Diagnostic value of double balloon enteroscopy for small-intestinal disease: experience from China.

BACKGROUND: Diseases of the small intestine include, among others, ulceration, chronic inflammation, Meckel's diverticula, vascular deformities, and cancer. OBJECTIVE: To study the diagnostic value of double balloon enteroscopy (DBE) for small-intestinal disease in a Chinese patient cohort. DESIGN: DBE was performed via the mouth, anus, or both approaches to diagnose small-intestinal disease. PATIENTS: We studied 155 patients with clinically suspected small-intestinal disease: 110 men and 45 women. Their age ranged from 6 to 75 (mean 41). There were 92 cases with small-intestinal hemorrhage, 39 with abdominal pain, 7 with diarrhea, 13 with abdominal distention, 3 cases with malnutrition, and 1 with diarrhea and refractory hypoalbuminemia. RESULTS: Among the 155 patients, lesions were found in 126 (81.3%). These lesions found were small-intestinal ulcers (including Crohn's disease), chronic inflammation, Meckel's diverticulae, vascular deformities, and carcinoma. Eighty-five of the 92 patients with suspected intestinal hemorrhage were confirmed, with a positive rate of 92.4%. Also confirmed were 24 of the 39 patients with abdominal pain (positive rate of 61.5%); 16 of the 23 patients with diarrhea, abdominal distention, or malnutrition (positive rate of 69.6%); and 1 patient with refractory hypoalbuminemia. Among the 126 patients with positive findings, the lesions were located in the small intestine in 116 patients, in the stomach and duodenum in 9 patients, and in the colon in 1 patient. In the 45 patients with small-intestinal ulcer, 29 patients had recurrent hemorrhage, 9 had abdominal pain, 4 had abdominal distention, 2 had malnutrition, and 1 had diarrhea. Ulcers were located in the jejunum in 20 patients, in the ileum in 20 patients, and in both the jejunum and ileum in 5 patients. For 7 patients with small-intestinal ulceration diagnosed as Crohn's disease, the concordance rate of diagnosis between preoperative and postoperative diagnosis was 57.1%, lower than other diseases (P < .01). One patient had a perforation. CONCLUSION: DBE is effective and safe for the diagnosis of small-intestine disease in a Chinese patient cohort.

[The diagnostic value of double balloon endoscopy in small intestine disease].

OBJECTIVE: To study the diagnostic value of double-balloon endoscopy (DBE) for small intestinal diseases. METHODS: 155 patients clinically suspicious of small intestinal diseases were studied. 110 of them were male and 45 female. Their age ranged from 6 to 75 and with an average of 41 years. They consisted of 92 cases of small intestinal hemorrhage, 39 abdominal pain, 7 diarrhoea, 13 abdominal distention, 3 malnutrition and one diarrhoea as well as refractory hypoalbuminemia. In the procedure, the operator manipulated and advanced the endoscope and the assistant helped to advance the over tube. RESULTS: Among the 155 cases lesions were found in 125 cases, with positive results accounting for 80.6%. These lesions mainly consisted of small intestinal ulcer (including Crohn's disease), chronic inflammation, Meckel's diverticulum, interstizialoma, vascular deformity and carcinoma of small intestine. In 84 of the 92 patients suspicious of intestinal hemorrhage the lesions were confirmed with a positive rate of 91.3%. In 24 of the 39 patients with abdomen pain the etiologies were confirmed with a positive rate of 61.5%. In 16 of the 23 patients with diarrhoea, abdominal distention and malnutrition the positive rate was 69.6%. The cause of the only one case with refractory hypoalbuminemia was confirmed. Among the 155 cases, 9 had lesions located in stomach and duodenum, 115 in small intestine and one in large bowel, no lesion was found in 30 cases. Among the patients, 43 were found to have small intestinal ulcer. In the 43 patients, 12 patients were with single intestinal ulcer and 31 with multiple. For cases of Meckel's diverticulum, interstizialoma, carcinoma, vascular deformity and intestinal adhesion of small intestine in this series, diagnoses made by DBE combined with morphology were completely consistent with those found in operation. However, for ulcer lesions (mainly Crohn's disease), there was diversity in the diagnoses between the two methods, the coincidence was 57.1%. Two patients had complication, one perforation of small intestine and the other acute intestinal stasis. CONCLUSIONS: DBE is efficient and safe for the diagnosis of small intestinal diseases, especially in confirming the lesions. However, for ulcer of small intestine, this method even combined with biopsy is sometimes unable to determine its nature, so surgery may be beneficial in this condition.

