Establishment and application of a flow cytometry-based method for detecting histone acetylation levels.

Histone deacetylase inhibitors, which have also received attention in AIDS and other diseases, are a new class of anticancer drugs developed in recent years. However, there is still a lack of a unified and reliable method for detecting histone acetylation levels in basic and clinical research. In this study, we developed a flow cytometry-based method to detect histone acetylation levels by comparing different sample processing temperature (on ice vs. room temperature), permeabilization method (intracellular vs. nuclear), antibody dose (antibody titration) and antibody incubation time (time gradient) using whole blood and peripheral blood mononuclear cells. In addition, we applied this optimized method in in vitro experiment and clinical trial of Chidamide (the only China FDA approved HDACi), the result of which confirmed that the flow cytometry-based method for detecting histone acetylation levels is a reliable, fast and convenient method which can be used in basic and clinical research.

Deciphering the Plastomic Code of Chinese Hog-Peanut (Amphicarpaea edgeworthii Benth., Leguminosae): Comparative Genomics and Evolutionary Insights within the Phaseoleae Tribe.

The classification and phylogenetic relationships within the Phaseoleae tribe (Leguminosae) have consistently posed challenges to botanists. This study addresses these taxonomic intricacies, with a specific focus on the Glycininae subtribe, by conducting a comprehensive analysis of the highly conserved plastome in Amphicarpaea edgeworthii Benth., a critical species within this subtribe. Through meticulous genomic sequencing, we identified a plastome size of 148,650 bp, composed of 128 genes, including 84 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. Comparative genomic analysis across seven Glycininae species illuminated a universally conserved circular and quadripartite structure, with nine genes exhibiting notable nucleotide diversity, signifying a remarkable genomic variability. Phylogenetic reconstruction of 35 Phaseoleae species underscores the affinity of Amphicarpaea with Glycine, placing Apios as a sister lineage to all other Phaseoleae species, excluding Clitorinae and Diocleinae subtribes. Intriguingly, Apios, Butea, Erythrina, and Spatholobus, traditionally clumped together in the Erythrininae subtribe, display paraphyletic divergence, thereby contesting their taxonomic coherence. The pronounced structural differences in the quadripartite boundary genes among taxa with unresolved subtribal affiliations demand a reevaluation of Erythrininae's taxonomic classification, potentially refining the phylogenetic contours of the tribe.

Efavirenz-induced exfoliative dermatitis.

Individuals with a human immunodeficiency virus (HIV) infection are at higher risk of developing adverse drug reactions. Multiple drugs are usually prescribed to patients with HIV infection for preventing the replication of HIV and for the treatment of the associated opportunistic infections. We report here the first case of an HIV-1-infected patient who developed an exfoliative dermatitis induced by efavirenz, a non-nucleoside reverse transcriptase inhibitor. Physicians should be aware of the possible occurrence of efavirenz-induced skin eruptions from the start of antiviral treatment of HIV infection.

Changes in levels of T cell subpopulations to monitor the response to antiretroviral therapy among HIV-1-infected patients during two years of HIV-1 replication suppression.

