In vitro evaluation of the role of antibodies against Helicobacter pylori in inhibiting adherence of the organism to gastric cells.

BACKGROUND: Once Helicobacter pylori infection is established, it is difficult to eradicate despite a persistent systemic and local immune response. It is not known whether immunisation can be used to prevent H pylori infection in humans. AIMS: To evaluate the effect of the human immune response on adherence of H pylori to gastric cells. METHODS: Human milk from a woman infected with H pylori and milk from a non-infected woman were each fractionated by chromatography on DEAE cellulose. Bacteria were incubated with either serum, human milk, human milk fractions, or secretory IgA before incubation with Kato III cells (cells from a gastric adenocarcinoma cell line). Bacterial adherence to the cells was assessed using flow cytometry. RESULTS: Serum from both the H pylori infected and non-infected women killed H pylori. This resulted from the action of complement as heating the serum to 56 degrees C for 30 minutes abolished the bactericidal activity. Immunoglobulin fractions from serum of both infected and non-infected women did not inhibit H pylori adherence to Kato III cells. Human milk from the woman infected with H pylori and from the non-infected woman inhibited binding of H pylori to Kato III cells by 50 to 70%. Secretory IgA isolated from human milk had minimal inhibitory effect on adherence and this was notably less than the inhibitory effect of whole human milk. CONCLUSIONS: Human milk inhibits adherence of H pylori to Kato III cells and this inhibition is independent of whether or not the donor is infected with H pylori. Secretory IgA has minimal inhibitory effect on H pylori adherence.     

Virulence and potential pathogenicity of coccoid Helicobacter pylori induced by antibiotics

AIM: To explore the virulence and the potential pathogenicity of coccoid Helicobacter pylori (H. pylori) transformed from spiral form by exposure to antibiotic. METHODS: Three strains of H. pylori, isolated from gastric biopsy specimens of confirmed peptic ulcer, were converted from spiral into coccoid from by exposure to metronidazole. Both spiral and coccoid form of H. pylori were tested for the urease activity, the adherence to Hep-2 cells and the vacuolating cytotoxicity to Hela cells, and the differences of the protein were analysed by SDS-PAGE and Western blot. The mutation of the genes including ureA, ureB,hpaA, vacA and cagA, related with virulence, was detected by means of PCR and PCR-SSCP. RESULTS: In the coccoid H. pylori,the urease activity, the adherence to Hep-2 cells and the vacuolating cytotoxicity to Hela cells all decreased. In strain F44, the rate and index of adherence reduced from 70.0% +/- 5.3% to 33% +/- 5.1% and from 2.6 +/- 0.4 to 0.96 +/- 0.3 (P < 0.01), respectively. The invasion of coccoid H. pylori into Hep-2 cell could be seen under electronmicroscope. SDS-PAGE showed that the content of the protein with the molecular weight over Mr 74000 decreased, and the hybriditional signal in band M(r) 125000 weakened, while the band M(r)110000 and M(r)63000 strengthened in coccoid H.pylori as shown in Western blot. The results of PCR were all positive, and PCR-SSCP indicated that there may exist the point mutation in gene hpaA or vacA. CONCLUSION: The virulence and the proteins with molecular weight over M(r)74000 in coccoid H.pylori decrease, but no deletion exists in amplification fragments from ureA, ureB, hpaA, vacA and cagA genes, suggesting that coccoid H.pylori may have potential pathogenicity.     

