Effects of killing Helicobacter pyloriquadruple therapy on peptic ulcer： A randomized double-blind clinical trial

AIM: To study the therapeutic efficacy of a Chinese and Western integrated regimen, killing Helicobacter pylori quadruple therapy on H pylori-associated peptic ulcers (PU). METHODS: With prospective and double-blind controlled method, seventy-five active PU patients with H pylori infection were randomized to receive one of the following three regimens: (1) new triple therapy (group A: lansoprazole 30 mg qd, plus clarithromycin 250 mg bid, plus amoxycillin 500 mg tid, each for 10 d); (2) killing Hp quadruple therapy(group B: the three above drugs plus killing H pylori capsule 6 capsules bid for 4 wk) and (3) placebo(group C: gastropine 3 tablets bid for 4 wk). H pylori eradication and ulcer healing quality were evaluated under an endoscope 4 wk after treatment. The patients were followed up for 5 years. RESULTS: Both the healing rate of PU and H pylori eradication rate in group B were significantly higher than those in group C (100% and 96.4% vs20% and 0%, respectively,P<0.005), but there was no significant difference compared to those in group A (88% and 92%, P>0.05). The healing quality of ulcer in group B was superior to that in groups C and A (P<0.05). The recurrence rate of PU in group B (4%) was lower than that in group A (10%) and group C (100%,P<0.01). CONCLUSION: Killing Helicobacter pylori quadruple therapy can not only promote the eradication of H pylori and healing quality of ulcer but also reduce recurrence rate of ulcer.     

幽门螺杆菌抗体检测分析

目的 对昆明地区感染幽门螺杆菌(H.pylori)人群检测血清CasA、VacA、Ure、Hsp60、RdxA五种IgG抗体,了解其与消化性溃疡、慢性胃炎的关系.方法 抽静脉血3 mL离心取血清,用H.pylori抗体芯片检测CagA、VacA、Ure、Hsp60、RdxA五种IgG抗体.结果 ①CagA抗体总阳性率为79.7%,十二指肠球部溃疡(DU)、慢性胃炎(CG)、胃溃疡(GU)的CagA抗体阳性率分别为88.0%、60.9%、83.3%,DU的CagA抗体检出率明显高于CG(P=0.008),而DU与GU(P=0.744)、CG与GU(P=0.633)之间的差异无统计学意义;②VacA抗体总阳性率为20.3%,DU、CG、GU的VacA抗体阳性率分别为14.0%、30.4%、33.3%.三者之间的差异无统计学意义(P=0.12);③Ure抗体总阳性率为97.5%,DU、CG、GU的Ure抗体阳性率分别为98%、95.7%、100%,三者之间的差异无统计学意义(P=0.534);④Hsp60抗体总阳性率为6.25%,DU、CG、GU的Hsp60抗体阳性率分别为4.0%、13.0%、0%,三者之间的差异无统计学意义(P=0.317);⑤RdxA抗体总阳性率为17.5%,DU、CG、GU的RdxA阳性率分别为14.0%、30.4%、0%,三者之间的差异无统计学意义(P=0.179).结论 昆明地区H.pylori感染人群CagA抗体阳性率为79.7%,与DU的关系更加密切;CasA、VacA抗体不能单独作为H.pylori感染阳性的标志物;Hsp60抗体水平对于H.priori感染的检测价值不大;RdxA抗体水平不能标志甲硝唑耐药的水平;而Ure抗体阳性率高达97.5%,可作为H.priori感染阳性的标志物,我们认为检测H.pylori血清抗体用于H.pylori感染的流行病学调查,应该以Ure抗体为主,辅以CagA抗体和VacA抗体.     

幽门螺杆菌对胎儿胃粘膜组织粘附作用的研究

目的 了解不同幽门螺杆菌(Helicobacter pylori)的粘附能力与粘附特征,探讨Hp的粘附机理及其致病。方法 用10株临床分离Hp对四个6～8月龄胎儿胃粘膜组织进行粘附试验。结果 不同Hp的粘附能力有很大差别。存在强粘附铢和弱粘附株;不同Hp对不同胎儿胃粘膜组织各个部位的粘附特征亦有差别。结论 Hp可分泌多种粘附素,机体分泌的粘附素受体亦多样,它们表达的种类以及数量影响粘附结果。     

