Expression of Helicobacter pylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine delivery system against H. pylori infection in mice

The use of Lactococcus lactis as an antigen delivery vehicle for mucosal immunisation has been proposed. To determine whether L. lactis could effectively deliver Helicobacter pylori antigens to the immune system, a recombinant L. lactis expressing H. pylori urease subunit B (UreB) was constructed. Constitutive expression of UreB by a pTREX1 vector resulted in the intracellular accumulation of UreB to approximately 6.25% of soluble cellular protein. Five different oral regimens were used to vaccinate C57BL/6 mice and the immune response measured. One regimen, which consisted of four weekly doses of 10(10) bacteria, followed after an interval of approximately 4 weeks by three successive daily doses, was able to elicit a systemic antibody response to UreB in the mice, although subsequently, a similar regimen produced a significant antibody response in only one out of six mice. The other three regimes, in which mice were vaccinated with two or three sets of three consecutive daily doses of recombinant bacteria over 30 days, failed to elicit significant anti-UreB serum antibody responses. In three regimens, the immunised mice were then challenged by H. pylori strain SS1 and no protective effect was observed. These findings suggest that any adjuvant effects of L. lactis are unlikely to be sufficient to produce an effective immune response and to protect against H. pylori challenge, when used to deliver a weak immunogen, such as UreB.     

Poliovirus replicons encoding the B subunit of Helicobacter pylori urease elicit a Th1 associated immune response.

The development of a vaccine for Helicobacter pylori is a key strategy for reducing the worldwide prevalence of H. pylori infection. Although immunization with recombinant B subunit of H. pylori urease (ureB) has yielded promising results, for the most part, these studies relied on the use of strong adjuvant, cholera toxin, precluding the use in humans. Thus, the development of new vaccine strategies for H. pylori is essential. Previous studies from our laboratory have described a vaccine vector based on poliovirus in which foreign genes are substituted for the poliovirus capsid genes. The genomes encoding foreign proteins (replicons) are encapsidated into authentic poliovirions by providing the capsids in trans. To test the utility of replicons as a vaccine vector for H. pylori, a replicon was constructed which encodes ureB. Expression of ureB in cells from the replicon was demonstrated by metabolic labeling followed by immunoprecipitation with anti-urease antibodies. To investigate the immunogenicity of the replicons, mice containing the transgene for the receptor for poliovirus were immunized via the intramuscular route. Mice given three doses of replicons did not develop substantial antibodies to ureB as determined by Western blot analysis using lysates from H. pylori. In contrast, mice given two doses of replicon followed by a single injection of recombinant ureB developed serum antibodies to ureB which were predominately IgG2a. Splenic lymphocytes from mice immunized with replicons alone, or replicons plus recombinant ureB produced abundant interferon-gamma and no detectable interleukin-4 upon stimulation with recombinant ureB. These results establish that poliovirus replicons encoding H. pylori ureB are immunogenic and induce primarily a T helper 1 associated immune response.     

A plasmid immunization construct encoding urease B of Helicobacter pylori induces an antigen-specific antibody response and upregulates the expression of beta-defensins and IL-10 in the stomachs of immunized mice

The objectives of this study were to investigate the efficacy of a prototype DNA immunization construct encoding the urease B subunit enzyme of Helicobacter pylori (H. pylori) for inducing adaptive and innate immune responses in mice immunized via intramuscular or subcutaneous routes and to further explore the adjuvant effects of the CpG motifs in the vector. Antibody, cytokine, and beta-defensin profiles were assessed in the stomachs of immunized animals: experiments were terminated 3 months after immunization because there was a significant increase in the anti-H. pylori urease B antibody response at Week 6 in mice immunized with the urease B construct. A long lasting expression of IL-10 mRNA was noted. Furthermore, a marked and sustained increase in the mRNA expression of beta-defensins was also observed, particularly beta1. This study demonstrates that an H. pylori urease B DNA construct can induce innate as well as adaptive immune responses in the stomachs of immunized mice. Upregulation of beta-defensin gene expression followed immunization and we believe that this is the first report of a DNA vaccine inducing innate anti-microbial responses. Such complex molecular interactions that modulate both innate and adaptive immune responses may be of critical importance in the control of mucosal pathogens, such as H. pylori.     

Immunization of rhesus monkeys with a mucosal prime, parenteral boost strategy protects against infection with Helicobacter pylori.