以白细胞介素-2为免疫佐剂的幽门螺杆菌尿素酶B亚单位核酸疫苗的构建及其免疫保护作用

背景：细胞因子具有免疫佐剂效应，但将其用作幽门螺杆菌（H．pylori）核酸疫苗佐剂的研究报道尚少。目的：构建同时含H．pylori尿素酶B亚单位（ureB）基因和小鼠白细胞介素-2（IL-2）基因的重组活减毒鼠伤寒沙门菌核酸疫苗，体外鉴定其表达蛋白的免疫原性，体内检测其对H．priori感染的免疫保护作用。方法：以聚合酶链反应（PCR）扩增H．priori ureB基因和小鼠IL-2基因，分别插入pUCmT载体，测序，通过一系列酶切、连接反应分别克隆人真核表达载体pIRES，酶切、PCR鉴定；重组质粒pIRES-ureB和pIRES-ureB-IL-2分别转化减毒鼠伤寒沙门菌LB5000，抽提质粒，进一步转化终宿主菌SL7207，反复传代培养。以Lipofectamine^TM2000将重组质粒分别体外转染COS-7细胞，蛋白质印迹法检测表达蛋白的免疫原性。以疫苗菌经口接种小鼠，4周后予H．pylori攻击，攻击后4周鉴定H．pylori感染状况。结果：测序结果显示扩增出的ureB和IL-2基因序列与GenBank中的H．pylori ureB和小鼠IL-2序列一致，酶切、PCR鉴定证实ureB和IL-2基因已克隆人pIRES载体，并成功构建了稳定的含H．pylori ureB和小鼠IL-2基因的重组活减毒鼠伤寒沙门菌核酸疫苗。蛋白质印迹法显示，pIRES-ureB-IL-2转染的COS-7细胞表达特异性UreB和IL-2蛋白。体内实验显示，疫苗接种组小鼠的免疫保护率显著高于PBS对照组（P〈0．01），其中ureB-IL-2疫苗组又显著高于ureB疫苗组（87．5％对62．5％，P〈0．05）。结论：成功构建了编码H．pylori ureB和免疫佐剂IL-2基因的重组活减毒鼠伤寒沙门菌核酸疫苗，体外实验证实其可表达具有免疫原性的抗原蛋白和佐剂蛋白，体内实验证实其对小鼠H．pylori感染具有免疫保护性，免疫佐剂IL-2可提高核酸疫苗的免疫保护率。     

Systemic and local immune responses against Helicobacter pylori urease in patients with chronic gastritis: distinct IgA and IgG productive sites

BACKGROUND: Helicobacter pylori urease is a major target for immune responses among various bacterial components in H pylori infected patients. AIMS: To analyse the relation between systemic and local humoral immune responses to H pylori urease and grades of chronic gastritis. PATIENTS: Seventy five patients with chronic gastritis associated with H pylori infection were classified into three groups (grade I, superficial gastritis; II, atrophic gastritis, quiescent; or III, atrophic gastritis, active). METHODS: Anti-H pylori urease specific antibodies in the serum, gastric juice, and biopsy specimens were determined by ELISA or western blotting analysis. The sites for H pylori urease and its specific antibody producing B lymphocytes were confirmed by immunohistochemical analysis. RESULTS: In the sera of patients with grade I gastritis, weak IgG but relatively strong IgG responses to H pylori urease were observed; dominant strong IgG responses were detected in grade II gastritis. In grade III gastritis, significant IgG and IgA responses were obtained. A similar pattern of IgA and IgG responses was detected in gastric juice and tissue. H pylori urease specific, antibody producing B cells were not found in the gastric mucosa of patients with grade I gastritis despite the presence of such B cells in the duodenal bulb. Specific B cells were observed in the gastric mucosa of patients with grade II and III gastritis with atrophy. CONCLUSIONS: Purified H pylori urease, together with localisation of its specific antibody producing B cells, are useful for serological testing and histopathological analysis for determining the stage of chronic gastritis and studying the pathogenesis of H pylori infection.     

In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly.

The formation of active urease in Klebsiella aerogenes requires the presence of three structural genes for the apoprotein (ureA, ureB, and ureC), as well as four accessory genes (ureD, ureE, ureF, and ureG) that are involved in functional assembly of the metallocenter in this nickel-containing enzyme. Slow and partial activation of urease apoprotein was observed after addition of nickel ion to extracts of Escherichia coli cells bearing a plasmid containing the K. aerogenes urease gene cluster or derivatives of this plasmid with deletions in ureE, ureF, or ureG. In contrast, extracts of cells containing a ureD deletion derivative failed to generate active urease, thus highlighting a key role for UreD in the metallocenter assembly process. Site-directed mutagenesis methods were used to overexpress ureD in the presence of the other urease genes, and the UreD protein was found to copurify with urease. A molecule of native urease apoprotein is capable of binding 0, 1, 2, or 3 molecules of UreD, consistent with a trimeric structure of urease catalytic units. The UreD-urease apoprotein complexes are competent for activation by nickel, with the level of activity obtained being directly related to the number of UreD molecules bound per urease molecule. Activation of the UreD-urease complexes is rapid and accompanied by UreD dissociation. We propose that UreD is a chaperone protein which stabilizes a urease apoprotein conformation that is competent for nickel incorporation.     

