| Objective1. To investigate the level of CD226 expression on T lymphocytes (mCD226 ) and NK cells from HIV/AIDS individuals, and its characteristics in comparing with other immune activation markers.2. To demonstrate the correlation between soluble CD226 ( sCD226) and mCD226 expression in HIV /AIDS individuals.3. To make sure the clinical significants of mCD226/sCD226 in evaluating immune status and disease progression of HIV /AIDS patients.Materials and Methods1. Reagents and instrumentsFluorescein isothiocyanate (FITC) conjugated monoclonal antibody (mAb) against GD226 ( LeoAl) was kindly provided by prof. Jin from Fourth Military Medical University. Phycoerythrin ( PE) and Peridinin chlorophylla protein (PerCP) conjugated anti- CD3, CD4, CD8, CD16 mAb , CD4 T lymphocyte absolute count kit ( TruCount ) and flow cytometry ( FACSCalibur) were all products from Becton Dickinson immuno-cytometry system ( USA). The kits used to detect HIV-1/2 antibodies and HFV-1 antigen (p24) was purchased from Or-ganon Teknika Company ( Holland) , Western Blot ( WB ) test reagents were products of Genelabs Diagnostics Company (Singapore). RPMI-1640 was from GIBCO Company, PHA and rIL-2 were from Sigma Company.2. Study Subjects34 of HIV-1 positive individuals included in the study were screened out byusing the antibody detection kits, and confirmed through WB test in our AIDS research center. 26 males and 8 females, range from 18 to 40 years old, Ac-ccording to the United States center for disease control (CDC) criteria for HIV infection disease (1993 ) , HIV seropositive individuals should be stratified a-symptomatic stage ( AS) ( CD4+ T lymphocyte greater than 200/ul) and AIDS stage (CD4+T less than 200/ul) , 25 cases were in AS, 9 cases in AIDS stage , they were infected through blood transfusion, drug abuse and sex course,no receiving anti-retroviral therapy before the studying. 26 healthy adult volunteers were selected as the control, including 16 males and 10 females, age is from 18 to 42 years old. Peripheral venous blood was drawn from HIV infected /AIDS patients and healthy control, and put into EDTA-K2 containing tubes.3. Peripheral blood mononuclear cells ( PBMC) infected by HIV-1 strain in vitroHIV-Isr33 strain and MT-4 cell line ware kind gifts from Reference Laboratory in CDC China, The infectious liter (TCID^ ) was examined according to routine procedure. PBMC were separated from healthy individuals, cells (106yjV ml ) were divided into several groups for further test, one group was mixed with HIV-1SF33( 125 TCIDso) culture for2h, wash twice, such cells were cultured, at the mean time establish control groups with or without HIV-1 and /or PHA mixed. Determined HIV-1 p24 antigen by collecting cultural supernatant to make sure the infectious effect on PBMCs.4. Immunofluorescence Stain and analysis by flow cytometry (FCM)To perform CD4 + T lymphocyte absolute counts; using a no-wash procedure with TruCOUNT kit, analysis by MultiSET software. To analysis NK related phenotypes by using a lyse procedure: different fluorochrome-conjugated mAbs were combinated as following: CD226/CD16/CD3, CD226/CD8/CD3, CD226/CD4/CD3. Adding 6ul of each mAb into Falcon tubes as the combination shown above, 80ul whole blood was added, after a 30min incubation at room temperature, treating the stained sample with Lysing Solution to lyse eryth-rocytes, then wash the sample twice with PBS to remove excess antibody and debris, resuspended in 0. 5ml phosphate buffered saline (PBS) solution consist of 0.5% polyformaldehyde, Finally, analyze the stained cells by FCM CellQuestsoftware, calculate the percentage ( % ) of antigen expression and specific subset absolute count ( cells/ mm3 ).5. Double antibody sandwich ELISA for the detection of sCD226Microtitre plates were coated with mAb-LeoAl, incubated at 4t for 24 h, wash plates two times. Add the test sample/standard CD226 Fc protein, add HRP-conjugated mAb, incubate at room temperature for 45 mins. Add freshly prepared...
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