bba1  (R&D Systems)

Journal: Arthritis Research & Therapy

Article Title: Suppression of endothelial cell activity by inhibition of TNFα

doi: 10.1186/ar3812

Figure Lengend Snippet: Certolizumab pegol inhibits TNFα-induced adhesion molecule expression on human dermal microvascular endothelial cells . E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are expressed on human dermal microvascular endothelial cells (HMVECs), inhibited by certolizumab pegol. Cell surface ELISAs were performed to determine the peak expression time of each adhesion molecule on HMVECs induced by TNFα (25 ng/ml). (A) E-selectin, (B) ICAM-1, and (C) VCAM-1 expression is significantly greater on TNFα-stimulated HMVECs compared with the nonstimulated (NS) group. HMVEC expression of E-selectin peaks at 6 hours, while ICAM-1 and VCAM-1 peak at 24 hours (* P < 0.05). Peak expression time points for each adhesion molecule were then used to examine whether certolizumab pegol can inhibit TNFα adhesion molecule induction on HMVECs. (D) E-selectin, (E) ICAM-1, and (F) VCAM-1 expression on HMVECs was stimulated with TNFα (25 ng/ml) in the presence or absence of different concentrations of certolizumab pegol (0.0001 to 10 μg/ml) or mouse-IgG (Ms-Ig) control for 6 hours for E-selectin and for 24 hours for ICAM-1 and VCAM-1 expression, when ECs protein lysates were collected. (G), (H) Western blot results indicate that E-selectin, ICAM-1 and VCAM-1 expression are entirely blocked by higher concentrations of certolizumab pegol (0.1 to 10 μg/ml). Blots are representative of three samples. The results are shown as the fold change of treatment samples to the NS group ± standard error of the mean. n , number of experiments.

Article Snippet: Mouse anti-human-E-selectin and anti-human-ICAM-1 antibodies and goat anti-human-VCAM-1 and anti-human-JAM-A antibodies (R&D, Minneapolis, MN, USA) were used at 2.5 μg/ml and plates were read using an ELISA reader (Bio-Rad, Hercules, CA, USA) set at 450 nm.

Techniques: Expressing, Western Blot

Journal: Arthritis Research & Therapy

Article Title: Suppression of endothelial cell activity by inhibition of TNFα

doi: 10.1186/ar3812

Figure Lengend Snippet: Certolizumab pegol inhibits HL-60 cell-human dermal microvascular endothelial cell adhesion induced by TNFα . TNFα-stimulated adhesion-molecule-mediated human promyelocytic leukemia (HL-60) cell-human dermal microvascular endothelial cell (HMVEC) adhesion is blocked by certolizumab pegol. Adhesion assays were performed using HL-60 cells and TNFα-preincubated HMVECs in the presence of anti-E-selectin or anti-intercellular adhesion molecule-1 (anti-ICAM-1) or anti-vascular cell adhesion molecule-1 (anti-VCAM-1) antibody, or of different concentrations of certolizumab pegol or mouse-IgG (Ms-Ig) control for 8 hours. (A) TNFα (25 ng/ml) significantly induces HL-60 adhesion to HMVECs, and anti-E-selectin but not anti-ICAM-1 or anti-VCAM-1 antibody block this interaction (* P < 0.05). (B) HL-60 cell adhesion to HMVECs is significantly inhibited by certolizumab pegol (0.005 to 1 μg/ml, from 10 to 57% of TNFα-induced binding; * P < 0.05). Results are presented as the percentage of TNFα-induced adhesion ± standard error of the mean. n , number of individual experiments. NS, nonstimulated.

Article Snippet: Mouse anti-human-E-selectin and anti-human-ICAM-1 antibodies and goat anti-human-VCAM-1 and anti-human-JAM-A antibodies (R&D, Minneapolis, MN, USA) were used at 2.5 μg/ml and plates were read using an ELISA reader (Bio-Rad, Hercules, CA, USA) set at 450 nm.

Techniques: Blocking Assay, Binding Assay

Journal: Arthritis Research & Therapy

Article Title: Suppression of endothelial cell activity by inhibition of TNFα

doi: 10.1186/ar3812

Figure Lengend Snippet: Certolizumab pegol inhibits HL-60 adhesion to rheumatoid arthritis synovial tissue vessels . Stamper-Woodruff assay and immunofluorescence were performed using frozen rheumatoid arthritis (RA) synovial tissue (ST) sections and fluorescence-labeled human promyelocytic leukemia (HL-60) cells. Synovial vessels are marked by von Willebrand factor antibody in red; calcein AM-labeled HL-60 cells appear as green dots; and cell nuclei are stained blue with 4',6-diamidino-2-phenylindole. HL-60 cells adhere to the synovial vessels (×200) when treated with: (A) mouse-IgG (Ms-Ig; negative control); (B) certolizumab pegol (10 μg/ml); or (C) anti-E-selectin antibody (Ab; positive control). The arrows in (A) to (C) point to HL-60 cells bound to vasculature. The adhesion ratio of each section was determined as the sum of adherent HL-60 cells divided by the sum of blood vessels in up to 10 fields of each section. (D) Adhesion results of the different treatments given as the percentage of Ms-Ig binding, defined as the adhesion ratio of the test group divided by the adhesion ratio of Ms-Ig-treated sections. Results represent the percentage of Ms-Ig binding ± standard error of the mean. n , number of RA patients. * P < 0.05.

Article Snippet: Mouse anti-human-E-selectin and anti-human-ICAM-1 antibodies and goat anti-human-VCAM-1 and anti-human-JAM-A antibodies (R&D, Minneapolis, MN, USA) were used at 2.5 μg/ml and plates were read using an ELISA reader (Bio-Rad, Hercules, CA, USA) set at 450 nm.

Techniques: Immunofluorescence, Fluorescence, Labeling, Staining, Negative Control, Positive Control, Binding Assay