Objective To investigate the relationship of the killer cell immunoglobulin-like receptor (KIR) gene polymorphism with Hashimoto′s thyroiditis(HT). Methods One hundred HT patients and 260 randomly matched healthy controls were enrolled to detect the KIR genotype. The genomic DNA were extracted, and 15 selected KIR genes, KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, KIR3DS1 and pseudogene KIR2DP1, were determined by a polymerase chain reaction using sequence-specific primers (PCR-SSP). Results The frequency of KIR2DL5 gene was significantly lower of the patient group than that of the control group (0.200 vs 0.312, RR=0.64, P<0.01). Conclusion There may be an association between pathogenesis of HT and KIR2DL5 gene.

Objective To study the association of CD4+CD8+Foxp3+ regulatory T cells with the HIV long term non-progressors(LTNP) in China. Methods Seventy-four HIV-1 infected patients (LTNP group, HIV group and AIDS group)and 16 normal controls were enrolled and the frequency of CD4+CD25+Foxp3+ regulatory T cells were detected by flow cytometry. To study the correlation between CD4+CD25+Foxp3+ regulatory T cells and disease progression, the absolute CD4+ T cells, viral load, apoptosis and activation of T cells were also examined. Results The frequency of CD4+CD25+Foxp3+ regulatory T cells in LTNP group was significantly lower than that in HIV and AIDS group (P<0.05). The frequency of CD4+CD25+Foxp3+ regulatory T cells was inversely related to CD4+ T cells and closely related to viral load and CD38, CD95 expression on CD4, CD8+ T cells (P<0.05). Conclusion The frequency of CD4+CD25+Foxp3+ regulatory T cells of HIV infected LTNP is significantly lower than typical progressors, which indicates that alternation of regulatory T cells may play a protective role in LTNP.

Objective To study the hepatocyte cells infected by hepatitis C virus (HCV) positive serum. Methods Human hepatocyte 7701 was incubated with HCV RNA-positive and HCV antibody(Ab) negative sera BP52. Then, the expression of HCV antigen and the presence of HCV-RNA in cell and supernatant were assayed by RT-PCR, sequence analysis, immunofluorescent staining, Western blot, confocal laser microscopy. The ultrastructural changes of infected cells were observed by electro-microscopy. Results Plus-strand RNA and minus-strand RNA were intermittently detected in cell and/or supernatant on day 7-45 after infection. Sequence analysis demonstrated that the positive DNA nucleic acids were identified with HCV 5′-non-coding region(NCR) sequence. HCV core and NS3 protein were expressed in cytoplasm of infected cells. After 2 or 3 weeks, obvious intracellular ultrastructural changes and virus-like particles were observed. Conclusion human hepatocyte 7701 could support replication of HCV in vitro, which could be a useful tool for setting up cell model of HCV infection and studying the mechanism of HCV infection.

Objective To investigate the relationships of the serum level of interleukin-1β(IL-1β), the single nucleotide polymorphism(SNP) of IL-1B and interleukin-1 receptor antagonist (IL-1RN) genes with the gastric cancer or the gastric cancer infected by Helicobacter pylori(Hp). Methods The SNP of the IL-1B(-31C/T and -511C/T) was determined by gene chip and the variable number of tandem repeat(VNTR) of IL-1RN were detected by agarose gel electrophoresis. The sera level of IL-1β and the concentrations of IgG, IgM and IgA of Hp antibodies were measured by ELISA. Results The serum level of IL-1β increased significantly in patients with gastric cancer than that in control group(P<0.001). Hp infection was detected in 69.2% of 260 patients and 46.5% of 284 controls[P<0.001, odds ratio (OR)=2.59]. Frequency of genotype IL-1B-31TT or IL-1B-511TT in patients with gastric cancer were significantly higher than that in healthy controls (P<0.01, OR=1.95; P<0.05, OR=1.62), respectively. Frequency in Hp+ gastric cancer group was higher than that in Hp- group (P<0.05, OR= 2.00), and frequency of haplotype T-T in patients group was significantly higher than that in healthy control(χ2=4.45, P<0.05). The serum level [(802±148) ng/L] of IL-1β of the gastric cancer group was significantly higher than that of the control group [ (501±125) ng/L, P<0.01]. The serum level of IL-1β in patients with -31T or -511T allele was (845±156) ng/L or (871±148) ng/L, significantly higher than that without -31T [(555±116) ng/L] and -511T allele [(581±128) ng/L]. Furthermore, The serum level of IL-1β in Hp+ group with T allele were significantly higher than that in Hp- group (P<0.001). There was no association of IL-1RN gene and other IL-1B gene with gastric cancer or Hp+ gastric cancer. Conclusion IL-1B-31TT genotype was related to gastric cancer. IL-1B-511TT genotype was related to gastric cancer or with Hp+ gastric cancer. Both IL-1B-31T and -511T are associated with IL-1B gene. The haplotype T-T may be the genetic susceptible factor to gastric cancer.

