Moreover, up-regulation of Smad6 and Smad7, inhibitors of BMP signaling, occurs in HFE-HH, identifying these molecules as potential aggravators of disease pathogenesis which may act by preventing appropriate induction

of hepcidin in the setting of hepatic iron overload. ALT, alanine aminotransferase; BMP, bone morphogenic protein; HAMP, selleck chemical hepcidin antimicrobial peptide; HH, hereditary hemochromatosis; Id1, inhibitor of differentiation 1; pSmad, phosphorylated Smad; RT-PCR, reverse transcription polymerase chain reaction; Smad, small mothers of decapentaplegic; SNP, single-nucleotide polymorphism; TfR, transferrin receptor; TGFβ, transforming growth factor β. Ethical approval for this study was obtained from the Research Ethics Committee of the Mater Misericordiae University Hospital, Dublin, Ireland. Informed written consent was obtained from all patients involved. Liver tissue was collected from 20 male C282Y selleck screening library homozygotes with HH prior to venesection therapy. Control liver tissue was obtained from four donor livers at time of transplant and three biopsies were from patients undergoing liver biopsy for investigation of abnormal liver function tests that demonstrated no inflammation or fibrosis, and were negative for iron staining. The immunohistochemical component of this study was extended with the

addition of a further 10 untreated C282Y homozygotes and three individuals with medchemexpress non-HFE hepatic hemosiderosis associated with chronic viral hepatitis, all of whom had hepatic iron concentrations measured. All study subjects were male and had no cirrhosis. Controls were negative for the HFE mutations C282Y and His63Asp (H63D). Following an overnight fast, blood samples were obtained from all patients with HH for serum ferritin, transferrin saturation,

iron, total iron binding capacity, full blood count, and liver function tests (including alanine aminotransferase [ALT]). HFE genetic analysis for C282Y and H63D mutations was performed using LightCycler technology (Roche Diagnostics) with Genes-4U ToolSets. Hepatic iron concentration was measured as described.34 Liver biopsies were independently evaluated by a single histopathologist (A.F.) for grading of hepatocellular iron staining (Perl’s Prussian blue stain) and fibrosis (METAVIR score).35 Liver samples were snap-frozen or placed in RNAlater and stored at −80°C prior to use. Total RNA was extracted using the RNAeasy kit (Qiagen, UK). Reverse transcription was performed using the high-capacity complementary DNA reverse transcription kit (Applied Biosystems [AB], Carlsbad, CA). Gene expression analysis for hamp(hepcidin antimicrobial peptide), bmp6, smad4, smad6, smad7, and Id1 was performed using AB gene expression assay systems, using AB 7000 sequence detector.