Rhesus monkeys were immunized with recombinant Helicobacter pylori urease vaccine given solely by the parenteral route or preceded by a priming dose given by the oral route. Two groups of monkeys received parenteral urease with either a synthetic glycolipid adjuvant (Bay) or aluminum hydroxide (alum) as adjuvants. A third group of monkeys received a priming dose of oral urease given with the mucosal adjuvant LT (Escherichia coli heat labile enterotoxin), followed by parenterally administered booster doses of urease adsorbed to alum. Monkeys receiving placebo served as controls. The monkeys received a total of 4 doses of vaccine with the first 3 doses given every 3 weeks and the last booster dose administered 14 weeks later. The monkeys were challenged orally with H. pylori one week after the last vaccine dose and euthanized 10 weeks after challenge, at which time, their stomachs were collected for determination of bacterial colonization and histopathology. Monkeys primed with the oral vaccine and boosted with the parenteral vaccine showed a statistically significant reduction in bacterial colonization when compared to sham-immunized control animals (P = 0.05; Wilcoxon rank sums test). Monkeys receiving parenteral only regimes of urease plus Bay or alum showed no difference in bacterial colonization compared with sham-immunized controls (P = 1.00 and P = 0.33, respectively). The mucosal prime-parenteral boost regime did not cause gastropathy. There was no difference in any of the 3 treatment groups with respect to gastric epithelial changes compared to control animals. There was also no difference in the type and extent of gastric inflammatory cell infiltrates between animals vaccinated by the mucosal prime-parenteral boost strategy and sham immunized controls. However, monkeys receiving the two parenteral-only regimens had slightly elevated gastritis scores.     

Oral vaccination with liposome-encapsulated recombinant fusion peptide of urease B epitope and cholera toxin B subunit affords prophylactic and therapeutic effects against H. pylori infection in BALB/c mice

A new fusion peptide CtUBE of cholera toxin B subunit and Helicobacter pylori urease B subunit epitope was expressed in Escherichia coli. With this fusion peptide, an oral liposome vaccine against H. pylori infection was prepared and evaluated in BALB/c mice. Based on the results of urease tests, quantitation of culturable bacteria colonies in mice stomachs and histological identification of gastritis, the mice were protected significantly after intragastric vaccination with this CtUBE liposome vaccine, which increased the content levels of specific anti-urease serum IgG and mucosal IgA for both prophylactic and therapeutic vaccination protocols. These results showed that the fusion peptide CtUBE retained immunogenicity and could be used as antigen in the development of an oral vaccine against H. pylori infection.     

Oral immunization of mice with a multivalent therapeutic subunit vaccine protects against Helicobacter pylori infection

Helicobacter pylori is a human class I carcinogen and no effective prophylactic or therapeutic H. pylori vaccine has yet been marketed. H. pylori can escape the host immune response, but the precise immune protection mechanisms in humans remain unknown. In this study, we developed a multivalent, subunit H. pylori vaccine candidate by formulating three commonly used H. pylori antigens, neutrophil-activating protein (NAP), urease subunit A (UreA) and subunit B (UreB) with the mucosal adjuvant, a double-mutant heat-labile toxin (dmLT) from Escherichia coli, and evaluated its immunogenicity and therapeutic efficacy in a mouse model of H. pylori infection. We found that oral immunization of H. pylori-infected mice significantly reduced gastric bacterial colonization at both 2 and 8 weeks after immunization. The reduction in bacterial burdens was accompanied with significantly increased serum antigen-specific IgG responses and mucosal IgA responses. Moreover, oral immunization also induced Th1/Th17 immune responses, which may play a synergistic role with the specific antibodies in the elimination of H. pylori. Thus, our vaccine candidate appears able to overcome the immune evasion mechanism of H. pylori, restore the suppression of Th2 immune responses with the induction of a strong humoral immune response. These results lay the foundation for the development of an optimized oral therapeutic H. pylori vaccine with increased immunogenicity of UreA and UreB, as well as providing long-term immunity.     

Identification of H-2d restricted Th epitopes in Urease B subunit of Helicobacter pylori

CD4+ T cells play important roles in protection against Helicobacter pylori (H. pylori) infection. In order to better understand the immune responses of H. pylori infection and improve immune interventions against this pathogen, we identified the Th epitopes in UreB of H. pylori, an excellent vaccine candidate antigen. By using the RANKPEP prediction algorithm, we have identified and characterized three Th epitopes within the UreB antigen, which can be recognized by CD4+ T cells from BALB/c (H-2d) mice. They were U(546-561), U(229-244), and U(237-251). These epitopes have important value for studying the immune response of H. pylori infection and for designing effective vaccine against H. pylori.     

