Differences between timepoints were examined using the Student’s t test or Mann-Whitney U test, where appropriate, or one-way analysis of variance (ANOVA) with Kruskal-Wallis test and Dunn’s multiple
comparison test. Correlations were performed using Pearson’s correlation or the Spearman Rank method, where appropriate. Direct logistic regression was performed to assess for variables associated with treatment outcomes, whereas predictors of the change in HCV RNA or iron levels over 24 hours were examined using hierarchical multiple linear regression, inputting variables associated with the dependent Torin 1 concentration variable at univariate analysis with a cutoff of significance of P < 0.1. Data analysis was performed using PASW Statistics 18.0 and Graphpad Prism 4.0. P < 0.05 was deemed significant. Serum hepcidin increased significantly upon PEG-IFN-α/RBV treatment, peaking at
12 hours (Fig. 1A; 5-fold average increase; T = 0 versus T = 12, P < 0.0001). Notably, serum hepcidin was undetectable at all timepoints in one patient with hereditary hemochromatosis who had undergone therapeutic venesection prior to HCV treatment (Supporting Fig. S1). Indeed, venesection is known to strongly suppress hepcidin production in these patients.20 Although the liver is the predominant source of hepcidin, hepcidin production by human lymphocytes upon iron or cytokine stimulation was recently reported.21 VX-765 in vivo Hepcidin mRNA expression was therefore examined in PBMCs by qPCR and was found to increase significantly from baseline to 12 hours (Fig. 1B; P = 0.01). Despite Florfenicol this induction, a significant negative correlation between PBMC hepcidin mRNA expression and serum hepcidin levels at
12 hours was seen (Fig. 1C; rho = −0.5, P = 0.005), suggesting hepatic hepcidin production may negatively regulate that of monocytes. Moreover, a trend toward a significant negative correlation between pretreatment PBMC hepcidin mRNA expression and serum ferritin was seen (rho = −0.334, P = 0.077). For ethical reasons, it was not possible to obtain serial liver biopsies in order to examine hepatic hepcidin expression over the 24-hour time period. Accompanying the rise in serum hepcidin were dramatic alterations in serum iron parameters, with an ≈50% reduction in both serum iron levels (SI) and transferrin saturation (TS) following the initial PEG-IFN-α/RBV dose (Fig. 2A,B; Time 0 versus T = 12 or T = 24, P < 0.001). Iron changes did not differ according to HCV genotype or IL28b gene polymorphisms (Supporting Fig. 2). Serum hepcidin increase correlated closely with the fall in SI and TS (Fig. 2C,D; P < 0.0001), and similarly changes in hepcidin were highly predictive of changes in serum iron, controlling for age and gender (R square 0.58; coefficient B 0.78, SE 0.66, 1.40; P < 0.