[Clinical value of monitoring CD14+ monocyte human leukocyte antigen (locus) DR levels in the early stage of sepsis].

OBJECTIVE: To explore the relationship of monitoring CD14(+) monocyte human leucocyte antigen (locus) DR (HLA-DR) and the outcome in the early stage of sepsis. METHODS: Thirty-six definitely diagnosed septic patients in intensive care unit (ICU) were included. CD14(+) monocyte HLA-DR levels were detected by flow cytometer on the first day of the study, and acute physiology and chronic health evaluation II (APACHE II) scores were evaluated. Their clinical values in predicting the outcome of the disease were assessed through correlation analysis. RESULTS: Among 36 sepsis patients CD14(+) monocyte HLA-DR level<30% was found in 6 patients (16.67%). The average APACHE II score was 24.17+/-4.45 (r=0.212, P=0.687), all of them die, CD14(+) monocyte HLA-DR level <40% was 27.78% (10/36), the scores of APACHE II score was 23.50+/-4.30 (r=-0.0251, P=0.484), and the mortality rate was 80% (8/10). CONCLUSION: CD14(+) monocyte HLA-DR level <30% is an immunosuppressive index. In predicting the outcome of sepsis, it might be better than APACHE II scores. Immunosuppression is primarily found in the early stage of sepsis, suggesting that the classical compensatory anti-inflammatory response syndrome (CARS) hypothesis needs to be revised and improved.

[Clinical classification of Peutz-Jeghers syndrome].

OBJECTIVE: To propose the clinical classification of Peutz-Jeghers syndrome (PJS). METHODS AND RESULTS: Retrospective analysis of 52 patients with PJS admitted in Nanfang Hospital from 1980 to 2003 was conducted. Twenty-four patients were found to have family history of PJS, who had a mean age of 19 years. In the PJS patients, the incidence of gastric polyps was 64.4%, colorectal polyps 76%, and small bowel polyps 95%. The number of polyps was above 50 in 19 of the 31 patients with gastric polyps, in 18 of the 38 patients with colorectal polyps, and in 8 of the 19 patients with small bowel polyps. The pathology of the majority of the polyps (63/108) was characterized by hamartomas, and the incidence of malignancy was 13.5% in the PJS patients. CONCLUSIONS: PJS can be classified according to family history and location, pathology, and number of the polyps. As most patients with over 50 polyps require surgical intervention, 50 polyps is recommended as the criteria for PJS classification. Endoscopic surgery may suffice for management of patients with fewer polyps (<50), while in patients with more polyps or small bowel polyps, open surgery combined with intraoperative endoscopic surgery is recommended.

[Preparation oral liposome-encapsulated recombinant Helicobacter pylori heat shock protein 60 vaccine for prevention of Hp infection].

OBJECTIVE: To prepare oral liposome-encapsulated recombinant Helicobacter pylori (Hp) heat shock protein 60 (Hsp60) vaccine and investigate its effect against Hp infection in mice. METHODS: The recombinant vector PET-22(+)/Hsp60 was transformed into BL21(DE3) E.coli. The recombinant protein was purified with Ni-NTA agrose resin and the oral liposome-encapsulated vaccine was prepared with phosphatidyl choline and cholesterols using film method, with the size distribution of the folate liposomes measured by transmission electronic microscopy. BALB/c mice were divided into 5 groups and immunized by intragastric administration of PBS, liposome, rHsp60 plus choleratoxin (CT), liposome-encapsulated rHsp60, and liposome-encapsulated rHsp60 plus CT, respectively, given once a week for 4 weeks. All the mice were challenged by Hp for 3 times within two weeks following the last immunization and sacrificed 3 weeks after the last challenge. Hp detection was performed by fast urease test. Semi-quantitative assessment of the bacterial colonization density observation of the inflammation severity and gastric histopathology were carried out. RESULTS: The soluble expression product accounted for 27% of the total bacterial protein. The purity of recombinant fusion protein was about 95% after purification. The mean size of the folate liposomes was 0.7+/-0.4 mum. PBS or liposome alone showed no immune-enhancing effect, and rHsp60 plus CT, liposome-encapsulated rHsp60 and liposome-encapsulated rHsp60 plus CT had the protective rates against Hp infection of 73.3%, 66.7% and 86.7%, respectively. The latter 3 preparations effected significantly reduced Hp infection and alleviated the inflammation in the gastric mucosa of the mice challenged with Hp. CONCLUSION: The oral liposome may serve as a potential adjuvant for Hp vaccine in preventing Hp infection.

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 mug/mL ampicillin overnight at 37 degrees. 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 beta-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.

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.

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.

