Objective To affirm the expression of Toll like receptor 4 (TLR4) on the surface membrane of platelet and to explore the immunomodulatory factors[(interlukine-8(IL-8),β-thromboglobulin(β-TG), soluble CD40 ligand(sCD40L)] released by platelets after platelets stimulated by TLR4 ligand.Methods TLR4 expressed on the platelet was detected by flow cytometry. Monoclonal anti-human FcγRⅡantibody(Ⅳ.3)-treated human platelets were cultured with LPS in the presence or absence of blocking monoclonal antibody to human TLR4. The release of IL-8, β-TG, sCD40L were measured by specific enzymelinked immunosorbent assay. Results Human platelets could express functional TLR4. The detection rate of TLR4 on platelets were decreased after LPS involvement(P<0.01). It was noted that sCD40L and β-TG were present in large concentration in the release of platelets stimulated by TLR4 ligand but the release of IL-8 was independent of platelet activation after TLR4 engagement. The concentration of sCD40L and β-TG had no statistical difference between 1-5 μg/ml LPS. The effects of LPS on the modulation of secretory factors were attenuated by preincubation of platelets with an anti-TLR4 monoclonal antibody. Conclusion The TLR4 on platelet could recognize and link LPS, induce the release of sCD40L, β-TG by platelet, but could not influence IL-8.

Objective To Study on CXCR1/CXCR2 antagonist G31P anti-inflammatory reaction mediated by neutrophils.Methods Detect whether G31P can block chemotaxis of neutrophils induced by human IL-8 and inhibit the release of IL-8 by epithelia of segmental bronchus;establish HEK293 cell line transfected by pcDNA3.0-CXCR1 ,2,4 and detect the chemotaxis of IL-8 for HEK293 ;establish the experi-mental model of pneumonia induced by the P.aeruginosa,take count of the nucleated cells in the bronchoal-veolar lavage fluid(BALF),analyze myeloperoxidase(MPO) of lung tissue and observe the histopathology changing of it.Results G31P can inhibit the chemotaxis for neutrophils and transfected HEK293 cell line,inhibit the A549 releasing of inflammatory mediators;the proportion of neutrophils declines in G31P treat-ment group,pathology examination appears clear discrepancy.Conclusion G31P can block the chemotaxis of chemotactic factor with ELR+ CXC to neutrophils,block the combination of chemotactic factor with its re-ceptor CXCR2,block the CXCR2 on the surface of alveolar epithelia and vascular endothelial cells.Accordingly,neutrophils recruiting to topoinflammation can be prevented.

Objective To investigate the changes of CD4~+ T cell subset in the role of immuno-pathogenesis of type 1 diabetes mellitus(T1DM). Methods Real-time PCR was used to evaluate the ex-pression levels of transcriptional factors (T-bet, GATA-3, Foxp3, ROR-γt), cytokines (IFN-γ, IL-4, IL-10, IL-17A) and negative regulatory factors (CTLA-4, GITR) mRNA from CD4~+ T cells. The proportions of Th1, Th2, Tr and Th17 cells in peripheral blood were detected by flow cytometric analysis. Plasma cyto-kine (IFN-γ, IL-4, TGF-β and IL-6) concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Results (1) The proportions of Th1 cells in peripheral blood from children with T1DM were siguificanfly increased than that of healthy controls, and proportions of Th2 were decreased (P < 0.01). There were no significant differences between diabetic patients and healthy controls regarding the proportions of Tr cells and Th17 cells(P >0.05). (2) Transcription levels of T-bet and IFN-γ mRNA were significantly up-regulated, while GATA3 and IL-4 were significantly down-regulated in children with T1DM. The mRNA expression levels of Tr negativity regulatory factors such as IL-10, CTLA-4 and GITR were lower in CD4~+ T cells from children with TIDM compared with the controls(P <0.01). There were no statistically differences to be observed in mRNA expression levels of ROR-γt and IL-17A genes between two groups(P > 0.05).(3) In comparison with controls, serum concentrations of IFN-γ or IL-4 were remarkable increased or de-creased respectively (P < 0. 01), while TGF-β and IL-6 did not change in children with T1DM (P > 0.05). Conclusion The Th1/Th2 imbalance might be play an important role in immunopathogenesis of T1DM. Functional deficiency of Tr cell might further exacerbate Th1/Th2 imbalance and lead to disturbance of cellu-lar immune response.

