A blocking peptide (BP), previously proven to inhibit CD81–E2 interaction,21 sequence CSPQYWTGPAC [OH], and control scrambled peptide CPWSAGYTQPC [OH] were prepared by the Organic Synthesis Core, Royal College of Surgeons, Ireland (purity >98%). HuT 78 cells (American Type Culture Collection, Rockville, MD) were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, Paisley, UK) containing supplements.16 Peripheral blood mononuclear cells (PBMCs), Tigecycline mw obtained from healthy volunteers, were separated on Ficoll-Histopaque density gradient (Fresenius

Kabi Norge AS, Oslo, Norway). The effect of HCV infectious serum on IL-2 production was tested using PBMCs from normal donors stimulated with plate-bound anti-CD3 or anti-CD3 and anti-CD28 (Pharmingen, San Diego, CA). For these experiments, normal/PCR+/PCR− serum (100 μL in a final volume of 500 μL serum-free medium) was incubated with cells for 1 hour prior to stimulation. HCVcc was generated as described.22 Briefly, RNA was transcribed in vitro from full-length genomes using the Megascript T7 kit (Ambion, Austin, TX) and electroporated into Huh-7.5 cells. High-titer stocks were generated

by 2 serial passages through naïve Huh-7.5 cells. Supernatants were collected at 72 and 96 hours after infection, pooled, concentrated, and stored at −80°C. Permission was received from the Ethics Committees of both St Vincent’s and St James’s Hospitals, Dublin, for all work on human tissue. Informed consent was obtained from all subjects.

Normal liver wedge biopsies were obtained from donor organs. HCV-infected this website liver was obtained at time Interleukin-3 receptor of transplantation for end-stage liver disease. Liver samples were immediately washed three times in Hank’s balanced salt solution and snap-frozen in liquid nitrogen, powdered using the Braun Mikrodismembrator II (Braun Apparate, Melsungen, Germany). Protein was extracted from ≈100 mg powdered tissue using 300 μL of lysis buffer (1% detergent Igepal, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate in phosphate-buffered saline) containing protease inhibitors. The extract was passaged several times through a 21-gauge needle (Beckton Dickinson) in lysis buffer, incubated on ice for 30 minutes, and centrifuged at 10,000g for 10 minutes at 4°C. Supernatant was harvested and total protein was quantified using the BCA Protein Assay Kit (Pierce, Rockford, IL). Formalin-fixed paraffin-embedded explant liver sections were immunostained with anti-CD3 or isotype-matched immunoglobulin G (DAKO) using the avidin-biotin complex immunoperoxidase method (Vectrastain Elite ABC Kit, Vector Laboratories, Burlingame, CA). Sections were microwaved for antigen retrieval in a 0.1 M sodium citrate buffer for 12 minutes prior to staining. Sections were evaluated for the presence of CD3+ cells using an Olympus light microscope by two independent observers.