Objective To examine the proliferative effect of synthetic cyclic human cartilage glycoprotein-39 (HCgp39) on T cell of collagen-induced arthritis (CIA) rat,and to explore the role of HCgp39 in rheumatoid arthritis (RA).Methods We established the rat model of the collagen-induced arthritis (CIA).The T lymphocytes were isolated and incubated with HCgp39.Proliferation of T cells was determined by cell counting kit-8.Results Two weeks after the first immunization,T cell response to HCgp39 was more significant in CIA groups than in controls( P＜0.01 ),and the response was associated with disease course ( r =0.732,P＜0.01 ) and anti- HCgp39 antibody ( r =0.460,P＜0.01 ).A strong correlation between T cell proliferation and pannus ( r =-0.516,P＜0.01 ),synovium score ( r =-0.346,P＜0.01 ) was also observed.Besides,the levels of anti- HCgp39 antibody and comp in each CIA group were significantly higher than in controls( P＜0.01 ),and the anti- HCgp39 antibody strongly correlated with disease course( r=0.346,P＜0.01 ) and comp( r =0.235,P＜0.01 ).Conclusion The proliferative response of T cell to HCgp39 was found in the early stage of CIA rat,and the HCgp39 peptide antibody was detected in serum,suggesting that the HCgp39 antigen plays an important role in the pathogenesis of early RA.

Objective To explore the effect of HTLV-1 (human T-cell leukemia virus type 1 ) Tax protein on the DcR3 gene expression in T cells.Methods The construction of DcR3 (-1010 bp to +114 bp) luciferase reporter gene; MT2,TaxP,and Jurkat E6-1 cells were transfected with DcR3 luciferase reporter gene (pGL3-DcR3-1uc) using liposomes according to the manufacturer's instructions.For the control group,pGL3-basic replaced it respectively.At 48 h after incubation,luciferase activity was measured with a luciferase assay system; Jurkat cells were transfected with pCMV-Tax-Bam using liposomes,and total RNA was extracted from the cells at 48 h after incubation.Reverse transcription was performed using standard protocols.Then real time PCR with primers DcR3 and 3-actin was conducted;The expression of DcR3 protein was detected by flow cytometry in MT2,TaxP,and Jurkat cells.Results The construction of DcR3 luciferase reporter gene is identified correctly; The detection of luciferase activity showed that the luciferase activity of experimental group in MT2 cells was increased by (32.07±12.43)-fold,of which in TaxP cells was increased by ( 13.27±4.04)-fold,and the luciferase activity of experimental group in Jurkat cells was increased by ( 1.26±0.49 ) -fold.And there is statistic significance about the relative luciferase activity of experimental group in MT2 cells and TaxP cells compared to the relative lueiferase activity of experimental group in Jurkat cells respectively ( P＜0.01 ) ; The result of real-time PCR showed that the level of DcR3 mRNA in the experimental group was higher compared with the control group(P＜0.05) ; The result of FCM shows the expression of DcR3 protein in MT2,TaxP cells is higher than that in Jurkat cells( P＜0.05 ).Conclusion Tax protein can promote the expression of DcR3 gene in T cells.

Objective To investigate the invasion,internalization and the organelles damage of the cultured dendritic cells ( DC2.4 strain) during Vibrio vulnificus (Vv) infection.Methods The study model was the cultured DCs infected by Vv 1.1758 strain.Electron microscopy was used to observe the localization of bacteria in different time point of infection,cell morphology and the process of organelles changes.The cytoskeleton structure including the microfilaments and the microtubules rearrangement was examined by the fluorescence microscope.Results The Vv were pinocytosed into the DC cells through double-sides,and localized at 1-2 μm of the inner side membrane.It cost 1.27,1.87,3.43 hours reaching the infection ratio of 25％,50％,75％,respectively.Using electron microscopy,the DCs had been observed the phagosome formation within 1h,chromatin activation within 2 h,chromatin aggregation 4 h,and the significant cytoskeleton structure disruption within 6 h.Endoplasmic reticulum,mitochondria and lysosomes became swollen.In DCs,the protruding filaments gradually reduced,and their shape changed from the point-like to the linearlike aggregation at the inner side of the plasma membrane,extended microtubules disappeared,the microtubules at the outside nuclear membrane striking rearranged.Conclusion After DC was infected by Vv,the bacteria were pinocytosed into the inner side of DC membrane,and the microfilaments were observed to move from the cytoplasm to cell membrane.In addition,the microtubules moved from the synapse and the cell membrane to the nuclear membrane.The high lethality of Vv could provoke to the DCs cytoskeleton rearrangements.