Objective To study the activation induced cell death (AICD) of CD4+ T cells in primary biliary cirrhosis(PBC)murine model induced by poly I∶C. Methods Thirty female C57BL/6 mice were divided into model and control group randomly, and the former were injected with 5 mg/kg of poly I∶C, the later with PBS. PBC mice were detected 16 weeks after injection. CD4+ T cells isolated from spleen were stimulated in vitro by Con A and anti-CD3, and the apoptosis were determined by Annexin-V and PI staining. The expression of Fas, FasL and TRAIL were assayed by relative quantitative real-time PCR. Bcl-2 was detected by Western blot. Results Compared with control group, the portal areas of mice in model group were infiltrated with mononuclear cells obviously. The positive rate of serum antimitochondrial antibody(AMA) and the level of alkali phosphatase (ALP) were higher than that in control group (P<0.001). AICD of splenic CD4+ T cells in model group was lower than that of control group (P<0.001). The mRNA of FasL and TRAIL in model mice was down-regulated. Simultaneously, the anti-apoptosis protein Bcl-2 was up-regulated in model group. Conclusion These observations suggest that a defect in AICD of auto-reactive TH1 cells may contribute to the pathogenesis of PBC model. Furthermore, this defect in AICD may results from the change of Fas/FasL, TRAIL pathway and the up-regulation of Bcl-2.

Objective To determine the effect of Mycobacterium tuberculosis spp. inducing transformation of THP-1 cells to epithelioid cells (EC) and the involved signaling pathways and their regulations. Methods THP-1 cells infection models respectively infected with M. tuberculosis strains H37Rv, bovis and phlei were established. Indirect immunofluorescent staining assays were used to detect the expressions of monocyte/macrophage differentiation antigen CD115 and EC differentiation antigen CD82 of the THP-1 cells before or after infection. By Sandwich ELISA Kits, the phosphorylation levels of p38MAPK, Akt1 and STAT3 of the THP-1 cells before or after infection were measured. The alterations of CD115 and CD82 expression levels were examined when the associated signaling pathways were blocked with specific blocking agents. Results CD115 expression was weakened and CD82 expression was strongly increased in all the THP-1 cells infected with the three strains. A temporal up-regulation of the p38MAPK phoshporylation level but no obvious alteration of Akt and STAT3 phosphorylation levels after THP-1 cells infected by strain H37Rv or bovis. The THP-1 cells infected with anyone of the three strains continuously expressed CD115 after MAPK, PI3K/Akt or JAK/STAT of the cells was blocked. Although JAK/STAT was blocked, the THP-1 cells respectively infected with the three different strains still expressed CD82. However, CD82 expressed in THP-1 cells infected by the strain H37Rv or bovis was disappeared when p38MAPK and PI3K/Akt pathways of the cells were blocked. Conclusion Strain H37Rv and bovis can induce the infected THP-1 cells transforming to EC and p38MAPK and PI3K/Akt signaling pathways participate and regulate the transformation procedure. Of the two signaling pathways p38MAPK seems to be more important.