Screening and identification of a novel B-cell neutralizing epitope from Helicobacter pylori UreB

Urease plays a crucial role in the survival and pathogenesis of Helicobacter pylori (H. pylori), and antibody neutralizing the urease activity may be implicated for the protection against H. pylori infection. Previously, a neutralizing monoclonal antibody (MAb) 6E6 against UreB of H. pylori was developed. In this work, we try to identify the B-cell epitope recognized by neutralizing MAb 6E6. Following screening a series of truncated proteins of UreB, an epitope was primarily localized in the aa 200-230 of UreB. Subsequently, we screened the overlapping synthetic peptides covering the aa 200-230 and identified a novel B-cell epitope (U(211-225), IEAGAIGFKIHEDWG) that was recognized by specific MAb 6E6. The newly identified epitope may help understanding of the protective immunity against H. pylori and be implicated for vaccine development.     

Immunization with recombinant Helicobacter pylori urease decreases colonization levels following experimental infection of rhesus monkeys

Rhesus monkeys, naturally colonized with H. pylori as indicated by culture and histology were immunized with either 40 mg recombinant H. pylori urease administered orally together with 25 microg Escherichia coli heat-labile enterotoxin (LT) or immunized with LT alone. An initial 6 doses were administered over an 8 week period. All five vaccinated monkeys had a greater than two-fold rise in urease-specific serum IgG and IgA level and urease-specific salivary IgA was induced in 3 of 5 vaccinated animals after 6 or 7 doses of vaccine. Vaccination had no measurable therapeutic effect on H. pylori colonization. H. pylori was eradicated from these monkeys with a course of antimicrobials plus omeprazole, a 7th vaccine dose was given (10 months after the 6th dose) and they were rechallenged with H. pylori. Necropsy was performed 23 weeks after rechallenge and H. pylori colonization was determined by histological examination of 12 individual gastric sites. A significant reduction in colonization (p < or = 0.0001; Friedman's analysis of variance) was found in the vaccinated animals. Histopathologic examination of necropsy tissues also revealed a trend towards reduced gastritis and epithelial alterations in the vaccinated group compared to animals receiving LT alone. This study provides the first evidence for effective vaccination of nonhuman primates against H. pylori, and preliminary evidence that a reduction in bacterial density attributable to immunization may lessen gastric inflammation.     

Oral immunization with recombinant Helicobacter pylori urease confers long-lasting immunity against Helicobacter felis infection

Recombinant Helicobacter pylori urease (rUre) has been shown to confer protection against challenge with Helicobacter felis in mice. The purpose of the present study was to examine duration of the immune response and long-term protective efficacy of immunization with rUre. Swiss Webster mice were orally immunized four times at weekly intervals with 100 microg rUre plus 5 microg heat-labile enterotoxin of Escherichia coli (LT) adjuvant, or with LT only. At 4, 10, 20 or 40 weeks post immunization, 25 rUre-immunized mice and control mice were challenged with H. felis and sacrificed at 2 or 10 weeks post-challenge. H. felis infection was assessed by gastric urease assay and by histology. Anti-H. pylori urease specific antibody levels were measured in serum and saliva both pre- and post-challenge. Over the 40 week time period, the infection rates in rUre-immunized mice were significantly lower than those in controls (p < 0.05) as assessed by gastric urease activity. Protection ranged from 79 100% at 2 weeks post-challenge and 63-78% at 10 weeks post-challenge. Gastric bacterial density in rUre-immunized mice was significantly lower than that of controls (p < 0.03) as determined by histologic assessment. Anti-urease antibody levels remained elevated in the serum and mucosal compartments at 39 weeks following immunization. This study shows that immunization with rUre plus LT results in long-lasting protective immunity against challenge with H. felis.     

