role of IFNs control of HIV replication, it is of importance to determine the specific impact of opiates on IFN signaling pathway and the mechanisms responsible for the actions. Materials and Methods Cell culture Peripheral blood was purchased from the Center for AIDS Research at the University of Pennsylvania. The protocol used to isolate blood from donors, purify the blood components, and distribute this material to the investigators was approved by the IRB of the Center for AIDS Research. These blood samples were screened for all normal blood-borne pathogens and certified to be pathogen free. Monocytes were purified from peripheral blood of three healthy adult donors according to our previously described technique. Freshly isolated monocytes were cultured in 48well culture plates at a density of 2.56105 cells/well in Dulbecco modified Eagle medium containing 10% fetal calf serum. Macrophages refer to 7-day-cultured monocytes in vitro. SIV was added to a cocktail containing poly, oligo, MgCl2, and dTTP and incubated for 20 h at 37uC. Then, 30 ml of the cocktail was spotted onto DE81 paper, dried and washed five times with 26 saline-sodium citrate buffer and once with 95% ethanol. The filter paper was then air-dried. Radioactivity was counted in a liquid scintillation counter. RNA extraction and real-time RT-PCR Total RNA from macrophages was extracted with Tri-Reagent as previously described. Total RNA was subjected to RT using the RT system with random primers for 1 h at 42uC. The reaction was terminated by incubating the reaction mixture at 99uC for 5 min, and the mixture was then kept at 4uC. The resulting cDNA was then used as a template for real-time PCR quantification. Real-time PCR was performed with 1/10 of the cDNA with the iQ SYBR Green Supermix as previously described. The amplified products were visualized and analyzed using the software MyiQ provided with the thermocycler. The oligonucleotide primers were synthesized by Integrated DNA Technologies, Inc. and sequences will “1635054 be available upon request. The cDNA was amplified by PCR and the products were measured using SYBR green I. The data were normalized to glyceraldehyde-3phosphate dehydrogenase and presented as the change in induction relative to that of untreated control cells. Reagents Morphine MEDChem Express 1173097-76-1 sulfate was obtained from National Institute on Drug Abuse. Naltrexone was obtained from Sigma. Mouse anti-HIV p24 monoclonal obtained from the AIDS Research and Reference Reagent Program. Mouse anti-SIV p28 monoclonal antibody was purchased from Fitzgerald Industries. Alexa Fluor 488 goat antimouse IgG ” and Hoechst 33342 were purchased from Invitrogen. Immunofluorescence assay Morphine and/or naltrexone treatment Seven-day-cultured macrophages were 
treated with or without morphine at different concentrations for different time points. To investigate whether naltrexone antagonizes the morphine action, we used naltrexone to treat macrophages for 1 h followed by morphine treatment. There were no cytotoxic effects of morphine and naltrexone treatment on macrophages as demonstrated by trypan blue dye staining. The macrophages infected with HIV Bal or SIV DeltaB670 strain were cultured on glass coverslips at adensity of 2.56105/well in 48-well plates. The macrophages were washed with 16 cold PBS twice. Cells were fixed at 4uC in 4% paraformaldehyde-4% sucrose in PBS for 20 min followed by 0.2% Triton X-100 for additional 10 min. Cells were blocked in Block Solution for 1 h at room temp
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