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Objective To investigate the effect of retinoblastoma binding protein 4 (RBBP4)in Sp1 -mediated HIV long terminal repeat(LTR)transcription.Methods RBBP4 expression vector and Sp1 expression vector were respectively co-transfected into 293 T cells with HIV promoter pHIV-LTR-Luc or Sp1 site mutated pHIV-LTR-sp1 -mut by liposome transfection,and the transfected cells were examined by dual luciferase reporter assay system.The effect of RBBP4 on the binding of Sp1 to LTR was further studied by chromatin immunoprecipitation (ChIP)and electrophoretic mobility shift assay (EMSA).Results The relative firefly luciferase activity activated by Sp1 was decreased from 62.5 to 16 at the dose of 500 ng of RBBP4 expression vector (t =14.52,P <0.01 ).When the Sp1 binding sites were mutated,the effects of 100,300 or 500 ng of RBBP4 expression vector on the firefly luciferase activity of HIV LTR were not statistically significance (t =1 .897,2.357 and 3.162,all P <0.05).ChIP results showed that when the binding of RBBP4 on HIV LTR increased,the binding of Sp1 on HIV LTR increased significantly (t =11 .93,P <0.01 ),while the reduced binding of RBBP4 on HIV LTR significantly attenuated the binding of Sp1 onto LTR(t =11 .38,P <0.01 ).The effect of RBBP4 on the binding of Sp1 to DNA in ChIP assays was further verified by EMSA assays.Conclusion RBBP4 can inhibit the Sp1 -mediated HIV LTR transcription in 293 T cells.
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Objective To investigate the role and mechanism of retinoblastoma protein-associated proteins 48 (RBBP4) in HIV-1 latency.Methods CEM-Bru cells latently infected with HIV-1 were stimulated with 25 ng/ml of tumor necrosis factor alpha (TNF-α) in combination with 10 ng/ml of interleukin-2 (IL-2).Chromatin immunoprecipitation (ChIP) was performed to detect the changes in RBBP4 and in histone deacetylases 1 and 2 (HDAC1/2) binding to long terminal repeat (LTR).Binding activities of HDAC1/2 and RNA polymerase Ⅱ (RNA Pol Ⅱ) to LTR and acetylated histone H3 at LTR region were detected by ChIP after partially interfering the expression of RBBP4 in CEM-Bru cells with electroporation.Initiating and elongated transcripts were measured by RT-PCR.Results The binding activities of RBBP4 and HDAC1/2 to LTR in HIV-1 latently infected cells were enhanced significantly as compared with those in TNF-α and IL-2 co-stimulated cells.Fewer RBBP4 and HDAC1/2 bound to LTR following the interference of RBBP4 expression, which was accompanied with enhanced histone acetylation and strengthened binding activity of RNA Pol Ⅱ to LTR.Moreover, more initiating transcripts were detected in HIV-1 latently infected cells after the RBBP4 expression was interfered by electroporation.Conclusion RBBP4 contributes to the maintenance of HIV-1 latency, in which HDAC1 and HDAC2 might be involved.
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Objective To explore the mechanism of latent human immunodeficiency ciency virus type 1 (HIV-1) infection is unclear, especially in dendritic cells (DC).We hypothesized that DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds with HIV-1 may activate HIV-1 provirus.Methods We generated a model by transfecting 293T cells with a DC-SIGN expression plasmid and a HIV-1 5'long terminal repeat (LTR) reporter plasmid, and then stimulated the 293T cells with HIV-1 gp120 protein, wild-type HIV-1 and VSV-G-pNL4.3 pseudotype virus ( without gp120 protein).CEM-Bru cells were transfected with the DC-SIGN expression plasmid and stimulated by HIV-1 gp120 protein.Then HIV-1 replication was detected.The involvement of the ERK, p38 and NF-κB pathways signaling in this response were determined by inhibiting the pathways specifically and detecting the phosphorylation of the signaling kinase.Results The HIV-1 5'LTR was reactivated by HIV-1 gp120 in DC-SIGN-expressing 293T cells.After HIV-1 gp120 protein stimulation of the mold of CEM-Bru cells, the increasing expression of HIV-1 Tat mRNA and HIV-1 p24,which implies early and late HIV-1 provirus replication was reactivated by the HIV-1 gp120/DC-SIGN stimulation.HIV-1 gp120/DC-SIGN stimulation reactivates latent HIV-1 provirus via the NF-κB signal pathway.Conclusion HIV-1 gp120/DC-SIGN stimulation reactivates latent HIV-1 provirus via the NF-κB signal pathway.
