Objective: To compare the expression levels of FOXP3 mRNA in the peripheral blood mononuclear cells(PBMC) of hepatitis B patients and healthy persons.Methods: Real-time fluorescence relative quantitative RT-PCR was used to determine the FOXP3 mRNA levels in the PBMCs from 25 patients with chronic hepatitis B and 11 healthy subjects.Results:The FOXP3 mRNA levels were significantly higher in the hepatitis B patients than in the normal subjects(P0.05).Conclusion: The high expression of FOXP3 might be an important factor for the persistence of HBV infection.

Immune suppression is a very important mechanism in alleviating immunologic injury,and recent study has showed that CD4~(+)CD25~(+) regulatory T cell is an important component of the immune suppression system.This T cell subset has an obvious immune-suppressing effect and produces its effect not by secreting cytokine but contacting cell directly.In this review,we describe the biological characteristics of CD4~(+)CD25~(+) regulatory T cell and its functions in infection immunity.

Objective To investigate the expression of T-cell subset,changes of TNF-?,IL-6,IL-8,sIL-2R levels in the sera and its role in development of chromic viral hepatitis B,in order to understand viral replication and clinical significance in patients with chronic hepatitis B(CVHB).Methods Alkaline phosphatase/antialkaline Phosphatase(APAAP) and ELISA techniques were used to detect the levels of T-cell subset in peripheral blood and serum TNF-?,IL-6,IL-8,sIL-2R in 290 patients with chronic hepatitis B.Results In the count of CD 4 + cell and CD 4 +/CD 8 + were significanyly lower,and in the count of CD 8 + cell and levels of TNF-?,IL-6,IL-8,sIL-2R in CVHB patients group were higher than in normal control group(P

Objective To explore the effect of the superantigens staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) on the level and positive sero-conversion rate of anti-HBs antibody induced by HBsAg. Methods Sixty BABL/C mice were randomly divided into 5 groups. The mice in group 1 were administered 4?g recombinant HBsAg by subcutaneous injection in the groin once a week for three weeks. The manner of HBsAg administration in the other four groups was the same as that in group 1. One day after the first administration, the mice were administered 25?g SEA (group 2) or 25?g SEB (group 3) by subcutaneous injection in contralateral groin. In the second week after the first administration, the mice were administered 25?g SEA (group 4) or 25?g SEB (group 5) by subcutaneous injection in contralateral groin when HBsAg were given. Serum levels of anti-HBs antibody in the five groups were measured once a week until the end of experiment using ELISA. Results On the third week, the positive sero-conversion rate and serum level of anti-HBs in group 1 were 41.7% and 0.758?0.126, respectively. At the same time, the anti-HBs sero-conversion rate in the group 2 and group 4 was 100%, and there was significant difference in the anti-HBs level compared with group 1(P0.05). Conclusion SEA could remarkably elevate the level and positive sero-conversion rate of anti-HBs after injection of HBsAg, and significantly enhance the humoral immune response induced by HBsAg in mice.

Objective Analysis of drug resistance mutations in the protease and reverse transcriptase gene and in the protease substrate cleavage sites of HIV-1 from AIDS pediatric plasma samples after long term treatment with protease and reverse transcriptase inhibitors for long time. Methods The protease, reverse transcriptase, gag and partial pol genes were amplified using Nest RT-PCR from 7 pediatric plasma samples. The PCR products of protease and reverse transcriptase gene were used directly for nucleotide sequencing. The PCR products of gag and pol gene were cloned into pCR2.1-TOPO TA cloning vector to be sequenced. The resulting nucleotide sequences were aligned and analyzed using Sequencing Analysis and HIV-1 Genotyping system software of PE company. Results Drug resistance mutations were found in the protease and reverse transcriptase genes from 6 patients' plasma samples. There are drug resistance mutations in the protease substrate cleavage sites in only 2 patients' plasma samples. Conclusions After long time(33～106 months) treatment of protease and reverse transcriptase inhibitors, drug resistance mutations in the protease and reverse transcriptase genes can be found in the most of the patients plasma samples(6/7), However, much less patients (2/7) were found to be with drug resistance mutations of the protease substrate cleavage sites.

Objective To detect circulating CD4 + CD25 + regulatory T cells (Treg) and Toll-like receptor(TLR)2 and TLR4 expression on the peripheral blood mononuclear cells (PBMCs) of patients with HBV-related liver cirrhosis (LC), and to explore the correlation between them. Methods PBMCs isolated from 30 LC patients, 21 chronic hepatitis B (CHB) patients and 16 normal controls(NC) were stained with fluorescent labeling anti-TLR2-PE, anti-TLR4-APC, anti-CD14-FITC monoclonal antibodies and anti-CD4-PerCP, anti-CD25-FITC, anti-CD127-PE. Samples were detected by flow cytometry. Statistic analysis be-tween groups was performed by Kruskal-Wallis H test. Spearman rank correlation was used to analyze the correlation of Treg and TLR2, TLR4. Results The expression of TLR2 and TLR4 were significantly up-reg-ulated in patients with LC than those in the controls (TLR2 : 200.3 ± 96.8 vs 94.1 ± 17.6, P < 0.05 ; TLR4:32.1 ±7.2 vs 17.8 ±3.9, P<0.05). The expression of TLR4 was significantly increased in pa-tients with LC than those in patients with CHB (TLR4 : 32. 1 ± 7.2 vs 25.2 ± 8.3, P < 0.05), but there were no differences of TLR2 expression between LC and CHB(200.3 ± 96.8 vs 214.0 ± 72.6, P > 0.05). Treg/CD4+ T cells were 5.07% ±1.43%, 5.88% ±1.66%, 4.21% ±1.24% in patients with LC, CHB and NC, respectively. Treg/CD4+ T cells were significantly increased in patients with CHB than those in pa-tients with NC(P<0. 05) and LC(P <0.05), but there were no differences between LC and NC(P > 0.05). TLR4 expression and Treg were positive correlation (r = 0. 469, P = 0. 032) and TLB2 expression were negative correlation in patients with LC (r = -0.428, P = 0.021). Conclusion The expression of TLR2 and TLR4 were up-regulated on PBMCs in patients with LC. It seems to be expression of TLR2 and TLR4 in-volved in the pathogenesis of LC.