Construction of recombinant attenuated Salmonella typhimurium DNA vaccine expressing H pylori ureB and IL-2

AIM: To construct a recombinant live attenuated Salm-onella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo. METHODS: H pylori ureB and mouse IL-2 gene fragments were amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified ureB and IL-2 genes was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions resulting in pIRES-ureB and pIRES-ureB-IL-2. The recombinant plasmids were used to transform competent E. coli DH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-ureB and pIRES-ureB-IL-2 were used to transform LB5000 and the recombinant plasmids extracted from LB5000 were finally introduced into the final host SL7207. After that, recombinant strains were grown in vitro repeatedly. In order to detect the immunogenicity of the vaccine in vitro, pIRES-ureB and pIRES-ureB-IL-2 were transfected to COS-7 cells using LipofectamineTM2000, the immunogenicity of expressed UreB and IL-2 proteins was assayed with SDS-PAGE and Western blot. C57BL/6 mice were orally immunized with 1 × 108 recombinant attenuated Salmonella typhimurium DNA vaccine. Four weeks after vaccination, mice were challenged with 1 × 107 CFU of live H pylori SS1. Mice were sacrificed and the stomach was isolated for examination of H pylori 4 wk post-challenge. RESULTS: The 1700 base pair ureB gene fragment amplified from the genomic DNA was consistent with the sequence of H pylori ureB by sequence analysis. The amplified 510 base pair fragment was consistentwith the sequence of mouse IL-2 in gene bank. It was confirmed by PCR and restriction enzyme digestion that H pylori ureB and mouse IL-2 genes were inserted into the eukaryotic expression vector pIRES. The experiments in vitro showed that stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying ureB and IL-2 genes was successfully constructed and the specific strips of UreB and IL-2 expressed by recombinant plasmids were detected through Western blot. Study in vivo showed that the positive rate of rapid urease test of the immunized group including ureB and ureB-IL-2 was 37.5% and 12.5% respectively, and was significantly lower than that (100%) in the control group (P < 0.01). CONCLUSION: Recombinant attenuated Salmonella typhimurium DNA vaccine expressing UreB protein and IL-2 protein with immunogenicity can be constructed. It can protect mice against H pylori infection, which may help the development of a human-use H pylori DNA vaccine.     

Celecoxib inhibits Helicobacter pylori colonization-related factors

AIM:To investigate the effect of celecoxib,a selective COX-2 inhibitor,on Helicobacter pylori(H.pylori) colonization-related factors and its mechanism.METHODS:After co-incubation with celecoxib,morphology of H.pylori strain 26695 was observed under a transmission electron microscope.Flagella motility was assessed by stab agar motility test.Adherence of H.pylori to AGS cells was determined by enzyme linked immunosorbent assay.Levels of mRNA expression in flagellar genes(flaA,flaB),urease genes(ureA,ureB)and ...     

Adherence and invasion of mouse-adapted H pylori in different epithelial cell lines

AIM： To assess the adhesion and invasion abilities of different mouse adapted H py/or/strains in different cell lines in vitro and investigate their effects on the virulence factors cagA and vacA. METHODS： The adherence and invasion abilities of different N pylori strains in different epithelial cell lines were examined by the gentamycin protection assay. The null mutants of cagA and vacA were processed by direct PCR mutation method. The morphologic changes of different cell lines after N pylori attachment were examined by microscopy.
RESULTS： The densities of adherence to and invasion into cells in vitro were different from those in the mouse infection experiments. 88-3887 strain could invade and adhere to cells stronger than SS1 and X47. All tested strains had better adhering and invasive abilities in SCG-7901 cell. CagA and vacA minus mutants had the same invasion and adherent abilities as their wild types. In all strains and cell lines tested, only AGS cell had the significant hummingbird phenotype after inoculation with the 88-3887 wild-type.
CONCLUSION： Both the host cells and the bacteria play important parts in the invasion and adhesion abilities of Hpylori. CagA and VacA are not related to the ability of invasion and adhesion of Hpylori in different cell lines in vitro.     