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Objective To establish and assess a new screening method for anti-HIV-1 drugs.Methods JLTRG cells were co-cultured with different proportions of H9/HTLV-Ⅲ B cells for 24,48,72 and 96 h.Intensity and density of green fluorescent protein were observed under fluorescence microscope,and were tested using flow cytometry.The optimal proportion of cells in co-culture system and the culture time were determined.The effectiveness of Enfuvirtide (T20) and Efavirenz (EFV),and their half maximal inhibitory concentrations (IC50) were determined by using cell co-culture system and half life of drugs.HIV load was detected using RT-PCR for HIV-1 p24 antigen,and its correlations with drug concentration and mean fluorescent intensity were analyzed by Spearman rank correlation analysis.Results Experiments demonstrated that JLTRG cells co-cultured with H9/HTLV-ⅢB cells at the proportion of 10 ∶ 1 for 72 hours was the best.Along with the concentrations of T20 and EFV changed,JLTRG cells were infected with HIV-1 in different degrees,and the IC50s of T20 and EFV were 10 nmol/L and 5 nmol/L,respectively.The concentrations of T20 and EFV were negatively correlated with mean fluorescent intensity and viral load (r =-1,-0.986 and-1,-1,P ＜ 0.01); and mean fluorescent intensity was positively correlated with viral load (r =0.986 and 1,P ＜ 0.01).Conclusion The drug screening method established in this study is efficient and easy to operate,which provides a new option for anti-HIV-1 drug screening.
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Objective To detect the signal pathways through which IL-4 regulates expression of DC-SIGN in THP-1 cells.Methods We used phorbol 12-myristate 13-acetate(PMA) differentiated THP-1 cells as the in vitro model of monocyte/macrophage cells to study the signal pathways involved in IL-4 regulated expression of DC-SIGN.DC-SIGN mRNA expression was detected by RT-PCR.Cytoplasmic DC-SIGN protein was tested by Western blot.Flow cytometry was used to detect cell surface expression of DC-SIGN.Cytoplasm and nuclear protein of PMA stimulated THP-1 cells induced by IL-4 for 0,10,20,30,60 and 120 min was extracted and detected by Western blot for signal pathway signaling protein and phosphoprotein.Results We found that a high expression of DC-SIGN could be induced by IL-4 at the levels of mRNA and cell surface protein.Up-regulated expression of DC-SIGN was almost completely blocked by the specific inhibitor of ERK pathway,and partly reduced by the specific inhibitors of JAK-STAT and NF-κB pathways.The activation of the three signaling pathways was directly confirmed by testing the phosphorylation of protein kinase within the cytoplasm and nucleus over time.Conclusion Multiple signaling pathways are involved in IL-4 induced high expression of DC-SIGN on THP-1 cells,in which ERK pathway is the main signal pathway.
