Aliquots were stored at ?20ï¿½ï¿½C until further processing. Surface levels of Icam-1 [RR1/1, fluorescein isothiocyanate (FITC); eBioscience, Vienna, Austria], ICAM-3 (CBR-IC3/1, FITC; eBioscience), CD11a/LFA-1 (25ï¿½ï¿½3, FITC; alpha-L subunit of LFA-1) and CD49d/VLA-4 (clone HP2/1, FITC; alpha-4 subunit of VLA-4) on seven immune cell subsets [CD3+ T cells (clone UCTH1, ECD), CD3+CD4+ T cells (SFCI12t4D11, PC7), CD3+CD8+ T cells (B9ï¿½ï¿½11, PC5), CD19+ B cells (J4ï¿½ï¿½119, PC7), CD14+ monocytes (RMO52, PC5), CD56+CD16+ NK cells (clones NKH-1 and 3G8, both phycoerythrin (PE)] and CD56+CD16+CD3+ NK?T cells expressed as relative fluorescence intensities (RFI) were analysed by five-colour flow cytometry (Cytometrics FC500; Beckman Coulter, Vienna, Austria), as described recently . For improved inter- and intra-individual comparability, RFI levels were calculated from median fluorescence intensities (MFI) of the single immune cell subpopulations by correcting them for the MFI VE822 of negative isotype-matched antibodies [immunoglobulin (Ig)G1-FITC/PE (clone ZX-3, Exalpha, Watertown, MA, USA), IgG-ECD/PC5/PC7 (clone 679ï¿½ï¿½1Mc7)] and relating them to the RFI of the positive controls [CD45-FITC/PE/ECD/PC5/PC7 (clone J33)] . The calculation was as follows: MFI (test sample)???MFI (isotype control)/MFI (positive control)???MFI (isotype control)?ï¿½ï¿½?1000 (annotation: multiplication by 1000 in reference to the log scale). Unless specified otherwise, all antibodies were obtained from Beckman Coulter. The human adhesion 6plex FlowCytomix Multiplex kit (eBioscience) was used for measuring serum concentrations of soluble AM [E-selectin (endothelial leucocyte adhesion molecule-1, 3-Methyladenine manufacturer ELAM-1), sICAM-1, sICAM-3, sPECAM-1 (CD31), P-selectin (CD62P, GMP-140) and sVCAM-1 (CD106)], according to the manufacturer's instructions. The prespecified detection limits were as follows (in ng/ml): sICAM-1 (5ï¿½ï¿½3), SE-selectin (1ï¿½ï¿½2), sICAM-3 (4ï¿½ï¿½8), sPECAM-1 (0ï¿½ï¿½8), sVCAM-1 (0ï¿½ï¿½2) and sP-selectin (5ï¿½ï¿½7). The GraphPad Prism version 5ï¿½ï¿½0 program (GraphPad Prism Software Inc., San Diego, CA, USA) was used for statistics and preparation of graphs. As none of the data sets were distributed normally, non-parametric tests were used for all Ibrutinib analyses. A P-value?ï¿½ï¿½?0ï¿½ï¿½05 was considered to represent a statistically significant difference. The four cell-bound AM (ICAM-1, ICAM-3, LFA-1, and VLA-4) were detected on all seven investigated immune cell subsets, although at different levels. To substantiate the proposed disease-related deregulation of AM expression patterns in MS, we compared AM levels of immune cells from HC (n?=?19) with those from treatment-naive MS patients (n?=?15, Fig.?1). In MS patients, ICAM-1 levels were increased significantly on the surface of B cells (P?0ï¿½ï¿½05), ICAM-3 on NK cells (P?0ï¿½ï¿½01), LFA-1 (P?0ï¿½ï¿½01) on CD4+ T cells [which was also reflected in the CD3+ T cell population, both (P?0ï¿½ï¿½01)] and on B cells (P?0ï¿½ï¿½05).