OBJECTIVES: The aim of this study was to compare the effect of 2 y of antiretroviral therapy (ART) on the percentage of activated CD38⁺CD8⁺ T cells and human leukocyte antigen (HLA)-DR⁺CD8⁺ T cells, and the expression of the co-stimulatory molecule CD28 on CD4⁺ and CD8⁺ T cells in the peripheral blood of HIV-infected adults, and to assess the use of immune activation markers to predict the virological response to ART in a cohort of HIV-1-infected patients in the north-western part of China. METHODS: We analyzed changes in the CD4⁺ T cell count, viral load, and the percentages of CD38⁺CD8⁺ T cells, HLA-DR⁺CD8⁺ T cells, CD28⁺CD4⁺ T cells, and CD28⁺CD8⁺ T cells in 48 patients with HIV diseases during 2 y of suppressive highly active antiretroviral therapy (HAART). Good virological responders (n = 20) were defined as those who had suppressed and maintained a plasma viral load below the detection limit of the assay for at least 12 months. Poor virological responders (n = 28) were defined as those with a detectable viral load at 6 and 12 months after beginning HAART. RESULTS: Among the 20 good responders, baseline median levels of CD38⁺CD8⁺ T cells were elevated, but had decreased significantly at 24 months of therapy (p < 0.0001). Median levels of HLA-DR⁺CD8⁺ T cells also decreased at 24 months of therapy (p < 0.0001). Levels of expression of CD28⁺CD4⁺ T cells rose steadily to 6 months (p = 0.03), and smoothly reached levels observed among HIV-negative blood donors during the 24 months of therapy (p > 0.05). Levels of expression of CD28⁺CD8⁺ T cells increased at 24 months (p = 0.04). Among the 28 poor responders, median levels of CD38⁺CD8⁺ T cells decreased significantly at 24 months (p < 0.0001). Levels of HLA-DR⁺CD8⁺ T cells also decreased at 24 months (p < 0.001). Levels of CD28⁺CD8⁺ T cells and levels of CD28⁺CD4⁺ T cells increased at 24 months remained unchanged. The percentage of CD38⁺CD8⁺ T cells appeared to provide a sensitive estimate of the overall immune recovery in comparison with the percentage of HLA-DR⁺CD8⁺ T cells, although this lacked specificity for the determination of early virological drug failure and did not appear to be a reliable surrogate for RNA viral load. CONCLUSIONS: We show that HAART can be used successfully in Chinese populations with elevated baseline immune activation markers and that the percentage of CD38⁺CD8⁺ T cells may be an additional parameter to the current criteria for estimating the antiretroviral response with HAART.

A human immunodeficiency virus-seronegative acquired immunodeficiency syndrome patient with opportunistic infections: A case report.

BACKGROUND: In rare cases, people living with chronic human immunodeficiency virus (HIV) infection do not develop antibodies despite demonstrable infection. Delayed or missed diagnosis of HIV infection leads to a lack of timely therapy, resulting in rapid disease progression with opportunistic infections or malignancies. CASE REPORT: A 44-year-old Chinese man presented with sore throat, oral leukoplakia, fever, dyspnoea and diffuse ground glass-like lesions in both lungs. Serum cytomegalovirus DNA was detectable, and CD4+ T-cell count was low. The patient was suspected of being a person living with HIV despite of the repeatedly negative HIV antibody tests using enzyme-linked immunsorbent assay and Western blot. Subsequently, high-plasma HIV RNA viral load was found on two repeated tests, while HIV DNA was also positive. Thus, the patient was confirmed as presenting with HIV-seronegative acquired immunodeficiency syndrome (AIDS). The symptoms improved in response to effective anti-fungal and anti-retroviral therapy after diagnosis. CONCLUSION: This is the third reported case of an HIV-seronegative AIDS patient in China, which are also rarely reported globally. HIV nucleic acid testing is important to screen out HIV infection, especially in those who present with severe immunodeficiency but remain HIV serogenative.

Efficacy and safety of the long-acting fusion inhibitor albuvirtide in antiretroviral-experienced adults with human immunodeficiency virus-1: interim analysis of the randomized, controlled, phase 3, non-inferiority TALENT study.

