Objective To investigate mutation patterns in core promoter(CP)region of hepatitis B virus(HBV).Methods HBV DNA was extracted from sera of patients with chronic HBV infection.The CP sequence was amplified by polymerase chain reaction(PCR)and cloned into pMD19 T vector.The positive clones were then sequenced.The sequences were compared with known HBV genome in GenBank to identify the mutation sites and patterns of patients with chronic HBV infection.Results There were 74 clones from 21 patients with chronic HBV infection which were sequenced.The sequence comparisons showed that there was a 234-nucleotide deletion in CP region of HBV genome in 54 clones and a 245-nucleotide deletion in one clone.These deletion regions included CP,HBeAg initiation codon and direct repeat sequence(DR)Ⅰ regions,which named CP deletion(CPD).A1585T replacement mutation was also found in HBV strain with CPD,which indicated that there was linkage between these two mutations.Conclusions A novel mechanism of HBeAg negative chronic hepatitis B is observed,which includes deletions of CP and HBeAg initiation codon.Meanwhile,a simple and useful PCR method is developed to detect CPD.

Objective To detect the expression of type Ⅰ interferon in monocyte-derived dendritic cells(MoDCs)after Toll like receptor(TLR)3 triggered in patients with chronic hepatitis B(CHB),and to evaluate immune responses of CHB patients and its roles in the mechanisms of persistent infection of hepatitis B virus(HBV)and chronicity of hepatitis.Methods Peripheral blood mononuclear cells(PBMCs)were isolated and purified using magnetic beads(plasma was saved simultaneously)from 26 CHB patients and 18 healthy volunteers(HV).Dendritic cells(DCs)were induced and proliferated in a culture medium with recombinant human granulocyte macrophage colony stimulating factor(rhGM-CSF)and recombinant human interleukin(rhIL-4).EX3s were stimulated with Poly Ⅰ:C and the supernatants were collected at 0 h and 24 h after stimulation.Type Ⅰ interferon(IFN-α and IFN-β)in plasma and supernatants were examined by enzyme linked immunosorbent assay (ELISA).Results The levels of type Ⅰ interferon in plasma were not significantly different in groups of HV and CH B.IFN-α and IFN-β expressions in supernatants before Poly Ⅰ:C stimulation were(80.00±16.15)ng/L,(36.39±13.90)ng/L in CHB group and(76.76±15.90)ng/L,(37.14±13.68)ng/L in HV group,respectively.And there were no statistical differences between two groups(t=1.651,t=0.178;both P＞0.05).IFN-α expressions in supernatants at 24 h after stimulation in two groups were both higher than those before stimulation(at 0 h),but there were no statistical differences(t=1.534,t=1.243;both P＞0.05).IFN-β expressions in supernatants at 24 h after stimulation in HV group was(54.57±16.80)ng/L,which was significantly higher than that at 0 h(37.14±13.68)ng/L(t=4.061,P＜0.05).However,there was no significant difference at 24 h than tht at 0 h in CHB group(t=1.796,P＞0.05).At 24 h after stimulation.IFN-β level was(54.57±16.80)ng/L in HV group,which was significantly higher than that[(41.64±12.57)ng/L]in CHB group(t=2.921,P＜0.05).Conclusions Functions of MoDCs from CHB patients are impaired and MoDCs could not express type Ⅰ interferon normally.Expression of type Ⅰ interferon after TLR3 triggered in CHB patients is mainly IFN-β.

Objective To investigate the inhibitive activities of hepatitis B immunoglobulin(HBIG)poly(butylcynaoacrylate)nanoparticles(HBIG-PBCA-NP)to hepatitis B surface antigen(HBsAg)and hepatitis B virus(HBV)DNA secretions using HBV infected cell model in vitro.Methods HepG 2.2.15 cells were cultured with media containing HBIG-PBCA-NP or HBIG for several days,or cultured with HBIG-PBC-NP and HBIG for 2 days and without HBIG-PBCA-NP and HBIG from day 3.The supernatants at different time points were collected for quantitative detection of HBsAg and HBV DNA.The comparisons between groups were done by variance analysis.Resalts Secretions of HBsAg and HBV DNA in supernatants of HepG2.2.15 cultured with 0.1-10.0 IU/mL of HBIG-PBCA-NP and HBIG were inhibited significantly compared with control group.HBsAg titers and HBV DNA levels in supernatants of HBIG-PBCA-NP group and HBIG group cultured with media without HBIG-PBCA-NP and HBIG kept decreasing at day 5 and 7,then rebounded at day 9 and 11.HBsAg titera in supernatants of 0.1,1.0,5.0 IU/mL HBIG-PBCA-NP group were all significantly different from those in HBIG group at day 9[(31.31±1.98)μg/L vs(40.62±2.99)μg/L,(23.79±1.31)μg/L vs(36.51±2.12)μg/L,(19.91±1.74)μg/L vs(33.03±1.65)μg/L;F=412.24,P＜0.01].Couclusion HBIG-PBCA-NP can inhibit secretions of HgsAg and HBV DNA in vitro,which is more effective than HBIG.