Objective To predict and screen the B-cell epitopes on nucleoprotein(NP)of human avian H5N1 virus strain.Methods As NP nucleotide sequences of strain A/GD/01/06(H5N1)were sequenced,B-cell epitopes were predicted by the analysis of the flexible regions of secondary structure for NPprotein and by screening on B-cell epitopes for NP protein with methods of Hydrophilicity Plot by KyteDoolittle,Surface Probability Plot by Emini,and Antigenic Index by Jameson-Wolf.Then a screening method was established by Hierarchical cluster,Bivariate correlate and Quartiles in SPSS13.0.and the variation of amino acids in NP protein was appraised in epitope prediction.Resuits The computer-predicted most possible epitopes for NP protein were located within or nearby its N-terminal 317-326,452-457,467-473,367-370,491-498,375-378,171-177,48-53,245-250.76-104 and so on.NP protein in A/GD/01/06(H5N1)increased a glycoprotein domain(NES368-370)by the substitution of N370S and changed the bio-features.Conclusion Stepwise prediction of the B-cell epitopes for the NP protein based on multiple parameters is helpful for the molecular-immunology and drug-screening,and the substitution of N370 S in NP of A/GD/01/06(H5N1)varied its antigenicity but didn't change the epitope size(SNEN367-370).

Objective To obtain purified ClpP produced by prokaryotic expression system,and to evaluate the protection effect elicited by ClpP in animal protection test.Methods The template DNA was isolated from the culture of TIGR4 Streptococcus pneumoniae.The complete ClpP open reading frame(ORF)was cloned into pET-32a expression vector by gene recombination technology in vitro.After prokaryotic expression,purification and sequence identification,the recombinant ClpP were innoculated into mice,and at the same time a group of mice were inoculated with the antibody to ClpP.We monitored the survival time of the innoculated mice after being challenged intraperitoneally with,TIGR4.Results We obtained high-expressed recombinant antigen protein which was then identified by Western blot,and we have got the recombinant antigen protein with a purity of more than 90%after being purified through Ni2+ affinity chromatography and processed by dialysis.The sunrvival time of mice immunized with ClpP or ClpP antibody were significantly langer than that of the mice received PBS.Conclusion The recombinant ClpP antigen protein can elicit protection to the invasive S.pneumoniae infection in mice,which might make ClpP as a candidate of S.pneumoniae vaccine.

Objective To detect the mecA gene and tst gene of toxic shock syndrome toxin 1 (TSST-1)of Staphylococcus aureus by using PCR and to learn the carrier condition of tst gene.Methods The mecA gene and tst gene of Staphylococcus aureus strains that isolated from clinical sources in our hospital during August 2006 to May 2007 were amplified in vitro using PCR,and to establish the rapid,specific,and sensitive method of detecting tst gene of methicillin resistant Staphylococcus aureus(MRSA).Results The mecA gene and tst gene were detected,and were made the gene sequencing successfully.Forty-one of 84 strains had mecA gene(48.81%),16 of 84 strains had tst gene(19.05%),10 of 84 strains had both of them,and the positive rate was 24.39%(10/41).Conclusion The proportion of tst gene positive strains of MRSA iS high in clinic,and it must be paid more attention.

Objective To construct the human IL-24 recombined adenovirus(Ad-IL24)and to observe the effect of Ad-IL24 on ex vivo culture of HL-60 cells.Methods pAdTrack-CMV-IL24 was constructed by PCR with pcDNA3.0-IL24 recombined plasmid as a template,enzyme digestion and ligation.The pAdTrack-CMV-IL24 lineared by Pme Ⅰ was co-transformed into BJ5183 with pAdEasy-1.The pAdEasy-1-pTrack-CMV-IL24 recombined adenovirus vector was lineared with Pac Ⅰ and then transfected in to QBI-293A cells.The Ad-IL24 was obtained and used to infect HL-60 ceils,and the effect on HL-60 cells was tested by LSCM,Mrrr,FCM,ELISA and immunohistochemistry staining.Results The pAdEasy-1-pTrack-CMV-IL24 vector was constructed and the Ad-IL24 was successfully obtained.The effect of inhibiting growth of HL-60 cells and inducing cell apoptosis by IL-24 was proved by LSCM,MTT and FCM.IL-24 down-regulated the expression of anti-apoptosis factor Bcl-2 and increased the secretion of IFN-γ,TNF-α of HL-60 cells.Conclusion Ad-IL24 can inhibit the growth of HL-60 cells and induce cell apoptosis through down-regulating expression of anti-apoptosis factor and increasing the secretion of IFN-γ,TNF-α of HL-60 cells.The human Ad-IL24 may provide a basic study for the future target therapy to tumors.