Objective To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori(H.pylori) and to study the immunogenicity of adhesin AlpA. Methods The adhesin AlpA DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b(+)and expressed in the BL21(DE3) E.coli strain. Adhesin AlpA immunogenicity was studied by Western blot test. Results DNA sequence analysis showed that the sequence of adhesin AlpA DNA was almost the same as that of the published by GenBank. The adhesin AlpA recombinant protein accounted for 31.9% of the total bacterial protein. Western blot analysis of rAlpA confirmed that it could be specially recognized by serum from rabbit immunized with AlpA itself and H.pylori infected patients. Conclusion Cloning and high-expression of adhesin AlpA and primary confirmation of its immunogenicity lay the foundation for H.pylori gene engineering vaccine preparation and adhering mechanism research.

Objective To evaluate the expression of Fas Ligand (FasL) on gastric T cell during H.pylori infection and its cytotoxicity to gastric epithelial cells. Methods FasL protein expressions on mono nuclear cells of gastric mucosa with and without H.pylori infection were examined by immunohistochemistry assay. FasL mRNA expressions were detected in gastric T cell lines isolated from H.pylori infected and uninfected gastric biopsies by RT PCR. The function of FasL expressed by gastric T cells to induce apoptosis in Fas bearing cells was determined by a co culture bioassay (JAM), while the Fas mediated apoptosis of gastric epithelial cells induced by gastric T cells were also evaluated by the same assay. Results FasL was expressed by the mononuclear cells accumulated in gastric mucosa with H.pylori infection, whereas the few mononuclear cells presented in uninfected gastric tissues were negative for FasL. FasL mRNA was detected in gastric T cells isolated from H.pylori infected gastric biopises, while that of uninfected gastric T cell lines was either negative or weak positive. Gastric T cells were capable of inducing apoptosis of Fas positive jurkat cells, while this could be blocked by Fas blocking antibody, ZB4. Conclusions T cells presented in gastric mucosa during H.pylori infection could damage gastric epithelium through Fas/FasL interaction.

To find novel antihepatitis drugs, a series of nitrate-oleanolic acid (OA) hybrids (10a, 10b, 11a-11e and 12a-12c) were designed and synthesized on the basis of previous studies using OA as lead compound, which is widely found in natural plants and liver-specific metabolism. In the present study, ten novel NO-releasing derivatives of OA were synthesized by connecting nitrate to the OA-3-OH through varying lengths of linkers containing antioxidants which were designed to increase the ability of these target compounds to scavenge free radicals. The structures of these objective compounds were determined by IR, MS, 1H NMR and elemental analysis. Their protective effects on anti-Fas mediated HepG2 cell apoptosis were in vitro evaluated by LDH assay. Compound 12a is the most potent inhibitor. Its effect on anti-Fas mediated HepG2 cell apoptosis and amount of NO-releasing in vitro are similar to those of positive control NCX-1000.

Objective To study the role of cytidine-phosphate-guanosine oligodeoxynucleotide(CpG ODN) on the development of Plasmodium liver stage.Methods Plasmodium yoelii BY265 18S rRNA was cloned,and the TaqMan real-time PCR was established on P.yoelii BY265 18S rRNA and mouse GAPDH as quantitative analysis model.The model was tested by the level of liver Plasmodium load with the liver cDNA in BALB/c mice infected by salivary gland sporozoites for 42 hours.Twelve BALB/c mice were randomly divided into CpG group,CpG control group and PBS control group which were injected respectively by ODN1826 30 ?g,ODN1826 control 30 ?g and 0.01 mol/L PBS 200 ?l via vena caudalis.Twenty-four hours later,each mouse was inoculated with 100 sporozoites.Mice were sacrificed in 42 hours after infection,and the liver load of Plasmodium was analyzed by TaqMan real-time PCR.Results The cloned Py BY265 18S rRNA gene showed 98% similarity to Py 17XNL.The quantitative analysis model consisted by 18S rRNA and GAPDH showed positive correlation between the level of liver Plasmodium load and the sporozoite inoculation dose to mice.The Plasmodium load in CpG ODN pre-treated mice was reduced to one fifth of the control group(0.28/1.33)(P