Cloning and expression and immunogenicity of Helicobacter pylori BabA2 gene.

AIM: To construct a recombinant strain which expresses BabA of Helicobacter pylori (H pylori) and to study the immunogenicity of BabA. METHODS: BabA2 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+) and expressed in the BL21 (DE3) E.coli strain. Furthermore, BabA immunogenicity was studied by animal test. RESULTS: DNA sequence analysis showed the sequence of BabA2 DNA was the same as the one published by GenBank. The BabA recombinant protein accounted for 34.8% of the total bacterial protein. The serum from H pylori infected patients and Balb/c miced immunized with BabA itself could recognize rBabA. CONCLUSION: BabA recombinant protein may be an potential vaccine for control and treatment of H pylori infection.

Development of an ELISA kit using monoclonal antibody to Clostridium difficile toxin A.

AIM: To establish an ELISA kit using monoclonal antibodies against Clostridium difficile (C. difficile) toxin A. METHODS: An indirect sandwich ELISA was described using the purified rabbit monospecific antiserum as capturing antibody. After the polystyrene microtitre plates with 96 flat-bottomed wells were coated with rabbit antiserum, the wells were blocked with 100 g/L BSA in PBS-T. C. difficile toxin A or culture filtrates were added to each well and then monoclonal antibodies IgG-horseradish peroxidase conjugate was added as detecting antibody, tetramethylbenzidine was used as substrate and A450 of the stopped reacting product was recorded in an automated plate reader. RESULTS: The tested specimens included culture filtrates of 2 strains of toxigenic C. difficile, 2 strains of non-toxigenic C. difficile, 26 strains of E. coli, 2 strains of S. dysenteriae, 1 strain of Bif. infantis, 5 strains of V. cholera, 2 strains of S. typhi, 7 strains of C. botulinum, 1 strain of toxigenic C. sordllii, and 1 strain of C. butyricum. A total of 47 strains of culture filtrates were all negative except for 2 strains of toxigenic C. difficile. The detective limitation of toxin A was 0.1 ng/mL. CONCLUSION: An ELISA kit with high specificity and excellent sensitivity for the rapid detection of C. difficile toxin A was established. It will be a useful tool for diagnostic test of C. difficile toxin A.

Simplified purification method for Clostridium difficile toxin A.

AIM: To establish the purification method for Clostridium difficile (C. difficile) toxin A. METHODS: C. difficile VPI 10463 filtrate was cultured anaerobically by the dialysis bag methods. And then the toxin A was purified by precipitation with 500 g/L (NH4)2SO4 and acid precipitation at pH 5.5, followed by ion-exchange chromatography on DEAE-Toyopearl. RESULTS: Purified toxin A exhibited only one band on native polyacrylamide gel electrophoresis (native-PAGE) and Ouchterlony double immunodiffusion. The molecular weight of toxin A was estimated to be 550,000. The purified toxin A had a protein concentration of 0.881 mg/mL. The minimum lethal dose was 1X10(6) MLD/mL (i.p.mice). The cytotoxic titer was 10(7) CU/mg. The haemagglutinate activity was at a concentration of 1.72 microg/mL. The ratio of fluid volume (mL) accumulated to the length (cm) of the loop was 2.46. CONCLUSION: The modified method for purification of toxin A of C. difficile was simple and convenient. It may be even more suitable for purification of toxin A on large scales.

[Preparation and characterization of monoclonal antibody against Clostridium difficile toxin A].

AIM: To prepare monoclonal antibodies (mAbs) against Clostridium difficile toxin A and identify their properties. METHODS: BALB/c mice were immunized with C.difficile toxin A. The splenocytes from immunized mice were fused with myeloma cells Sp2/0. The hybridoma cells were screened by indirect ELISA and limiting dilution method. The titer and relative affinity of ascitic mAbs were determined by ELISA. Specificity of mAbs was analyzed by Western blot. RESULTS: Six hybridoma cell lines (2H7, 3E9, 4B5, 5C10, 6G8 and 8A1) secreting mAbs against C.difficile toxin A were obtained. The Ig classes and subclasses of mAbs 2H7, 3E9 and 6G8 were IgM, mAbs 4B5 and 8A1 were IgG1, and mAb 5C10 was IgG2a. All 6 mAbs had no neutralization activity. Epitope recognized by 5 mAbs(2H7, 4B5, 5C10, 6G8 and 8A1) differed from that by mAb 3E9. Relative affinities of mAbs 8A1 and 4B5 were all above 10(5), and those of other 4 mAbs were 10(4). Western blot analysis no-denatured PAGE showed that all 6 mAbs reacted to C.difficile toxin A with M(r) being 55 x 10(4), and under the condition of denatured SDS-PAGE, Western blot analysis showed that all 6 mAbs reacted to subunits of C.difficile toxin A with M(r) being 5 x 10(4)-24 x 10(4). CONCLUSION: Six mAbs against C.difficile toxin A with high titers were obtained successfully with satisfactory specificity and relative affinity, which will be useful for detection of C.difficile toxin A.

