[Epidemic analysis of HIV-1 incidence using BED-CEIA assay in Tianjin, China, 2010].

OBJECTIVE: To explore the epidemic characteristics of HIV-1 recent infection among high-risk populations in Tianjin and to provide reasonable evidence for intervention strategy. METHOD: Blood samples were detected using BED-CEIA to identify recent HIV-1 infection, and HIV/AIDS cases in Tianjin 2010 were collected to analyze epidemic data. RESULTS: 349 of reported HIV/AIDS cases were confirmed from total 777 207 people. ALL of 87 HIV-1 recent infections were males and mainly detected from men who have sex with men (MSM), voluntary counseling and testing (VCT) and STD outpatients, the HIV-1 incidence of each group was 1.48%, 0.38%, 0.17% respectively. There were not significant difference in recent HIV-1 infection rate between other areas and locals. The homosexual transmission was 80.46% among recent HIV-1 infections. CONCLUSION: The results of this study showed that the homosexual contact was primary route of HIV transmission in Tianjin. The infection of HIV was serious among MSM, effective interventions should be taken for HIV/AIDS control and prevention urgently.

[Survey on recent infection of human immunodeficiency virus among men who have sex with men in Tianjin during 2008 - 2009].

OBJECTIVE: To study the situation of HIV infections among men who have sex with men (MSM) in Tianjin during 2008 - 2009 and to provide reasonable evidence for intervention strategy. METHODS: Transect investigations in MSM were conducted three times during 2008 - 2009. Blood samples were collected and detected to identify the recent HIV infection with IgG-capture BED-enzyme immunoassay (BED-CEIA) before HIV incidence was estimated. RESULTS: 1799 specimens were tested and the HIV prevalence rates of each study were 6.7%, 8.6% and 6.2%, while the incidence rates were 2.7%, 2.5% and 2.8%, respectively. The estimated incidence rates among these testees were 5.36% and 5.52% per year in 2008 and 2009. RESULTS: of this study showed that the HIV incidence stabilized at high level among MSM in Tianjin, calling for the effective interventions be taken for HIV/AIDS control and prevention.

A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads.

AIM: To develop a novel high-throughput format assay to monitor the integrase (IN) strand transfer (ST) reaction in vitro and apply it to a reaction character study and the identification of antiviral drugs. METHODS: The donor DNA duplex, with a sequence identical to the U5 end of HIV-1 long terminal repeats, is labeled at its 5' end with biotin (BIO). The target DNA duplex is labeled at its 3' end with digoxin (DIG). IN mediates the integration of donor DNA into target DNA and results in a 5' BIO and 3' DIG-labeled duplex DNA product. Streptavidin-coated magnetic beads were used to capture the product, and the amount of DIG was measured as the ST reaction product. The assay was optimized in 96-well microplate format for high-throughput screening purpose. Moreover, the assay was applied in a ST reaction character study, and the efficiency of the assay in the identification of antiviral compounds was tested. RESULTS: The end-point values, measured as absorbance at 405 nm was approximately 1.5 for the IN-mediated ST reaction as compared with no more than 0.05 of background readings. The ST reaction character and the half maximal inhibitory concentration (IC50) values of 2 known IN inhibitors obtained in our assay were similar to previously reported results using other assays. The evaluation parameter Z' factor for this assay ranged from 0.6 to 0.9. CONCLUSION: The assay presented here has been proven to be rapid, sensitive, and specific for the detection of IN ST activity, the reaction character study, as well as for the identification of antiviral drugs targeting IN.

High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction.

AIM: To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3'-processing reaction in vitro and apply it to inhibitor screening. METHODS: The recombinant human immunodeficiency virus (HIV)-1 integrase (IN) is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV-1 long terminal repeats (LTR). Based on the fluorescence properties of molecular beacons, the substrate is designed to form a stem-loop structure labeled with a fluorophore at the 5' end and a quencher at the 3' end. IN cleaves the terminal 3'-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal parameters were obtained. Moreover, 2 IN inhibitors were employed to test the performance of this assay in antiviral compound screening. RESULTS: The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3'-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recombinant HIV-1 IN, 400 nmol/L substrate, and 10 mmol/L Mn(2+). The IN 3'-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC50 values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay. CONCLUSION: Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3'-processing reaction and that it can be used for inhibitor screening.

