Journal: International Journal of Molecular Sciences

Article Title: Increased Expression of Ephrins on Immune Cells of Patients with Relapsing Remitting Multiple Sclerosis Affects Oligodendrocyte Differentiation

doi: 10.3390/ijms22042182

Figure Lengend Snippet: Ephrin expression levels on immune cells of patients with relapsing-remitting multiple sclerosis (RR-MS) and of healthy controls. Immune cells of patients with relapsing-remitting multiple sclerosis (MS) and of healthy controls (HC) were analyzed by flow cytometry for the percentages of expression of ephrin-A1 ( A ), ephrin-A2 ( B ), ephrin-A3 ( C ) and ephrin-B3 ( D ) on total PBMC, CD3 + T cells, CD19 + B cells and CD14 + monocytes. * p < 0.05, ** p < 0.01.

Article Snippet: Recombinant human Fc-IgG fragments or ephrinA2-Fc fragments (R&D, Minneapolis, MN, USA) were mixed with or without anti-ephrin-A2 blocking antibody (AF607; R&D) and incubated at 37 °C for 1 h prior to stimulation.

Techniques: Expressing, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Increased Expression of Ephrins on Immune Cells of Patients with Relapsing Remitting Multiple Sclerosis Affects Oligodendrocyte Differentiation

doi: 10.3390/ijms22042182

Figure Lengend Snippet: Ephrin means fluorescence intensity on different immune cells.

Article Snippet: Recombinant human Fc-IgG fragments or ephrinA2-Fc fragments (R&D, Minneapolis, MN, USA) were mixed with or without anti-ephrin-A2 blocking antibody (AF607; R&D) and incubated at 37 °C for 1 h prior to stimulation.

Techniques: Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Increased Expression of Ephrins on Immune Cells of Patients with Relapsing Remitting Multiple Sclerosis Affects Oligodendrocyte Differentiation

doi: 10.3390/ijms22042182

Figure Lengend Snippet: Ephrin expression levels on a subset of T cells of patients with RR-MS patients and of healthy controls. T cells of patients with RR-MS and HC were analyzed by flow cytometry for the percentages of expression of ephrin-A1 ( A ), ephrin-A2 ( B ), ephrin-A3 ( C ) and ephrin-B3 ( D ) on CD4 + T cells, CD8 + T cells, Tregs, Th1, Th2 and Th17 cells. * p < 0.05.

Article Snippet: Recombinant human Fc-IgG fragments or ephrinA2-Fc fragments (R&D, Minneapolis, MN, USA) were mixed with or without anti-ephrin-A2 blocking antibody (AF607; R&D) and incubated at 37 °C for 1 h prior to stimulation.

Techniques: Expressing, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Increased Expression of Ephrins on Immune Cells of Patients with Relapsing Remitting Multiple Sclerosis Affects Oligodendrocyte Differentiation

doi: 10.3390/ijms22042182

Figure Lengend Snippet: Ephrin mean fluorescence intensity on different T cell subtypes.

Article Snippet: Recombinant human Fc-IgG fragments or ephrinA2-Fc fragments (R&D, Minneapolis, MN, USA) were mixed with or without anti-ephrin-A2 blocking antibody (AF607; R&D) and incubated at 37 °C for 1 h prior to stimulation.

Techniques: Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Increased Expression of Ephrins on Immune Cells of Patients with Relapsing Remitting Multiple Sclerosis Affects Oligodendrocyte Differentiation

doi: 10.3390/ijms22042182

Figure Lengend Snippet: Immune cells of patients with RR-MS increased EphA-receptor phosphorylation in a co-culture bioassay. HEK-293T cells were unstimulated or stimulated with recombinant proteins or co-cultured with immune cells of HC or RR-MS patients and immunostained to phosphorylated Eph A2+A3+A4-receptors (p-Eph-r) and DAPI nuclear staining. ( A ) Representative confocal microscopy images of HEK-293T cells unstimulated or stimulated with 0.5 µg/mL of recombinant human Fc-IgG negative control (hrFc IgG), 0.5 µg/mL human recombinant ephrin-A2 (hrEphrin-A2) protein, and pre-incubated with or without 20 µg/mL anti-ephrin-A2 blocking antibody against ephrin-A2 (anti-ephrin-A2). Magnification ×20; scale = 50 µm. ( B ) Average of the Eph-receptor phosphorylation analysis shown in A. p < 0.05, * p value of hrEphrin-A2 vs. unstimulated (US), ** p value of hrEphrin-A2 vs. hrFc IgG, *** p value of hrEphrin-A2 vs. hrEphrin-A2+ anti-ephrin-A2. ( C ) Representative confocal microscope images of immunofluorescence staining for p-Eph-r of 293T-HEK cells co-cultured with immune cells of HC or RR-MS patients −/+ anti-ephrin-A2 antibody. Magnification ×20; scale = 50 µm. ( D ) Average of the Eph-receptor phosphorylation analysis shown in ( C ). * p value without vs. with anti-ephrin-A2, ** p value of MS vs. US, *** p value of MS vs. HC.

Article Snippet: Recombinant human Fc-IgG fragments or ephrinA2-Fc fragments (R&D, Minneapolis, MN, USA) were mixed with or without anti-ephrin-A2 blocking antibody (AF607; R&D) and incubated at 37 °C for 1 h prior to stimulation.

Techniques: Phospho-proteomics, Co-Culture Assay, Bioassay, Recombinant, Cell Culture, Staining, Confocal Microscopy, Negative Control, Incubation, Blocking Assay, Microscopy, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Increased Expression of Ephrins on Immune Cells of Patients with Relapsing Remitting Multiple Sclerosis Affects Oligodendrocyte Differentiation

doi: 10.3390/ijms22042182

Figure Lengend Snippet: Immune cells of a patient with RR-MS inhibited the differentiation of oligodendrocytes precursor cells to maturing oligodendrocytes. ( A ) Representative confocal microscopy images of HEK-293T cells unstimulated or stimulated with 0.5 µg/mL of recombinant human Fc-IgG negative control (hrFc IgG), or 0.5 µg/mL human recombinant ephrin-A3 (hrEphrin-A3) protein, and pre-incubated with or without 30 µM, 60 µM or 90 µM Eph-receptor inhibitor KYL peptide (KYL) Scheme 20. scale = 50 µm. ( B ) Average of Eph-receptor phosphorylation analysis shown in A. * p < 0.05, ** p < 0.01. OPCs were unstimulated or co-cultured with PBMCs of patients with RR-MS with or without a pre-incubation with 60 µM inhibitor KYL peptide in oligodendrocyte differentiation medium for 4 days. ( C ) Representative confocal microscopy images of OPCs cells immunostained by anti- A2B5 and DAPI nuclear staining after 4 days of differentiation. Magnification ×25; scale = 50 µm. ( D ) Average of A2B5 expression level analysis. ( E ) Representative confocal microscopy images of OPCs immunostained by GalC and DAPI nuclear staining after 4 days of differentiation. Magnification ×25; scale = 50 µm. ( F ) Average of the GalC expression levels analysis shown in D.

Article Snippet: Recombinant human Fc-IgG fragments or ephrinA2-Fc fragments (R&D, Minneapolis, MN, USA) were mixed with or without anti-ephrin-A2 blocking antibody (AF607; R&D) and incubated at 37 °C for 1 h prior to stimulation.

Techniques: Confocal Microscopy, Recombinant, Negative Control, Incubation, Phospho-proteomics, Cell Culture, Staining, Expressing