Phenotypic and genotypic characterization of Helicobacter pylori from patients with and without peptic ulcer disease

BACKGROUND: Helicobacter pylori plays an important role in peptic ulcer disease, although not all H. pylori-infected persons will develop a peptic ulcer. Currently, H. pylori strains cannot be divided into commensals and pathogens. METHODS: Fifty H. pylori strains were cultured from patients divided into five groups on the basis of upper endoscopic findings: gastric ulcer, duodenal ulcer, gastritis, esophagitis, or normal. The ultrastructural adherence pattern in vivo, autoagglutination, hemagglutination, adhesion to human gastric adenocarcinoma (AGS) cells, and the lipopolysaccharide (LPS) profile of H. pylori strains were recorded; randomly amplified polymorphic DNA (RAPD) and urease gene typing were performed and correlated with diagnostic groups. RESULTS: Electron micrographs showed that H. pylori strains from patients with gastric ulcers adhered more frequently through filamentous strands and were less frequently found free in mucus than any other diagnostic group (P < 0.0001). Neither median hemagglutination titer nor median adhesion capacity to a human gastric adenocarcinoma cell line was related to endoscopic findings. Nevertheless, H. pylori strains from patients with gastric ulcers were more prone to autoagglutinate than were strains from the other diagnostic groups (P = 0.03). H. pylori strains from gastric ulcer patients were found to be more homogeneous, as determined by RAPD and urease gene typing, than strains from the other diagnostic groups (P < 0.01). In addition, a positive correlation was found between a patient's age and the adhesion to AGS cells of the patient's H. pylori strain (P = 0.006). CONCLUSION: A combination of an H. pylori autoagglutination test, RAPD, and urease gene typing may be useful in separating gastric ulcer-related strains from duodenal ulcer-related and non-ulcer dyspepsia-related strains.     

表达幽门螺杆菌尿素酶B亚单位基因减毒鼠伤寒沙门疫苗菌的构建和鉴定

目的构建表达幽门螺杆菌(Helicobacter     

Urease-positive bacteria other than Helicobacter pylori in human gastric juice and mucosa

BACKGROUND AND AIM: Many bacteria carry the urease enzyme in different human ecosystems, but Helicobacter pylori is the only known bacterium showing urease activity in gastric ecosystems. For this reason, the rapid urease test (RUT) on gastric biopsies and urea breath test (C-UBT) are used to detect H. pylori infection.The aim of this study was to evaluate the presence of urease-positive bacteria other than H. pylori in gastric juice and mucosa in hypochlorhydric subjects. METHODS: Twenty-five hypochlorhydric and 10 normochlorhydric patients were analyzed for the presence of H. pylori and bacterial overgrowth both in gastric juice and on the mucosa. During upper gastrointestinal endoscopy at 8.00 a.m. gastric juice samples and biopsy specimens were taken from the antrum and corpus. All samples were analyzed using standard microbiological procedures like aerobic/anaerobic growth, gram-staining, gas chromatography, API test, 96-clone method, and selective medium to search for specific bacteria. In addition, all strains isolated were screened for urease activity using the CP-test. Urease positive strains were tested for the capacity to survive in an acid environment with or without urea (10 mM/L), at pH 7, 4, 3, and 2, respectively, at different times (0, 20, 30, and 60 min). RESULTS: Six hypochlorhydric patients had 10 strains of urease-positive non-H. pylori bacteria among which Staphylococcus capitis urealiticum showed the strongest urease activity. CONCLUSIONS: Hypochlorhydric patients present many urease-positive bacteria other than H. pylori. The strong urease activity may be responsible for false positive results at RUT or UBT test in patients with suspected H. pylori infection.     

