with -511C/T had a significantly lower OR. Chance may have influenced the significance of the result. Moreover, because this study was cross-sectional, changes in cigarette smoking and alcohol consumption habits were not involved in the analyses. Thus, the changes from previous habits may have affected the ORs.

Lipopolysaccharide (LPS)-stimulated IL-1β expression by whole blood leukocytes in vitro was lower in subjects with -31T and -511C[37,38]. Since IL-1β inhibits gastric acid secretion, thereby providing a favorable environment for H pylori to survive in the stomach[12], the results of the previous Japanese study and the moderate drinkers of our study were compatible to the in vitro IL-1β expression studies.

The IL6-634C/G (denoted -572G/C in reference 20) polymorphism was not associated with H pylori seropositivity among Jamaicans[20]. In our study, the polymorphism was also not associated with H pylori seropositivity among our entire group of subjects. However, current smokers with the -634C/G genotype had a significantly higher OR for H pylori infection.

Persons with the C allele of the IL6-174G/C polymorphism are common among Caucasians, but extremely rare among East Asians[39,40]. However, persons with the G allele of the IL6-634C/G polymorphism are common among East Asians, and this genotype significantly relates to recurrent pregnancy loss[39], bone mineral density[41], and diabetic nephropathy[42]. Additionally, the -634G allele is associated with an elevated production and secretion of IL-6 by peripheral blood mononuclear cells in vitro[42].

In a study of young and healthy Caucasians, the IL-6 polymorphism was not associated with the IL6-174 genotypes in non-smokers, but in smokers where the -174C allele was associated with a higher number of leukocytes, lymphocytes, and monocytes[43]. In our study, the smokers with the -634G/G genotype had no significant results, perhaps because of a smaller sample size. In contrast, we found that the impact of the -634G allele on CRP elevation was greater in non-smokers than in current smokers (in press at Hypertens Res). Thus, the effect of IL6 gene polymorphisms and the gene-environment interactions on H pylori infection should also be further elucidated.

In our study, the IL10-1082C/G polymorphism was not associated with H pylori infection. As previously mentioned, negative results were reported for Jamaicans[20] and Italians[19]. Other IL10 promoter polymorphisms, such as -819C/T and -592C/A, have been reported[19] and a Japanese study showed that the combination of the IL8-251T/A and IL10-819C/T polymorphisms was significantly associated with H pylori infection, but the IL10-819C/T polymorphism alone did not have a statistically significant effect[44]. Furthermore, associations between IL10-1013A/A[45] and -819C/T[16] genotypes on non-cardia gastric cancer were reported. An experiment in mice showed that increased IL-10 levels may reduce the inflammation of H pylori infection[16]. Unfortunately, we did not evaluate other IL10 promoter polymorphisms or the IL8-251T/A polymorphism; further studies are required to clarify how these polymorphisms effect H pylori infection.

Because this study examined IgG antibodies to H pylori, which can reflect a previous infection, IgG seropositivity to H pylori may not reflect active infection. However, the relative sensitivity, specificity, and rates of agreement between the results obtained using the enzyme immunoassay employed in the present study (i.e., the E plate) and those obtained by the culture/rapid urease test have been reported to be 100%, 80.0%, and 97.1%, respectively[29]. Strains isolated from Japanese gastric ulcer patients were used as antigens to prepare the E plate. Thus, this serological method to detect H pylori infection in Japanese is a suitable method for this type of genotype-associated study.

In summary, we observed the TNF-857T/T genotype significantly reduced the OR for H pylori seropositivity. Because the ORs for the subjects with both TNF-1031T/T and -857T/T genotypes were the same as the subject who was classified with the TN-857T/T genotype alone, the combination of genotypes also revealed a reduction in the OR. In the entire group of subjects analyzed, the promoter region polymorphisms of IL1B, IL6, and IL-10 had no association with H pylori infection. After stratification according to cigarette smoking and alcohol consumption, never-smokers with the IL1B-511C/T genotype and 1-5 times/week drinkers with the IL1B-511T/T, IL1B-31C/T, and -31T/T genotypes, had a significant association with H pylori infection. Among current smokers, the IL6-634C/G genotype also had a significant association. However, interactions terms between the aforementioned genotypes and lifestyles were not statistically significant. Further studies are required to clarify the effects of the pro- and anti-inflammatory cytokine polymorphisms and the gene-environmental interactions on H pylori infection.

