Figure 5. GXMGal effect on Th1 response.

Activated PBMC (A, B and D) (5×10⁶/ml) from Control and RA were incubated for 20 min, 2, 18 and 72 h in the presence or absence (NS) of GXMGal (10 µg/ml), MTX (10 ng/ml) or DEX (10 nM). After 20 min of incubation, cell lysates were analyzed by western blotting. Membranes were incubated with Ab to T-bet. Actin was used as an internal loading control. Normalization was shown as mean ± SEM of five independent experiments. *, p<0.05 (triplicate samples of 5 different Control and RA; treated vs untreated cells) (A). Culture supernatants were collected after 2, 18 and 72 h to test IFN-γ, IL-12p70 (B) and IL-8 (D) levels by specific ELISA assays. *, p<0.05 (triplicate samples of 7 different Control and RA; RA GXMGal-treated vs untreated cells); ^†, p<0.05 (triplicate samples of 7 different Control and RA; RA MTX-treated vs untreated cells); ‡, p<0.05 (triplicate samples of 7 different Control and RA; RA DEX-treated vs untreated cells). Activated purified CD4⁺ T cells (1×10⁶/ml) (C) from RA were stimulated as above described and intracellular stained for T-bet and IFN-γ. The percentage of T-bet⁺/IFN-γ⁺ CD4⁺ T cells from RA after 18 h of GXMGal or MTX treatment was shown as mean ± SEM of five independent experiments. *, p<0.05 (triplicate samples of five different RA; treated vs untreated cells).