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Objective To study Kaposi's sarcoma-associated herpesvirus(KSHV)infection in chronic hepatitis B(CHB)patients and its correlation with hepatitis B virus(HBV)replication and treatment-related factors.MethodsEnzyme-linked immunosorbent assay(ELISA)with recombination protein KSHV ORF65 was employed to detect the KSHV antibody and real-time polymerase chain reaction(PCR)was performed to detect KSHV DNA and HBV DNA in CHB patients.Age,HBV replication and licorice preparation treatment of patients were further analyzed.Comparison of rates was done using X~2 test.Results KSHV ORF65 antibody positive rates were 27.3% in 161 male CHB patients and 30.0% in 50 female patients(X~2=0.135,P＞0.05).The KSHV infection rates were increased with age,but this tendency was not obvious in patients older than 40 years old.The highest infection rate was in age group of 31-40 years old which was 37.1%.The positive rate of HBV DNA in CHB patients with KSHV infection was 73.5%,which was 56.3% in uninfected patients(X~2=3.969,P＜0.05).The average plasma level of KSHV DNA in patients treated with licorice preparations was 204.7 copy/mL and that in patients without licorice preparation treatment was 533.9 copy/mL.Eight patients were KSHV DNA positive(KSHV DNA＞ 100 copy/mL)in 16 patients treated with licorice preparations and 23 were positive in 33 patients without licorice preparation treatment.Conclusions The KSHV infection rates are increased with age of CHB patients.KSHV infection may interfere with HBV replication and licorice preparations may suppresss KSHV replication in vivo.
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Objective To evaluate the immune reconstitution of HIV-1 infected individuals after long-term highly antiretroviral therapy (HAART). Methods Twenty-five HIV-1 infected individuals receiving HAART, 17 without HAART and 15 healthy controls were included in the study. CD4 +T, CD8 +T,CD8/human leukocyte antigen DR (HLADR) + T, CD8/CD38 +T cells, and the expression of CD127 on CD3 +T cells from peripheral blood samples were measured by flow cytometry. IL-7 in peripheral blood was measured by enzyme-linked immunosorbort assay (ELISA) in HAART group. t test was performed to compare the measurement data among the groups. Results Before HAART, the count of CD4 + T cells in HIV-1 infected group was lower than that of the healthy control (t =9. 12, P ＜0. 01 ), while the counts of CD8 + T,CD8/HLADR+T, and CD8/CD38 +T cells were higher than those of the healthy control (t = 4.48, 4.89 and 3.88, P＜0. 01 ). Ater 7 years' antiviral therapy, CD4 +T cells increased, CD8 +T cells decreased, but both of them didn' t reach to the normal levels ( t = 2.66 and 2.43, P ＜ 0.05 ). While the counts of CD8/HLADR+T cells and CD8/CD38 +T cells almost reached to the normal levels (t = 0. 86 and 1.39, P ＞0.05). Before HAART, the concentration of IL-7 in HIV-1 infected group was higher than that in healthy controls (t =5.31, P ＜0.01 ). It decreased with HAART, but was still higher than the normal level (t =2. 81, P ＜ 0. 05 ). The expression of CD127 on CD3 + CD8 + T cells in non-HAART group was significantly lower than that in healthy control ( t =- 6.01, P ＜ 0.01 ), while that in HAART group was higher ( t = 2.32,P ＜0.05), but still not reached to the normal level ( t = 4.49, P ＜ 0. 05 ). CD127 expression on ( CD45RA + ) CD3 + CD8 + T cells almost increased to the normal level ( t= 0. 28, P ＞ 0. 05 ), while that on ( CD45RO + ) CD3 + CD8 + T cells was still remarkably lower than the normal ( t = 4. 86, P ＜ 0. 05 ). Conclusion Long-term HAART can partially restore the count and function of lymphocyte subsets in HIV-1 infected individuals, and the abnormal immune activation can be inhibited.
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Objective To detect the role of AP-1 or ETS-1 transcription factor binding sites (TFBS) in activity of DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) pro-moter. Methods Genomic DNA was extracted from peripheral blood. Complete DC-SIGN promoter and those without AP-1 or ETS-1 TFBS were amplified by PCR using designed primers. The PCR products were digested with MLu Ⅰ and Bgl Ⅱ and then ligated into MLu Ⅰ and Bgl Ⅱ digested pGL-3/Basic and pGL-3/En-hancer vectors. Transfection in Hacat and 293 cells was accomplished with Trans Fast liposome. Activity of luciferase was detected after 48 h. Results The DC-SIGN promoters without AP-1 or ETS-1 TFBS and the recombined pGL-3 vectors were correctly constructed. The activity of DC-SIGN promoters without AP-1 was reduced 20% (293) and 10% (Hacat), which was 40%-50% with enhancer. The activity of DC-SIGN pro-moters without ETS-1 was nearly vanished, no matter with or without enhancer. Conclusion ETS-1 TFBS, not AP-1 TFBS, plays an important role in activity of DC-SIGN promoter.