Expression of Helicobacter pylori AlpA protein and its immunogenicity

AIM: To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA by PCR technique, was sequenced and the biological information was analyzed, and inserted into the Nco I and Not I restriction fragments of the expression vector pET-22b(+) using T4 DNA ligase. The resulting plasmid pET-AlpA was transformed into competent E.coli BL21(DE3) cells using ampicillin resistance for selection. Recombinant strains were incubated in 5 mL LB with 100 μg/mL ampicillin overnight at 37 ℃. Sonication of BL21(DE3)pET-22b(+)/AlpA was analyzed by Western blot to detect AlpA immunogenicity. RESULTS: The gene encoding AlpA protein was amplified by PCR with chromosomal DNA of H pylori Sydney strain (SS1) as templates. It revealed that AlpA DNA fragment amplified by PCR had approximately 1 500 nucleotides, compatible with the previous reports. The recombinant plasmid pET-22b(+)/AB was successfully constructed. DNA sequencing showed one open reading frame with the length of 588 bp. It encoded seven conservative regions that showed good antigenicity and hydrophobicity by Parker and Welling method. Furthermore, INTERNET EXPASY, NNPREDICT and ISREC predicted that it was a porin-like structure consisting of β-pleated sheets that were embedded in the outer membrane. BLAST analyzed 836 767 protein sequences and found that the similar sequences were all belonging to H pylori OMP sequences. SDS-PAGE and scan analysis showed that the molecular weight of AB was 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein, among which dissolved expression amounted to 21.9% of sonicated supernatant. The rAB purity amounted to 96% through affinity chromatography. Western blot analysis of rAB confirmed that it could be specially recognized by serum form rabbit immunized with AlpA and H pylori infected. CONCLUSION: Adhesin AlpA recombinant protein may be a potential vaccine for control and treatment of H pylori infection.     

Frequencies of the expression of main protein antigens from Helicobacter pylori isolates and production of specific serum antibodies in infected patients

AIM: To investigate the frequencies of the expression of main protein antigens of Helicobacter pylori (H py/ori) isolates, such as UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB and the production of specific antibodies in sera from H pylori-infected patients, and to understand the correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori. METHODS: H pylori strains in biopsy specimens from 157 patients with chronic gastritis and peptic ulcer were isolated and serum samples from the patients were also collected. The target recombinant proteins rUreB, rVacA, rCagAl, rHpaA, rNapA, rFlaA and rFlaB expressed by the prokaryotic expression systems constructed in our previous studies were collected through Ni-NTA affinity chromatography. Rabbit antisera against rUreB, rVacA, rCagAl, rHpaA, rNapA, rFlaA and rFlaB were prepared by using routine subcutaneous immunization. By using ultrasonic lysates of the isolates as coated antigens, and the self-prepared rabbit antisera as the first antibodies and commercial HRP-labeling sheep anti-rabbit IgG as the second antibody, expression frequencies of the seven antigens in the isolates were detected by ELISA. Another ELISA was established to detect antibodies against the seven antigens in sera of the patients by using the corresponding recombinant proteins as coated antigens, and the sera as the first antibody and HRP-labeling sheep anti-human IgG as the second antibody respectively. Correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori were statistically analysed. RESULTS: In the 125 isolates of H pylori, the positive rates of UreB, VacA, CagAl, HpaA, NapA, FlaA and FlaB were 100%, 65.6%, 92.8%, 100%, 93.6%, 100% and 99.2% respectively. In the 125 serum samples from the H pylori infected patients, the positive rates of antibodies against recombinant UreB, VacA, CagA1, HpaA, NapA, FIaA and FlaB were 100%, 42.4%, 89.6%, 81.6%, 93.6%, 98.4% and 92.8% respectively. H pylori strains were isolated from 79.6% (125/157) of the biopsy specimens, but no close correlations among the H pylori infection frequencies and different types of chronic gastritis and peptic ulcer could be found (P>0.05, x2 = 0.01-0.87). The VacA positive rate (82.40%) in the strains isolated from the specimens of patients with peptic ulcer and the anti-VacA positive rate (54.3%) in the sera from the patients were significantly higher than those (51.5%, 32.3%) from the patients with chronic gastritis (P<0.01, x2= 13.19; P<0.05, x2= 6.13). When analysis was performed in the different types of chronic gastritis, the VacA in the strains isolated from the specimems of patients with active gastritis showed a higher expression frequency (90.0%) than those from superficial (47.9%) and atrophic gastritis (30.0%) (P<0.05, x2 = 5.93; P<0.01,x2 = 7.50). While analysis was carried out in the strains isolated from the specimens with superficial (93.8%) and active gastritis (100%), NapA showed a higher expression frequency compared to that from atrophic gastritis (60.0%) (P<0.01, x2 = 8.88; P<0.05, X2=5.00). CONCLUSION: The types of chronic gastritis and peptic ulcer and their severity are not associated with H pylori infection frequency but closely related to the infection frequency of different virulent H pylori strains. The optimal antigens for developing vaccine and diagnostic kit are UreB, FlaA, HpaA, FlaB, NapA and CagAl, but not VacA.     