Footnotes

S- Editor Zhu LH L- Editor Alpini GD E- Editor Yin DH

References
1.  Epidemiology of, and risk factors for, Helicobacter pylori infection among 3194 asymptomatic subjects in 17 populations. The EUROGAST Study Group. Gut. 1993;34:1672-1676.  [PubMed]  [DOI]
2.  Asaka M, Kimura T, Kudo M, Takeda H, Mitani S, Miyazaki T, Miki K, Graham DY. Relationship of Helicobacter pylori to serum pepsinogens in an asymptomatic Japanese population. Gastroenterology. 1992;102:760-766.  [PubMed]  [DOI]
3.  Kusters JG, van Vliet AH, Kuipers EJ. Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev. 2006;19:449-490.  [PubMed]  [DOI]
4.  Saijo Y, Utsugi M, Yoshioka E, Horikawa N, Sato T, Gong Y, Kishi R. Relationship of Helicobacter pylori infection to arterial stiffness in Japanese subjects. Hypertens Res. 2005;28:283-292.  [PubMed]  [DOI]
5.  Franceschi F, Leo D, Fini L, Santoliquido A, Flore R, Tondi P, Roccarina D, Nista EC, Cazzato AI, Lupascu A. Helicobacter pylori infection and ischaemic heart disease: an overview of the general literature. Dig Liver Dis. 2005;37:301-308.  [PubMed]  [DOI]
6.  Suzuki T, Matsushima M, Masui A, Watanabe K, Takagi A, Ogawa Y, Shirai T, Mine T. Effect of Helicobacter pylori eradication in patients with chronic idiopathic thrombocytopenic purpura-a randomized controlled trial. Am J Gastroenterol. 2005;100:1265-1270.  [PubMed]  [DOI]
7.  Szlachcic A. The link between Helicobacter pylori infection and rosacea. J Eur Acad Dermatol Venereol. 2002;16:328-333.  [PubMed]  [DOI]
8.  Noach LA, Bosma NB, Jansen J, Hoek FJ, van Deventer SJ, Tytgat GN. Mucosal tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-8 production in patients with Helicobacter pylori infection. Scand J Gastroenterol. 1994;29:425-429.  [PubMed]  [DOI]
9.  Jung HC, Kim JM, Song IS, Kim CY. Helicobacter pylori induces an array of pro-inflammatory cytokines in human gastric epithelial cells: quantification of mRNA for interleukin-8, -1 alpha/beta, granulocyte-macrophage colony-stimulating factor, monocyte chemoattractant protein-1 and tumour necrosis factor-alpha. J Gastroenterol Hepatol. 1997;12:473-480.  [PubMed]  [DOI]
10.  Lindholm C, Quiding-Järbrink M, Lönroth H, Hamlet A, Svennerholm AM. Local cytokine response in Helicobacter pylori-infected subjects. Infect Immun. 1998;66:5964-5971.  [PubMed]  [DOI]
11.  Bodger K, Crabtree JE. Helicobacter pylori and gastric inflammation. Br Med Bull. 1998;54:139-150.  [PubMed]  [DOI]
12.  Beales IL, Calam J. Interleukin 1 beta and tumour necrosis factor alpha inhibit acid secretion in cultured rabbit parietal cells by multiple pathways. Gut. 1998;42:227-234.  [PubMed]  [DOI]
13.  Saperas E, Taché Y. Central interleukin-1 beta-induced inhibition of acid secretion in rats: specificity of action. Life Sci. 1993;52:785-792.  [PubMed]  [DOI]
14.  Crabtree JE, Shallcross TM, Heatley RV, Wyatt JI. Mucosal tumour necrosis factor alpha and interleukin-6 in patients with Helicobacter pylori associated gastritis. Gut. 1991;32:1473-1477.  [PubMed]  [DOI]
15.  Lindholm C, Quiding-Järbrink M, Lönroth H, Svennerholm AM. Induction of chemokine and cytokine responses by Helicobacter pylori in human stomach explants. Scand J Gastroenterol. 2001;36:1022-1029.  [PubMed]  [DOI]
16.  Sutton P, Kolesnikow T, Danon S, Wilson J, Lee A. Dominant nonresponsiveness to Helicobacter pylori infection is associated with production of interleukin 10 but not gamma interferon. Infect Immun. 2000;68:4802-4804.  [PubMed]  [DOI]