Objective To study the effect of p50 on IKKα at IL-1β promoter in LPS tolerant cells and to reveal the mechanism of the inhibition of IL-1β mRNA by pS0. Methods THP-1 human promono-cyte model of endotoxin tolerance that simulates the sepsis leukocyte phenotype was used. Chromatin immu-noprecipitation assay(CHIP) and real-time PCR were applied to quantify the binding of p50 and IKKα to IL-1βpromoter. IL-1β mRNA transcription was studied after knocking-down of p50 and/or IKKα. Results With LPS stimulation, p50 binding did not reduce but somewhat increased at IL-1β promoter in tolerant THP-1 cells. Knocking-down of p50 increased the transcription of IL-1β mRNA, which revealed the inhibi-tory effect of p50 in tolerant cells. In contrast, the accumulation of IKKα to IL-1β promoter decreased with LPS stimulation in tolerant cells; However, IKKα binding increased after p50 gene knock-down. In the meantime, IL-1β mRNA transcription increased; At last, IL-1β mRNA decreased again after double-knoc-king down of p50 and IKKα. Conclusion p50 is an inhibitory protein at IL-1β promoter in tolerant THP-1 cells. The unresponsiveness of IL-1β mRNA transcription to LPS at least partly results from the inhibition of IKKα binding to IL-1β promoter by p50.

Objective To clarify the serological characteristics and predictive value of HIV antibody indeterminant and to evaluate the efficiency of 3 assays to discriminate HIV antibody indeterminant.Methods Three hundred and ninety-four HIV antibody indeterminant serum samples were collected and the Western blot pattern were analyzed.Ninety-seven HIV antibody indeterminant individuals were followed up,and the development of HIV antibody were observed.The initial serum samples of 67 followed individuals were tested by viral load,line immunoblot assay and ELISA for HIV-1 p24,with the golden standard of follow up,the efficiency of 3 kinds of assay to discriminate HIV antibody indeterminant were evaluated.Results There were 38 patterns among 394 HIV antibody indeterminant,the proportions of env,pol and gag indeterminant were 37.54%,4.04%and 58.37% respectively.Five HIV antibody indeterminant cases were converted to HIV antibody positive among 97 followed individuals,they were all env indeterminant and HIV antibody developed rapidly.HIV viral load was an ideal assay to discriminate HIV antibody indeterminant with best sensitivity.Conclusion The indeterminant of gag were most common,but were unspecific reaction.Env indeterminant were with the greatest predictive value of HIV infection,especially the gp160p24 and gp160.Viral load assay can be applied to discriminate HIV antibody indeterminant.

Objective To elucidate the molecular basis on the differences of infectivity in infected cells between Sindbis virus(SINV:YN87448 virus)and Sindbis like virus(SINLV:XJ-160 virus).Methods Compare the E(glycoprotein)gene sequence and secondary structure of YN87448 virus and XJ-160 virus by bioinformatics analysis.Analyze the contribution of E gene to the biological differences between SINV and SINLV by constructing recombinant virus.Results By bioinformatics analysis,YN87448 virus and XJ-160 virus have the same genomic structure,which has 11 717 nt and 11 626 nt respectively.There are 82 amino acid differences between E gene of these two viruses,and showed scattered distribution.The main peak is basically the same for the hydrophobic of the E gene protein,but in some region existing small differences.The recombinant virus which exchanged the E gene of XJ-160 virus with YN87448 virus totally showed the biological character of YN87448 virus,either in the showing time of CPE,plaque forming time and plaque diameter,or in expression of functional proteins.Conclusion E gene plays a major role in the differences of infectivity in infected cells between SINV and SINLV,this result provide the molecular biological evidences for elucidating the biological differences between SINV and SINLV.

