Effects of fucosylated milk of goat and mouse on Helicobacter pylori binding to Lewis b antigen

AIM:To evaluate the effects of animal milk containing fucosylated antigens on Helicobacter pylori (H pylon) binding to Lewis b antigen.METHODS:A mammary gland expression vector containing human α1-3/4-fucosyltransferase cDNA sequences was constructed. Transient expression of human(α1-3/4-fucosyltransferase cDNA in goat mammary cell and establishment of transgenic mice were performed. The adhesion inhibitory properties of milk samples were analyzed by using Hpylori.RESULTS: Goat milk samples were found to inhibit bacterial binding to Lewis b antigen. The highest inhibition was observed 42 h after injection of the plasmid. The binding activity of Hpylori to Lewis b antigen reduced mostly, by 83%, however milk samples from transgenic mice did not inhibit Hpylori binding to Lewis b antigen.CONCLUSION: The use of “humanized“ animal milk produced by the transgenic introduction of fucosylated antigen can perhaps provide an alternative therapy and preventive measure for Hpylori infection.     

Lewis antigen expression and stability in Helicobacter pylori isolated from serial gastric biopsies

The expression of Lewis antigens by the gastric pathogen Helicobacter pylori in serial biopsy isolates was investigated to assess antigen distribution and stability. A total of 26 asymptomatic subjects were given various doses of 3' sialyllactose for up to 56 days. Gastric biopsies were performed during the dosing period, as well as 30 days after dosing, which provided 127 H. pylori isolates that were examined by use of ELISA and immunoblot. A large proportion of subjects (14/26) yielded sequential H. pylori isolates, which appeared genetically identical but had variable Lewis antigen expression. The proportion of subjects with H. pylori isolates not expressing any Lewis antigens was greater than that previously reported (10/26). Thus, the expression of the Lewis antigens by H. pylori does not appear to be a requirement for colonization, whereas the antigen expression after human infection is more variable than the previously reported rate observed with in vitro cultures.     

Expression of Lewis(b) blood group antigen in Helicobacter pylori does not interfere with bacterial adhesion property

AIM: The finding that some Helicobacter pylori strains express Lewis b (Le(b)) blood group antigen casts a doubt on the role of Le(b) of human gastric epithelium being a receptor for H. pylori. The aim of this study was to determine if expression of Le(b) in H. pylori interferes with bacterial adhesion property. METHODS: Bacterial adhesion to immobilized Le(b) on microtitre plate was performed in 63 H. pylori strains obtained from Singapore using in vitro adherence assay. Expression of Lewis blood group antigens was determined by ELISA assay. RESULTS: Among 63 H. pylori strains, 28 expressed Le(b) antigen. In vitro adhesion assay showed that 78.6 % (22/28) of Le(b)-positive and 74.3 % (26/35) of Le(b)-negative H. pylori isolates were positive for adhesion to immobilized Le(b) coated on microtitre plate (P=0.772). In addition, blocking of H. pylori Le(b) by prior incubation with anti-Le(b) monoclonal antibody did not alter the binding of the bacteria to solid-phase coated Le(b). CONCLUSION: The present study suggests that expression of Le(b) in H. pylori does not interfere with the bacterial adhesion property. This result supports the notion that Le(b) present on human gastric epithelial cells is capable of being a receptor for H. pylori.     

Construction of attenuated Salmonella typhimurium Strain expressing Helicobacter pylori conservative region of adhesin antigen and its immunogenicity