Pathogen DNA as target for host-generated oxidative stress: role for repair of bacterial DNA damage in Helicobacter pylori colonization

Helicobacter pylori elicits an oxidative stress during host colonization. This oxidative stress is known to cause lesions in the host DNA. Here we addressed the question as to whether the pathogen DNA is subject to lethal or mutational damage by the host-generated oxidative response. H. pylori Hpnth mutants unable to repair oxidized pyrimidines from the bacterial DNA were generated. H. pylori strains lacking a functional endonuclease III (HpNth) showed elevated spontaneous and induced mutation rates and were more sensitive than the parental strain to killing by exposure to oxidative agents or activated macrophages. Although under laboratory conditions the Hpnth mutant strain grows as well as the wild-type strain, in a mouse infection the stomach bacterial load gradually decreases while the population in the wild-type strain remains stable, showing that endonuclease III deficiency reduces the colonization capacity of the pathogen. In coinfection experiments with a wild-type strain, Hpnth cells are eradicated 15 days postinfection (p.i.) even when inoculated in a 1:9 wild-type:mutant strain ratio, revealing mutagenic lesions that are counterselected under competition conditions. These results show that the host effectively induces lethal and premutagenic oxidative DNA adducts on the H. pylori genome. The possible consequences of these DNA lesions on the adaptability of H. pylori strains to new hosts are discussed.     

Diagnosis and genotyping of Helicobacter pylori by polymerase chain reaction of bacterial DNA from gastric juice

BACKGROUND: Efficient and accurate detection of Helicobacter pylori infection as well as identification of virulence-associated alleles are important for the treatment of gastroduodenal diseases caused by this gastric pathogen. The present study was performed to test the efficiency of gastric juice polymerase chain reaction (PCR) method for the rapid detection of H. pylori infection and to determine the bacterial genotypes without the need for culture, which is often not feasible especially in developing countries. METHODS: DNA was extracted from gastric juice samples collected from 45 subjects and was used to amplify urease B gene (ureB) for H. pylori. Results obtained from this method were further confirmed by rapid urease test (RUT), histology and culture. Genotypes of the infected strains predicted from gastric juice PCR were compared to the genotype data obtained from the isolated strains. RESULTS: Among 45 cases, 32 were positive by RUT, 37 by histological examination, 25 by gastric juice PCR method, while culture yielded positive results for 19 samples. Except for one case, all the 19 culture-positive strains gave the same genotype with the gastric juice PCR result. It was found that the gastric juice PCR is more efficient for detection of multiple-strain infection as compared to genotype data obtained from strains isolated as pooled culture. CONCLUSIONS: This moderately sensitive technique could be employed with good efficiency, particularly in cases where it is difficult to obtain biopsy. Moreover, with this method bacterial genotype could be obtained.     

Virulence of water-induced coccoid Helicobacter pylori and its experimental infection in mice

AIM: To explore the virulence and the infectivity of coccoid Helicobacter ppylori(H.pylori) transformed from spiral form by exposure to sterile tap water.METHODS: Three strains of H.pylori, isolated from gastric biopsy specimens of confirmed peptic ulcer, were converted from spiral into coccoid form by exposure to sterile tap water.Both spiral and coccoid forms of H.pylori were tested for the urease activity, and the adherence to Hep-2 cells. The presence of flagella was examined under electron microscopy. In the experimental animal infection, the spiral and coccoid forms of H.pylori originated from the same strain F49 were inoculated intragastrically into BALB/c mice respectively four times at a 3-day interval. Half of the mice from each group were sacrificed at Day 21 and Day 28 after the last inoculation. Histology and H.pylori colonization were detected by urease test of gastric mucosa, cultures of H. pylori,and electron microscopy and so on.RESULTS: The urease activity and the ability of adherence to Hep-2 cells were found to be lower in coccoid H.pylori than that in its spiral form. For example, the transformation in strain F44 led to a significant decrease of the adherence rate and adherence index from 70.0±5.3 % to 30.2±3.5 %(P＜0.01), and from 2.6±0.4 to 0.86±0.3 (P＜0.01),respectively. The flagella of coccoid H.pylori were observed under electron microscope. In the experimental infection in mice, the positive rate of gastric mucosa urease test was 93.8 % (15/16) in the group infected by spiral H.pylori and 50 % (8/16) in the group infected by coccoid H. pylori,and the estimated coccoid H.pylori colony number was 1.75 vs0,56. The positive rates of H. pyloriculture were 87.5 %(14/16) in spiral H. pylori group and 68.8 % (11/16) in coccoid H.pylorigroup. There was no significant difference in either urease test or bacterial culture rate between the groups examined at Day 21 and Day 28 after inoculation. Electron microscopic examination of the samples taken from both groups showed the adherence of H. pylori in spiral,bacillary and coccoid shapes to the epithelial cells of gastric wall. Histological examination showed the occurrence of gastric mucosal injury as indicated by various degrees of erosion, ulcer, and inflammatory cell infiltration. Mucosal injury was slighter in the mice infected by coccoid H. pylori.No positive result was obtained in the control group that received intragastrical administration of sterile tap water.     