Objective To evaluate the quality of four domestic and three imported fourth-generation HIV diagnostic reagents.Methods The specificity and sensitivity of these assays were analyzed when testing HIV negative samples and HIV-1 RNA positive samples.The relative seroconversion sensitivity index was analyzed when testing BBI seroconversion panels.Results The sensitivity of seven 4th-generation assays were 100％ (95％ CI:99.86％-100％ ),and one sample at the window period of HIV-1 infection were detected as positive.Of the seven assays,one imported assay exhibited the relative largeδ + value (1.0892),and the small δ+ value were found on the remaining six assays (0.0836-0.3003 ).For the samples negative for HIV antibody,varying degrees of false positives were observed on the seven assays ( specificity:97.80％ -99.60％,δ- value:-1.3803 to -0.4778).When testing the BBI seroconversion panels,the relative seroconversion sensitivity index of domestic assays were -0.500-0,however,which of imported assays were -0.600 and -0.700.Conclusion The seven reagents exhibited high sensitivity and specificity.The 4th generation HIV assays can be used as blood screening reagents to find the samples at window period of HIV-1 infection,thus indicating the certain meaning in reducing the transmission risk of HIV-1 for fourth-generation HIV diagnostic reagents.However,the better efficiency to detect HIV-1 early infection was observed on the imported assays than on the domestic assays.

Objective To discuss the prevalence of non-EV71,non-CA16 virus strains of hand,foot and mouth disease(HFMD) in Guangdong province between 2008 and 2009,and analyze the genetic evolution of these non-EV71,non-CA16 virus strains.Methods Isolated viruses from stool samples collected from outpatient and in-patient cases of HFMD between 2008 and 2009 by human rhabdomyosarcoma(RD) cell and HEp-2 cell,cultures that exhibited a characteristic enterovirus cytopathic effect were evaluated by RT-PCR.Those strains which identified non-EV71,non-CA16 were analyzed by VP1 sequencing and then were identified by BLAST program.A phylogenetic tree was constructed using the Neighor-Joinning method in the MEGA 4.0 software.Results Twenty-two virus strains of non-EV71,non-CA16 were obtained,and nine of the twenty-two virus strains in 2008 were classified into CA2,CA4,and CB3 by BLAST; thirteen of the twenty-two virus strains in 2009 were classified into EV80,Echo13,Echo30,CBS,Echo24,CA10,CA6,and poliovirus 1 by BLAST.The honology of all strains was low,and all the strains belonged to CA,CB,Echoviruses,Enterovirus and poliovirus subgroup.Conclusion Except for EV71 and CA16 was a major causative agent in prevail of HFMD in Guangdong province between 2008 and 2009,there also existed other subgroup Enterovirus.The other twenty-two strains respectively belonged to CA,CB,Echoviruses,Enterovirus and poliovirus subgroup,and none of those strains was predominant.Muti-species Enterovirus occurred concomitantly.

Objective To identify the efficacy small interfering RNA against Pseudomonas aeruginosa expressing MexA-MexB-OprM efflux pumps.Methods Four siRNA ( siRNA1,siRNA2,siRNA3 and siRNA4) against mexB gene were designed and prepared by electricity transference in vitro.MICs of antibiotic combined with efflux pump inhibitors against multiple resistant strain PAO1 and PAO3 were determined by E-test method.The mRNA expression levels of efflux pump gene (mexB) were quantified by real time fluorescent quantitative PCR.Results siRNA expression vectors were constructed success by enzyme cut method.48 after PAO1 and multiple drug resistant PAO3 transfected with siRNA4,the sensibilities to antibiotic were enhanced.48 after PAO1 and multiple drug resistant PAO3 transfected with siRNAl,siRNA2 and siRNA3,the sensibilities to antibiotic didn't change obviously.48 after PAO1 and multiple drug resistant PAO3 ttransfected with siRNA4,the expression level of mexB was decreased obviously (P ＜ 0.05 ).48 after PAO1 and multiple drug resistant PAO3 transfected with siRNA1,siRNA2 and siRNA3,the expression level of mexB didn't change obviously.Conclusion siRNA against Pseudomonas aeruginosa expressing MexA-MexB-OprM efflux pumps enhanced the sensibility to antibiotic and inhibited the expression of mexB gene.Our results demonstrate the using RNAi may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.

