Plasma was stored at −20°C until assay. CCL2 plasma level measurements were assayed by the enzyme-linked immunosorbent assay (ELISA) Proteasome inhibitor using the commercially available Quantikine assay system (R&D Systems, Abingdon, UK). This assay had a sensitivity of 5 pg/ml. A snap-frozen fragment of each liver biopsy was stored at −80°C.
Snap-frozen liver biopsies were crushed with a MagNalyser (Roche Diagnostics, Vilvoorde, Belgium). PolyA-mRNA was extracted using Magnapure (Roche Diagnostics) according to the manufacturer’s instructions, including DNase treatment. RNA was quantified using a Lightcycler 480 system (Roche Diagnostics) with a one-step quantitative reverse transcription–polymerase chain reaction (qRT–PCR). Hypoxanthine–guanine phosphoribosyltransferase (HPRT) was used as a housekeeping gene. Primers and probes were designed with primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA): CCL2 sense 5′-ACTTCACCAATAGGAAG ATCTCAGT-3′; anti-sense 5′-TGAAGATCACAGCTTCTTTGG-3′; probe 5′-(6Fam)-TCGGGAGCTATAGAAGAATCACCAGCA-(Tamra)-3′; IL-8 sense 5′-CTCTCT TGGCAGCCTTCCT-3′; anti-sense 5′-TCTAAGTTCTTTAGCACTCCTTGG-3′; probe 5′-(6Fam)-TCTGCAGCTCTGTGTGAAGGTGCA-(Tamra)-3′. GSK458 research buy Copy numbers were calculated as described previously [19]. Paraffin-embedded liver biopsy sections were stained with haematoxylin and eosin
and Sirius red. AH was defined by the presence of hepatocytes with ballooning degeneration with or without Mallory’s hyaline surrounded by polymorphonuclear leucocytes [17,18]. Paraffin-embedded formalin-fixed liver biopsies were deparaffinized in xylene and rehydrated in graded alcohol and water. Tissue slides were incubated with monoclonal anti-myeloperoxidase (MPO) (clone 59A5, 1/200; Leica-Ménarini, Florence, Italy), anti-CD3 (clone PS1, 1/300; Leica-Ménarini) and anti-CD68 (clone KP1,1/1000; Dako, Glostrup, Denmark) antibodies for detection of neutrophils, T lymphocytes and macrophages, respectively. Diaminobenzidine (DAB) (Dako) was used as chromogen. Immunohistochemistry for IL-17 was performed as described previously [20]. The numbers of positive cells for different staining were counted by two independent Astemizole investigators
(D.D, L.V.) in a blinded manner on 20 fields per sample (original magnification ×400). Granulocyte pellets obtained after a Ficoll gradient of blood from ALD patients were depleted of erythrocytes by hypotonic saline lysis (NH4CL 15 mM, NaHCO310 mM, ethylenediamine tetra-acetic acid 0·1 mM, pH 7·4). Neutrophils were identified by their light-scattering properties and expression of CD15 and CD16. Surface staining was performed with phycoerythrin-labelled anti-CD15, fluorescein-isothiocyanate-labelled anti-CD16 and Alexa fluor-647-labelled anti-CCR2 (clones HI98, 3G8 and 48607, respectively; BD Biosciences, Erembodegem, Belgium) mouse anti-human antibodies. Cell analysis by flow cytometry was performed using FACScalibur (BD Biosciences).