幽门螺杆菌尿素酶B亚单位在大肠杆菌中的融合表达与鉴定

目的 通过对幽门螺杆菌（Helicobacter pylori,Hipylori)尿素酶B亚单位在大肠杆菌中表达的研究，探索其免疫反应性。方法 用高保真PCR方法扩增H.pylori ureB基因，并定向克隆人pNEB193中，然后经酶切回收后插入原核表达载体pMAL-C2X进行融合表达。采用蛋白印迹法对表达产物进行鉴定。结果 特异PCR和酶切鉴定证实融合基因ureB克隆人表达载体，SDS-PAGE结果显示ureB在大肠杆菌中以融合蛋白形式MBP-ureB高效表达，约占细胞总蛋白量的26.7%。该融合蛋白可与H.pylori阳性病人血清发生特异反应。结论 成功构建了高效表达H.pylori ureB的重组菌，为Hp基因工程疫苗的进一步研究奠定了基础。     

Pathogenicty and immune prophylaxis of cag pathogenicity island gene knockout homogenic mutants

AIM: To clarify the role of cag pathogenicity island (cagPAI) of Helicobacter pylori (H pylori ) in the pathogenicity and immune prophylaxis of H pylori infection. METHODS: Three pairs of H pylori including 3 strains of cagPAI positive wildtype bacteria and their cagPAI knockout homogenic mutants were utilized. H pylori binding to the gastric epithelial cells was analyzed by flow cytometry assays. Apoptosis of gastric epithelial cells induced by H pylori was determined by ELISA assay. Prophylaxis effect of the wildtype and mutant strains was compared by immunization with the sonicate of the bacteria into mice model. RESULTS: No difference was found in the apoptasis between cagPAI positive and knockout H pylori strains in respective of the ability in the binding to gastric epithelial cells as well as the induction of apoptosis. Both types of the bacteria were able to protect the mice from the infection of H pylori after immunization, with no difference between them regarding to the protection rate as well as the stimulation of the proliferation of splenocytes of the mice. CONCLUSION: The role of cagPAI in the pathogenicity and prophylaxis of H pylori infection remains to be cleared.     

H pylori colocalises with MUC5AC in the human stomach

BACKGROUND: The bacterium Helicobacter pylori is able to adhere to and to colonise the human gastric epithelium, yet the primary gene product responsible as a receptor for its adherence has not been identified. AIMS: To investigate the expression of the gastric mucins MUC5AC and MUC6 in the gastric epithelium in relation to H pylori colonisation in order to examine their possible roles in the binding of H pylori. PATIENTS: Seventy two consecutive patients suspected of having H pylori infection. METHODS: MUC5AC, MUC6, and H pylori were detected in single sections of antral biopsy specimens using immunohistochemical triple staining. RESULTS: MUC5AC was expressed in the superficial epithelium and the upper part of the gastric pits. MUC6 expression was detected in the lower part of the gastric pits. The expression of both mucins in the epithelium was complementary. In each patient, there was a sharply delineated transition between MUC5AC and MUC6 producing cell populations. In all H pylori positive patients there was a striking colocalization of H pylori and MUC5AC; more than 99% of the bacteria were associated with either extracellular MUC5AC or the apical domain of MUC5AC producing cells. CONCLUSIONS: H pylori is very closely associated with extracellular MUC5AC and epithelial cells that produce MUC5AC. This indicates that MUC5AC, but not MUC6, plays a role in the adhesion of H pylori to the gastric mucosa.     