A novel method for preparation of tissue microarray.

AIM: To improve the technique of tissue microarray (tissue chip). METHODS: A new tissue microarraying method was invented with a common microscope installed with a special holing needle, a sampling needle, and a special box fixing paraffin blocks on the microscope slide carrier. With the movement of microscope tube and objective stage on vertical and cross dimensions respectively, the holing procedure on the recipient paraffin blocks and sampling procedure of core tissue biopsies taken from the donor blocks were performed with the refitted microscope on the same platform. The precise observation and localization of representative regions in the donor blocks were also performed with the microscope equipped with a stereoscope. RESULTS: Highly-qualified tissue chips of colorectal tumors were produced by a new method, which simplified the conventional microarraying procedure, and was more convenient and accurate than that employing the existing tissue microarraying instruments. CONCLUSION: Using the refitted common microscope to produce tissue microarray is a simple, reliable, cost-effective and well-applicable technique.

[Electron microscopic observation of primary cultured laterally spreading tumor cell line].

OBJECTIVE: To observe the microscopic characteristics of laterally spreading tumor (LST) cell line in primary culture. METHODS: The cells isolated from a rectum LST specimen obtained by endoscopic mucosal resection was primary cultured, followed by observation with scanning and transmission electron microscope in comparison with the cells of adenocarcinoma and normal mucosa of the rectum. RESULTS: Scanning and transmission electron microscopes both revealed numerous microvilli covering the surface of the LST cells, and the cytoplasm contained large quantity of lysosomes, mitochondria and phagosomes. Obviously heterogeneous cell nuclei were present with abnormal nuclear fossa and huge nucleoli. CONCLUSION: The cultured LST cells are highly malignant.

Expression of Helicobacter pylori Hsp60 protein and its immunogenicity.

AIM: To express Hsp60 protein of H pylori by a constructed vector and to evaluate its immunogenicity. METHODS: Hsp60 DNA was amplified by PCR and inserted into the prokaryote expression vector pET-22b (+), which was transformed into BL21 (DE3) E.coli strain to express recombinant protein. Immunogenicity of expressed Hsp60 protein was evaluated with animal experiments. RESULTS: DNA sequence analysis showed Hsp60 DNA was the same as GenBank's research. Hsp60 recombinant protein accounted for 27.2% of the total bacterial protein, and could be recognized by the serum from H pylori infected patients and Balb/c mice immunized with Hsp60 itself. CONCLUSION: Hsp60 recombinant protein might become a potential vaccine for controlling and treating H pylori infection.

[Regulation of Fas expression in gastric epithelium: one of the auto-immune mechanisms of Helicobacter pylori pathogenesis].

OBJECTIVE: To investigate the pathogenic mechanism of Helicobacter pylori (H.pylori)-mediated gastric epithelial cell damage. METHODS: Fas expressions in gastric epithelial cell lines and freshly isolated gastric epithelial cells with or without H.pylori infection were evaluated by flow cytometry. The modulation of Fas expression in gastric epithelial cells by H.pylori or by Th1 cytokines present in H.pylori-infected gastric mucosa was assessed in vitro. The function of Fas expressed by the gastric epithelial cells to induce cell apoptosis was determined by enzyme-linked immunosorbent assay (ELISA) after incubation of the cells with anti-Fas antibody. RESULTS: All of the three gastric epithelial cell lines, AGS, N87 and kato-III, expressed detectable Fas protein when examined by flow cytometry. The percentage of positive cells and the amount of Fas protein on cell surface were larger in freshly isolated gastric epithelial cells with H.pylori infection than in uninfected cells (P<0.05). H. pylori alone or in combination with Th1 cytokines (IFN-gamma and TNF-alpha) significantly increased the expression of Fas in gastric epithelial cell lines in vitro. After incubation with IgM anti-Fas mAb, Fas-bearing gastric epithelial cells underwent apoptosis, and this effect of Fas was enhanced by IFN-gamma. CONCLUSION: Th1 cells accumulated in the gastric mucosa during H.pylori infection is involved in the damage of gastric epithelium through the modulation of Fas/Fas ligand interaction.

