in mice with coxsackievirus B3 (CVB3)-induced viral myocarditis through a comparative study with Carve-dilol.Methods 150 BALB/c male mice were divided into four groups including control group (n=30), myocarditis group (n=40), Ivabradine treatment group (n=40) and Carvedilol treatment group (n=40). Viral myocarditis was induced by intraperitoneal injection of CVB 3 in experimental mice , while mice in the control group were injected with PBS accordingly .The mice in four groups were respectively administered with PBS, PBS, Ivabradine and Carvedilol after 24 h of infection for 14 consecutive days .Heart specimens were collected from 8 mice of each group on days 4, 7 and 14 after measuring their heart rates .The patho-logical changes in heart tissues were observed through hematoxylin e-osin staining .Semi-quantitative RT-PCR and ELISA were performed to detect the expressions of MCP -1, IL-6 and TNF-αat mRNA and protein lev-els.CVB3 RNA was quantified by semi-quantitative RT -PCR as well .Results Compared with myocarditis group, the histopathological damages in myocardium were significantly alleviated in both Ivabradine group and Carvedilol group on days 7 and 14.The expressions of MCP-1,IL-6 and TNF -αat mRNA level were up-regulated in mice treated with Ivabradine and Carvedilol as compared with those in control group .Compared with myocarditis group , the expressions of TNF-αon day4 and IL-6 on day 7 at mRNA level were reduced in Ivabradine and Carvedilol treatment groups .MCP-1 expression at mRNA level was only down-regulated in Iv-abradine group on day 7.Concluison Ivabradine treatment could alleviate histopathological damages in my -ocardium of mice with CVB3-induced viral myocarditis , which was similar to the effects of carvedilol treat-ment.The treatment effects might be associated with the down-regulation of expressions of IL-6, TNF-αand MCP-1 at mRNA and protein levels .

Objective To investigate the mechanism of acquisition of HLA-G1 antigen by NK cells.Methods K562 cells stably expressing HLA-G1 antigen (K562-G1) were constructed.K562-G1 cells, K562 cells and shed HLA-G1 were respectively co-cultured with NK-92MI cells to observe the acquisi-tion of HLA-G by NK cells.To further investigate the mechanism , NK-92MI cells with blockage HLA-G re-ceptors were further co-cultured with K562-G1 cells and HLA-G1 proteins expressing on K 562-G1 cells were blocked and then co-cultured with NK-92MI cells. Acquisition of HLA-G 1 by NK-92MI cells was analyzed by flow cytometry and fluorescence microscopy .The effects of HLA-G1 expression on the cytotoxicity of NK-92MI cell were evaluated by flow cytometry analysis based on CD 107a labeling.R esults NK-92MI cells could quickly acquire HLA-G1 from K562-G1 cells in co-culture experiments .Blockade of HLA-G1 or its re-ceptors KIR2DL4 and ILT2 with specific mAbs did not affect the acquisition of HLA-G1 by NK-92MI cells. Moreover, HLA-G1 could significantly inhibit the cytotoxicity of NK cell ( P<0.01).Conclu sion NK-92MI cells acquire HLA-G1 from K562-G1 cells via trogocytosis , which is not associated with affinity be-tween receptor and ligand , extracellular domain of HLA-G1 or passive adhesion .

Objective To investigate the regulatory effects of miR-146a on inflammation factors production in C ryptococcus neoformans treated THP-1 cells.Methods Cryptcoo ccus neoformans strains were heat killed .Fluorescence quantitative RTP-CR and ELISA were used to detect the levels of IL -1βand TNF-αin THP-1 cells treated with heat-killed Cryptococcus neoformans.In addition, the production of IL-1βand TNF-αwere analyzed before and after Dectin-1or TLR 4 blocked .THP-1 cell lines that were respectively transfected with miR-146 a mimics and inhibitors were constructed and the production of IL-1βand TNF-αin these cell lines were analyzed after interfered with Cryptococcus neoformans.Results With the interference of heat-killed Cryptococcus neoformans, the expression of miR-146a was up-regulated in THP-1 cells, but was down-regulated after Dectin-1 or TLR4 blocked.The expressions of IL-1βand TNF-αinduced by heat-killed Cryptococcus neoformans were enhanced in miR-146a mimics transfected THP-1 cells, but was inhibi-ted in inhibitors transfected THP-1 cells.Conclusion Heat-killed Cryptococcus neoformans could up-regu-late miR-146a expression in THP-1 cells via Dectin-1 and TLR.miR-146a could negatively regulate the ex-pressions of IL-1βand TNF-αinduced by Cryptococcus neoformans.