Objective To study a functional variable fragment of heavy chain(VH)antibody against the terminal protein(TP)region of hepatitis B virus(HBV)polymerase introduced by human immunodeficiency virus Tat protein transduction domain(TAT)and the inhibitive activity of TAT-VH on the replication of HBV in vitro.Methods The gene encoding TAT-VH was cloned into prokaryotic expression vector pET28a(+).Recombinant plasmid was transduced into E coli BL21(DE3)LysS,then the protein was expressed and purified.The purified TAT-VH fusion protein was added into HepG2.2.15 cell culture.The transduction efficiency was evaluated by indirect fluorescence assay(IFA).The cytotoxicity of TAT-VH was detected by Methabenzthiazuron(MTT)assay.HBV DNA level in HepG2.2.15 cell culture was measured using quantitative polymerase chain reaction(PCR).The data were analyzed by one-factor analysis of variance and t test.Results TAT-VH fusion protein was successfully expressed and purified.It was confirmed by IFA and MTT assay that TAT-VH was introduced into HepG2.2.15 cells and the cell growth was not affected.The level of HBV DNA in supernatant of HeDG2.2.15 cell culture with 5 000 nmol/L TAT-VH was(1.211±0.132)lg copy/mL,which was significantly lower than control group[(5.325±0.041)lg copy/mL,t=72.91,P＜0.05].Meanwhile,the level of intracellular HBV DNA was(3.521±0.411)lg copy/mL,which was significantly lower than control group[(8.532±0.132)lg copy/mL.t=28.41,P＜0.05].Conclusion The HBV replication is inhibited by anti-TP TAT-VH antibodies in vitro,which provides valuable experimemal basis for developing therapy of HBV infection with intracellular antibody.

Objective To investigate the change of T helper 17 cells (Th17) in the peripheral blood mononuclear cell (PBMC) in patients with liver cystic echinococcosis. Methods Fifty-six subjects were divided into three groups: healthy controls (HD, n = 20), patients with cystic echinococcosis (CE, n= 18) and patients with cystic echinococcosis combined with bile fistula (BF,n= 18). The frequency of Th17 cells in CD4+ T lymphocytes was detected by flow cytometry. Th17-related cytokines including interleukin (IL)-17 and IL-23 were measured by enzyme-linked immunosorbent assay (ELISA). The data were analyzed by t test and Pearson correlation analysis.Results The frequency of Th17 in the peripheral blood was significantly lower in CE group compared to BF group and HD group [(0. 23±0. 11)% vs (0. 76±0.43)% vs (0.52±0.50)%; t=2. 225 and4. 077 respectively, both P＜0.05), while there was no statistical difference between BF group and HD group (t=1. 931, P＞0.05). The levels IL-17 and IL-23 were (12.1±3.7) ng/L and (84.4±46.0) ng/L respectively in CE group, which were lower than those in BF group [(15.5±4.1) ng/L and (138.6±37. 9) ng/L, respectively; t=2. 515 and 3. 649 respectively, both P＜0.05] and those in HD group [(14.8±4.4) ng/L and (138.1±48. 7) ng/L, respectively; t=2. 401 and 3. 706 respectively,both P ＜0.05], while there was no statistical difference between BF group and HD group (t=0. 534,P ＞0.05). Serum concentrations of IL-17 were all positively correlated with the concentrations of IL23 in these three groups (r=0. 657, P＜0.05). Conclusion The frequeny of Th17 cells in PBMC and the serum concentrations of IL-17, IL-23 are significantly reduced in patients with cystic echinococcosis.