Protective efficacy of vaccines based on the Helicobacter suis urease subunit B and γ-glutamyl transpeptidase

Helicobacter suis causes gastric lesions in pigs and humans. This study aimed to evaluate the protective efficacy of immunization with combinations of the H. suis urease subunit B (UreB) and γ-glutamyl transpeptidase (GGT), both recombinantly expressed in Escherichia coli (rUreB and rGGT, respectively). Mice were intranasally immunized with rUreB, rGGT or a combination of both proteins, administered simultaneously or sequentially. Control groups consisted of non-immunized and non-challenged mice (negative controls), sham-immunized and H. suis-challenged mice (sham-immunized controls), and finally, H. suis whole-cell lysate-immunized and H. suis challenged mice. Cholera toxin was used as mucosal adjuvant. All immunizations induced a significant reduction of gastric H. suis colonization, which was least pronounced in the groups immunized with rGGT and rUreB only. Consecutive immunization with rGGT followed by rUreB and immunization with the bivalent vaccine improved the protective efficacy compared to immunization with single proteins, with a complete clearance of infection observed in 50% of the animals. Immunization with whole-cell lysate induced a similar reduction of gastric bacterial colonization compared to rGGT and rUreB in combinations. Gastric lesions, however, were less pronounced in mice immunized with combinations of rUreB and rGGT compared to mice immunized with whole-cell lysate. In conclusion, vaccination with a combination of rGGT and rUreB protected mice against a subsequent H. suis infection and was not associated with severe post-vaccination gastric inflammation, indicating that it may be a promising method for control of H. suis infections.     

Novel methods of quantitative real-time PCR data analysis in a murine Helicobacter pylori vaccine model

Monitoring of Helicobacter pylori in the stomach is important to assess the efficacy of new vaccines against the pathogen. To realise the full potential of quantitative real-time PCR (q-PCR), this technology has to offer accurate and easy models of post-PCR data analysis. In this work, we used a variety of absolute and relative approaches of q-PCR data analysis to monitor the H. pylori infection in the stomach of immunized mice. Relative quantification was performed with Ct-based methods, with the DART program, and with two methods based on the mathematical analysis of raw fluorescence kinetics, the LinReg program and the Sigmoidal Curve Fitting Method. The different calculation methods were validated in mice immunized with cell lysates of Lactococcus lactis expressing the H. pylori urease subunit B in combination with cholera toxin. The H. pylori load was found to be reduced in immunized mice by a factor of 50-144, depending on the calculation method employed. We found that relative quantification using DART, LinReg and Sigmoidal Curve Fitting methods generated similar results (infection ratios of 54-58) with absolute quantification results (54-65). Results were very different to those using relative quantification Ct-based methods without a correction for PCR efficiency (ratio of 92-144) and with results based on conventional culture method (ratio of 34). Overall, this study demonstrates that q-PCR associated with a relative quantification analysis is a powerful tool for the monitoring of microorganisms in tissue. It could be used as an alternative to standard curve approach especially for the investigation of microbial load in vaccine models.     

Immunization with a recombinant fusion protein protects mice against Helicobacter pylori infection

More than 50% of the world's population is infected with the bacterium Helicobacter pylori. If left untreated, infection with H. pylori can cause chronic gastritis and peptic ulcer disease, which may progress into gastric cancer. Owing to the limited efficacy of anti-H. pylori antibiotic therapy in clinical practice, the development of a protective vaccine to combat this pathogen has been a tempting goal for several years. In this study, a chimeric gene coding for the antigenic parts of H. pylori FliD, UreB, VacA, and CagL was generated and expressed in bacteria and the potential of the resulting fusion protein (rFUVL) to induce humoral and cellular immune responses and to provide protection against H. pylori infection was evaluated in mice. Three different immunization adjuvants were tested along with rFUVL: CpG oligodeoxynucleotides (CpG ODN), Addavax, and Cholera toxin subunit B. Compared to the control group that had received PBS, vaccinated mice showed significantly higher cellular recall responses and antigen-specific IgG2a, IgG1, and gastric IgA antibody titers. Importantly, rFUVL immunized mice exhibited a reduction of about three orders of magnitude in their stomach bacterial loads. Thus, adjuvanted rFUVL might be considered as a promising vaccine candidate for the control of H. pylori infection.     