Objective To analyze the antibiotic resistance of the carbapenem no-susceptibility Enterobacteriaceae isolated from pediatric patients and the resistant genes of carbapenemase-producing.Methods In all,46 carbapenem no-susceptibility Enterobacteriaceae strains were isolated from patients at Beijing Children's Hospital between January 2008 and December 2010.Agar dilution method recommended by the Clinical and Laboratory Standards Institute was used to examine the minimum inhibitory concentrations (MICs) of 14 antimicrobial agents.Phenotypic testing for carbapenemase-producing was conducted using Hodge test and double-disk synergy test.PCR was used to detect the expression of the carbapenemase-related genes KPC,GES,IMI/NMC-A,SME,IMP,VIM,GIM,SPM,SIM and OXA.WHONET5.6 was used to perform resistance analysis.Results Among 46 carbapenem no-susceptibility Enterobacteriaceae strains,26 (56.5％) were Klebsiella pneumoniae strains,13(28.3％ ) were Enterobacter cloacae and 7( 15.2％ ) were Escherichia coli.The rates of imipenem and meropenem no-susceptibility Klebsiella pneumoniae were 69.2％ and 80.8％,Enterobacter cloacae were 76.9％ and 100％ and Escherichia coli were 85.7％ and 100％,respectively.40(87.0％ ) strains were positive of Hodge test.41 (89.1％) strains were positive of doubledisk synergy test.38 (82.6％) were positive for the IMP genotype.The carbapenemase-related genes were not found in other 8 strains.Conclusion The prevalence of carbapenem no-susceptibility Enterobacteriaceae strains in Klebsiella pneumoniae isolates is relatively high in children.Resistance to imipenem was lower than that to meropenem from Klebsiella pneumoniae,Enterobacter cloacae and Escherichia coli strains.Many carbapenem no-susceptibility Enterobacteriaceae isolated from pediatric patients carry the blaIMP gene.No the KPC gene was found.

Objective To construct a qseC-deleted mutant strain of E.coli by Red recombination and to study the effect of qseC gene on biofilm formation in the mutants.Methods The chloramphenicolresistant gene flanked by homologues of target genes was amplified by PCR and electro-transformed into E.coli MC1000.When induced by L-arabinose,the plasmid pKD46 could express three recombinant proteins of λ-prophage,which led to the replacement of target gene(qseC) with chloramphenicol-resistant gene.Then the chloramphenicol-resistant gene was eliminated by FLP-promoted recombination events.The biofilm formation of wild-type and mutant strain was detected by crystal violet staining.Results The qseC-deleted mutant of E.coli was confirmed by various PCR and DNA sequencing.Gene qseC was completely deleted.There was no significant difference in growth ability between the qseC mutant strain and the wild-type strain MC1000.The biofilm formation of wild-type and mutant strain was quantified by crystal violet staining.The absorbance determined with a plate reader at 570 nm was 1.00±0.15 and 0.47±0.10 respectively.Conclusion The qseC-deleted mutant of E.coli was constructed successfully.And the qseC gene plays an important role in regulation of biofilm formation in E.coli.

Objective To explore the effect of hepatitis B virus(HBV) on the expression of high sensitive C-reaction protein(hs-CRP) and its clinical implication.Methods mRNA expression of hs-CRP in HepG2 and HepG2.2.15 cells was measured by RT-PCR,serum hs-CRP levels in patients with HBV infection and in healthy individuals were measured by biochemical analyzer Olympus5400,the expression of hs-CRP difference among patients with chronic hepatitis B,liver cirrhosis and hepatocellular carcinoma were analyzed.Results Expression of hs-CRP mRNA was higher in HepG2.2.15 cells than in HepG2 cells,serum hs-CRP levels was much higher in HBV patients as compared to healthy individuals ( P＜0.05 ),hs-CRP was detected at higher levels in patients with liver cirrhosis or hepatocellular carcinoma than those with chronic hepatitis B.Conclusion HBV can upregulated the expression of hs-CRP,which is associated with the disease progression.

ObjectiveTo investigate the effects of nicotine on the expression of Th1/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured CD4+ T of rat sensitized by ovalbumin.Methods Two weeks after immunization by ovalbumin,splenic CD4+ T cells of Wistar rat were purified using CD4+T cell enrichment kit.Purified CD4+ T cells of rat were cultured and divided into 4 groups:a control group,1 μg/ml nicotine stimulated group,10 μg/ml nicotine stimulated group,100 μg/ml nicotine stimulated group.These cells,in their groups,were stimulated with or without nicotine and were all challenged simultaneously with OVA.Supernatants and cell pellets were harvested after being stimulated for 24 h.The concentration of IFN-γ and IL-4 in supernatants were measured by enzyme-linked immunosorbent assay (ELISA).Real-time PCR was used to determine the mRNA expression of T-bet and GATA-3 in CD4+T cells.Results ( 1 ) IFN-γproduction was significantly decreased in all nicotine treated groups [ ( 113.78±6.06) ng/L,(70.31±7.26) ng/L,(20.00±2.14) ng/L] compared with the control group[ (142.30± 5.89) ng/L],and the level of IL-4 was significantly increased in all nicotine treated groups [ (69.49±3.91) ng/L,(93.63±4.56) ng/L,(50.97±3.07) ng/L] compared with the control group[ (36.91±3.24) ng/L].(2) Expression of T-bet mRNA in all nicotine treated groups(0.73±0.03,0.57±0.04,0.31 ±0.00) was lower than that in the control group(0.98±0.09),but expression of GATA-3 mRNA in all nicotine treated groups (4.31±0.26,5.16±0.23,1.56±0.14) was significantly higher than that in the control group(1.00±0.07).Conclusion Nicotine may play a key role in the development of Th2-type allergic inflammation in asthma by promoting over-expression of GATA-3 mRNA and downregulating the expression of T-bet mRNA.