AIM: To construct a non-resistant and attenuated Salmonella typhimurium (S. typhimurium) strain which expresses conservative region of adhesion AB of Helicobacter pylori (H pylori) and evaluate its immunogenicity. METHODS: The AB gene amplified by PCR was inserted into the expression vector pYA248 containing asd gene and through two transformations introduced into the delta Cya, delta Crp, delta Asd attenuated Salmonella typhimurium strain, constructing balanced lethal attenuated Salmonella typhimurium strains X4072 (pYA248-AB). Bridged ELISA method was used to measure the expression of AB antigen in sonicate and culture supernatant. According to the method described by Meacock, stability of the recombinant was evaluated. Semi-lethal capacity test was used to evaluate the safety of recombinant. The immunogenicity of recombinant was evaluated with animal experiments. RESULTS: The attenuated S. typhimurium X4072 (pYA248-AB) which expresses AB was successfully constructed. Furthermore, bridged ELISA assay showed that the content of AB in recombinant X4072 (pYA248- AB) culture supernatant was higher than that was in thallus lytic liquor. And after recombinant X4072 (pYA248- AB) was cultured for 100 generations without selection pressure, the entire recombinant bacteria selected randomly could grow, and the AB antigen was defected positive by ELISA. The growth curve of the recombinant bacteria showed that the growth states of X4072 (pYA248) and X4072 (pYA248-AB) were basically consistent. The survival rate of C57BL/6 was still 100%, at 30 d after mice taking X4072 (pYA248-AB) 1.0 x 10(10) cfu orally. Oral immunization of mice with X4072 (pYA248-AB) induced a specific immune response. CONCLUSION: In vitro recombinant plasmid appears to be stable and experiments on animals showed that the recombinant strains were safe and immunogenic in vitro, which providing a new live oral vaccine candidate for protection and care of H pylori infection.     

幽门螺杆菌黏附素基因alpA的克隆及序列分析

目的　获取幽门螺杆菌黏附素基因alpA ,并将它克隆到质粒 pET - 2 2b(+)中进行核苷酸序列分析。 方法　利用PCR技术扩增alpA ,并将其定向插入pET - 2 2b(+)载体中通过DNA序列分析仪进行核苷酸分析。 结果　DNA序列分析表明 ,所克隆的alpA基因序列与GenBank公布的基本一致。 结论　本研究获得了序列正确的alpA基因 ,为其重组表达及其相关研究奠定了良好基础。     

粪便抗原检测诊断幽门螺杆菌现症感染

目的 评价应用免疫酶联吸附试验(ELISA)检测粪便中幽门螺杆菌(Helicobacter pylori)抗原诊断H.pylori现症感染的敏感性和特异性。方法 应用^14C呼气试验以及幽门螺杆菌粪便抗原(HpSA)试验，对100例因上消化道不适就诊，怀疑有H.pylori感染的患者进行检测，观察两种检查的符合率。结果 ^14C呼气试验和HpSA同时阳性者38例，^14C呼气试验阳性而HpSA阴性者4例；^14C呼气试验和HpSA同时阴性者57例，^14C呼气试验阴性而HpSA阳性1例。以^14C呼气试验作为金标准计算，HpSA检测方法的敏感性为90．48％，特异性为98．28％。结论 幽门螺杆菌抗粪便原检测与^14C呼气试验有较高的符合率，而且简便易行，不需特殊设备，解决了无法进行呼气试验的婴幼儿和有肺部疾患者的非侵人性幽门螺杆菌现症感染诊断问题，是一种非侵入性幽门螺杆菌现症感染诊断的新方法。     

幽门螺杆菌重组VacA-HpaA IgY的制备

目的:采用基因工程技术大量表达、提纯幽门螺杆菌(H pylori)细胞空泡毒素毒性片段,和黏附素片段的融合蛋白,以此为抗原,制备高效价的抗VacA-HpaA蛋黄抗体(IgY)．方法:大量培养重组菌pQE30-VacA-HpaA- DH5α,诱导表达获得融合蛋白VacA—HpaA, Ni2 -NTA树脂纯化．将纯化的重组蛋白免疫鸡,水稀释法提取IgY,硫酸铵沉淀法纯化浓缩VacA—HpaA IgY．ELISA法测定抗体产生的时间-效价变化,SDS—PAGE分析纯度, Bradford法测定蛋白含量,Western blot检测其分别对VacA和HpaA抗原的特异性,ELISA法检测效价．结果:重组蛋白主要以包涵体形式表达,免疫鸡后提取的IgY可与该蛋白发生免疫反应, IgY效价总体上随免疫时间增加而升高．经纯化、浓缩后,获得纯度为60％左右,效价为1: 12 800的VacA-HpaA IgY,蛋白浓度为22 g／L, Westem blot显示在Mr27 000和Mr30 000处分别有相应条带,ELISA检测与VacA和HpaA反应的效价分别为1:3200和1:6400(P<0．01)．结论:成功制备了高浓度、高效价的抗重组VacA-HpaA的特异性IgY．     