Helicobacter pylori and Microcirculation

Microcirculation is one of the susceptible targets in the pathogenesis of disorders caused by exogenous pathogens, such as the well‐known bacterium, Helicobacter pylori (Hp). While extensive studies have been published on the kinetics of Hp infection, only a few have dealt with the microcirculatory aspects of Hp‐induced disorders. This review summarizes the recent developments in the elucidation of the pathophysiology of disorders caused by Hp infection, focusing on the microcirculatory aspect. Animal and human studies were systematically reviewed to identify published data on the microcirculatory aspects of Hp infection. This updated summary of the microcirculatory aspects of Hp‐induced disorders illustrates potential approaches that might be used to clarify the pathophysiology of the gastric and extragastric sequelae of Hp infection and the therapeutic strategies that could be adopted.     

外膜蛋白疫苗对幽门螺杆菌感染的免疫保护作用

研究幽门螺杆菌(H.pylori)外膜蛋白(OMP)加大肠杆菌不耐热内毒素(LT)经口免疫对小鼠H.pylori感染的保护作用。探讨定量细菌培养在疫苗免疫效果评价中的应用。方法从H.pyloriNCTC11637菌株制备OMP和全菌超声粉碎抗原,LT作为粘膜免疫佐剂。通过灌胃分别以H.pylori全菌超声粉碎抗原1mg加LT10μg(A组)、H.pyloriOMP0.5mg加LT10μg(B组)和生理盐水(C组)免疫小鼠(n=30),每周1次,共4次。免疫完成后4周以H.pyloriSydneyStrain1菌株攻击小鼠1次(1×107CFU/只),攻击后4周处死小鼠,取胃分别作快速尿素酶试验、病理检查及定量细菌培养,观察H.pylori定植情况。结果C组小鼠Hpylori定植密度为2.40×107CFU/g胃组织,A组和B组小鼠分别为2.03×105CFU/g和6.76×105CFU/g胃组织,免疫组的定植密度明显降低。结论口服HpyloriOMP加LT对小鼠H.pylori感染具有一定免疫保护作用,但不能完全预防感染,需进一步筛选更为有效的抗原和佐剂。定量细菌培养可以更敏感、更客观地反映胃粘膜H.pylori定植量和评价疫苗免疫效果。     

幽门螺杆菌的长丝体变异及返祖

我们采用改良Hp培养基(HBSV培养基)对Hp进行自然诱导,发现Hp球形变异体在含有适量蛋黄和血清的培养中容易返祖及传代,部分培养超过5天的菌落中有长丝体形成,或在球变体两侧形成长短不等的菌丝。长丝体变异的菌落与一般无异,但形态多较大,长丝体约为典型菌的3～10倍以上,菌体弯曲不规则或缠绕,粗细不均,胞壁缺陷,但可在普通渗透压的平板上生长。长丝体不稳定,接种于Hp或HBSV培养基返祖成典型的“S”形Hp。本研究提出这样的假说:Hp变异由典型“S”形→球变→长丝体和巨球体变异→返祖成典型Hp,Hp的这种变异可能存其传染和感染复发中起重要作用。     

Helicobacter pylori and its role in vocal folds' minimal lesions

Background: Chronic laryngitis and/or vocal fold minimal lesions (VFMLs) are common associations with gastro esophageal reflux disease. Helicobacter pylori (HP) is a Gram‐negative spiral organism accused of being a common cause of gastritis, gastroesophageal reflux disease and peptic ulcer. HP has been recently isolated from tonsils, adenoids, sinus and middle ear mucosa in patients with chronic sinusitis or chronic middle ear effusion. Objective: The objective of this study is to assess the presence of HP in VFMLs. Methods: The study included 14 patients with VFMLs [six cases with vocal fold polyps and four cases with vocal fold nodules, and four cases with posterior granulomas; one of them associated with right vocal fold (VF) nodule]; all underwent carbon‐13 urea breath test, esophago‐gastro‐duodenoscopy with gastric biopsy and direct laryngoscopy with microlaryngosurgery to extract the VF lesions. Biopsies were subjected for two tests: detection of the 23S ribosomal RNA gene of HP by real‐time polymerase chain reaction (RT‐PCR) and immunohistochemical reactions (IHC). Results: HP was detected by RT‐PCR in 10 of 14 patients with VFML; HP was also detected by IHC in the same number of VFML and gastric mucosa specimens. Conclusion: HP is a common finding in cases of VFML; its eradication should be considered when dealing with a patient with VFML. Please cite this paper as: Tiba M, Fawaz S and Osman H. Helicobacter pylori and its role in vocal folds' minimal lesions. Clin Respir J 2010; 4: 237–240.     