Objective To explore the relationship between expressions of C-type lectin receptors on natural killer(NK) cells and infant human cytomegalovirus (HCMV) infection. Methods Seventynine cases of HCMV infection infants and 39 cases of HCMV non-infection control infants admitted during January 2006 to June 2008 were recruited in this study. According to HCMV pp65 antigenemia levels in the peripheral blood, 79 cases of HCMV infection infants were divided into two groups: 48cases of active HCMV infection and 31 cases of inactive HCMV infection. The 48 cases of infants with active HCMV infection were treated with ganciclovir for 2 weeks. The expressions of NKG2A,NKG2C, and NKG2D receptors on NK cells in the peripheral blood were examined by flow cytometry.Data analysis was done using SPSS 17.0 software. Comparisons among 3 groups were performed by Kruskal-Wallis nonparametric test for independent samples and comparisons between groups were done by Mann-Whiteney nonparametric test for paired samples. Results There was no difference of the inhibitory receptor NKG2A expression on NK cells among groups of active HCMV infection, inactive HCMV infection and HCMV non-infection controls (x2 = 3. 95, P＞0. 05). However, there was obvious difference of activating receptors of NKG2C and NKG2D expressions on NK cells among the three groups (x2 =24.91 and x2 =47. 80, respectively; both P＜0.01). The expressions of NKG2C and NKG2D on NK cells in the HCMV infection group were both higher than the control group (Z=-4.72 and Z=-5.15, respectively; both P＜0.01). The expression of NKG2D on NK cells in the active HCMV infection group was higher than that in the inactive HCMV infection group (Z= -5.08,P＜0.01). The expression of NKG2D on NK cells decreased after ganciclovir treatment (Z= - 1.34,P=0. 07). Conclusion Expressions of NKG2C and NKG2D on NK cells might play a significant role in regulating NK cell function and anti-HCMV immunity in infants.

Objective To investigate the phenotype, frequency of Th17 cells and the association between Th17 cells and viral clearance in patients with H1N1 influenza A. Methods Three groups including 70 confirmed patients with H1N1 influenza A, 30 patients with seasonal influenza as well as 68 healthy subjects as controls were enrolled in this study. The percentages of Th1, Th2, Treg and Th17 lymphocytes in the peripheral blood were determined by intracellular staining and flow cytometry. The levels of interferon-γ (IFN-γ), transforming growth factor-beta (TGF-β),interleukin-6 (IL-6) in plasma and supernatant of the peripheral blood mononuclear cell (PBMC)culture were quantified by enzyme-linked immunosorbent assay (ELISA). Viral load in nasopharyngeal swabs was detected by real time quantitative reverse transcription-polymerase chain reaction (RTPCR). Data were analyzed by one way ANOVA and liner correlation analysis. Results The percentage of Th17 cells in H1N1 influenza A patients was (2. 740±0. 210)%, which the percentage of was significantly decreased compared to healthy subjects (3. 443 ±0. 154)% and seasonal influenza patients (3. 443±0. 277) % (F=4. 242, P＜0. 05); while the percentage of Thl, Th2 and Treg cells were not significantly different among these groups. Moreover, the TGF-β level in plasma of H1N1 influenza A patients was (10±8) ng/mL, which was significantly lower than healthy subjects (43 ±32 ) ng/mL and seasonal influenza patient ( 18 ± 10) ng/mL ( F= 17.72, P＜0.01 ). The TGF-β level in the supernatant of PBMC culture of H1N1 influenza A patients was (782 ± 736) pg/mL, which was significantly lower than healthy subjects (1462±315) pg/mL and seasonal influenza patients (1481 ±348) pg/mL (F=5. 730, P＜0.01). Additionally, the viral clearance period was inversely correlated with the percentage of Th17 cells (r=-0.38, P=0.02). Conclusions The proportion of Th17 cells in patients with H1N1 influenza A is significantly decreased, which is closely correlated with the level of TGF-β. This decrease may results in the delayed viral clearance.