Accuracy of Helicobacter pylori stool antigen for the detection of Helicobacter pylori infection in dyspeptic patients

AIM: The premier platinum Helicobacter pylori (H pylori) stool antigen (HpSA) test is an enzyme immunoassay (EIA) that detects an H pylori antigen present in human stools. However, at present there is no uniformity about the cut off level required to consider the test as positive or negative. So we need the cut off level for our local population. The aim of this study was to evaluate the HpSA for the detection of H pylori infection in dyspeptic patients and to determine the sensitivity, specificity of the HpSA test in the diagnosis of H pylori infection, as compared to other standardized diagnostic techniques. METHODS: Sixty-three dyspeptic patients were selected from patients who came to the Division of Gastrointestinal Clinic in Cipto Mangunkusumo Hospital, Jakarta, Indonesia. H pylori infection was confirmed in all patients by histology and rapid urease test (CLO test). Positive results for H pylori were based on positive results from both rapid urea test and microscopic detection of H pylori. Stool specimens were analyzed for H pylori antigen using HpSA immunoassay. RESULTS: A total 63 patients consisted of 31 (49.2%) males and 32 (50.8%) females ranging in ages between 16 and 73 years with a mean age of 42.4+/-15 years. The mean age of men was 43.2+/-15.7 years and women was 41.6+/-14.4 years. Endoscopic findings in this study included gastric cancer 1.6%, peptic ulcer 4.8%, duodenal ulcer 7.9%, esophagitis 6.3%, gastritis 77.7%, and gastroduodenitis 4.8%. According to the predefined study criteria, 6 (9.5%) of 63 patients were positive for H pylori. In the diagnosis of infection, the area under the receiver operating characteristic (ROC) curve for the HpSA test was 0.722 (95% CI, 0.518-0.927). Using a cut-off value of 0.274 instead of 0.16 (as recommended by the manufacturer) the sensitivity and the specificity were 66.7% and 78.9% respectively. CONCLUSION: The HpSA stool test, using a cut-off value of 0.274, may be useful for the primary diagnosis of H pylori infection, its specificity is similar to other standard tests but its sensitivity was lower.     

幽门螺杆菌5种候选疫苗抗原基因的克隆、表达及抗原性的鉴定

目的:构建含人Hpylori5种候选疫苗抗原Lpp20,HspA,UreaseA,CagA,UreaseB的编码基因的重组质粒并研究其抗原性.方法:应用PCR技术从Hpylori染色体中扩增编码Lpp20,HspA,UreaseA,CagA,UreaseB的基因片段,将其T-A克隆和测序,并与GenBank公布的其他Hpylori菌株基因序列比较,再将目的基因克隆至融合表达载体pGEX-4T-1上中进行表达,用GST亲和层析对其进行纯化,纯化产物用于对29株小鼠抗Hpylori-全菌单克隆抗体(mAb)的鉴定及与Hpylori感染患者血清进行Westernblot分析.结果:扩增的Lpp20,HspA,UreaseA,CagA,UreaseB基因全长分别为528bp,351bp,675bp,855bp,1704bp(GenBank登录号分别为DQ106902,DQ141574,DQ141577,DQ141575,DQ141576),与GenBank公布的其他菌株的核酸序列的同源性在95%-99%,表达Lpp20,HspA,UreaseA,CagA,UreaseB融合蛋白的相对分子质量分别约为48000,41000,52000,60000,91000Da,29株小鼠抗Hpylori全菌mAb中针对Lpp20,HspA,UreaseA,CagA,UreaseB抗原的分别为4,5,5,1,6株,5种抗原的纯化产物均可被Hpylori感染患者血清特异性识别.结论:重组表达的Lpp20,HspA,UreaseA,CagA,UreaseB均具有较好的抗原性.     

