He received remuneration for the assembly of educational material from Novartis. was verified by comparison with healthy controls and was characterized by up-regulation of LFA-1 (CD3+CD4+ T cells, B cells), VLA-4 (CD3+CD8+ G6PD activator AG1 T cells, NK cells), ICAM-1 (B cells) and ICAM-3 (NK cells). Effects of GA treatment were most pronounced after 6 months and included attenuated levels of LFA-1 (CD3+CD4+) and VLA-4 (CD3+CD4+, CD3+CD8+, NK, NK?T, monocytes). Further effects included lowering of ICAM-1 and ICAM-3 levels in almost all immune cell subsets. Soluble AM levels in RRMS did not differ from healthy controls and remained unaltered after GA treatment. The deregulated pro-migratory expression profile of cell-bound AM is usually altered by GA treatment. While this alteration may contribute to the beneficial action of the drug, the protracted development and unselective changes indicate more secondary immune regulatory phenomena related to these effects. to GA treatment were recruited [for 10?min. Aliquots were stored at ?20C until further processing. Determination of cell-bound AM on immune cell subsets Surface levels of ICAM-1 [RR1/1, fluorescein isothiocyanate (FITC); eBioscience, Vienna, Austria], ICAM-3 (CBR-IC3/1, FITC; eBioscience), CD11a/LFA-1 (253, 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 (B911, PC5), CD19+ B cells (J4119, 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 circulation 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 of unfavorable isotype-matched antibodies [immunoglobulin (Ig)G1-FITC/PE (clone ZX-3, Exalpha, Watertown, MA, USA), IgG-ECD/PC5/PC7 (clone 6791Mc7)] 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)??1000 (annotation: multiplication by 1000 in reference to the log scale). Unless specified normally, all antibodies were obtained from Beckman Coulter. Determination of soluble AM The human adhesion 6plex FlowCytomix Multiplex kit (eBioscience) was utilized for measuring serum concentrations of soluble AM [E-selectin (endothelial leucocyte adhesion molecule-1, 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 (53), SE-selectin (12), sICAM-3 (48), sPECAM-1 (08), sVCAM-1 (02) and sP-selectin (57). Statistics The GraphPad Prism version 50 program (GraphPad Prism Software Inc., San Diego, CA, USA) was utilized for statistics and preparation of graphs. As none of the data sets were distributed normally, non-parametric tests were utilized for all analyses. A em P /em -value??005 was considered to represent a statistically significant difference. Results Surface expression of AM in RRMS and HC 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 ( em n /em ?=?19) with those from treatment-naive MS patients ( em n /em ?=?15, Fig.?1). In MS patients, ICAM-1 levels were increased significantly on the surface of B cells ( em P /em ? ?005), ICAM-3 on NK cells G6PD activator AG1 ( em P /em ? ?001), LFA-1 ( em P /em ? ?001) on CD4+ T cells [which was also reflected in the CD3+ T cell populace, both ( em P /em ? ?001)] and on B cells ( em P /em ? ?005). VLA-4 levels increased significantly on G6PD activator AG1 the surface of CD8+ T cells ( em P /em ? ?005) and CD19+ B G6PD activator AG1 cells ( em P /em ? ?005). No differences in AM expression levels were observed on NK?T cells or monocytes from MS patients and HC. Open in a separate window Physique 1 Cell-bound adhesion molecules (AM) on different mononuclear cell populations from treatment-naive patients with relapsingCremitting multiple sclerosis ( em n /em ?=?15) and healthy controls ( em n /em ?=?19) (aCh). The box-plots represent medians and the errors bars the interquartile range (25th and 75th percentiles). * em P /em ? ?005, ** em P /em ? ?001; mo.: months. Effects of GA on AM expression levels in RRMS To determine the potential impact of GA therapy on cell-bound AM, the surface expression of ICAM-1, ICAM-3, LFA-1 and VLA-4 were studied around the immune cell subsets at different time-points (15, 6, 9 and 12 months from treatment initiation) during a period of 12 months and related to baseline GPM6A levels (Fig.?2). A decrease in the expression levels of ICAM-3 was found on all immune cell subsets following treatment with GA. Comparable results were observed for ICAM-1 with GA treatment, with B cells, CD8+ T cells and monocytes being the only exceptions. ICAM-1 expression was unaltered on B cells over the entire observation.