A study of Helicobacter pylori infection in patients with pernicious anemia

INTRODUCTION: Chronic atrophic gastritis presents with atrophy of the gastric mucosa, hypochlorhydria or achlorhydria and unstable gastrin level. Type A chronic atrophic gastritis associated with hypergastrinemia is regarded as the principle causative factor for pernicious anemia. AIM: The study aimed at evaluation of the incidence of H. pylori infection in patients with pernicious anemia and analyze its relation to the severity of gastritis. MATERIAL AND METHODS: Forty patients with pernicious anemia (group 1) were examined for presence of H. pylori infection. Sex- and age-matched patients with gastric ulcer (group 2) and chronic superficial gastritis (group 3) were used as controls. Three antral forceps biopsies were obtained from all patients during videogastroscopy. The presence of H. pylori was verified by urease test, histological and microbiological examination. RESULTS: All patients with pernicious anemia had chronic atrophic gastritis and several times lower incidence of H. pylori infection than the patients with gastric ulcer. Chronic atrophic gastritis was not diagnosed in group 3 patients. Statistically significant difference in Helicobacter pylori infection was found between groups 1 and 2 (P < 0.001) but not between groups 1 and 3 (P > 0.05). CONCLUSIONS: Atrophic gastritis was diagnosed in all patients with pernicious anemia. These patients showed significantly lower incidence of H. pylori infection than the gastric ulcer patients. The patients with pernicious anemia had lower gastritis index and quantitatively less expressed infection than the other two groups.     

Oral immunization with HpaA affords therapeutic protective immunity against H. pylori that is reflected by specific mucosal immune responses

In the present study, we evaluated the capacity of Helicobacter pylori adhesin A (HpaA), a H. pylori specific colonization factor, to induce therapeutic protection against H. pylori infection in mice. We found that oral immunization of H. pylori infected mice with HpaA induced protection, i.e. significant reduction in bacterial load in the stomach. This was even more pronounced when a combination of HpaA and urease was used. The protection was strongly related to specific mucosal CD4+ T cell responses with a Th1 profile as well as to mucosal IgA responses locally in the stomach. These findings suggest that HpaA is a promising vaccine candidate antigen for use in a therapeutic vaccine against H. pylori.     

不同形式胃泌素在幽门螺杆菌致病中的作用

  目的  探讨幽门螺杆菌(Helicobacter pylori, H.pylori)感染对血清胃泌素及外泌体源性胃泌素的影响。  方法  随机收集79例慢性胃炎患者血清,13C呼气试验判断H.pylori感染情况,ELISA法测定血清胃泌素和外泌体源性胃泌素浓度。  结果  血清胃泌素及外泌体源性胃泌素在非萎缩性胃炎(non-atrophic gastritis, NAG)与萎缩性胃炎(atrophic gastritis, AG)两组患者中差异均无统计学意义(t=-0.197, P=0.844; t=0.221, P=0.826)。在NAG患者中,H.pylori阳性亚组血清胃泌素及外泌体源性胃泌素均低于H.pylori阴性亚组(t=2.488, P=0.017; t=2.655, P=0.012)。而在AG患者中,血清胃泌素浓度在H.pylori阳性与阴性两亚组中差异无统计学意义(t=1.888, P=0.082);但外泌体源性胃泌素在H.pylori阳性亚组高于H.pylori阴性亚组(t=2.394, P=0.029)。  结论  H.pylori感染早期、胃黏膜发生萎缩前,血清胃泌素下降;当H.pylori持续感染导致萎缩后,血清外泌体源性胃泌素分泌增加,胃泌素多以外泌体形式存在,并可能参与了H.pylori感染致病分子机制。     

Improvement in lipid and haemostasis patterns after Helicobacter pylori infection eradication in type 1 diabetic patients

Helicobacter pylori has been implicated in the cardiovascular risk of diabetic patients. The aim of our study was to investigate whether the Helicobacter pylori infection plays a role in the lipid and haemostasis patterns of type 1 diabetic patients. Twenty nine patients with type 1 diabetes mellitus and H. pylori infection were enrolled (Chlamydia pneumoniae negative). The H. pylori infection status was assessed by serology and urease breath test. In all patients levels of total cholesterol, triglyceride, HDL cholesterol, LDL cholesterol, lipoprotein (a) (Lpa) C reactive protein (CRP), fibrinogen, thrombin/antithrombin III complex (TAT), plasminogen activator inhibitor type 1(PAI-1), tissue plasminogen activator (t-PA) and von Willebrand antigen were measured. All patients were evaluated before and after H. pylori eradicating treatment with amoxicillin, clarithromycin and omeprazole. Twenty two patients were eradicated and seven remained infected.In H. pylori eradicated patients, HDL cholesterol increased (59.7+/-18.9 mg/dl vs 65.2+/-15. 9 mg/dl, P < 0.05), after treatment. After H. pylori eradication, the levels of CRP and TAT decreased (48+/-0.7 ng/l vs 3.3+/-0.4 ng/l;P < 0.05), (27.7+/-44.7 microg/ml vs 2.1+/-1.4 microg/ml, P < 0.05), respectively. The decrease in TAT was higher in the group of H. pylori (+) patients with higher levels of TAT (TAT > 20 ng/ml, 92.8+/-41.6 ng/ml vs 1.9+/-2.0 ng/ml, P < 0.005; TAT 4Eth 20 ng/ml; 10.1+/-5.2 ng/ml vs 2.2+/-0.6 ng/ml, P < 0.05). These changes did not occur in patients without H. pylori eradication. Eradication of H. pylori infection in type 1 diabetic patients modifies some parameters of lipid and haemostasis patterns, (increase of HDL-cholesterol, reduction of Lpa and decrease of CRP and TAT) and so contributes to improvement of cardiovascular risk factors in these patients.     