A comparison of Lewis x and Lewis y expression in Helicobacter pylori obtained from children and adults

There are no reports, to our knowledge, on the expression of Lewis (Le) antigens in Helicobacter pylori isolates from children. The aim of this study was to compare the expression of Le antigens by H. pylori isolates from children and from adults. Totals of 278 clones from 22 children with recurrent abdominal pain and 293 clones from 22 adults with (n=10) or without (n=12) duodenal ulcer were studied. Expression of Le(x) and Le(y) antigens was determined by ELISA, using monoclonal anti-Le antibodies. The Le phenotype of the patients was determined in gastric juice with a hemagglutination assay. Clones expressing Le(x) were more common in children than in adults (55.4% vs. 33.4%, respectively; P<.001), and Le(y) was more common in adults than in children (81.6% vs. 66%, respectively; P<.01). A trend analysis showed a significant decline in frequency of clones expressing Le(x) with age (P=.021). In this community, expression of Le antigens differs in H. pylori isolates obtained from children versus adults.     

幽门螺杆菌粘附素AlpA基因克隆、表达及免疫原性研究

目的 构建表达幽门螺杆菌(Hp)黏附素AlpA的候选菌株并研究其免疫原性。方法 利用PCR技术扩增粘附素AlpA基因，将其定向插入pET-22b( )载体，在BL21(DE3)大肠杆菌中表达并通过免疫印迹实验研究其免疫原性。结果 克隆的粘附素AlpA基因序列与基因库公布的基本一致，粘附素AlpA重组蛋白表达量占菌体总蛋白的31．9％，经免疫印迹证实该重组蛋白可被AlpA免疫兔血清和Hp,感染患者血清所识别。结论．AlpA克隆、高效表达及其免疫原性的初步证实，为Hp基因工程疫苗的研制和粘附机制的研究打下了基础。     

Discrepancy between Helicobacter pylori stool antigen assay and urea breath test in the detection of Helicobacter pylori infection.

BACKGROUND: The reference diagnostic methods available for detection of Helicobacter pylori infection are either invasive (histology) or expensive and highly sophisticated (Urea Breath Test). A new enzyme immunoassay, which can be easily performed in any laboratory, has been developed to detect Helicobacter pylori in stool specimens (HpSA-Meridian Diagnostics, Cincinnati, USA). Aim of the study was to compare HpSA to Urea Breath Test. PATIENTS AND METHODS: A total of 125 patients (52 never treated for Helicobacter pylori infection and 73 after Helicobacter pylori eradication therapy) referring to our Department, underwent both tests within two weeks. RESULTS: Contrasting results between the two tests were found in 30% of cases: in 19% of the untreated patients and in 37% of the treated patients (p<0.001). The main discrepancy consisted in positive HpSA associated with negative Urea Breath Test. Mean HpSA value in such conditions was 0.273 optical density, while in patients with both positive tests, it was 1.192 optical density. In untreated, but not in treated patients, raising the HpSA cut off value significantly decreased the percentage of conflicting results. CONCLUSIONS: Some disagreement was detected between HpSA and Urea Breath Test results, especially in treated patients. Possible explanations for our findings are a low HpSA cut off value together with the identification of Helicobacter pylori coccoid forms by the immunoassay but not by the urease based Urea Breath Test. The higher percentage of discrepancy detected in treated patients might support this hypothesis.     

幽门螺杆菌粪便抗原检测在体检人群中的应用价值

[目的]探讨中老年人群幽门螺杆菌(HP)感染情况及HP粪便抗原(HPSA)在体检中的应用价值.[方法]对1 774例≥40岁人群粪便标本进行HPSA检测,分析HP感染及其与胃病症状的关系.[结果]1774例中HP阳性者251例,HP阳性率14.15％; 40～～50岁者HP阳性率最高(19.71％),＞80岁者阳性率最低(9.04％);HP感染与吸烟饮酒的生活习惯及胃部症状无关.[结论]生活条件良好的中老年群体HP阳性率呈收敛趋势,与吸烟、饮酒无明显关系,无症状人群应重视HPSA监测,HP阳性人群应行胃镜检查.     

