A positive assay for identification of cagA negative strains of Helicobacter pylori

A new PCR protocol was developed for the positive identification of cagA negative Helicobacter pylori strains. Amplification of a portion of the genome across the insertion point of the cag pathogenicity island (the ES-"empty site") generated a 106-bp fragment, which produces a positive signal for cagA negative strains. Combined with the results of the cagA assay, the signals for ES allowed the complete characterization of the patients' cagA status. DNA sequencing analysis confirmed the identity of the ES fragment. The new protocol and cagA assay were applied to 22 DNA preparations isolated from stools from H. pylori infected adult patients and to 21 DNA preparations isolated from stools from H. pylori infected children. The same analysis was also performed on nine colonies of H. pylori derived from gastric biopsies of nine of the adult patients. The total number of cagA positive cases from adult patients was 14 or 63.6% (11 mono- and 3 mixed) and of the cagA negative cases (or ES positive) was 9 or 40.9% (6 mono- and 3 mixed). Of the 21 stool DNA samples from children, 6 (28.6%) were cagA positive, 12 (57.1%) were cagA negative and 3 (14.3%) were positive for cagA and for the ES simultaneously. The proportions of mixed cagA positive and cagA negative H. pylori infections were almost equal in adults and children (13.6% and 14.3%, respectively). No reaction products of the proper fragment sizes for cagA or the empty site (ES) were obtained from any of the stool DNA samples of 10 H. pylori uninfected subjects (100% specificity). This noninvasive assay discriminates consistently cagA negative cases from cagA positive strains and from amplification failures. It can be a useful tool for clinical and epidemiological studies of H. pylori infection.     

Orogastric vaccination of guinea pigs with Helicobacter pylori sonicate and a high dose of cholera toxin lowers the burden of infection

Guinea pigs were vaccinated orogastrically with Helicobacter pylori cell sonicate (CS) and 10 microg or 100 microg cholera toxin (CT) or CT only. Nai;ve animals were used as a control. In both experiments, vaccination primed the local IgG and IgA response, irrespective of the CT dose. After challenge, only the group of animals immunised with CS and 100 microg CT had a significantly lower number of H. pylori in the antral region of the stomach, but vaccination did not prevent H. pylori infection. This protective effect was not associated with a switch in IgG subclass, which remained predominantly IgG2. The levels of specific antibodies in serum and the gastric mucosa which were similar to naive unprotected animals. In conclusion, the ability of mucosal adjuvants such as CT to induce a protective immune response may be host dependent and findings in the Helicobacter-mouse model should be interpreted with caution.     

Eradication of Helicobacter pylori infection equally improves chronic urticaria with positive and negative autologous serum skin test

BACKGROUND: The aim of the study was to examine effects of Helicobacter pylori eradication on chronic idiopathic urticaria (CU) with and without positive aulogous serum skin test (ASST). METHODS: Seventy-eight patients with CU were checked for the positivity ASST and H. pylori urea (13)C-urea breath test ((13)C-UBT). Twenty-one patients were with both positive ASST and positive (13)C-UBT (group A), and 24 patients were with negative ASST and positive (13)C-UBT (group B). All patients with positive (13)C-UBT received a 14-day, open treatment with amoxicillin 1 g b.i.d., clarithromycin 500 mg b.i.d., and omeprazole 20 mg b.i.d. H. pylori eradication was assessed by a second (13)C-UBT after 8 weeks. In control group, 33 patients with CU were included. The effect of H. pylori eradication on CU was evaluated by urticaria activity score (UAS), measured at study entry and at 8 and 16 weeks. RESULTS: At week 8, baseline UAS reduced from 4.7 +/- 1.1 to 2.4 +/- 1.4 (p = .027) in group A and from 4.3 +/- 1.5 to 2.3 +/- 1.2 (p = .008) in group B, without statistically significant difference between the two groups. In control group and in six patients with H. pylori eradication failure, no changes of UAS were noted. CONCLUSION: Eradication of H. pylori infection by triple therapy significantly and equally reduces UAS in CU patients with positive and negative ASST.     