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DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is specific receptor on Dendritic cells, and plays a pivotal role on antigens presentation. Uptodate, the clear regulation mechanisms for DC-SIGN expression are not available.IL-4 is one of the most important cytokines inducing DC-SIGN production, while, NF-κB is an important transcription factor controlling signaling transduction. Both IL-4 and NF-κB are closely related to DC-SIGN regulation. NF-κB and IL-4 actions on DC-SIGN promoter activity, DC-SIGN expression as well as interactions between IL-4 and NF-κB were investigated in THP-1 cell. It was found that the mutation of NF-κB binding site in DC-SIGN promoter results in DC-SIGN promoter activity decrease about 50%.NF-κBp50 stimulates DC-SIGN expression in THP-1 cells. IL-4 upregulates DC-SIGN expression on THP-1 cells as well as NF-κB production. These data reveal that NF-κB is associated with IL-4 induced DC-SIGN expression.
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Objective To optimize the PCR primer sets for Simian virus 40 (SV40) detection and establish an assay method for SV40 which is of high sensitivity, strong specificity, broad applicability. Methods Two pairs of PCR primers were designed of based on 21 different SV40 strains genome by Primer Premier 5.00 software, and the features of two pairs of PCR primers were analyzed by Oligo software (version 6.71), conservative nucleotide of two pairs of PCR primers and the PCR amplification product were analyzed by DNAMAN software (version 6.0.40). Two pairs of new-built PCR primers were compared with those derived from China pharmacopoeia (Clip) in these aspects. The detection sensitivity of four pairs of PCR primers were analyzed using different SV40 DNA diluent as PCR template. The detection specificity of four pairs of PCR primers were analyzed using sterile water, Vero cell DNA, SV40 DNA as PCR template, respectively. Results The sequences of the new PCR primer sets VP1 and T are conservative for 21 Strains. The sequences of PCR primer sets GCVP1 and GCT are conservative for SV40 strains whose accession No. is J02400, NC_001669, AF316139 and AF316141. As far as the same diluent SV40 DNA template is concerned, the PCR amplification efficiency of PCR primer set VP1 and T is higher than that of PCR primer set GCVP1 and GCT. There are non-specific band in nucleic acid electrophoresis for amplification products of PCR primer sets GCVP1 and GCT, whereas there are no non-specific band in nucleic acid electrophoresis for amplification products of PCR primer sets VP1 and T. Conclusion The new assay method for SV40 nucleic acid sequence has many better qualities than those in Chp such as high sensitivity, strong specificity, broad applicability, conservation of primers and their amplification products and so on.