BACKGROUND: Albuvirtide is a once-weekly injectable human immunodeficiency virus (HIV)-1 fusion inhibitor. We present interim data for a phase 3 trial assessing the safety and efficacy of albuvirtide plus lopinavir-ritonavir in HIV-1-infected adults already treated with antiretroviral drugs. METHODS: We carried out a 48-week, randomized, controlled, open-label non-inferiority trial at 12 sites in China. Adults on the World Health Organization (WHO)-recommended first-line treatment for >6 months with a plasma viral load >1000 copies/mL were enrolled and randomly assigned (1:1) to receive albuvirtide (once weekly) plus ritonavir-boosted lopinavir (ABT group) or the WHO-recommended second-line treatment (NRTI group). The primary endpoint was the proportion of patients with a plasma viral load below 50 copies/mL at 48 weeks. Non-inferiority was prespecified with a margin of 12%. RESULTS: At the time of analysis, week 24 data were available for 83 and 92 patients, and week 48 data were available for 46 and 50 patients in the albuvirtide and NRTI groups, respectively. At 48 weeks, 80.4% of patients in the ABT group and 66.0% of those in the NRTI group had HIV-1 RNA levels below 50 copies/mL, meeting the criteria for non-inferiority. For the per-protocol population, the superiority of albuvirtide over NRTI was demonstrated. The frequency of grade 3 to 4 adverse events was similar in the two groups; the most common adverse events were diarrhea, upper respiratory tract infections, and grade 3 to 4 increases in triglyceride concentration. Renal function was significantly more impaired at 12 weeks in the patients of the NRTI group who received tenofovir disoproxil fumarate than in those of the ABT group. CONCLUSIONS: The TALENT study is the first phase 3 trial of an injectable long-acting HIV drug. This interim analysis indicates that once-weekly albuvirtide in combination with ritonavir-boosted lopinavir is well tolerated and non-inferior to the WHO-recommended second-line regimen in patients with first-line treatment failure. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02369965; https://www.clinicaltrials.gov.Chinese Clinical Trial Registry No. ChiCTR-TRC-14004276; http://www.chictr.org.cn/enindex.aspx.

Therapeutic effect of RANTES-KDEL on inhibition of HIV-1 in CD34(+) human hematopoietic stem cells (hHSC).

Cell surface receptors, such as the CCR5 chemokine receptors, represent key determinants of the human immunodeficiency virus type 1 (HIV-1) entry into target cells. The CC-chemokine, RANTES (regulated upon activation, normal T-cell expressed and secreted), a ligand for CCR5, have been targeted to the lumen of endocytoplasmic reticulum (ER) using a KDEL (ER-retention signal) fusion termed RANTES-KDEL and this construct was found to prevent effectively transport of newly synthesized CCR5 to the cell surface. Lentiviral vectors have emerged as potent and versatile tools of gene transfer for basic and applied research are able to transduce nondividing cells and maintain sustained long-term expression of transgenes. For this reason, an HIV-based lentiviral vector expressing RANTES-KDEL, pLenti6/V5-R-K, was constructed and then cotransfected with the ViraPower Packaging Mix (pLP1, pLP2, and pLP/VSVG) into 293FT cells to produce a replication-incompetent lentivirus stock. The lentiviral stock was titrated using HeLa cells, and the expression of the gene of interest, RANTES, was detected by indirect immunofluorescence. Based on the above results, the lentiviral stock was transduced into CD34(+) human hematopoietic stem cells (hHSC) separated magnetically from the cord blood (the purity was 96.8% evaluated by flow cytometry). Finally, the levels of p24 in the cultures of pLenti6/V5-R-K-transduced CD34(+) hHSC were detected after infection by HIV-1 DP1 (a R5-tropic HIV-1 strain, which was isolated by the Centers for Disease Control and Prevention of China in Henan province in 2000 from a Chinese man who had asymptomatic HIV-1 infection with a history of blood transfusions). It was shown that pLenti6/V5-R-K transduction inhibited expression of the DP1 p24 antigen by 51%, 58% and 60% on the 4th, 7th and 10th day respectively (P<0.05).

The short hairpin RNA driven by polymerase II suppresses both wild-type and lamivudine-resistant hepatitis B virus strains.