cagA+ Helicobacter pylori induces greater levels of prostaglandin E2 than cagA- strains

cagA+ Helicobacter pylori (HP) infection is associated with an increased risk of distal gastric cancer. Previous studies investigating the effect of HP infection on prostaglandin E2 (PGE2) levels have not differentiated between cagA+ and cagA- strains and consequently have produced contradictory results. The aim was to investigate the effect of cagA+ strains on PGE2 and enhance the understanding of the mechanisms leading to gastric diseases. Hundred patients without peptic ulcers and not on medication were recruited (one later excluded) from endoscopy clinics: six biopsies were obtained from each patient. PGE2, colonization density and histology were determined. In addition, HP status was assessed by histology, CLOtest and culture with cagA+ being determined by PCR. Sixty-nine patients were HP- and 30 HP+ (10 cagA+, 18 cagA-, 2 undetermined). In age and sex-matched patients, PGE2 was significantly greater (P = 0.04) in HP+ (37.2 +/- 1.2 pg/mg per 20 min) than in HP- (22.6 +/- 1.2). In patients without atrophy, those infected with cagA+ had significantly higher (P = 0.03) PGE2 levels (53 +/- 1.1) than HP- patients (22.6 +/- 1.1) and greater levels (P = 0.29) than cagA- patients (35 +/- 1.3). In conclusion, the increased levels of PGE2 in the presence of cagA+ infection could be an important factor by which cagA+ strains enhance the gastric mucus layer protective functions leading to established colonization, gastritis and increased risk of gastric cancer. However, further evaluation with a large-scale multi-centre study is required to substantiate this hypothesis.     

cagA gene variants in Malaysian Helicobacter pylori strains isolated from patients of different ethnic groups

Helicobacter pylori infection of a distinct subtype of cagA may lead to different pathological manifestation. The aim of this study is to determine the presence of cagA gene and its variants in H. pylori infection among different ethnic groups and its effect on gastroduodenal diseases. Overall detection of cagA among the 205 clinical isolates of H. pylori was 94%. Variations in size of the 3' region of cagA gene were examined among 192 Malaysian H. pylori cagA-positive strains. Results showed that three cagA variants differing in fragment length of PCR products were detected and designated as type A (621-651bp), type B (732-735bp) and type C (525 bp). Although there was no association between any of the cagA subtypes with peptic ulcer disease (p>0.05), an association between cagA subtypes with a specific ethnic group was observed. Specific-cagA subtype A strains were predominantly isolated from Chinese compared to Malays and Indians (p<0.0005), and cagA subtype B strains were predominantly isolated from Malays and Indians compared to Chinese (p<0.05). The cagA type A strains of H. pylori is commonly found in the Chinese patients who have a higher risk of peptic ulcer disease, thus indicating that it could be used as an important clinical biomarker for a more severe infection.     

幽门螺杆菌cagA克隆表达

克隆表达4株幽门螺杆菌的cagA基因,以方便地获得大鼠CagA蛋白和重组表达质粒,为临床诊断CagA阳性幽门螺杆菌感染,以及进一步研究不同类型CagA功能及其与疾病关系提供材料。PCR扩增幽门螺杆菌的cagA基因,克隆至PinPoint^TMXa-1T载体,酶切鉴定连接方向,IPTG诱导正向连接克隆表达CagA融合蛋白并进行SDS－PAGE和Western blots鉴定。结果显示PCR扩增得到3.5-3.8kb的CagA基因,PCR及酶切鉴定得到正向连接的重组克隆,SDS－PAGE及Western blots证实正向连接的重组克隆表达CagA融合蛋白。构建了4种cagA的重组表达质粒,通过转化同一宿主菌可研究不同CagA的功能和致病性差异;通过亲和层析纯化融合蛋白可获大量CagA蛋白,用于血清学诊断CagA阳性幽门螺杆菌感染,及不同抗原性CagA与疾病之间的关系。     