Objective To construct S6K1 shRNA gene recombinant adenovirus(S6K1 Ax)and evaluate its gene silencing effects on mouse cell lines and C57 BL/6J mice level.Methods Three S6K1 shRNA gene sequences were designed and spliced from pcPUR plasmid,pcDNA3.1 plasmid to cosmid plasmid and transfected into adenovirus.S6K1 Ax which has best gene silencing effect was selected and proliferated in 293 cell.Silencing effect of S6K1Ax was checked on mice AML12,C2C12,3T3-L1 cell lines.C57BL/6J liver was obtained after S6K1 Ax was injected into mice tail vein six days later.S6K1 was evaluated by qRT-PCR and Western blot.Alanine transferanse(ALT)was examined before and after S6K1 Ax injected.Results S6K1Ax can silence S6K1 expression of mouse AML12,C2C12 and 3T3-L1 cell lines and liver of C57 BL/6J mice on Western blot.S6K1 mRNA expression of C57BL/6J liver were control group 1.39±0.21 vs S6K1Ax group 0.63±0.09,t=6.132.P<0.01.ALT of mice hepatic function did not change after S6K1Ax injected:before(15.15±4.43)U/L,after(17.32±4.22)U/L,t=1.451,P>0.05.Conclusion Construction of shS6K1 Ax can knockdown S6K1 gene on mice cell lines and C57BL/6J mice liver,it provides a good tool to study the function of S6K1.

Objective To investigate the sequences of the 3' end of genomes from enterovirus 71 isolated from pediatric patients with different symptoms in Beijing during 2008-2009.Methods Clinical specimens were collected from pediatric patients suffering from hand,foot and mouth disease(HFMD)and/or with neurological complications who visited the affiliated Children's Hospital during the epidemic seasons of 2008 and 2009 in Beijing.The samples were inoculated into the Vero cell line,and the virus isolates were further identified by nested reverse transcription-nested PCR(RT-nPCR)assay using universal enterovirus primers,type specific primers for EV71 and CA16.The 3' end of genomes(including 3D and 3' UTR regions)from 10 EV71 strains derived from various clinical presentations were amplified and sequenced.Results Analysis of the nucleotide sequences of the amplified fragments showed that the 3' end of genomes of 10 EV71 isolates include the 3D region of 1386 nucleotides(nt)encoding the 3D polymerase(3Dpol,462 amino acids),the terminator codon TGA and 3' UTR of 81 nt.Nucleotide sequence identities among 3D regions from these 10 EV71 isolates were in the range of 95.8%-99.6%,while the nucleotide sequence identities for 3' UTR were 96.3%-100%.The majority of nucleotide changes were located at the third codon positions which caused silent mutations,thus the amino acid sequence changes of 3Dpol among those 10 EV71isolates were scanty.The residues 140 and 263 which were R and 1 were substituted by K and V,respectively in 3 of 4 neurovirulent strains,whereas only 1 of 6 strains from mild cases had these 2 amino acid changes.The sequences of the 3D and 3' UTR regions of 10 EV71 isolates were compared to the representative strains of known genotypes from the GenBank.The nucleotide and amino acid sequences of 10 EV71 isolates in the 3D region exhibited highest homology to the subgenotype C4 of EV71(92.7%-94.2%and 96.8%-97.6%.respectively).However,3' UTR of 10 EV71 isolates shared the highest nucleotide identity with CA16/G10(88.9%-91.4%).The phylogenetic analysis based on the 3D regions demonstrated that 10 EV71 isolates had the closest genetic relationship with the representative isolate of CA sub-genotype of EV71 and shared more homology with CA16/G10 than other known genotypes of EV71.Conclusion Genetic analysis of the 3' end of genomes from 10 EV71 strains indicated that the 3' end of genome may play a role in the evolution of EV71.