Objective To study the gene expression change of circumsporozoite protein (CSP) and erythrocyte binding protein (MAEBL) in the Plasmodium yoelii sporozoites from oocyst and salivary gland at different phases during Plasmodium yoelii development in mosquitos. Methods The total RNA of plasmodium was extracted from the abdomen on day 5, 7, 10 and the thoraces on day 12, 15, 18 after Anopheles stephensi was infected by Plasmodium yoelii, then the expression of CSP and MAEBL mRNA was analyzed by semiquantitative RT-PCR with rRNA 28S as control. Results The transcription of CSP and MAEBL of plasmodium sporozoite were obviously up-regulated during the mosquito stage (P

Objective To evaluate the clinical manifestations, endoscopic features and the clinical pathological characteristics of H. heilmannii-associated gastritis, and to compare these variables with those of H. pylori-ussociated gastritis. Methods The clinical data, endoscopic findings and pathologic characteristics of 3107 patients, who underwent endoscopy from 2005 to 2007, were retrospectively analyzed. Results Twenty-five cases of H. heilmannii infection were identified, the infection rates of H. heilmannii and H. pylori were 0.80% (25/3107) and 4.12% (1060/3107) respectively. Three cases were mixed infections. Of 25 patients, 20 showed such gastroenterologic symptoms to a greater or less extent as abdominal distending pain,nausea and anorexia, and other 5 cases were asymptomatic. All 25 patients showed chronic gastritis by en-doscopy, including chronic superficial gastritis (7/25, 28% ), erosion ( 3/25, 12% ), chronic atrophic gastritis (4/25, 16%), bile reflux(1/25, 4%), ulcer (1/25, 4%), polyp (1/25, 4%) and duodenal bulbar inflammation (2/25, 8% ). In rapid urease test, 3 cases were hyper-positive, 3 cases positive, 7 ca-ses mild-positive and 12 cases negative. According to histological observation, H. heilmannii scattered or ac-cumulated within the gastric pits, glandular lumen or mucus. The organism was observed in parietal cells with cell damage in one case. Sporadic lymphatic and plasmic infiltration were found in all patients with H.heilmannii infection, infiltration of neutrophils (12/25), gland atrophy and intestinal metaplasia (4/25)and lymphoid follicles (6/25) were also observed. Compared with H. pylori-associated gastritis, H. heilman-nii-associated gastritis showed less inflammation, less helicobacter density, mononuclear cell infiltration and neutrophilic activity ( P < 0.05 ). Conclusion H. heilmanaii mainly induces chronic gastritis, which is less severe than H. pylori-associated gastritis.

Objective To understand the ethics conflict situations between the pre-hospital patients and ambulance staff's determinations. Methods Taking a survey among the pre-hospital emergency physicians(80 people)and nurses(248 people)by Questionnaire of ethics conflicts during pre-hospital emergeney service,to investigate the ethics conflict situations between the pre-hospital patients and ambulance staff's determinations. Resulls (8.046±6.990)%of the patients who needed treatments refused to be treated completely,and(14.544±10.558)%of them refused partially.(14.451±14.747)% of the patients who needed ambulance transport refused to be delivered.In the patients who refused treatments and transportation.payment problem accounted for(23.52±19.79)%,(22.22±20.84)%of them did not believe they needed.(5.77±4.47)%of them wished to die,(19.44.4±18.65)%of them were hard to be idenfified.Other reasons accounted for(30.08±25.78)%.(20.31.4±16.66)% of the patients refused the ambulance crews' judge for some state.(29.66.4±24.02)%of the patients who got the pre-hospital emergency service were not necessary to call an ambulance.(22.1 l±19.52)%of the patients' demand conflicted with pre-hospital emergency services network management system.Conclusions There exists some conflicts between the pre-hospital patients and ambulance crews' determinations.