Non-urease producing Helicobacter pylori in chronic gastritis

Background: Helicobacter pylori infection is the commonest cause of gastritis. Different patterns of immune response to H. pylori infection and characteristics of bacteria are considered to contribute to clinical outcomes. Aim: To determine characteristics of the host H. pylori relationship in subjects with non‐ulcer dyspepsia and a histological diagnosis of gastritis. Methods: Thirty‐five subjects with chronic gastritis undergoing endoscopy (mean age 53 years, range 24–82, 14 male and 21 female) were studied, none of whom was on nonsteroidal anti‐inflammatory drugs or antibiotics. H. pylori infection was determined by rapid urease test (CLOtest), culture, antibody and RT‐PCR for Ure C, Cag A and 26 kDa gene and histology. Cytokine production of mucosal IL‐6 and IL‐8 were measured by ELISA. Results: Fifteen subjects were positive by CLOtest and/or bacterial culture. In these subjects histology showed numerous helical forms of H. pylori (Group I). Nine subjects were negative by CLOtest, bacterial culture, and mRNA for urease C fragment, but positive by PCR for the 26 kDa protein encoding gene. Histology in these subjects showed the presence of either coccoid forms (four), or scant helical forms (two), or mixed coccoid/helical forms (three) (Group II). Eleven subjects were negative by all methods of detection (Group III). IgG and IgA antibody levels in serum (p<0.05) and gastric tissue culture supernatant (p<0.001) were significantly higher in Group I than those in Group II or III. There were significant differences in the IgG serum and IgA supernatant antibody levels (p<0.01 and p<0.05) when Group II was compared to Group III. Supernatant IL‐6 levels were significantly higher in Group I (p<0.01) than those from Groups II and III. EL‐8 levels were higher in Group I (p<0.01) and Group II (p<0.05) when compared to Group III. Conclusions: ‘H. pylori‐negative’ gastritis can be associated with a non‐urease producing form of H. pylori, with a reduction in both local and systemic antibody levels and mucosal pro‐inflammatory cytokines.     

Epidemiology of Helicobacter pylori infection and gastric cancer in Asia

In Asia, the prevalence of Helicobacter pylori (H. pylori) infection varies markedly in different countries. Higher prevalence rates are found in developing Asian countries while lower rates have been reported in more industrialized and developed countries. Within a country, the seroprevalence rates may vary between distinct geographic regions. H. pylori infection is an important etiological factor for the occurrence of non‐cardia gastric adenocarcinoma. The incidence rate of gastric adenocarcinoma in Asia tends to mirror the seroprevalence rate of H. pylori infection; however, there are populations with high seroprevalence rates of H. pylori infection that paradoxically have low incidence rates of gastric adenocarcinoma. These diverse clinical outcomes are related to bacterial virulence factors, concomitant environmental factors, host susceptibility and immune response. This review summarizes the current epidemiology of H. pylori infection in Asia and analyzes these data in the context of gastric cancer epidemiology.     

Advances in vaccination against Helicobacter pylori

A vaccination against Helicobacter pylori may represent both prophylactic and therapeutic approaches to the control of H. pylori infection. Different protective H. pylori-derived antigens, such as urease, vacuolating cytotoxin A, cytotoxin-associated antigen, neutrophil-activating protein and others can be produced at low cost in prokaryote expression systems and most of these antigens have already been administered to humans and shown to be safe. The recent development by Graham et al. of the model of H. pylori challenge in humans, the recent published clinical trials and the last insight generated in animal models of H. pylori infection regarding the immune mechanisms leading to vaccine-induced Helicobacter clearance will facilitate the evaluation of immunogenicity and efficacy of H. pylori vaccine candidates in Phase II and III clinical trials.     

Identification and eradication of Helicobacter pylori infection in haemophilic patients

The aim of this study was to identify and eradicate H. pylori infection in patients with haemophilia. Patients were screened for IgG antibodies against H. pylori ; active infection was determined using a ¹³C-urea breath test and infected patients were given combination therapy with antibiotics to eradicate infection. Seventy-eight of 219 (36%) patients with haemophilia were found to have an elevated serum antibody titre against H. pylori ; of 36 antibody-positive patients with confirmatory testing, 14 were found to have active H. pylori infection. H. pylori infection was successfully eradicated in every infectedpatient using acombination of ranitidine plus two antibiotics (usually amoxycillin and metronidazole). It is concluded that eradication of H. pylori infection is likely to be a cost-effective screening strategy in patients with haemophilia, to prevent complications of peptic ulcer disease.     