17.  Katsuda N, Hamajima N, Tamakoshi A, Wakai K, Matsuo K, Saito T, Tajima K, Tominaga S. Helicobacter pylori seropositivity and the myeloperoxidase G-463A polymorphism in combination with interleukin-1B C-31T in Japanese health checkup examinees. Jpn J Clin Oncol. 2003;33:192-197.  [PubMed]  [DOI]
18.  Katsuda N, Hamajima N, Matsuo K, Saito T, Ito LS, Inoue M, Takezaki T, Tajima K, Tominaga S. Association between the interleukin 1B (C-31T) polymorphism and Helicobacter pylori infection in health checkup examinees. Nihon Koshu Eisei Zasshi. 2001;48:604-612.  [PubMed]  [DOI]
19.  Zambon CF, Basso D, Navaglia F, Belluco C, Falda A, Fogar P, Greco E, Gallo N, Rugge M, Di Mario F. Pro- and anti-inflammatory cytokines gene polymorphisms and Helicobacter pylori infection: interactions influence outcome. Cytokine. 2005;29:141-152.  [PubMed]  [DOI]
20.  Tseng FC, Brown EE, Maiese EM, Yeager M, Welch R, Gold BD, Owens M, Cranston B, Hanchard B, El-Omar E. Polymorphisms in cytokine genes and risk of Helicobacter pylori infection among Jamaican children. Helicobacter. 2006;11:425-430.  [PubMed]  [DOI]
21.  Hamajima N, Shibata A, Katsuda N, Matsuo K, Ito H, Saito T, Tajima K, Tominaga S. Subjects with TNF-A-857TT and -1031TT genotypes showed the highest Helicobacter pylori seropositive rate compared with those with other genotypes. Gastric Cancer. 2003;6:230-236.  [PubMed]  [DOI]
22.  Atsuta Y, Ito LS, Oba-Shinjo SM, Uno M, Shinjo SK, Marie SK, Goto Y, Hamajima N. Associations of TNF-A-1031TT and -857TT genotypes with Helicobacter pylori seropositivity and gastric atrophy among Japanese Brazilians. Int J Clin Oncol. 2006;11:140-145.  [PubMed]  [DOI]
23.  Ogihara A, Kikuchi S, Hasegawa A, Kurosawa M, Miki K, Kaneko E, Mizukoshi H. Relationship between Helicobacter pylori infection and smoking and drinking habits. J Gastroenterol Hepatol. 2000;15:271-276.  [PubMed]  [DOI]
24.  Brenner H, Berg G, Lappus N, Kliebsch U, Bode G, Boeing H. Alcohol consumption and Helicobacter pylori infection: results from the German National Health and Nutrition Survey. Epidemiology. 1999;10:214-218.  [PubMed]  [DOI]
25.  Gikas A, Triantafillidis JK, Apostolidis N, Mallas E, Peros G, Androulakis G. Relationship of smoking and coffee and alcohol consumption with seroconversion to Helicobacter pylori: a longitudinal study in hospital workers. J Gastroenterol Hepatol. 2004;19:927-933.  [PubMed]  [DOI]
26.  Brenner H, Rothenbacher D, Bode G, Adler G. Relation of smoking and alcohol and coffee consumption to active Helicobacter pylori infection: cross sectional study. BMJ. 1997;315:1489-1492.  [PubMed]  [DOI]
27.  Hamajima N, Matsuo K, Saito T, Tajima K, Okuma K, Yamao K, Tominaga S. Interleukin 1 polymorphisms, lifestyle factors, and Helicobacter pylori infection. Jpn J Cancer Res. 2001;92:383-389.  [PubMed]  [DOI]
28.  Uno M, Hamajima N, Ito LS, Oba SM, Marie SK, Shinjo SK, Onda H, Saito T, Takezaki T, Tajima K. Helicobacter pylori seropositivity and IL-1B C-31T polymorphism among Japanese Brazilians. Int J Mol Med. 2002;10:321-326.  [PubMed]  [DOI]
29.  Kawai T, Kawakami K, Kudo T, Ogiahara S, Handa Y, Moriyasu F. A new serum antibody test kit (E plate) for evaluation of Helicobacter pylori eradication. Intern Med. 2002;41:780-783.  [PubMed]  [DOI]
30.  Sata F, Yamada H, Suzuki K, Saijo Y, Yamada T, Minakami H, Kishi R. Functional maternal catechol-O-methyltransferase polymorphism and fetal growth restriction. Pharmacogenet Genomics. 2006;16:775-781.  [PubMed]  [DOI]