Objective To investigate the association of two single nuclear peptides(SNPs)polymorphisms(rs11209026 and rs11805303)which lies in interleukin-23 receptor(IL23R)gene with susceptibility to inflammatory bowel disease(IBD).Methods The target SNPs were directly sequenced by polymerase chain reaction(PCR)and gene polymorphisms of 50 healthy and 81 patients with IBD (Crohn's disease in 41 patients and ulcerative colitis in 40 patients)were analyzed using chromassoftware.Results The geno-type frequency and allelic frequency of rs11209026 were 7.3%and 3.7%in patients with Crohn's disease respectively,15.0%and 7.5%in patients with ulcerative colitis respectively as well as 14.0%and 7.0%in normal population respectively(all P value＞0.05).The geno-type frequency and allelic frequency of rs11805303 were 22.0%and 52.4%in patients with Crohn's disease respectively,15.0% and 41.2% in patients with ulcerative colitis respectively as well as 34.0%and 59.0%in normal population respectively(all P value＞0.05).But in allelic frequency there was significant difference between ulcerative colitis patients and normal population(P=0.018).The polymorphisms of rs11805303 loci did not correlate with age,gender,disease duration.activity and site in patients with ulcerative colitis.Conclusions IL23R gene polymorphism is not associated with the susceptibility to Crohn's disease.rs11805303 allele may be related with susceptibility to ulcerative colitis.But no correlation was found between the SNP polymorphisms and the clinical characteristic of ulcerative colitis.

Objective To explore the effects of polymorphisms of Crohn's disease related NOD2 gene and human beta-defensin 2 (hBD-2) on transcription of hBD-2 gene and its mechanism. Methods HEK293T cells were transfected with hBD-2 gene and NOD2 eukaryotic expression plasmid, and were then stimulated with LPS, TNF-α, or BAY 11-7082 (antagonist of NF-κB), respectively. Transcriptional activity of hBD-2 was detected afterwards. Results LPS could suppress transcription of hBD-2 (P=0. 020), which was increased by TNF-α in a dose-dependent manner (P =0. 004). In the presence of LPS, there was sig-nificant difference in transcriptional activity of hBD-2 between wild-NOD2 transfected group and mutated NOD2 (P268S) transfected group (P=0. 008), but there was no significant difference between wild hBD-2 transfected group and mutated hBD-2 transfected group (P=0. 053). With the stimulation of TNF-α (5 ng/ml), there was a significant difference between mutated hBD-2 transfected group and wild hBD-2 transfected group (P=0. 006), but no significant difference between wild-NOD2 transfected and mutated NOD2 transfected group was defected (P = 0. 064). Pretreatment with BAY 11-7082 before TNF-α (5 ng/ml) significantly inhibited the transcriptional activity of hBD-2 (P < 0. 001). Conclusion The poly-morphism of NOD2 affects the innate expression of hBD-2, the polymorphism of site in hBD-2 promoter (-233) may lead to significant decline of the inducible expression of hBD-2, and NF-κB might be a key pathway that NOD2 protein mediates the expression of defensin.

OBJECTIVETo prepare the polyclonal antibody against methyl-accepting chemotaxis signal transduction protein (MCP) of Helicobacter hepaticus (H.hepaticus).

METHODSThe recombinant plasmid pET22b+/MCP was transformed into E.coli BL2l(DE3) to express the fusion protein His-rhMCP under the induction of IPTG. The fusion protein was purified and the antibody was obtained by immunizing rabbits. The titer of the polyclonal antibody was tested by indirect ELISA, and the specificity of the antibody was identified based on Western blotting using the prepared cell surface proteins (CSPs) of the bacteria.

RESULTSThe fusion protein was successfully expressed, and the titer of the antibody reached 1:32 000. Western blotting indicated that the antibody could specifically bind to CSPs and His-rhMCP.

CONCLUSIONThe antibody with a high titer and specificity was prepared to facilitate further study of the pathogenicity and epidemiology of H.hepaticus in human.

Animals ; Antibodies, Bacterial ; biosynthesis ; genetics ; Antibody Specificity ; Bacterial Proteins ; immunology ; Helicobacter hepaticus ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Signal Transduction