The diversity of vacA and cagA genes of Helicobacter pylori in East Asia

It has been reported that Helicobacter pylori infection with the type I strain, which expresses the VacA and CagA antigens, is associated with duodenal ulcer. We examined the diversity of vacA and cagA genes in 143 isolates obtained from patients with duodenal ulcer or chronic gastritis in East Asia (two different areas of Japan, Fukui and Okinawa, and also in Hangzhou, China) by polymerase chain reaction (PCR) and sequence analysis. Diversities of cagA and vacA genes were detected in East Asia. The prevalence of cagA-positive H. pylori was significantly different between Fukui and Okinawa (P=0.0032). The prevalence of Western type CagA was significantly higher in Okinawa than in Fukui (P<0.0001). However, there was no significant association between the genotype of cagA and clinical outcome. In Japan, the predominant vacA genotype was s1c/m1b. In contrast, in Hangzhou, the predominant vacA genotype was s1c/m2, and they were all East Asian CagA-positive. These findings suggest that a distinct distribution of the vacA and cagA genotypes is present in East Asia, regardless of clinical outcome.     

Animal models of Helicobacter pylori infection and disease

The acceptance of Helicobacter pylori as a major human pathogen has necessitated the development of animal models to help elucidate the pathogenic mechanisms of this bacterium and aid in the development of improved strategies for the treatment of gastric disease. Appropriate models, utilising a range of animal species, have been developed to examine factors such as the influence of host responses and bacterial factors in disease development and the success of new therapeutic regimens, including vaccination, to cure infection.     

The cagA status of Helicobacter pylori isolates from dyspeptic children in Turkey

There are inconsistent reports regarding cytotoxin-associated gene A (cagA) status of Helicobacter pylori isolates and the severity of the mucosal lesions in children. The aim of this study was to determine the prevalence of cagA(+) strains and to evaluate its correlation with clinic and endoscopic findings. We examined 45 H. pylori strains that were grown on brain-heart infusion agar supplemented with 7% horse blood. Following 72 h of incubation colonies were harvested and bacterial DNA was extracted. Polymerase chain reaction primers F1 and B1 were used to amplify a 348-bp internal fragment of cagA. The prevalence of cagA in Turkish pediatric patients was 55.6%. No association was found between cagA status and the severity of gastro-duodenal lesions.     

Association of Helicobacter pylori infection with coronary heart disease.

The role of Helicobacter pylori (HP) as the main etiological factor in gastritis and peptic ulcer disease is undisputable. Gastric mucosal damage caused by HP involves various bacterial and host-dependent toxic substances that have been recently associated with an increased risk of coronary artery disease (CAD), possibly through the activation acute phase response and of procoagulant hemostatic factors. Recent studies showed a close and strong correlation between plasma increments of some cytokines such as IL-6 or TNFalpha and cardiovascular diseases. HP infection induces platelet activation and aggregation that could be the pathogenic explanation of the association between HP infection and CAD. The aim of this study was to determine the seroprevalence of HP infection and antibodies to CagA, an antigen that is expressed by the most virulent HP strains inducing an enhanced gastric inflammatory response, in patients undergoing routine coronary artery examination. We studied 76 patients with CAD and 81 healthy controls patients without significant change in coronary circulation. Angiograms were read by two independent experienced cardiologists blinded to the results of HP status. The presence of serum IgG antibodies to HP and to CagA and plasma interleukin-8 (IL-8) levels was measured by ELISA. In addition plasma C-reactive protein fibrinogen, total cholesterol and lipids levels were measured in all studied patients. Seropositivity to HP was found in 81.5 % of cases and in 51% of controls and the difference in prevalence was statistically significant, the odds ratio being 4.3 for Hp patients. Antibody to CagA protein was detected in 47.3% of CAD but only in 28% of healthy controls (OR = 2.3 vs OR = 10). C-reactive protein, plasma fibrinogen and total cholesterol were, respectively higher in patients with CAD than in controls. Present data show that there is significant link between CAD and HP infection. The HP infection significantly increases the risk of CAD, especially when both the anti-HP IgG and anti-CagA IgG are considered. Higher prevalence of cytotoxic HP strains might enhance the atherosclerotic process by inducing a persistent, low grade inflammatory response in arterial wall with enhanced synthesis of acute phase reactants.     

