Journal: Arthritis Research & Therapy

Article Title: Suppression of the inflammatory response by disease-inducible interleukin-10 gene therapy in a three-dimensional micromass model of the human synovial membrane

doi: 10.1186/s13075-016-1083-1

Figure Lengend Snippet: Gene expression in synovial micromasses after stimulation with LPS and TNF-α in the absence or presence of IL-10. Micromasses from synovial cell suspensions (three per group) were cultured until lining formation was evident. Subsequently, the micromasses were stimulated for 2 h or 4 h with medium containing 100 ng/ml LPS, 10 ng/ml TNF-α, 10 ng/ml IL-1β in the absence and presence of 10 ng/ml IL-10. Gene expression levels of SOCS3 at 2 h ( a ), TNF-α at 4 h ( b ), IL-1β at 4 h ( c ) and IL-6 at 4 h ( d ) were measured. Expression levels are depicted as threshold cycle (Ct) +/- SD, corrected for GAPDH expression. Statistical comparison within stimulation groups was performed by two-way ANOVA and between groups by one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001. IL-1β interleukin-1 beta, IL-6 interleukin-6, LPS lipopolysaccharide, SOCS3 suppressor of cytokine signaling 3, TNF-α tumour necrosis factor alpha

Article Snippet: Cytokine concentrations were determined by luminex multianalyte technology on the Bio-Plex 200 (Bio-Rad, Hercules, CA, USA) in combination with Bio-Plex pro human cytokine kits (Bio-Rad) according to the manufacturer’s protocol.

Techniques: Gene Expression, Cell Culture, Expressing, Comparison

Journal: Arthritis Research & Therapy

Article Title: Suppression of the inflammatory response by disease-inducible interleukin-10 gene therapy in a three-dimensional micromass model of the human synovial membrane

doi: 10.1186/s13075-016-1083-1

Figure Lengend Snippet: Cytokine production by stimulated micromasses treated with IL-10 viral vectors. Micromasses from synovial cell suspensions of five patients were transduced with lentiviral vectors coding for PGK-luciferase, PGK-IL10 or CXCL10p-IL10 after formation of a synovial lining. Subsequently, the micromasses were stimulated with medium containing 100 ng/ml LPS (all patients) or 10 ng/ml TNF-α (three patients). The concentrations of IL-1β ( a ), IL-6 ( b ) and TNF-α ( c ) in the supernatant after 24 h were measured using a multiplex ELISA assay. IL-1β and TNF-α could only be quantified in three patients. Because of high variations in IL-6 production between patients, the values were first normalized for every individual patient for PGK-luc unstimulated. The basal values (pg/ml) were 2.7 × 10 5 , 6.5 × 10 5 , 2.7 × 10 4 , 5.1 × 10 4 and 3.3 × 10 5 respectively. The medium, LPS- and TNF-α-stimulated groups were compared by t test. Significancies without a capped line were compared to PGK-luc. * P < 0.05, ** P < 0.01, *** P < 0.001. IL-1β interleukin-1 beta, IL-6 interleukin-6, LPS lipopolysaccharide, TNF-α tumour necrosis factor alpha

Article Snippet: Cytokine concentrations were determined by luminex multianalyte technology on the Bio-Plex 200 (Bio-Rad, Hercules, CA, USA) in combination with Bio-Plex pro human cytokine kits (Bio-Rad) according to the manufacturer’s protocol.

Techniques: Transduction, Luciferase, Multiplex Assay, Enzyme-linked Immunosorbent Assay