[Effects of the antibacterial peptide cecropins from Chinese oak silkworm, Antheraea pernyi on 1, 2-dimethylhydrazine-induced colon carcinogenesis in rats].

OBJECTIVE: To gain insight into the putative anticancer effect of the antibacterial peptides, cecropins, from Chinese oak silkworm, Antheraea pernyi, on the cancer cells and 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats. METHODS: Growth inhibitory effect of the cecropins on normal human gastric epithelial cell line (GES-1) and human colon adenocarcinoma cell line (LS-174T) was observed using a microculture tetrazolium (MTT) colorimetric methods. Male Wistar rats were divided into 4 groups. Group1 was given on a weekly basis cecropins from Antheraea pernyi (3,000 Ua/ml) by gavage at 2 doses of 10 ml/kg body weight and exposed to subcutaneous injection of DMH at the dose of 20 mg/kg body weight. Group 2 was received weekly DMH only. Group 3 was given the cecropins by gavage at the same dose as in group 1. Group 4 was weekly exposed to subcutaneous injection of EDTA (1 mmol/L). All treatments were completed in a course of 18 weeks and the experiment was finished at week 33. RESULTS: MTT assay showed selective cytotoxic activity of the cecropins against the human colon adenocarcinoma cells line. The viability of the cancer cells was about 54% and 100% for the normal cells. There was a significantly lower incidence of large intestinal tumors in rats gavaged with cecropins (65%, P<0.01), but the tumor burden (tumors/tumor-bearing animal) and tumor mass index were comparable between the groups (P>0.05). CONCLUSION: The cecropins possess effective anti-tumor activity with no cytotoxicity against normal eukaryotic cells, and impede the neoplastic process in murine large intestines.

[In vitro evaluation of the safety and biological activity of recombinant Helicobacter pylori blood group antigen-binding adhesin].

OBJECTIVE: To evaluate the safety and biological activity of recombinant Helicobacter pylori (Hp) blood group antigen- binding adhesin (rBabA ) in vitro so as to investigate the feasibility of using rBabA as a Hp vaccine. METHODS: ELISA was used to measure rBabA-specific antibody in the serum of Hp-infected patients, and the proliferation of T lymphocytes in response to rBabA was examined by MTT assay. T cell apoptosis induced by rBabA was detected by diphenylamine assay. The effect of rBabA on Hp binding into human gastric carcinoma cell line(MGC-803) was determined by light microscopy. RESULTS: rBabA did not induce T cell apoptosis in BabA antibody- negative patients and was capable of stimulating T cell proliferation in rBabA antibody-positive patients. In the serum samples from 38 Hp-infected patients, the rBabA antibody positivity rate was 18.4%. rBabA could partially inhibit the binding of Hp to gastric epithelial cells. Under light microscope, the adhesion of Hp to MGC-803 was significantly inhibited by rBabA in comparison with negative control with PBS pretreatment. CONCLUSION: rBabA proves to be a safe and immunogenic bacterial component of Hp, which stimulates humoral and cellular immunity and can be a hopeful antigen targeting at BabA2 gene-positive Hp strain for the development of Hp vaccine.

[Cloning and immunogenicity of conservative region of adhesin gene of Helicobacter pylori].

OBJECTIVE: To construct a candidate strain of Helicobacter pylori (Hp) that expresses the proteins of the conservative region of 4 adhesins (BabA2, AlpA, AlpB, and HopZ) and study its immunogenicity. METHODS: The DNA of Hp was extracted. Primers were designed according to the C-terminal structural gene sequence (called CB) of AlpA. The CB gene was amplified by PCR and inserted into the prokaryotic expression vector pET-22b (+) and expressed in BL21 (DE3) strain of Escherichia coli. The product of expression, CB, was purified by affinity chromatography and identified by Western blot analysis. ELISA assay was used to measure the CB-specific antibody in the specimens of serum of 55 Hp infected patients. Rapid urease test (RUT) was used on biopsy specimens collected by gastroscopy as parallel control. RESULTS: A recombinant plasmid pET-22b (+)/CB was constructed with the conservative region of the 4 adhesins. DNA sequencing showed one open reading frame of 588 bp encoding a polypeptide of 195 amino acids. The recombinant CB (rCB) protein, with a molecular weight of 22.5KD, amounted to 29% of the total bacterial protein. The purity of purified rCB was 96%. Western blot analysis showed that the rCB protein could be specifically recognized by the serum from Hp infected patients. The kappa coefficient was 0.76 for evaluation by ELISA and RUT results. CONCLUSION: CB has the potential to be used as a vaccine against Hp infection.