BACKGROUND: Chronic infection with hepatitis B virus (HBV) is widespread because of the limited availability of therapeutic treatments. Although previous reports have suggested that RNA interference has promise as a treatment for HBV infection, further studies of long-term and off-target drug effects on HBV, especially on drug-resistant strains of HBV, are needed. Therefore, seven vectors that express short hairpin RNAs (shRNAs), driven by the polymerase II promoter, pSilencer4.1/HBV, were constructed to target open reading frames (ORFs) of the HBV C and S genes from wild-type and drug-resistant strains. Treatment efficiency was also assessed. METHODS: The pSilencer4.1/HBV vectors were investigated in HepG2.2.15 cells and transgenic mice that consistently produce wild-type HBV. Additionally, vectors that produce a lamivudine-resistant strain of HBV were developed and cotransfected, along with pSilencer/HBV, into both HepG2 cells and mice. The effects of polymerase-II-driven pSilencer4.1/HBV were compared with those of polymerase-III-driven pSilencer3.1/HBV at both the gene and protein level. RESULTS: pSilencer4.1/HBV inhibited the expression of viral protein, DNA and HBV subtype ayw mRNA in both HepG2.2.15 cells and transgenic mice. Toxicity, as well as off-target effects, did not occur after a short- to medium-term examination. Moreover, an HBV strain resistant to lamivudine, subtype adr, was suppressed by shRNA in both HepG2 cells and mice. In contrast to polymerase III, vectors that used polymerase II could drive efficient silencing without off-target effects. CONCLUSIONS: Silencing by shRNA dramatically inhibited HBV expression and replication regardless of strain type. ShRNA could therefore be a promising treatment for HBV infection.

Quantitative hepatitis B core antibody level is a new predictor for treatment response in HBeAg-positive chronic hepatitis B patients receiving peginterferon.

A recent study revealed that quantitative hepatitis B core antibody (qAnti-HBc) level could serve as a novel marker for predicting treatment response. In the present study, we further investigated the predictive value of qAnti-HBc level in HBeAg-positive patients undergoing PEG-IFN therapy. A total of 140 HBeAg-positive patients who underwent PEG-IFN therapy for 48 weeks and follow-up for 24 weeks were enrolled in this study. Serum samples were taken every 12 weeks post-treatment. The predictive value of the baseline qAnti-HBc level for treatment response was evaluated. Patients were further divided into 2 groups according to the baseline qAnti-HBc level, and the response rate was compared. Additionally, the kinetics of the virological and biochemical parameters were analyzed. Patients who achieved response had a significantly higher baseline qAnti-HBc level (serological response [SR], 4.52±0.36 vs. 4.19±0.58, p=0.001; virological response [VR], 4.53±0.35 vs. 4.22±0.57, p=0.005; combined response [CR], 4.50±0.36 vs. 4.22±0.58, p=0.009)). Baseline qAnti-HBc was the only parameter that was independently correlated with SR (p=0.008), VR (p=0.010) and CR(p=0.019). Patients with baseline qAnti-HBc levels ≥30,000 IU/mL had significantly higher response rates, more HBV DNA suppression, and better hepatitis control in PEG-IFN treatment. In conclusion, qAnti-HBc level may be a novel biomarker for predicting treatment response in HBeAg-positive patients receiving PEG-IFN therapy.

[The relationship between cellular infection by Hantaan virus and expression of beta3 integrin].