幽门螺杆菌粪便抗原试验检测幽门螺杆菌感染的临床评价

目的 评价一种新的酶免疫法——幽门螺杆菌(HP)粪便抗原(HPSA)试验检测HP感染和监测HP根除治疗的可靠性。方法 未接受过抗HP治疗的患者分为2组，A组331例，无胃部手术史；B组65例，胃大部切除术后。2组患者因上消化道症状而接受胃镜检查，以胃黏膜活检标本快速尿素酶试验(RUT)和组织学检查(W-S染色)联合检测HP作为“金标准”，对HPSA试验的准确性进行评价，并与另一非侵入性的^13C-尿素呼气试验(^13C-UBT)加以比较。此外，A组中HP阳性的56例患者(C组)给予三联根除治疗1周，分别于停药后第1、7、14、21、28天收集粪便标本进行HPSA测定。于停药后第28天测定^13C-UBT，并以此为标准，评价HPSA试验的准确性。结果 A组患者经“金标准”诊断HP阳性175例，阴性156例。HPSA试验的敏感性为95，4％，特异性为91．0％，与^13C-UBT比较差异无统计学意义。B组患者中，经“金标准”诊断HP阳性30例，阴性35例。月psA试验敏感性为90．0％，^13C-UBT的敏感性为66．7％。HPsA试验的敏感性明显优于^13C-UBT(P＜0．05)。C组患者于停药后第28天经^13C-UBT诊断HP阳性16例，阴性40例。HPSA于停药后第1天54例阴性，此后随时间推移，未成功根除病例陆续转为阳性，而成功根除病例仍大部分保持在阴性范围，仅少数病例出现假阳性。停药后第28天的准确性最高(92．9％)。结论 HPSA试验是一种可靠的非侵入性检测方法，对于抗HP治疗前、后患者HP感染的诊断均有较高的准确性。对于胃大部切除术后的患者亦有较高的诊断价值。     

幽门螺杆菌表达LeWis B抗原不影响细菌的黏附

目的：研究幽门螺杆菌（Hp)表达LewisB抗原对细菌黏附的可能影响,以及Hp的babA2基因与消化性溃疡的关系,方法,采用体外黏附分子法检测细菌的黏附性,聚合酶反应法（PCR）检测Hp的babA2基因,结果：（1）在47例消化性溃疡Hp株中,18株为babA2基因阳性,而31例非溃疡性消化不良Hp株中,14株为babA2阳性（P＝0．427）,（2）在表达LewisB抗原的31株Hp中,24株细菌黏附阳性,而不表达LewisB抗原的47株Hp中,38株呈细菌黏附阳性（P＝0．463）,结论：（1）Hp的babA2基因与化性溃疡的发生无关,（2）Hp表达LewisB抗原不影响细胞的黏附,从而支持上皮LewisB是Hp的受体。     

Molecular analysis of Helicobacter pylori virulent-associated genes in hepatobiliary patients

Objectives

The Helicobacter pylori virulence-associated genes in hepatobiliary patients, including vacA, iceA, babA2, cagA and cagE, have not been reported. The aim of this study was to investigate these genes and the association of those and the clinical outcomes in hepatobiliary diseases.

Methods

Eighty H. pylori-PCR-positive cases were obtained from hepatobiliary patients, representing both cholangiocarcinoma (CCA) (n = 58) and cholelithiasis (n = 22). The diversity of virulence genes was examined by polymerase chain reaction and DNA sequencing. Phylogenetic analysis of cagA was determined using the maximum parsimony method.

Results

The vacAs1a + c/m1, iceA1 and babA2 genes were the most predominant genotypes in both CCA and cholelithiasis patients. The cagA and cagE genes were found significantly more frequently in patients with CCA than those with cholelithiasis (P < 0.05). The cagA positive samples were the Western-type cagA and showed that almost all of the detected sequences in Thai hepatobiliary and Thai gastric cancer patients were classified in the same cluster but separated from the cluster of Japan and other countries.

Conclusions

The cagA and cagE genes may be associated in the pathogenesis of hepatobiliary diseases, especially of CCA. Besides the bacterial variation, other host factors may be involved in the pathogenesis of hepatobiliary cancer.