Helicobacter pylori infection in patients with Hepatitis C Virus positive chronic liver diseases

BACKGROUND AND GOALS: One-third of patients with liver cirrhosis suffers from acute peptic ulcer, a disease strongly correlated with Helicobacter pylori (H. pylori) infection. We report the seroprevalence of antibodies to H. pylori in 179 patients with Hepatitis C Virus (HCV)-related chronic active hepatitis and cirrhosis. MATERIALS AND METHODS: Among patients, 135 (86 males and 49 females, mean age 51.2 +/- 13.28, range 27-77 years) had chronic active hepatitis (CAH) and 44 cirrhosis (28 males and 16 females, mean age 62.4 +/- 9.2, range 37-77 years). Serum antibodies to H. pylori were tested using a commercial enzyme immunosorbent assay. The control population consisted of 619 consecutive blood donors (523 males, 96 females, mean age 47 +/- 5.3 years, range 18-65). RESULTS: The overall prevalence of antibodies to H. pylori was 73.1% (131/179) among patients and 47% (291/619) among blood donors (p<0.0001; OR 3.08 [95%CI, 2.10-4.51]). 70.5% (24/34) of patients aged less than 40 years were seropositive for H. pylori versus 34.2% (90/263) of controls (p<0.0001; OR 4.61[95%CI, 2.0-10.85]). Among cirrhosis patients, the prevalence of antibodies to H. pylori was 79.5% (35/44) versus 47% (291/619) of controls (p<0.0001; OR 4.38 [95%CI, 1.98-9.98]). Overall seroprevalence among CAH patients was 71.1% (96/135) versus 47% (291/619) of blood donors (p<0.0001; OR 2.77 [95%CI, 1.82-4.24]). CONCLUSIONS: The high seroprevalence of antibodies to H. pylori in patients with HCV-positive liver diseases explains the elevated incidence of peptic ulcer, and warrants studies on the pathogenic role in human liver diseases of Helicobacter spp which is known to cause chronic hepatitis and hepatocellular carcinoma in mice.     

Emergence of recombinant strains of Helicobacter pylori during human infection

Genetic recombination can be important evolutionarily in speeding the adaptation of organisms to new environments and in purging deleterious mutations. Here, we describe polymerase chain reaction (PCR), hybridization and DNA sequence-based evidence of six such exchanges between two strains of Helicobacter pylori during natural mixed infection of a patient in Lithuania. One parent strain contained the 37 kb long, virulence-associated cag pathogenicity island (PAI), and the other strain lacked this PAI. Most H. pylori from the patient had descended from the cag ⁺ parent, but had become cag ⁻ during infection. This had resulted from transfer of DNA containing the 'empty site' allele from the cag ⁻ strain and homologous recombination, not from excision of the cag PAI without DNA transfer. Other cases of recombination involved genes for an outer membrane protein ( omp 5 and omp 29; also called HP0227 and HP1342) and a putative phosphoenolpyruvate synthase ( ppsA  ; HP0121). Replacement of a short patch of DNA sequence (36–124 bp) was also seen. As the chance of forming any given recombinant is small, the abundance of recombinants in this patient suggests selection for particular recombinant genotypes during years of chronic infection. We suggest that genetic exchange among unrelated H. pylori strains, as documented here, is important because of the diversity of this gastric pathogen and its human hosts. Certain H. pylori recombinants may grow better in a given host than either parent. The vigour of growth, in turn, could impact on the severity of disease that infection can elicit.     

The effect of Helicobacter pylori on neutrophil chemotaxis is independent of cagA

Sixteen Helicobacter pylori strains were studied in order to determine their neutrophil chemotactic activity and the association with the presence cagA gene. Neutrophil chemotactic activity was detected by a modified Boyden chamber method and the results were expressed in terms of chemotactic index (CI). The presence of cagA was determined by PCR. Of the 16 strains, eight were cagA+ and eight were cagA-. All of the isolated strains showed chemotactic activity. The mean value of CI of the patient group was significantly higher than the negative control (P < 0.01). The mean value of CI of zymosan-activated serum (P < 0.05) and the reference strain H. pylori NCTC 11637 (HP11637) (P < 0.01) was significantly higher than the patient group's mean value of CI. There were no statistical significance in the CI between cagA+ and cagA- strains (P > 0.05). It is concluded that H. pylori attracts neutrophils by chemotaxis, however, there is no association with cagA.     