[Experimental study of adoptive immunotherapy using CD3AK cells].

AIM: To analyze the immunological properties and biological activity of a monoclonal antibody (mAb) against CD3 molecule(yCD3), and to observe the tumor-suppressive activity of CD3AK cells in vitro and in vivo. METHODS: FCM was used to test the specificity of yCD3 and the immunological phenotype and cytokine production of CD3AK. 3H-TdR assay was used to measure the transformation of lymphocytes activated by yCD3. LDH assay was used to analyze the cytotoxic activity of CD3AK in vitro. Mice bearing tumors were used to observe the anti-tumor effect of CD3AK cells. RESULTS: yCD3 could bind specifically to T cells. 5 microg yCD3 could competitively inhibit 70% of standard anti-CD3 antibody to bind with CD3 molecules on cell membrane. 8 microg/L of yCD3 stimulated the proliferation of peripheral blood lymphocytes, which could be further boosted by IL-2 or anti-CD28 antibodies. Among activated CD3AK cells, CD3+, CD8+, and CD25+ cells increased. IL-2 and IFN-gamma producing CD3+ cells were also increased, to 3.29- and 2.47- fold, respectively, under the co-stimulation of anti-CD28 antibody. When the ratio of effective cells and target cells was 80:1, 57.54% target cells were killed. As compared with control, the percent of tumor inhibition in CD3AK cells treated tumor-bearing mice was 33.17%, and the inhibition rate of lung metastasis was 39.70%. The CD3AK cells treatment was more effective when combined with LAK cells. CONCLUSION: yCD3 could activate T cells and significantly induce the tumor-suppressive activity of CD3AK cells in vitro and in vivo, which lays a foundation for adoptive immunotherapy against tumors in clinical medicine.

[The application of SCGE-KIAS in monitoring of DNA damage in lymphocytes of tumor patients treated with cyclophosphamide].

Single cell gel electrophoresis assay (SCGE), also named as alkaline comet assay, was a simple, rapid and sensitive method to evaluate DNA damage. In this study SCGE technique was used to monitor DNA damage difference in tumor patients caused by chemotherapy, DNA damage distribution frequency and DNA damage characters were analyzed by komet image analysis system (KIAS). The results showed that cyclophosphamide greatly caused DNA damage in lymphocytes of tumor patients. There was significant difference of peripheral blood lymphocyte DNA damage between tumor patients and healthy controls. Tail length of lymphocytes were 33.69 +/- 7.56 micro m, and tail DNA% we re 31.51 +/- 5.4 6% in 10 cancer patients treated with cyclophosphamide, while Tail length were 1 6.2 +/- 1.5 micro m and tail DNA% were 7.46 +/- 1.15% in healthy controls. there was great significant difference on tail length and tail DNA% values between cancer patients and healthy controls (P < 0.01). In conclusion, the successful measurement of DNA damage caused by Cyclophosphamide treatment means that the alkaline comet assay as a valuable tool can be very useful in cancer epideminology study, and also be valuable to evaluate DNA damage status of patients in clinic.

[Construction of GPI-CTLA4Ig chimeric molecule and its expression on CHO-dhfr(-) cells].

AIM: To develop a novel immunosuppressant GPI-CTLAIg modified by glycosyl-phosphatidylinositol(GPI). METHODS: GPI-modified CTLAIg was produced by linking-up of CTLA4Ig with GPI-modification signal sequences from decay-accelerating factor (DAF). Chimeric molecule GPI-CTLA4Ig gene was cloned into eukaryotic expression vector pCI-dhfr. Using lipofectine-mediated gene transfer technique, pCI-GPI-CTLA4Ig was transfected into CHO-dhfr(-) cells, and the transfectants were screened by methotrexate (MTX). Expression of the recombinant protein was assessed by RT-PCR, ELISA, cell immunofluorescence staining and Western blot, and the purification of expressed protein was performed by protein A affinity chromatography. RESULTS: The chimeric molecule GPI-CTLA4Ig has been constructed and stablely expressed on CHO-dhfr(-) cells. CONCLUSION: GPI-modified CTLAIg will may be used as novel immunosuppressant for suppressing reaction in graft rejection.