OBJECTIVE: To investigate the relationship between cellular entry of Hantaan virus (HTNV) and expression of beta3 integrin in beta3-integrin-deficient and HTNV-insusceptible China hamster ovary (CHO) cells. METHODS: Eukaryotic expression vector encoding human integrin beta3 and eukaryotic expression vector harboring human integrin alphav or alphaIIb subunit cDNA were transfected into HTNV non-permissive CHO cells individually or collectively. Screening for stable transfectant clones was performed using G418 selective (culture medium. The exogenous gene expression was analyzed qualitatively and quantitatively by immunofluorescence assay (IFA) and flow cytometry (FCM). Various modified CHO cells and untransfected CHO cells were infected using HTNV A9. At various time points after infection, HTNV antigens in infected cells were detected qualitatively and quantitatively by IFA, FCM. RESULTS: Highly-effective surface expression of beta3 integrin was measured in CHO/alphavbeta3 and CHO/alphaIIbbeta3, while weaker surface expression was detected in CHO/beta3 (P < 0.05). Expression of alphav or alphaIIb integrin in the individually transfected group was significantly lower than in the cotransfected group (P < 0.01) and the sites of localization changed. In contrast, effective surface expression was not seen when pcDNA3 was transfected alone. The infection rate of CHO/alphavbeta3 (60.1%) and CHO/alphaIIbbeta3 (55. 9%) cells were significantly higher than that of CHO/beta3 (38.7%) cells, while the infection rate of CHO/beta3 was significantly higher than that of CHO/alphav, CHO/pcDNA3 and CHO cells respectively. There was a close relationship between the positive percentage of HTNV A9-infected cells and expression of beta3 integrin. CONCLUSION: These results indicated that cellular entry of HTNV was related to the expression of beta3 integrin.

Clinical characteristics and current management of hepatitis B and C in China.

AIM: To describe a population of outpatients in China infected by hepatitis B virus (HBV) and/or hepatitis C virus (HCV), and assess their current management status. METHODS: A multicenter, cross-sectional study of HBV- and/or HCV-infected patients was conducted from August to November, 2011 in western China. Patients ≥ 18 years of age with HBV and/or HCV infections who visited outpatient departments at 10 hospitals were evaluated, whether treated or not. Data were collected on the day of visit from medical records and patient interviews. RESULTS: A total 4010 outpatients were analyzed, including 2562 HBV-infected and 1406 HCV-infected and 42 HBV/HCV co-infected patients. The median duration of documented infection was 7.5 years in HBV-infected and 1.8 years in HCV-infected patients. Cirrhosis was the most frequent hepatic complication (12.2%), appearing in one-third of patients within 3 years prior to or at diagnosis. The HCV genotype was determined in only 10% of HCV-infected patients. Biopsy data were only available for 54 patients (1.3%). Antiviral medications had been received by 58.2% of patients with HBV infection and 66.6% with HCV infection. Nucleos(t)ide analogs were the major antiviral medications prescribed for HBV-infected patients (most commonly adefovir dipivoxil and lamivudine). Ribavirin + pegylated interferon was prescribed for two-thirds of HCV-infected patients. In the previous 12 mo, around one-fifth patients had been hospitalized due to HBV or HCV infection. CONCLUSION: This observational, real-life study has identified some gaps between clinical practice and guideline recommendations in China. To achieve better health outcomes, several improvements, such as disease monitoring and optimizing antiviral regimens, should be made to improve disease management.

Analysis of the immune response to Hantaan virus nucleocapsid protein C-terminal-specific CD8(+) T cells in patients with hemorrhagic fever with renal syndrome.

Hantaan virus (HTNV), the prototype member of the Hantavirus genus in the family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS), which is characterized by capillary leakage, hemorrhage, and renal injury, and is an important public health problem in China. Some kinds of immune cells, particularly CD8(+) T cells, are involved in the pathogenesis of Hantavirus infection. The nucleocapsid protein (NP) of the Hantavirus is the most conserved structural protein and the most abundant viral protein produced during infection. It is one of the important target antigens that induce the CD8(+) T-cell response. In this study, we examined the CD8(+) T-cell response to HTNV NP C-terminal polypeptides. We synthesized 23 overlapping C-terminal polypeptides and detected the antigen-specific CD8(+) T cell response in 15 patients with HFRS. The results demonstrated that there were NP-specific T-cell responses in bulk cultures of peripheral blood mononuclear cells (PBMCs) from 9 of 15 patients. The peptide 51 (aa 301-315: SPSSIWVFAGAPDRC), peptide 60 (aa 355-369: LRKKSSFYQSYLRRT), and peptide 70 (aa 415-429: DVKVKEISNQEPLKL) induced strong CD8(+) T-cell responses. Among them, peptide 70 induced CTL responses in donors 7, 9, and 11, and the strongest responses were seen in donor 11. Depletion of CD8(+) T cells from PBMCs completely abrogated the peptide-specific T-cell response, while depletion of CD4(+) T cells did not diminish the number of IFN-gamma spot-forming cells. These data suggest that infection with HTNV results in CTL responses to immunodominant regions on the NP.