Objective:To study the different effect of fused gene IL-2/Fc administrated at different time on immune responses induced by HBV preS_2S genetic vaccine.Methods:BALB/c mice were immunized with purified recombinant plasmids at 0, 2, 4 weeks. Two groups were designed. One group were injected with pcDNA3.1S_2S at first, then pcDNA3.1IL-2/Fc was administrated after three days. While the other group were injected two plasmids at the same time. We compared the immune responses through detecting the anti-HBs titers, CTL cytotoxicity level, proliferation of lymphocytes and cytokines levels secreted by cultural splenocytes.Results:The data demonstrated that the humoral and cellular immune responses induced by pcDNA3.1IL-2/Fc as adjuvant and administrated after HBV preS_2S DNA vaccine 3 days mice were dominant higher than other groups.Conclusion:Genetic adjuvant administrated after Ag priming can enhance its effect.

Objective To develop a reverse genetics system for Hantaan virus (HTNV) 84FLi strain by using RNA polymerase [ (pol Ⅰ)-mediated transcription. Methods Complementary DNA (cDNA) containing the coding sequence for chloramphenicol acetyhransferase (CAT) was inserted into the 5'-and 3'-terminal untranslated regions of HTNV 84FLi L segment. These chimeric cDNAs (pol Ⅰ expression cassette) were cloned into plasmids and between the human pol Ⅰ promoter and terminator to generate sense and anti-sense RNA pol Ⅰ transcription reporter plasmids. The reporter plasmids were transfeeted into 293T cells or the 1:1 combination of 293T and HTNV infected Vero cells. These cells were cotransfected with expression plasmids encoding Ⅰ. (RNA dependent RNA polymerase) and N (nucleoprotein) viral proteins, Cells were harvested 48 h post-transfection and the CAT activity was detected. The 293T cells were infected with the supernatant to explore the passage ability of CAT activity. ResultsThe reporter plasmids pLvRNA-CAT and pLcRNA-CAT were constructed successfully. CAT activity was detected in transfected cells and could also be serially passaged in the rescued virus minigenomes. Conclusion The RNA polymerase ]-driven reverse genetics system successfully rescues HTNV 84FLi minigenomes.

Objective To analyze of CD4+ CD25high regulatory T cells(Treg)in peripheral blood of chronic HBV patients and its correlation with multiple clinical indicators.Methods Thirty-five hepatitis B virus(HBV)infected patients in this study were divided into four different clinical types:HBsAb+group(n=5),inactive hepatitis group(n=8),chronic hepatitis group(n=12),and immune tolerance group(n=10).The number of CD4+CD25high Treg and related T cells subgroup in CD3/CD4/CD8 was thoroughly examined by flow cytometry in peripheral blood of HBV infected patients and the healthy contrast group(n=12).Serum HBV markers were determined by commercial ELISA kits.Serum HBV DNA was quantified by commercial real-time PCR kit.Statistical differences were studied to investigate the correlations between CD4+CD25high Treg and different clinical types of HBV infection and clinical indicators.Results The absolute counts of CD25high Treg and its frequency in CD4+ T cells were similar between HBV infected patients [(12.35±6.48)/μ,(1.82±0.87)%]and health controls[(8.91±3.11)/μl,(1.35±0.39)%],P＞0.05.The frequency of CD25high Treg in CD4+ T cells from the immune tolerance group was significantly higher than that of the HBsAb+ group,chronic hepatitis group,and the healthy contrast group(P＜0.05).The absolute counts of CD25high Treg from the immune tolerance group were significantly higher than the healthy control group(P＜0.05),and the frequency of CD25high Treg in CD4+ T cells is negatively correlated to the ALT level(r=-0.418,P=0.038),positively correlated to CD4/CD8 ratio(r=0.344,P=0.021),no correlation to the HBV DNA level(r=0.118,P＞0.05).The absolute counts of CD25high Treg were positively correlated to CD4/CD8 ratio(r=0.360,P=0.015),no correlation to ALT level and HBV DNA level(r=-0.211,r=0.060,P＞0.05).Conclusion CD4+ CD25high Treg may play a role in immunopathogenesis of chronic HBV infection.

The coding region of S genome segment of Hantaan virus (76/118 strain) was inserted into the eukarytic expression plasmidpVR1012. The recombinant expression plasmid pVRS22 was constructed. Vero-E6 cells were transiently transfected in vitro with pVRS22 plasmid. The transient expression of Hantaan virus nucleocapsid proteins in Vero-E6 cells was detected by indirect immunofluorescence assay (IFA) with monoclonal antibody 5H5 against Hantaan virus.