Deletion of cagA gene of Helicobacter pylori by PCR products

AIM: Cytotoxin-associated protein (antigen) A (CagA) plays an important role in Helicobacter pylori(H pylori) pathogenesis.Our aim was to obtain cagA mutant strains by a new mutation method so as to better understand the mechanism of CagA in epithelial cells. METHODS: In contrast with the traditional method using suicide plasmid,we constructed cagA- mutant strains directly with PCR products. The constructed mutant clones grew on selective media and allelic exchange was confirmed by Southern blot. Furthermore, two different transformation methods, electroporation, and natural transformation, were compared with regard to the efficiency of recombination. RESULTS:The mutation by PCR products could be completed within 3-5 d, and the recombination rate by electroporation and natural transformation was 4.02×10-8 and 1.03×10-9 respectively. Mutation rate by electroporation (4.02×10-8) was far higher than by natural transformation (1.03×10-9) (P= 0.000<0.005). CONCLUSION: cagA- mutant strains have been constructed, which is important for further study on the function of CagA in epithelial cells.A mutation method by directly using PCR products has been proved successful with a much higher mutation rate, and is easier,especially when in combination with electroporation.This method could be widely used in gene deletion of H pylori.     

胃黏蛋白在抗幽门螺杆菌感染中的作用

黏蛋白(MUCs)是特殊的上皮细胞分泌的大分子量的糖蛋白,是构成胃黏液凝胶的主要组成成分.多项研究显示幽门螺杆菌(H.pylori)通过其表面的黏附素分子或非黏附素分子与胃黏蛋白结合从而定植在胃黏膜上皮上.在H.pylori感染过程中,H.pylori导致了胃黏蛋白的表达发生了改变.反之,胃黏蛋白也因其特有的结构在抗H.pylori感染中也发挥了重要作用.此文就目前有关胃黏蛋白在抗H.pylori感染中的作用的研究现状及进展作一简要概述.     

Vaccinating against Helicobacter pylori in the developing world

Helicobacter pylori infects more than half the world’s population and in developing nations the incidence can be over 90%. The morbidity and mortality associated with H. pylori-associated diseases including ulcers and gastric cancer therefore, disproportionately impact the developing world. Mice have been used extensively to demonstrate the feasibility of developing a vaccine for H. pylori infection, and for testing antigens, routes of immunization, dose, and adjuvants. These successes however, have not translated well in clinical trials. Although there are examples where immune responses have been activated, there are few instances of achieving a reduced bacterial load. In vivo and in vitro analyses in both mice and humans demonstrates that the host responds to H. pylori infection through the activation of immunoregulatory mechanisms designed to suppress the anti-H. pylori response. Improved vaccine efficacy therefore, will require the inclusion of factors that over-ride or re-program these immunoregulatory rersponse mechanisms.     

Increased activity of Pgp multidrug transporter in patients with Helicobacter pylori infection

AIM: To determine whether local antibiotic resistance involves P-glycoprotein (Pgp)-mediated active drug out-pumping during Helicobacter pylori (H pylori) infection treatment with classic antibiotic therapy. METHODS: Pgp activity was determined in gastric mucosa biopsy specimens obtained from 53 patients with pathohistologically verified gastritis and microbiologically confirmed H pylori infection, and compared with the Pgp activity in 12 control subjects with normal endoscopic findings. The H pylori positive patients were treated with short-term 7-d therapy consisting of two antibiotics (amoxicillin and azithromycin/metronidazole and clarithromycin) and a proton pump inhibitor. Pgp activity was determined by flow cytometry in the test of rhodamine dye efflux and quantified as mean fluorescence ratio (RMF). RESULTS: Upon the first cycle, H pylori was successfully eradicated in 20 patients, whereas therapy was continued in 33 patients. In the course of antibiotic therapy, RMF increased (P＜0.05) and gastric cells showed higher rhodamine dye efflux. The mean pre-treatment RMF values were also higher (P＜0.0001) in patients with multiple therapeutic failure than in those with successful H pylori eradication and control subjects. CONCLUSION: Pgp might be one of the causes of therapy failure in patients with H pylori and antibiotic therapy could be chosen and followed up on the basis of the Pgp transporter local activity.