31.  Suzuki K, Sata F, Yamada H, Saijo Y, Tsuruga N, Minakami H, Kishi R. Pregnancy-associated plasma protein-A polymorphism and the risk of recurrent pregnancy loss. J Reprod Immunol. 2006;70:99-108.  [PubMed]  [DOI]
32.  Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005;21:263-265.  [PubMed]  [DOI]
33.  Hedrick PW. Gametic disequilibrium measures: proceed with caution. Genetics. 1987;117:331-341.  [PubMed]  [DOI]
34.  Van Heel DA, Udalova IA, De Silva AP, McGovern DP, Kinouchi Y, Hull J, Lench NJ, Cardon LR, Carey AH, Jewell DP. Inflammatory bowel disease is associated with a TNF polymorphism that affects an interaction between the OCT1 and NF(-kappa)B transcription factors. Hum Mol Genet. 2002;11:1281-1289.  [PubMed]  [DOI]
35.  Kamizono S, Hiromatsu Y, Seki N, Bednarczuk T, Matsumoto H, Kimura A, Itoh K. A polymorphism of the 5' flanking region of tumour necrosis factor alpha gene is associated with thyroid-associated ophthalmopathy in Japanese. Clin Endocrinol (Oxf). 2000;52:759-764.  [PubMed]  [DOI]
36.  Higuchi T, Seki N, Kamizono S, Yamada A, Kimura A, Kato H, Itoh K. Polymorphism of the 5'-flanking region of the human tumor necrosis factor (TNF)-alpha gene in Japanese. Tissue Antigens. 1998;51:605-612.  [PubMed]  [DOI]
37.  Wen AQ, Wang J, Feng K, Zhu PF, Wang ZG, Jiang JX. Effects of haplotypes in the interleukin 1beta promoter on lipopolysaccharide-induced interleukin 1beta expression. Shock. 2006;26:25-30.  [PubMed]  [DOI]
38.  Hall SK, Perregaux DG, Gabel CA, Woodworth T, Durham LK, Huizinga TW, Breedveld FC, Seymour AB. Correlation of polymorphic variation in the promoter region of the interleukin-1 beta gene with secretion of interleukin-1 beta protein. Arthritis Rheum. 2004;50:1976-1983.  [PubMed]  [DOI]
39.  Saijo Y, Sata F, Yamada H, Kondo T, Kato EH, Kishi R. Single nucleotide polymorphisms in the promoter region of the interleukin-6 gene and the risk of recurrent pregnancy loss in Japanese women. Fertil Steril. 2004;81:374-378.  [PubMed]  [DOI]
40.  Lim CS, Zheng S, Kim YS, Ahn C, Han JS, Kim S, Lee JS, Chae DW. The -174 G to C polymorphism of interleukin-6 gene is very rare in koreans. Cytokine. 2002;19:52-54 DOI : 10.1006/cyto.2002.1951.  [PubMed]  [DOI]
41.  Ota N, Nakajima T, Nakazawa I, Suzuki T, Hosoi T, Orimo H, Inoue S, Shirai Y, Emi M. A nucleotide variant in the promoter region of the interleukin-6 gene associated with decreased bone mineral density. J Hum Genet. 2001;46:267-272.  [PubMed]  [DOI]
42.  Kitamura A, Hasegawa G, Obayashi H, Kamiuchi K, Ishii M, Yano M, Tanaka T, Yamaguchi M, Shigeta H, Ogata M. Interleukin-6 polymorphism (-634C/G) in the promotor region and the progression of diabetic nephropathy in type 2 diabetes. Diabet Med. 2002;19:1000-1005.  [PubMed]  [DOI]
43.  Ortlepp JR, Metrikat J, Vesper K, Mevissen V, Schmitz F, Albrecht M, Maya-Pelzer P, Hanrath P, Weber C, Zerres K. The interleukin-6 promoter polymorphism is associated with elevated leukocyte, lymphocyte, and monocyte counts and reduced physical fitness in young healthy smokers. J Mol Med (Berl). 2003;81:578-584.  [PubMed]  [DOI]
44.  Hamajima N, Katsuda N, Matsuo K, Saito T, Hirose K, Inoue M, Zaki TT, Tajima K, Tominaga S. High anti-Helicobacter pylori antibody seropositivity associated with the combination of IL-8-251TT and IL-10-819TT genotypes. Helicobacter. 2003;8:105-110.  [PubMed]  [DOI]
45.  El-Omar EM, Rabkin CS, Gammon MD, Vaughan TL, Risch HA, Schoenberg JB, Stanford JL, Mayne ST, Goedert J, Blot WJ. Increased risk of noncardia gastric cancer associated with proinflammatory cytokine gene polymorphisms. Gastroenterology. 2003;124:1193-1201.  [PubMed]  [DOI]