Objective To investigate the function of phosphatidylinositol phospholipase C encoded by LB361 gene of L.interrogans ( L-PI-PLC) and its mechanism in inducing macrophage apoptosis .Meth-ods The PI-PLC domains in the sequence of LB 361 gene of L.interrogans serovar Lai strain were analyzed by bioinformatics method .Prokaryotic expression system was established to express the recombinant L -PI-PLC ( rL-PI -PLC).The enzymatic activity of rL-PI-PLC in hydrolyzing phosphatidylinositol -4,5-bisphos -phate (PIP2) substrate into inositol-1,4,5-trisphosphate (IP3) was determined by IP3 fluorescence polariza-tion assay.LB361gene expressions at mRNA and protein levels as well as the secretion of LB 361gene prod -ucts were detected by real-time fluorescent quantitative RT -PCR and Western blot assay after infection of hu-man THP-1 macrophages with L.interrogans serovar Lai strain.A LB361 gene-transfected THP -1cell line was generated for evaluation of the mechanism of LB 361 gene products in elevating intracellular free Ca 2+( [Ca 2+] i) concentration and inducing the apoptosis of transfected THP -1 cells with the use of laser confocal microscopy and flow cytometry.Re sul ts The rL-PI-PLC hydrolyzed PIP2 into IP3 with a Km of 199 μmol/L and a Kcat of 8.566×10-5 S-1 .The expressions of LB361gene at mRNA and protein levels were both signifi -cantly up-regulated after infection of THP-1 cells with L.interrogans serovar Lai strain .Moreover , the exter-nal secretion of L-PI-PLC was also found during infection .The concentrations of IP 3 and [ Ca2+] i in the LB361 gene-transfected THP-1 cells were significantly increased compared to those in the non-transfected THP-1 cells, resulting in a high [Ca2+]i-dependent apoptosis of partial THP-1 cells.Conclusion PI-PLC is encoded by the LB361 gene of L.interrogans, which could induce the apoptosis of macrophages through el-evating [ Ca2+] i concentration during infection of microphages with L.interrogans.

Objective To establish a standardized quantitative enzyme-linked immunosorbent as-say ( ELISA) recommended by WHO for the detection of human IgG antibodies specific for Streptococcus pneumoniae capsular polysaccharides ( Pn PS ELISA ) .Methods According to the WHO recommended standard Pn PS ELISA protocol , capsular polysaccharide concentrations of 13 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) of Streptococcus pneumonia for coating were optimized;the ELISA plates and AP conjugated goat anti-human IgG antibody for the detection were experimentally selected .Using the established assay parameters assured by testing quality control standards ( QCs) provided by WHO refer-ence laboratory , a panel of 16 LIBP ( Lanzhou Institute of Biological Products Co .Ltd.) QCs were measured for comparison analysis between WHO reference laboratory and LIBP laboratory .In the meantime , the range of IgG concentrations of an internal QC panel 907 for 13 serotypes of pneumococcal capsular polysaccharide were established for routine QC work in LIBP laboratory , and inter-assay precision within LIBP laboratory was assessed as well .Results The standardized Pn PS ELISA assay established in LIBP laboratory met the criteria required by WHO .There was a high correlation between the data collected by WHO reference labora -tory and LIBP laboratory (slope=0.94, coefficient of correlation r=0.97, P<0.05).Eighty one percent of IgG concentrations measured by LIBP laboratory were within the range of ±40%of those measured by WHO reference laboratory , which met the criteria of 75%data falling within the ambit of ±40%of assigned values commonly used for comparison .The ranges of IgG concentration in LIBP QC 907 for 13 serotypes had been established.The inter-assay precision in LIBP laboratory was high with coefficiency of variation ( CV) less than 30%.Conclusion LIBP laboratory has successfully established the standardized ELISA recommended by WHO for quantitative detection of human IgG antibodies against pneumococcal capsular polysaccharides (Pn PS ELISA).The range of IgG concentrations in LIBP QC 907 for 13 serotypes of pneumococcal capsular polysaccharide are also established for routine quality-control practice .

