The PBMCs were diluted to 1 × 106 cells/mL in RPMI (Roswell Park Memorial Institute-1640) supplemented with 10% FBS and incubated for 24 h in 5% CO2 at 37°C, then cultured for 18 h in the presence or absence of recombinant IFN-α2a at 1000 IU/mL (Roferon-A, Roche, Basel, Switzerland). Supernatants were harvested and stored at −20°C until analysis. Levels of G-CSF and CXCL-10 were measured in the cell culture supernatants
by this website enzyme-linked immunosorbent assay (ELISA) as directed by the manufacturer (DuoSet, R&D Systems, Minneapolis, MN, USA). The limit of detection of both assays was 32 pg/mL. Human PBMCs were obtained from the buffy coat preparations of healthy blood donors (National Blood Centre, Dublin, Ireland) by density gradient centrifugation. Monocytes (> 97–99% CD14+) were purified by CD14+ immunomagnetic-positive selection (Miltenyi Biotec, Bergisch Gladbach, Germany). PBMCs,
CP-868596 chemical structure CD14+ monocytes or CD14- cells were cultured at 1 × 106 cells/mL in RPMI supplemented with 10% FBS for 18 h stimulated with 1 µg/mL CL097 (a TLR7/8 agonist, Invivogen, San Diego, CA, USA) in the presence or absence of 1000 i.u./mL recombinant IFN-α2a (Roferon A). Previous reports have shown that CL097 could activate TLR7 mediated NFκB at concentrations of 0.1 µg/mL and TLR8 mediated NFκB at concentrations of 1 µg/mL.16 Supernatants were harvested and stored at −20°C until analysis. Levels of G-CSF and CXCL10 were determined in the supernatant by ELISA (R&D Systems). Statistical analysis
was carried out using GraphPad Prism version 5.02 for Windows (GraphPad Software, San Diego, CA, USA). Differences between G-CSF and CXCL10 secretion under multiple culture conditions were evaluated using anova with the Newman–Keuls post-hoc test. The Wilcoxon matched-pairs test was used for comparisons within patients between individual time points during IFN-α therapy. Differences between different patient groups were calculated Dimethyl sulfoxide using the Mann–Whitney U-test. Correlations were calculated using the Spearman rank order correlation. A P-value < 0.05 was considered statistically significant. Details of patients and controls are shown in Table 1. Fifty five patients and 16 healthy controls participated in total. In two patients, treatment was discontinued early during the course of IFN-α therapy because of side-effects, and their results were completely excluded. Thirty eight patients achieved sustained virologic response (SVR), while 15 did not respond to treatment (NR). The single pre-treatment predictor of response was non-1 viral genotype (Table 1). When PBMCs were thawed and viability determined, only 43 patients had sufficient viable cells in their pre-treatment samples. In vitro secretion of G-CSF by PBMCs obtained from patients prior to their commencement on anti-viral therapy did not predict the subsequent requirement for therapeutic G-CSF to treat IFN-α induced neutropenia (Fig.