利用杆状病毒表达幽门螺杆菌cagA基因

幽门螺杆菌cagA基因克隆到杆状病毒表达系统的pBlueBacHis2A转移载体中,将重组质粒pBlueBacHis2A-CagA与亲本病毒Bac-N-blue DNA共转染Sf9细胞,以空斑法纯化获得的重组杆状病毒.经PCR法鉴定后进行扩增培养,SDS-PAGE和Western bolt检测结果证实所表达的蛋白为CagA蛋白,间接ELISA分析表明,表达产物可与Hp感染者血清发生特异性的免疫反应.     

利用杆状病毒表达幽门螺杆菌cagA基因

幽门螺杆菌cagA基因克隆到杆状病毒表达系统的 pBlueBacHis2A转移载体中 ,将重组质粒 pBlueBacHis2A CagA与亲本病毒Bac N blueDNA共转染Sf9细胞 ,以空斑法纯化获得的重组杆状病毒。经PCR法鉴定后进行扩增培养 ,SDS PAGE和Westernbolt检测结果证实所表达的蛋白为CagA蛋白 ,间接ELISA分析表明 ,表达产物可与Hp感染者血清发生特异性的免疫反应     

Immunoproteomics of Helicobacter pylori infection and relation to gastric disease

The Gram negative bacterium Helicobacter pylori is a human pathogen which infects the gastric mucosa and causes an inflammatory process leading to gastritis, ulceration and cancer. A systematic, proteome based approach was chosen to detect candidate antigens of H. pylori for diagnosis, therapy and vaccine development and to investigate potential associations between specific immune responses and manifestations of disease. Sera from patients with active H. pylori infection (n = 24), a control group with unrelated gastric disorders (n = 12) and from patients with gastric cancer (n = 6) were collected and analyzed for the reactivity against proteins of the strain HP 26695 separated by two-dimensional electrophoresis. Overall, 310 antigenic protein species were recognized by H. pylori positive sera representing about 17% of all spots separated. Out of the 32 antigens most frequently recognized by H. pylori positive sera, nine were newly identified and 23 were confirmed from other studies. Three newly identified antigens which belong to the 150 most abundant protein species of H. pylori, were specifically recognized by H. pylori positive sera: the predicted coding region HP0231, serine protease HtrA (HP1019) and Cag3 (HP0522). Other antigens were recognized differently by sera from gastritis and ulcer patients, which may identify them as candidate indicators for clinical manifestations. The data from these immunoproteomic analyses are added to our public database (http://www.mpiib-berlin.mpg.de/2D-PAGE). This platform enables one to compile many protein profiles and to integrate data from other studies, an approach which will greatly assist the search for more immunogenic proteins for diagnostic assays and vaccine design.     

Helicobacter pylori iceA, clinical outcomes, and correlation with cagA: a meta-analysis

Background

Although the iceA (induced by contact with epithelium) allelic types of Helicobacter pylori have been reported to be associated with peptic ulcer, the importance of iceA on clinical outcomes based on subsequent studies is controversial. The aim of this study was to estimate the magnitude of the risk for clinical outcomes associated with iceA.

Methods

A literature search was performed using the PubMed and EMBASE databases for articles published through April 2011. Published case-control studies examining the relationship between iceA and clinical outcomes (gastritis, peptic ulcer, including gastric ulcer and duodenal ulcer, and gastric cancer) were included.

Results

Fifty studies with a total of 5,357 patients were identified in the search. Infection with iceA1-positive H. pylori increased the overall risk for peptic ulcer by 1.26-fold (95% confidence interval [CI], 1.09–1.45). However, the test for heterogeneity was significant among these studies. Sensitivity analysis showed that the presence of iceA1 was significantly associated with peptic ulcer (odds ratio [OR] = 1.25, 95% CI = 1.08–1.44). The presence of iceA2 was inversely associated with peptic ulcer (OR = 0.76, 95% CI = 0.65–0.89). The presence of iceA was not associated with gastric cancer. Most studies examined the cagA status; however, only 15 studies examined the correlation and only 2 showed a positive correlation between the presence of cagA and iceA1.

Conclusion

Our meta-analysis confirmed the importance of the presence of iceA for peptic ulcer, although the significance was marginal.