Objective To study the possibility of using heterogeneous antiserum in single radial immunodiffusion (SRID) for quantitative detection of HA in H7N9 influenza vaccine product when H7N9-specific antiserum is not available in order to establish a testing method for the detection of H 7N9 antigen in any urgent situation.Methods Antisera specific for H7N1, H7N2, H7N3 and H7N7 were obtained from NIBSC and used for SRID assay .Amino acid sequences of hemagglutinins were comparatively analyzed be-tween H7N9 virus and other viruses used to prepare heterogeneous antiserum .The titers of antisera against H7N9 and their homogenous antigens were detected by double immunodiffusion method .Based on the results of homology analysis and cross-reaction, a suitable antiserum was selected and its applicability was further validated by the SRID assay using H7N9 antigen.Results Influenza A virus subtype H7N3 that used for preparation of 07/278 antiserum showed the highest HA homology with H7N9 (97.14%).The titer of 07/278 antiserum against H7N9 antigen was 1 ∶8 as detected by double immunodiffusion assay .The H7N9 anti-gen and the 07/278 antiserum could form a clear precipitation line in SRID assay .The detection of H7N9 antigen in the range of 10 to 40μg/ml showed a good linearity in the standard curve .Conclusion The 07/278 antiserum from NIBSC can be used as an alternative reagent for the quantitative detection of hemaggluti -nin in H7N9 influenza virus vaccine .

Objective To investigate the role of recombinant Vibrio vulnificus cytolysin (rVvhA) in inducing THP-1 cells damage and study the pathway of associated calcium influx .Methods Inverted mi-croscope, CCK-8 cell proliferation kit, Fluo3/AM staining and caspase activity detection were performed to analyze the damage of THP-1 cells induced by rVvhA and the pathway of calcium influx .Results rVvhA had cytotoxic effects on THP-1 cells in a dose-dependent manner .The concentrations of extracellular K +and LDH were respectively up-regulated after 1 h and 6 h of 12 μg/ml rVvhA intervention .Verapamil , Mibe-fradil and SKF-96365 could not prevent the influx of free Ca 2+induced by rVvhA .The activities of caspase-3 and caspase-9 were singanificantly enhanced by rVvhA in a time-dependant manner .Conclusion rVvhA can induce THP-1 cells damage through triggering extracellular calcium influx via porous channel on cell membrane.Moreover, rVvhA might induce THP-1 cell apoptosis through activating caspase-9/3-dependent pathway .