Down-regulation of CXCR4 expression by SDF-KDEL in CD34(+) hematopoietic stem cells: An anti-human immunodeficiency virus strategy.

CXCR4 plays an essential role as the first discovered coreceptor for the entry of T cell tropic isolates of HIV-1. Blocking the surface expression of this receptor may be a potential strategy to prevent HIV-1 infection. A lentiviral vector, pLenti6/V5-S-K, expressing a SDF-KDEL fusion protein was constructed and a replication-incompetent lentiviral stock was produced. The lentiviral stock was transduced into CD34(+) hHSC and the transient expression of the recombinant protein, SDF-1, was assayed using indirect immunofluorescence. The surface expression of CXCR4 in CD34(+) hHSC pretreated with different amounts of recombinant lentiviral vectors was detected by flow cytometric analysis. A marked down-regulation of CXCR4 expression in the cells transduced with recombinant lentiviral vectors pLenti6/V5-S-K was observed by flow cytometry with PE-conjugated anti-human CXCR4 monoclonal antibodies which showed the percentages of the inhibition effects of CXCR4-SDF-1 mediated syncytium formation are presented by concentration. P24 antigen levels of cell culture supernatants were detected on the 4th, 7th, and 10th day, with 10(3) TCID50 HIV-1 infected CD34(+) hHSC to evaluate the inhibitory effect of pLenti6/V5-S-K transduction on HIV-1 infection. The cells transfected with pLenti6/V5-S-K had a significant reduction of HIV-1 DP27 infection compared to controls (P<0.05).

Toll-like receptor 4 siRNA attenuates LPS-induced secretion of inflammatory cytokines and chemokines by macrophages.

Toll-like receptor 4 (TLR4) is critical for activation of macrophages by Lipopolysaccharide (LPS). In this study, we investigated the silencing effects of TLR4-specific 21-nt small interfering RNAs (siRNA) on TLR4 expression in RAW264.7 cells. It was found that treatment with TLR4 siRNA down-regulated the TLR4 mRNA and protein expression in macrophage RAW264.7 cells, and reduced the sensitivity of the cells to LPS stimulation. Our findings also demonstrate that treatment with TLR4 siRNA significantly decreased the tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) expression induced by LPS. TLR4 siRNA treatment also impaired the signalling of mitogen-activated protein kinases (MAPK) induced by LPS in RAW264.7 cells. These data suggest that inhibition of TLR4 expression by TLR4 siRNA may be therapeutically beneficial in controlling the overall responses of immune cells to LPS.

[Identification of a functional ITAM-like sequence within G1 cytoplasmic tail of Hantaan virus].

The G1 cytoplasmic tail of Hantaan virus (HTNV) harbors a highly conserved region, which is homologous to immunoreceptor tyrosine-based activation motifs (ITAM) and is termed the ITAM-like sequence. To demonstrate the potential signal-transducing activity of G1 ITAM-like sequence resembling the canonical ITAM within immune and endothelial cells, a series of experiments were performed to define its interaction with cellular kinases. The synthesized G1 ITAM-like peptide was shown to coprecipitate with cellular phosphoprotein complexes by an immune-complex kinase assay. Mutational analyses showed that this ITAM-like sequence was a substrate for the Src family kinase Fyn, and two conserved tyrosine residues were required for coprecipitating Lyn, Syk, and ZAP-70 kinases. These findings demonstrated that HTNV envelope glycoprotein G1 contains a functional ITAM-like sequence in its cytoplasmic tail, which can bind critical cellular kinases that regulate immune and endothelial cell functions.