Iron deficiency and Helicobacter pylori infection in the United States

Using data from the current National Health and Nutrition Examination Survey (1999-2000), the authors assessed whether Helicobacter pylori infection is associated with iron deficiency and iron-deficiency anemia (IDA) in the United States. Iron deficiency was defined as at least two abnormal results out of three biomarkers of iron stores. IDA was defined as a low hemoglobin level in the presence of iron deficiency. H. pylori infection was measured by serology. Complex survey estimators were used in the analysis. For 7,462 survey participants aged >or=3 years, H. pylori infection was associated with decreased serum ferritin levels (percent change = -13.9%, 95% confidence interval (CI): -19.5, -8.0) but not with levels of free erythrocyte protoporphyrin, transferrin saturation, or hemoglobin (percent change = 1.5%, -2.8%, and -1.1%, respectively). Multinomial logistic regression analyses indicated that H. pylori infection was associated with the prevalence of IDA (prevalence odds ratio (POR) = 2.6, 95% CI: 1.5, 4.6) and, to a lesser degree, other types of anemia (POR = 1.3, 95% CI: 1.0, 1.7). H. pylori infection was associated with a 40% increase in the prevalence of iron deficiency (POR = 1.4, 95% CI: 0.9, 2.0) after controlling for relevant covariates. In the United States, H. pylori infection was associated with iron deficiency/IDA regardless of the presence or absence of peptic ulcer disease.     

幽门螺杆菌治疗中抗生素相关性腹泻的预防

目的 探讨双歧三联活菌胶囊（培菲康）对根除幽门螺杆菌（HP）治疗中所致抗生素相关性腹泻（AAD）的预防作用.方法 将128例HP阳性的胃及十二指肠溃疡患者随机分成A组（阿莫西林、甲硝唑、奥美拉唑、果胶铋四联治疗组）和B组（四联加培菲康治疗组）;两组抗生素疗程均为2周,奥美拉唑及果胶铋疗程为4周;抗生素治疗结束4周后复查13C-尿素呼气试验及尿素酶试验,判断根除HP疗效.结果 A、B组两组根除HP效果相近,根除率分别为92.19%和96.88%（P＞0.05）;AAD发生率B组明显低于A组（P〈0.05）,＞60岁患者AAD发生率明显高于〈60岁患者（P〈0.05）.结论 老年人AAD发生率高,培菲康能有效防治根除HP感染治疗中引起的AAD.     

Vitamin B12 status and its association with Helicobacter pylori infection in alcohol dependent patients

Both infection with Helicobacter pylori and alcohol abuse have been associated with low vitamin B12 serum levels. The interaction between both risk factors is unknown. The aim of this study was to determine whether Helicobacter pylori infection is associated with low vitamin B12 levels in alcohol dependent patients. Blood samples were obtained from adult alcohol dependent patients undergoing detoxification and analyzed for serum vitamin B12 levels. Helicobacter pylori infection was serologically measured. Patient characteristics, medication use and alcohol consumption at admission were assessed by interview. A total of 6 out of 89 patients included presented low vitamin B12 levels, all were sub clinical deficient (<250 pmol/L) and none were clinical deficient (<150 pmol/L). Infection with Helicobacter pylori was present in 29% of the patients. The average vitamin B12 levels in Helicobacter pylori seropositive and seronegative patients were 1,033 pmol/L (SD 741) and 971 pmol/L (SD 717), respectively. The relation between Helicobacter pylori infection and vitamin B12 deficiency was not of significance (OR=0.48; 95% CI [0.05-4.32]). In conclusion, Helicobacter pylori infection is not a risk factor for low vitamin B12 levels in alcohol dependent patients.