The Study On The Resistance Of CD4+T Lymphocytes From Chinese HIV Exposed Seronegative Individuals

ObjectiveThe study was to learn about the relative resistance of CD4 + T lymphocytes from ESN to HIV - 1 strains, therefor to study the mechanism of ESN in China.Methods1. Subjects; One male and ten female HIV - seronegative individuals confirmed by western blot were recruited from liaoning and Jilin province, with P24 - antigen seronegative , more than 10 times sexual exposed to HIV -1 strains without protection within recent 6 months, aging from 25 to 45. The control group consist of ten healthy individuals, One male and nine female , aging from 25 to 45.Sample collection: 40ml sterile whole blood were collected with heparin anticoagulant.The origin of HIV -1 stains : Seven HIV -1 strains were prepared . One was lab - virus strain SF33 ( donated by CDC in China ). Six were isolated in this work.HIV -1 virus isolated : HIV -1 virus was cultured by the method of mini -whole blood. PBMC(peripheral blood mononuclear cell) of healthy donators were isolated by standard Ficoll - hypaque density centrifugation. The cells were stimulated with PHA for 72h in RPMI -1640. Moderating PBMC concentration to 1 million per well. Adding 25, 50, 100ul whole blood respectively to per well and cultured in 37℃.. Collected supernatant per 3 days until 50 days and detected P24 and store in - 130℃.Virus tropism assay: Monocytes were separated from freshly isolated PBMC by adherence onto plastic for 2 h at 37℃. Monocytes changed to macrophages after culturing 5 days. Cocultured macrophages and MT2 cells with 100 TCID50 HIV -1 virus and observed cytopathogenic cells and detected p24 — antigen.HIV viral load assay: HIV - 1 viral load was measured in thawed plasma samples using a quantitative RT - PCR assay ( COBAS AMPLICOR, Roche Company, Switzerland) according to the instructions provided with the HIV -1 RNA monitor Test Kit version 1.5. The detectable range was 400 - 7. 5 × 10~5 copies/ml.CD4 + T cell isolates: PBMC of subjects were isolated by standard Ficoll -hypaque density centrifugation. Remove monocytes, Per 1 x 10 cells were treated with 20ul anti - CD4 immunomagenetic beads in 6℃ for 15 minutes. Wash at 300g for 10 minutes. 500ul cell suspend liquor went through LS column to positive selection for CD4 + T lymphocytes.Infection of CD4 + T cell with HIV - 1: Cultures of CD4 + lymphocytes from ESN and control subjects were inoculated with HIV - 1 of 600TCID50 in 37℃ for 4 hours. Washed with PBS twice at 300g for 10 minutes and cultured with RPMI - 1640 ( adding IL -2 by 100U/ml). Virus replication was assessed at regular interval over a 2 - week period by measuring P24 antigen in the culture supematants.2. Statistical Analysis: Nonparametric data for mean area under curve were analyzed using Mann Whitney U - test with SPSS 11.0 software.Results1. The characters of HIV - seropositive and HIV strains; All virus strains were B' isoforms. Four virus strains were NSI and two were SI.2. CD4 + T lymphocytes of ESN and control subjects infected by different HIV -1 strain : ESN group attain lower P24 replication than control group when infected by M - tropism HIV - 1 ( P <0.05). ESN group show lower P24 replication when infected by M - tropism HIV - 1 than infected by T - tropism HIV-1 (P <0. 05) . There are no difference in two group when infected by T -...