Adherence and internalization of Helicobacter pylori by HEp-2 cells.

Helicobacter pylori colonizes the mucous layer of the stomach and the surface of gastric mucous cells. Although H. pylori is not generally thought of as invasive, it has been observed in the lamina propria and within vacuoles in the cytoplasm of epithelial cells. The authors report that isolates of H. pylori can enter into the cytoplasm of tissue culture epithelial cell lines such as HEp-2 cells. Intracellular uptake of H. pylori by HEp-2 cells is rapid and appears to require both the N-acetylneuraminyllactose-binding adhesin and another factor present only in living bacteria. Uptake of H. pylori was inhibited by ammonium chloride and chloroquine at concentrations that did not effect either adherence or bacterial viability. Dansylcadaverine, an inhibitor of receptor clustering and internalization, also inhibited uptake but not adherence of H. pylori. Uptake was completely inhibited when H. pylori and HEp-2 cells were incubated at 4 degrees C under conditions that did not effect bacterial adherence. Cytochalasin B, an inhibitor of phagocytosis, did not inhibit uptake. It is concluded that H. pylori is internalized either by receptor-mediated endocytosis or by a closely related pathway.     

Adherence of Helicobacter pylori to HEp-2 cells.

The adherence of 25 strains of Helicobacter pylori was evaluated in HEp-2 cells. These bacterial isolates, obtained from Chilean patients with gastric disorders, were also tested for haemagglutination of human red blood cells. Adherence of HEp-2 cells was expressed as a common property of all strains, irrespective of whether the cultures were grown on semi-solid or in liquid media. Previous reports that haemagglutinating activity was present in cells grown only on semi-solid media were confirmed. Adherence to HEp-2 cells was suppressed when bacterial cells were pretreated with homologous or heterologous whole human serum, containing specific antibodies of H. pylori. Adherence remained unaltered when bacterial cells were similarly treated with normal serum lacking specific antibodies. These observations imply that adhesions are expressed in vivo and suggest that an adherence mechanism, not depending on the expression of specific haemagglutinin antigen, operates for H. pylori.     

Construction of prokaryotic expression system of ureB gene from a clinical Helicobacter pylori strain and identification of the recombinant protein immunity

AIM: To clone ureB gene from a clinical isolate of Helicobacter pyloriand construct a prokaryotic expression system of the gene and identify immunity of the expressed recombinant protein. METHODS: ureBgene from a clinical Hpyloristrain Y06 was amplified by the high fidelity polymerase chain reaction technique. The target DNA fragment amplified from ureB gene was sequenced after T-A cloning. Prokaryotic recombinant expression vector pET32a inserted with ureB gene (pET32a-ureB) was constructed. The expression of recombinant UreB protein (rUreB) in E. coli BL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations was examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of Hpylori and an immunodiffusion assay using a self-prepared rabbit anti-rUreB antibody were applied to determine immunity of the target recombinant protein. ELISA was used to detect the antibody against rUreB in sera of 125 Hpyloriinfected patients and to examine rUreB expression in 109 Hpylori isolates. RESULTS: In comparison with the reported corresponding sequences, the nucleotide sequence homology of the cloned ureB gene was from 96.88-97.82% while the homology of its putative amino acid sequence was as high as 99.65-99.82%. The rUreB output expressed by pET32a-ureB-BL21DE3 was approximate 30% of the total bacterial proteins, rUreB specifically combined with the commercial antibodies against whole cell of Hpylori and strongly induced rabbits to produce antibody with a 1:8 immunodiffusion titer after the animals were immunized with the recombinant protein. Serum samples from all Hpyloriinfected patients were positive for UreB antibody and UreB expression were detectable in all tested Hpyloriisolates. CONCLUSION: A prokaryotic expression system with high expression efficiency of Hpylori ureBgene was successfully established. The expressed rUreB showed qualified immunoreactivity and antigenicity. High frequencies of UreB expression in different Hpyloriisolates and specific antibody against UreBin sera of Hpyloriinfected patients indicatet hat UreB is an excellent antigen candidate for developing H pylori vaccine.