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<p><b>OBJECTIVE</b>To study the severe acute respiratory syndrome (SARS)-associated coronavirus genotype and its characteristics.</p><p><b>METHODS</b>A SARS-associated coronavirus isolate named ZJ01 was obtained from throat swab samples taken from a patient in Hangzhou, Zhejing province. The complete genome sequence of ZJ01 consisted of 29,715 bp (GenBank accession: AY297028, version: gi: 30910859). Seventeen SARS-associated coronavirus genome sequences in GenBank were compared to analyze the common sequence variations and the probability of co-occurrence of multiple polymorphisms or mutations. Phylogenetic analysis of those sequences was done.</p><p><b>RESULTS</b>By bioinformatics processing and analysis, the 5 loci nucleotides at ZJ01 genome were found being T, T, G, T and T, respectively. Compared with other SARS-associated coronavirus genomes in the GenBank database, an A/G mutation was detected besides the other 4 mutation loci (C:G:C:C/T:T:T:T) involved in this genetic signature. Therefore a new definition was put forward according to the 5 mutation loci. SARS-associated coronavirus strains would be grouped into two genotypes (C:G:A:C:C/T:T:G:T:T), and abbreviated as SARS coronavirus C genotype and T genotype. On the basis of this new definition, the ZJ01 isolate belongs to SARS-associated coronavirus T genotype, first discovered and reported in mainland China. Phylogenetic analysis of the spike protein gene fragments of these SARS-associated coronavirus strains showed that the GZ01 isolate was phylogenetically distinct from other isolates, and compared with groups F1 and F2 of the T genotype, the isolates of BJ01 and CUHK-W1 were more closely related to the GZ01 isolate. It was interesting to find that two (A/G and C/T) of the five mutation loci occurred in the spike protein gene, which caused changes of Asp to Gly and Thr to Ile in the protein, respectively.</p><p><b>CONCLUSION</b>Attention should be paid to whether these genotype and mutation patterns are related to the virus's biological activities,epidemic characteristics and host clinical symptoms.</p>
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Humans , Middle Aged , Genotype , Mutation , Severe acute respiratory syndrome-related coronavirus , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To confirm the close relationship of high co-infection rate between HIV and hepatitis virus in intravenous drug users (i.v.DUs).</p><p><b>METHODS</b>Anti-HIV, HBV and HCV were detected by ELISA in the serum from 35 scattered and 15 massed i.v.DUs. PCR and RT-PCR were performed to confirm the infection of HIV, HBV, HCV, HGV and TTV among the 15 massed intravenous drug abusers.</p><p><b>RESULTS</b>Among the 50 i.v.DUs, the positive rates of anti-HCV1 HBsAg, anti-HBe and anti-HBc were 92% (46/50), 12% (6/50), 10% (5/50) and 66% (33/50), respectively. In the samples of HBsAg positive, their HBeAg was also positive. Although the positive rate of serum markers was different in the massed i.v.DUs compared to the scattered i.v.DUs, no significant difference was shown. In the cases of massed i.v.DUs, the positive rates of HIV DNA, HBV-DNA, HCV-RNA, HGV-RNA, and TTV-DNA were 100% (15/15), 26.6% (4/15), 53.3% (8/15), 33.3% (5/15) and 26.6% (4/15), respectively. Among the 15 massed intravenous drug users, one was infected with HIV, HBV, HCV, HGV and TTV; two were infected with HIV, HBV, HCV and HGV; three were infected only with HIV; and the remaining had other forms of co-infection.</p><p><b>CONCLUSION</b>The co-infection rate of HIV, HBV, HCV, HGV and TTV in intravenous drug users is very high.</p>
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Humans , HIV Infections , Hepatitis, Viral, Human , Substance Abuse, IntravenousABSTRACT
Objective:To explore the change of serum interleukin 16 (IL-16) level and the effects of highly-active antiretroviral therapy (HAART) on IL-16 in patients with HIV infection.Methods:77 patients with HIV infection were studied,with fifteen normal subjects studied as controls.The patients were subdivided into stages according to the standards by US Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO).Of all the individuals,the CD4+T cells and CD8+T cells and the amout of IL-16 were measured at each stage to find the difference among normal and groups of the patests,and between the groups with and without HAART.Results:The level of CD4+ T cells in the experimental group were lower than that of the control group in of the patients (P
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Objective To investigate the molecular epidemiology as well as the subtypes of HIV in intravenous drug abusers, which can help pursue the source of infection in Zhejiang province and predict the epidemic strain in the future. Methods Such as ELISA, Western blot, nest PCR, DNA and sequencing technologies were used to analyze HIV subtypes of 15 strains isolated from intravenous drug abusers(IVDU) from Xinjiang autonomous region. Results All of the 15 IVDUs were infected with HIV 1, 2 of which were infected by subtype E and the others were infected by subtype C. Conclusions Subtype C is main HIV subtype infecting IVDUs. The epidemic subtype may have changed in China.
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5um)were more.The material middle diameters(MMD)of samples all surpassed 5?m.