Objective To investigate the pathogenic features and antibiotic resistance profile of nosocomial and community-acquired spontaneous bacterial peritonitis (SBP) in liver cirrhosis patients.Methods Two hundred and twenty-six cirrhotic patients with SBP who were admitted to Beijin Ditan Hospital from January 2001 to December 2008 were recruited into this study. The bacterial identification and drug susceptibility were performed. The data were analyzed by Chi square test and t test. Results Eighty-six(38.0% ) patients were diagnosed with nosocomial SBP and 140 (62.0%)were diagnosed with community-acquired SBP. The proportion of Child-Pugh Class C cases in patients with nosocomial SBP was higher than patients with community acquired SBP (97.7% vs. 82.8%; x2= 11. 489, P=0.001). Mortality rate in patients with nosocomiat SBP was also higher than patients with community acquired SBP (50. 0% vs. 30. 0%; x2 =9. 081,P=0. 003). Total 28 species (232strains) of bacteria were isolated from these patients. 77.5 % (69/89) of the nosomial SBP cases and 76.9% (110/143) of community-acquired SBP cases were caused by Gram-negative bacteria (mainly were Escherichia coli and Klebsiella pneumoniae). 19.1% nosocomial SBP cases and 21. 8%community-acquired SBP cases were caused by Gram-positive bacteria. Fungus infections accounted for 3.4% and 1.4% of these two population, respectively(P＞0.05). In patients with nosocomial SBP,19 out of 32 Escherichia coli stains and 5 out of 14 Klebsiella pneunmoniae strains were extended spectrum β-lactamase (ESBL) positive, while among 60 Escherichia coli stains and 32 Klebsiella pneunmoniae strains, only 11 Escherichia coli stains were ESBL positive (P＜0.05). The resistance rates of Gram-negative strains to cephalosporin and quinolone in nosocomial SBP patients were both higher than those in community-acquired SBP patients(P＜0. 05), but all Gram-negative isolates were sensitive to imipenem (P＞ 0. 05). No Gram-positive isolates resistant to vancomycin were found.Conclusions The liver cirrhosis patients with Child-Pugh Class C are vulnerable to nosocomial SBP and the prognosis is poor. Although the pathogenic spectrum are similar in cirrhotic patients with nosocomial and community-acquired SBP, which mainly are Escherichia coli and Klebsiella pneumoniae, the percentage of ESBL producing strains is higher in nosocomial SBP patients compared to that in community-acquired SBP patients.

Objective To analyze the immunogenicity of selected B-cell epitopes of Epstein-Barr virus (EBV) latent membrane protein-2 (LMP2). Methods Three potential dominant B-cell epitopes of LMP2199-209, LMP2318-322 and LMP2381-391 from EBV LMP2 had been predicted using bioinformaties methods. The gene fragments of three epitopes were cloned respectively into pET32a(+) vector and transformed into E. coli strain BL21 (DE3). After identification by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, the expression products were purified by Ni-NTA agarose affinity chromatography. BALB/c mice in immunized groups were immunized by multi-point intracutaneous injection with the three purified epitope proteins,respectively; and mice in control groups were injected with pET32a (+) protein or phosphate buffered saline(PBS), respectively. The sera from mice at week O, week 3 and week 6 of injection were collected for determination of epitope-specific antibody IgG by enzyme linked immunosorbent assay (ELISA) using epitope proteins as coating antigens. The ability of serum antibody recognizing nature EBV antigen was determined at week 6 of immunization. Results Three epitope proteins of LMP2199-209 ,LMP2318-322 and LMP2381-391 were successfully expressed in prokaryotic system. Epitopespecific antibodies IgG could be detected respectively in the sera of all immunized mice, and the levels of antibodies increased with immunized time increasing. The antibody levels in LMP2318-322 immunized group at week 3 and week 6 were significantly higher than that of pET32a (+) protein control group (F= 493.85 and 773.99, respectively; both P＜0. 05), and the antibody levels in LMP2381-391 immunized group at week 3 and week 6 were also significantly higher than that of pET32a (+) protein control group (F= 926.33 and 309.14, respectively; both P＜0.05). Antibody level in LMP2199-209 immunized group at week 6 was significantly higher than that of pET32a ( + ) protein control group (F=87.27, P＜0.05). The antibody IgG in serum from immunized mice with three epitope proteins could all recognize nature EBV antigens, especially LMP2199-209 and LMP2381-391 immunized groups.Conclusions Three possible dominant epitopes of LMP2199-209, LMP2318-322 and LMP2381-391 from EBV LMP2 are prepared by prokaryotic expression system and exhibit obvious immunogenicity, which could be used for further research of EBV infection and related tumor vaccine.

Objective To analyze the immunological characteristics of Trichinella spiralis secretory antigen P53 and to evaluate its value in diagnosis of trichinellosis. Methods An open read frame of secretory antigen P53 was cloned from Trichinella spiralis by reverse transcriptasepolymerase chain reaction (RT-PCR) and then sequenced. Bioinformatics analysis was performed to search for its homologues in other helminths and predict its potential linear B cell epitopes and T cell epitopes. The sequence coding mature peptide was inserted into prokaryotic expression vector pET28a(+) and the purified recombinant product was identified by Western blot using serum samples of patients infected with Trichinella spiralis or other helminth. Results Bioinformaties analysis results showed that there was no P53 homologue in other helminths, which indicated that there were many linear B cell epitopes and T cell epitopes in TsP53. The recombinant P53 antigen only reacted with the serum samples of patients infected with Trichinella spiralis without any cross-reaction with the serum of patients infected with other helminths. Conclusion P53 has strong immunogenicity and immunoreactivity, which may be a promising candidate for developing Trichinella spiralis specific diagnostic method.