Objective To investigate the role of regulatory B cells ( Breg ) in the immunological pathogenesis of Kawasaki disease ( KD) .Methods Twenty-eight children with acute KD and twenty-eight age-matched healthy subjects were enrolled in this study .Blood samples were collected before and after the treatment of intravenous immunoglobulin (IVIG).The proportions of CD19+CD24hiCD38hi Breg and CD4+CD25+Foxp3+regulatory T cells (Treg), and the expressions of co-stimulatory molecules (CD80, CD86 and PD-L1) were analyzed by flow cytometry .The concentration of IL-10 protein was measured by enzyme-linked immunosorbent assay .Real-time PCR was performed to evaluate the expressions of Foxp 3, CTLA4 and GITR in CD4+T cells and IL-10 in CD19+B cells at mRNA level.Results (1) The proportions of CD19+CD24hi CD38hi Breg in peripheral blood and the expressions of IL-10 at mRNA level in CD19+B cells from patients with KD were lower than those from healthy controls (P<0.05), but significantly increased after treated with IVIG (P<0.05).Upon stimulation of lipopolysaccharide for 48 hours, the concentrations of IL-10 protein in culture supernatant of B cells from patients with KD were still lower than those from healthy controls [(31.06±8.26) pg/ml vs.(46.32±13.91) pg/ml, P<0.05].(2) Compared with healthy controls , the expressions of CD80, CD86 and PD-L1 were remarkably down-regulated in patients with acute KD ( P<0.05).With the treatment of IVIG, the expressions of CD80 and CD86 were significantly up-regulated though lower than those of the control group (P<0.05).There was no significant difference in the expression of PD-L1 before and after treatment (P>0.05).(3) The proportions of CD4+CD25+Foxp3+Treg and the expressions of Foxp3, CTLA-4 and GITR at mRNA level were significantly decreased in KD group compared with those in control group (P<0.05), but were increased to some extent after IVIG treatment (P<0.05). In addition, a positive correlation between the proportion of CD 19+CD24hiCD38hi Breg and the proportion of CD4+CD25+Foxp3+Treg was found in patient with acute KD (r=0.62, P<0.05).Conclusion Breg cell deficiency and its impaired function might be one of the important factors causing immune dysfunction in pa -tients with acute KD .

Objective To establish a duplex fluorescence PCR for detection of HIV proviral DNA and to evaluate its application for early diagnosis of HIV infection in infants .Methods A duplex fluores-cence PCR system was set up based on TaqMan technology for detection of human ribonuclease P ( RNase P) gene and long terminal repeat ( LTR) region of HIV.A recombinant plasmid containing the targeted gene fragment , pTG19-T, was constructed by TA cloning technique and used as the template for evaluation of sen -sitivity of the assay .Blood samples from 11 healthy individuals and 98 HIV-infected patients were collected and detected to validate the assay specificity .The assay of duplex fluorescence PCR was then carried out to detect 96 infant blood samples collected from several maternal and child health hospitals in Zhejiang province from January 2011 to September 2012 for early diagnosis of HIV infection .The results were compared with those by using the Roche HIV DNA qualitative detection kit .Results The established duplex fluorescence PCR could specifically detect HIV proviral DNA with a specificity of 100%and a detection sensitivity of 100 cps per reaction .The coincidence rate between the established assay and the Roche HIV DNA qualitative de -tection kit was 100%in the detection of 96 blood samples .Conclusion The duplex fluorescence PCR as-say showed advantages of cost-effectiveness , convenience , good specificity and accuracy with high sensitivi-ty.It could be used for early diagnosis of HIV infection in infants and also as a general technical platform for the detection of HIV proviral DNA .

Objective To investigate the virulence role of ompT of Escherichia coli in the patho-genesis of neonatal meningitis .Methods Adhesive abilities of the parent strain E 44 and the isogenic ompT-deletion mutant strain ( E44 ∶ΔompT) to human brain microvascular endothelial cells were evaluated in in vitro model.Low-copy-number plasmid pST containing ompT locus and point mutant plasmid pST 85 were transferred into E44 ∶ΔompT to construct the complemented mutant strain , and its adhesive ability was ana-lyzed.Influences of ompT deletion on E44 strain in its ability of bacterial intestinal colonization and ability of penetrating the blood-brain barrier were determined . Results In comparison with the parent strain , E44 ∶ΔompT strain showed significantly impaired adhesive ability to human brain microvascular endothelial cells, which could be partly restored by inserting the complementary plasmids of pST and pST 85.Deletion of the ompT did not affect Escherichia coli K1 in normal intestinal colonization in in vivo model.E44 ∶ΔompT strain could induce bacteremia , which was similar to that induced by the parent strain , but its ability of crossing the blood-brain barrier was significantly declined .Conclusion The study demonstrate that ompT plays an important role as the virulence element of Escherichia coli in binding to brain microvascular endothe-lial cells and penetrating the blood-brain barrier .Further study should be performed to investigate the influ-ences of OmpT proteinase on the virulence of Escherichia coil.