L. could mediate robust NK\ADCC were recognized and compared with putative neutralizing epitopes. Materials and methods Study subjects A total NOS2A of 31 chronic HCV service providers and 49 healthy controls were recruited from a village in central China 9. Chronic HCV contamination was recognized by anti\HCV responses and detection of HCV RNA. The clinical and laboratory characteristics of the study subjects are summarized in Table 1. Plasma HCV antibodies were detected using the Architect anti\HCV assay Gambogic acid Gambogic acid (Abbott GmbH & Co KG, Wiesbaden, Germany) and confirmed by the HCV\recombinant immunoblot assay (RIBA) assay (Wantai Biological Pharmacy, Beijing, China). HCV RNA was detected using the Abbott actual\time HCV amplification kit (Abbott Molecular, Des Plaines, IL, USA), according to the manufacturer’s instructions. None of the hepatitis C patients received any form of anti\HCV therapy, and all participants were unfavorable for hepatitis A computer virus (HAV), hepatitis B computer virus (HBV), HIV and tuberculosis (TB). Plasma and peripheral blood mononuclear cells (PBMCs) were separated from ethylendiamine tetraacetic acid (EDTA) anti\coagulated whole blood specimens and stored at ?80C and ?180C, respectively. The study protocol was approved by the institutional review government bodies of Peking University or college Health Science Center. Informed consent was obtained from each individual enrolled in the study. Table 1 Characteristics of 31 chronic hepatitis C computer virus (HCV) service providers and 49 healthy controls
Number3149Female (%)* 18 (581)30 (612)Age (years) ? 48 (62???33)46 (58???34)BMI ? 232 (252???204)228 (250???206)Clinical dataanti\HCV S/CO value ? 1412 (103???162)NegativeHCV RNA (log10 IU/ml) ? 643 (660???591)NegativeHCV genotype ?1b21n.a. ?2a8n.a. ?others0n.a.ALT (IU/l) ? 48 (128???17)28 (34???15)AST (IU/l) ? 44 (109???18)26 (33???14)Total protein (g/l) ? 782 (852???706)768 (824???724)Albumin (g/l) ? 440 (513???365)456 (530???387)Total bilirubin (mol/l) ? 141 (172???112)118 (154???75)Direct bilirubin (mol/l) ? 43 (59???30)4.0 (5.9???2.7) Open Gambogic acid in a separate window *Number of cases (%). ?Mean (range). BMI?=?body mass index; n.a.?=?not available; S/CO?=?transmission/slice\off; ALT?=?alanine aminotransferase; AST?=?aspartate aminotransferase. Evaluation of the non\specific antibody\dependent NK cell responses by intracellular cytokine staining A novel non\specific ADCC assay based on intracellular cytokine staining (ICS) was used to detect ADCC responses by circulating CD56+ NK cells 10. Briefly, 1 105 P815 cells (a mouse leukaemic cell collection) were treated with medium or with a 1?:?100 dilution of polyclonal rabbit anti\mouse lymphocyte serum (Accurate Chemical and Scientific Corp., Westbury, NY, USA) for 1 h at 37C/5% CO2 in a volume of 200 l of R10 medium (RPMI\1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mmol L\glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin), and then washed twice with ice\chilly R10 medium; 1 106 peripheral blood mononuclear cells (PBMCs) were stimulated with R10 medium alone, uncoated P815 cells, antibody \coated P815 cells or phorbol myristate acetate (PMA) plus ionomycin (positive control) (Sigma\Aldrich, St Louis, MO, USA). Cells were cultured with CD107a\phycoerythrin\cyanin 5 (PE\Cy5) (clone H4A3; BD Biosciences, San Jose, CA, USA), Golgi\Quit (BD Biosciences) and brefeldin A (Sigma\Aldrich) for 6 h at 37C/5% CO2. After culture, PBMCs were stained with CD3\eFluor 450 (clone 17A2; eBioscience; San Diego, Gambogic acid CA, USA), CD16\allophycocyanin (APC)\Cy7 (clone 3G8; BD Biosciences) and CD56\PE\Cy7 (clone B159; BD Biosciences). Then, cells were permeabilized using 025% saponin (Thermo Fisher Scientific; Waltham, MA, USA), and ICS was carried out with IFN\ fluorescein isothiocyanate (FITC) (clone 25723.11; BD Biosciences) and TNF\\APC (clone 6401.1111; BD Biosciences). After staining, cells were washed in phosphate\buffered saline (PBS) and fixed with 2% paraformaldehyde (PFA). All data were acquired on a BD FACS Fortessa (BD Biosciences) and analysed using FlowJo software (Treestar, Ashland, OR, USA). NK cell purification Untouched NK cells were enriched from PBMCs using an NK cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). In brief, NK cells were negatively isolated by depleting non\NK cells (i.e. T cells, B cells, stem cells, dendritic cells, monocytes, granulocytes and erythroid cells) using a cocktail of biotin\conjugated antibodies, followed by streptavidin\coated microbeads. Isolation of highly real NK cells was achieved by depletion of magnetically labelled cells..