[Specific immune responses to human immunodeficiency virus type 1 Gag, Tat, Rev and Nef proteins].

AIM: To investigate the features of HIV-1-Gag-, Tat-, Rev- and Nef- specific cytotoxic T-lymphocyte (CTL) responses in infected individuals in China. METHODS: The HIV-1-specific CTL responses were analyzed with an IFN-gamma ELISPOT assay by using 220 overlapping peptides spanning the entire HIV-1 Clade B (HIV-1B) and C (HIV-1C) Gag, Tat, Rev and Nef proteins consensus sequences. RESULTS: For either HIV-1B or HIV-1C, Gag and Nef were preferentially targeted by HIV-1 specific CTLs, Rev and Tat proteins were also recognized to different extent. In comparison of the immune responses between HIV-1B and HIV-1C, the magnitude and frequency were roughly identical but there were some differences in the immunodominant regions. For HIV-1B, the highest response magnitude was detected in 288-313 amino acids of Gag p24, and for HIV-1C, in 155-181 amino acids of Gag p24. The most frequently recognized region was located in 106-143 amino acids of Nef either in HIV-1B or in HIV-1C (48.1%). CONCLUSION: HIV-1-specific CTLs mainly directed against HIV-1 Gag and Nef in Chinese, and there is some difference between HIV-1B and HIV-1C. There exists the cross-recognition between the Clade B and Clade C. These data suggest that the study on HIV-1-specific CTL responses in Chinese will provide strategies for the vaccine design in China.

[Experimental study on immune responses induced in mice by gp120 gene vaccine of Chinese HIV-1 strain].

AIM: To construct the gp120 DNA vaccine of Chinese HIV-1 strain and evaluate the immune responses induced with it in BALB/c mice. METHODS: The recombinant expression vector pVAX1-GP120 was constructed by inserting HIV gp120 gene into the eukaryotic expression vector pVAX1 and confirmed with EcoR I/Pst I and DNA sequencing. BALB/c mice were immunized with pVAX1-GP120 and pVAX1 respectively. The levels of serum anti-HIV antibody and IFN-gamma of the immunized mice were detected by ELISA. The proliferation of splenocytes was determined by MTT colorimetry and the specific cytotoxic T lymphocytes (CTLs) response by LDH assay. RESULTS: Restriction enzymes digestion analysis and DNA sequencing results revealed that the pVAX1-GP120 had been constructed successfully. The titer of anti-HIV antibody and the IFN-gamma level in mice immunized with the pVAX1-GP120 were higher than those in mice immunized with pVAX1 respectively (P<0.01). As compared with mice immunized with pVAX1 alone, the cytotoxic activity of specific CTLs and antigen-specific lymphoproliferative responses in mice immunized with pVAX1-GP120 were significantly enhanced (P<0.01). CONCLUSION: Specific cellular and humoral immune responses in mice can be induced with gp120 gene vaccine of Chinese HIV-1 strain, which lays the foundation for further development of therapeutic HIV vaccine against HIV-1 infection.

[Inhibition of momordica anti-HIV protein of MAP30 on HBeAg expression by laser scanning confocal microscopy].

AIM: To investigate the effect of momordica anti-HIV protein of M(r ) being 30 000 (MAP30) on HBV expression by laser scanning confocal microscopy. METHODS: HBV DNA-transfected hepatocarcinoma cells 2.2.15 were cultured in the presence of MAP30. Then quantitative analysis of HBeAg expression in the 2.2.15 cells by laser scanning confocal microscopy after indirect immunofluorescence staining was performed. RESULTS: The fluorescence intensity in 2.2.15 cells co-cultured with MAP30 was notably weaker than that in control cells. CONCLUSION: MAP30 can effectively inhibit HBeAg expression in the 2.2.15 cells.