Objective To investigate the prevalence of qnr and aac(6')-Ⅰ b-cr genes in water-borne environmental bacteria and clinical isolates of Citrobacter freundii, and the subtypes of qnr gene. Methods Environmental bacteria were isolated from surface water samples obtained from 10 distinct loca-tions in Hangzhou city, and clinical isolates of C. Freundii were isolated from several hospitals of 4 cities in China. Minimum inhibitory concentrations (MICa) of ciprofloxacin, levofloxacin and nalidixic acid were de-termined by agar dilution method, qnrA, qnrB, qnrS and aac(6')-Ⅰ b-cr genes were screened by PCR, and the genotypes were analyzed by DNA sequencing. Results Seventy-eight gram negative bacilli (including 33 Enterobacteriaceae, 21 Aeromonas spp., 10 Acinetobacter spp., 10 Pseudomonas app., 2 Alcaligenes app. , and 2 Plesiomonas app.) were isolated from water samples. Among these isolates, 8 of 10 C. Freundii were positive for qnrB gene. qnrS1 and aac (6')-Ⅰ b-cr were detected in two distinct Escherichia coil, and qnrS2 was detected in a Aeromonas punctata qnr and aac(6')-Ⅰ b-cr genes were present in 75 (72.8%) and 12 (11.6%) of 103 clinical isolates of C. Freundii, respectively. Three (2.9%) C. Freundii isolates were positive for qnrA1 gene, 65 (63.1%) qnrB, 1 (1.0%) qnrS2, 5 (4.8%) were positive for both qnrA1 and qnrB, and 1 was positive for both qnrS1 and qnrB. Within the subtypes of qnrB gene, qnrB9 predominated, followed by qnrB8 and qnrB6. Conclusion It was the first isolate of Aeromonas spp. Harboring qnrS2 gene outside Europe. The prevalence of qnrB in water-borne environmental and clinical isolates of C.freundii was particularly high and qnrB9, qnrB8 and qnrB6 were the most common subtypes, aac(6')-Ⅰ b-cr gene widely spread in clinical isolates of C. Freundii.

Objective To analyze the application of recombinant VCA-BALF4(S) and VCA-BFRF3 proteins in serological diagnosis of nasopharyngeal carcinoma (NPC). Methods DNA extracted from the B95-8 cells was used as the templates in a polymerase chain reaction(PCR). VCA-BALF4 1008 bp (S) (aa 287-623) and VCA-BFRF3 531 bp(177 as) were generated and inserted into pPICZaA vector.The recombinant plasmids were transformed into GS115 yeast by electroporation. The yeast transformants in-duced by methanol expressed recombinant proteins. The recombinant proteins synthesized were coated to mi-croplate for detection of EBV-IgA antibody in NPC patients by ELISA. Results We have successfully se-cretly expressed the recombinant VCA-BALF4(S) and VCA-BFRF3 protein in the Pichia pastoris. The mo-lecular weight of products were approximately 37×10~3 and 18×10~3, respectively. The recombinant proteins VCA-BALF4(S) and VCA-BFRF3 in the culture supematant showed good immunoreactivity with IgA anti-bodies to EBV by Western blot. A novel ELISA was established using Pichia pastoris-expressed VCA-BALF4 (S) and VCA-BFRF3 proteins. Serum samples were collected from patients with NPC and healthy controls and using this ELISA tested. The sensitivity of VCA-BALF4(S) and VCA-BFRF3 tests in the NPC sera were 88.7% and 71.0%, whereas the specificity of normal individuals are 96.4% and 91.8% separately. Con-clusion The recombinant proteins BALF4 (S) and BFRF3 were highly secretly expressed in Pichia pastoris,the diagnostic value of two recombinant proteins in screening for NPC patients were primary evaluated and the valuable pPICZa-BALF4(S) yeast strain was obtained.