Journal: Journal of Inflammation Research
Article Title: Actives from the Micro-Immunotherapy Medicine 2LMIREG ® Reduce the Expression of Cytokines and Immune-Related Markers Including Interleukin-2 and HLA-II While Modulating Oxidative Stress and Mitochondrial Function
doi: 10.2147/JIR.S445053
Figure Lengend Snippet: Actives from the micro-immunotherapy medicine MIM-7 reduce the expression of HLA-II in HUVEC cells and in human PBMCs-derived M1-macrophages. ( A ) HUVEC cells were treated with 20 ng/mL IFN-γ for 48 hours and the expression of HLA-DR was assessed by flow cytometry. The results are presented as the mean ± standard deviation (S.D.) of the median of fluorescence of n = 3 replicates. ( B ) HUVEC cells were treated with 20 ng/mL IFN-γ, concomitantly with either the Veh. (grey histogram), or MIM-7 (purple histograms) for 48 hours and the expression of HLA-DR was assessed by flow cytometry. The effects of two independent capsules of MIM-7 (#1 and #2) were assessed. The results are presented as the mean ± S.D. of the percentage of the median fluorescence of the Veh., for n = 3 replicates. ( C ) Representative scheme of the experimental protocol. Human PBMCs from two healthy donors were seeded at 500.000 cells/well in complete medium added with 2% decomplemented human serum and 50 ng/mL macrophage colony-stimulating factor (M-CSF) to promote macrophage survival. On day 0 (D0), the cells were treated either with the Veh., or MIM-7, added with 20 ng/mL IFN-γ. The medium/treatment was renewed on D3, and the cells were challenged with 100 ng/mL LPS on D5. On D6, the cells were analyzed by flow cytometry for their viability and their expression levels of HLA-DP. LPS: lipopolysaccharide; RPMI: Roswell Park Memorial Institute medium. ( D ) Cell viability of M1-macrophages isolated from two healthy donors, treated for 6 days with either the Veh. (grey dots), or MIM-7 (purple dots), in the presence of IFN-γ (20 ng/mL) and LPS (100 ng/mL). The data are presented as the mean ± standard error of the mean (S.E.M.) of the percentage of viable cells amongst the total cells, for n = 3 replicates per donor. ( E ) Expression of the membrane-marker HLA-DP in M1-macrophages isolated and treated as described in ( C ). The data are presented as the mean ± S.E.M. of HLA-DP expression for n = 3 replicates per donor. The results are displayed as percentages of the Veh. condition for the two individual donors analyzed. The dotted lines in ( D and E ) highlight the effect of MIM-7 compared with the Veh.
Article Snippet: Briefly, PBMCs were isolated from 2 donors (25- and 30-year-old females); then, on D0, the cells were plated in 96-well plate, at the density of 500.000 cells/well, and cultivated in complete Roswell Park Memorial Institute Medium (RPMI) medium (ref: P04-17500, batch number #7131121, PAN-Biotech GmbH), added with 2% decomplemented human AB serum (ref: #H4522, batch number #SLCC1483, Sigma-Aldrich, Saint-Louis, MO, USA) and 50 ng/mL M-CSF.
Techniques: Expressing, Derivative Assay, Flow Cytometry, Standard Deviation, Fluorescence, Capsules, Isolation, Membrane, Marker
