acid substitutions in the HCV core protein during clinical HCV infections, alters the response to interferon based HCV therapy [45], suggesting that HCV core plays a role in interferon mediated effects and possibly TRAIL mediated apoptosis. We propose a model of HIV/HCV mediate liver disease whereby co-incubation of HIV gp120 treated and HCV core expressing hepatocytes results in accelerated hepatocyte death which is TRAIL mediated. Therefore, untreated HIV infection would create a TRAIL sensitive hepatocyte with increased TRAIL-R2 expression. When a second insult is introduced into the liver, such as HCV infection, TRAIL production increases and there is TRAIL mediated hepatocyte death; importantly TRAIL antagonists reverse this process, suggesting a novel treatment strategy for liver disease associated with HIV/HCV co-infection. Methods Chemicals and Reagents The following chemicals and reagents were used for experi- ments in the manuscript: Dulbecco’s modification of Eagle’s medium (DMEM) (Mediatech Inc., Manassas, VA), Fetal bovine serum (FBS) (Atlanta Biologicals Inc., Lawrenceville, GA), Recombinant superkiller TRAIL (skTRAIL) (Alexis Biochemicals, San Diego, CA), Recombinant HIV-1 IIIB envelope glycoprotein 120 (gp120) and recombinant human T- cell receptor sCD4 (Immuno Diagnostics Inc., Woburn, MA), Bovine serum albumin (BSA) (Sigma Chemical Co, St. Louis, MO), anti-actin and anti- tubulin antibodies (Molecular Probes, Carlsbad, CA), Antibodies to TRAIL-R1, -R2, -R3, -R4 and isotype control, for flow cytometry (BD Bioscience, San Diego, CA) and TRAIL-R2 for Western blot (Capralogics Inc., Hardwick, MA), active caspase-3 (BD Biosciences), TRAIL-R2-Fc chimera (R & D system, Minneapolis, MN), His tagged soluble HCV core protein (PROSPEC, Ness Ziona, Israel). Cell Viability The human hepatocyte cell line Huh7 (ATCC) was cultured in DMEM with 10% FBS and 100 units/mL of penicillin, 100 mg/ mL streptomycin, and 200 mM glutamine. The Huh7 cells were plated at a density of 36105 cells/well in a 6 well plate. 5 mg/mL HIV glycoprotein 120 IIIB [46] was used to prime Huh7 cells for six hours, and then cells were incubated with 100 ng/mL of skTRAIL or with HCV core transfected Huh7 cells for thirty six hours. In some experiments, Huh7 cells were pre-incubated for 1 hour at 37 C with rhTRAIL-R2/Fc chimera (0.5 mg/ml) or control before co-incubation. Apoptosis was determined by staining for Active Caspase 3 and analyzing by flow cytometry, as described below. HCV Core Transfection Huh7 cells were co-transfected with a mixture of supercoiled pTet-on, pTts (Clontech) and pTRE2-191 and lipofectamine 2000 reagent (Invitrogen, CA). Transfected cells were grown in complete culture medium for 24 h. To induce the conditional expression of HCV-core protein, transfected cells were treated with Doxycycline 1 mg/mL for the indicated period and the expression of core was confirmed by western blot analysis. Western Blots Huh7 cells were treated with 5 mg HIV gp120 for 24 hours and lysed in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1% Triton-X100, 2 mg/ml aprotinin, 5 mg/ml leupep- tin, 1 mM PMSF and 1 mM Saponin) on ice. Jurkat cells and PHA and IL2 stimulated PBMCs were used for positive controls. HCV Hepatocytes Kill HIV Hepatocytes through TRAIL 30 mg of protein was resolved on a 15% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel and electrotransferred to poly- vinylidene fluoride (PVDF) membranes (Millipore Corporation, Bedford, MA). Membranes were blocked with 5% nonfat milk in TBST [Tris base (10 mM), NaCl (150 mM), Tween (0.1%), pH 7.5] for two hours at room temperature and probed with anti- TRAIL-R2 (1:1000) antibodies overnight at 4 C. Actin (Sigma, St. Louis, MO) was used as a loading control and intensity was measured using EL Logic 2200 imaging software system. Real Time Quantitative PCR Total RNA was isolated from control or GP120 (1 mg/ml) treated Huh7 cells for 48 h, using Qiagen RNeasy mini kit (Qiagen, Valencia, CA) and reverse transcribed using the ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) with 2.5 mM random oligos, and 1 mg of RNA. The cDNA was amplified in 25 ml of PCR buffer with Platinum SYBR Green qPCR SuperMix-UDG with ROX kit (Invitrogen, CA) in the presence of TRAIL-R2 or actin specific primers (sense primer for TRAIL-R2 59-AAGACCCTTGTGCTCGTTGT - 39, anti- sense primer 59-AGGTGGACACAATCCCTCTG - 39, actin sense primer 59 -GAAACTACCTTCAACTCCATC - 39) and antisense primer 59 –CGAGGCCAGGATGGAGCCGCC -39. The PCR conditionsincluded a hot-start by 95uC for 2 min followed by 40 cycles of 95uC for 15 s, 60uC for 1 min. Samples were run in triplicates on the ABI PRISM 7700HT sequences detection system (Applied Biosystems) to determine the threshold cycle (CT). To determine the variation on mRNA expression, we analysis according to the Delta-Delta threshold (Ct) method, each Ct value was first normalized to the respective beta-actin Ct value. Flow Cytometry Huh7 cells were harvested, washed in cold PBS, and fixed in 2% paraformaldehyde overnight at 4 C. Cells were blocked with 2% BSA/PBS and stained with phycoerythrin (PE) conjugated anti- human anti-TRAIL-R1,-R2, -R3, -R4 or isotype control mAb (1:40) or rabbit polyclonal anti-active caspase-3 antibodies (1:40) and then permeabilized with 0.1% NP-40 for 15 minutes or isotype control (Abcam Inc., Cambridge, MA) for one hour on ice, and analyzed by flow cytometry (FACScan flow cytometer, BD Biosciences, San Diego, CA). Immunohistochemistry With Mayo Clinic Institutional Review Board approval (IRB 1039-03), liver samples were obtained through the National Disease Research Interchange (NDRI, Philadelphia, PA) from 10 HIV infected, 10 HCV infected, 10 HIV/HCV infected and 10 uninfected individuals. All patients were HBV negative and died of unrelated traumatic causes. Anti-TRAIL and anti-TRAIL-R2 staining was performed in 10 mM citrate at pH 6.0 and 99 C for 20 min. To block non-specific antibody binding, sections were incubated with blocking solution 1 [PBS, BSA 20 mg/ml (Serva, Germany, Cat. No. 11924), human IgG 1 mg/ml Gamma-Venin, (Aventis Behring)] for 20 min. Sections were then incubated overnight at 4 C in blocking solution in the presence of the first antibody or isotype-matched antibodies IgG1 and IgG2b at the same concentration, both obtained from DakoCytomation GmbH. Sections were washed twice in PBS and incubated with blocking solution 2 [20% normal goat serum from Dianova GmbH (Jackson ImmunoResearch Laboratories, Inc.)] for 20 min. After blocking, sections were incubated with secondary biotiny- lated antibody for 30 min at room temperature, rinsed twice for 5 min in PBS and incubated for 30 min with streptavidin-alkaline phosphatase [Super-Sensitive Detection Kit, BioGenex San PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27171