Figure 1.

Immunomodulatory effects of PEGA on PLNC. Rats were immunized with myelin oligodendrocyte glycoprotein (MOG) 35-55+CFA (A–F) or with spinal cord homogenate (SCH) (G–N). PLNC were isolated on day 6 p.i. and stimulated with MOG35-55 (A–D), or with MBP (G–N), or with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of brefeldin A for 4 h before staining (L,M). CD4+ cells were purified from PLNC obtained on day 6 p.i. and stimulated with anti-CD3 and anti-CD28 antibodies (E,F). The cells were cultivated in the absence (0) or presence of PEGA for 24 h and cell viability was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (A), Cytokine levels were determined in cell culture supernatants by ELISA (B,C,E,F), 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) was measured by fluorimetry (D), Apoptosis was determined by Annexin V staining (G), proliferation was measured by Ki67 staining (H), activation was assessed by CD25 (I) and OX40 staining (J) in CD4⁺ T cells by flow cytometry. Treg were identified as CD4⁺CD25⁺Foxp3⁺ cells (K), Th17 as CD4⁺IL-17⁺ cells (M), and Th1 as CD4⁺IFN-γ⁺ cells (M) by flow cytometry. Relative levels of cytokines mRNA were determined by “real-time” RT-PCR (N). Individual values of 3 (E,F,H), 4 (B–D), 5 (L–N), or 6 (A,G,I–K) samples are presented. In addition, the mean values +/− SD are given (A–F) One-way ANOVA followed by Tukey’s multiple comparison test (A–F), paired Student’s t-test (G–M), and unpaired Student’s t-test (N), * p < 0.05, ** p < 0.01, *** p < 0.001, vs. 0.