Que Inhibits T Cell Proliferation Figure 3. Que decreases the number of infiltrating T cells in spinal cord. Panels display the number of infiltrating CD4/8 positive T cells in the EAE spinal cord with/without Que treatment. A–C: CD4+ cells were not observed in the spinal cord prior to immunization (A). An extensive infiltration of CD4+ cells was detected throughout the spinal cord and enriched in the white matter of the EAE mice (B). Que treatment significantly decreases the amount of infiltrating CD4+ T cells (C) (N = 5, *, P,0.05). E–G: CD8+ cells display a similar decrease after Que treatment (N = 5, *, p,0.05), but the overall extent of CD8+ cells detected was much less than that of CD4+ cells. Quantification of the staining is depicted in the bar graphs (D and H). Scale bar A–G = 0.5 mm, F’ = 50 mm. doi:10.1371/journal.pone.0042746.g003 an RNA PCR Kit (AMV) (Takara). The cDNA was analyzed by real- time PCR with the Rotor Gene6000 (Corbett Research, Australia) according to the protocol provided by the manufacturer and 22DDCt method. Briefly, PCRs were performed using SYBR premix Ex Taq (Takara) in a final volume of 20 ml. The thermal conditions were 95uC for 10 seconds followed by 40 cycles of 95uC for 5 seconds, 60uC for 15 seconds and 72uC for 15 seconds. The primers were IL-2 sense 59- CATTGACACTTGTGCTCCTTG-39, antisense 59- GGTTCCTGTAATTCTCCATCCTG-39, mouse actin-b sense 59-CGTGCGTGACATTAAGGAGAAG-39, antisense 59- GGAAGGAAGGCTGGAAGAGTG-39. Detection of IL-2 by ELISA The supernatants were obtained and examined by ELISA (eBioscience). In brief, serially diluted supernatants samples and internal standards samples (recombinant murine IL-2) were incubated with immobilized antibody. Antibody was detected with HRP-labeled rabbit anti-mouse IgG1 and IgG2a (Zymed Labora- tories), which were then detected with the substrate o-phenylene- diamine. The relative concentration of antibody was determined from a standard curve of known concentrations of unlabeled murine IL-2 antibody (Southern Biotechnology Associates). Statistical Analysis Data were expressed as means 6 SEM. Statistical analysis between experimental groups was evaluated using analysis of variance (ANOVA). A two-tailed paired Student’s t test was used for comparing individual treatment groups. A probability of p,0.05 was taken as a statistically significant difference. Results Que Administration Attenuates Clinical Signs and Protects Myelin from Breakdown in EAE Que treatment was initiated on day 16 post-immunization when mice attained a clinical score of 0.5, and mice were monitored for a total of 40 days (Fig. 1A). The untreated EAE mice continued to deteriorate with increased clinical scores that reached a value of approximately 3, whereas mice treated with Que reached a value of 1, then stabilized at that level and did not display further deterioration (Fig. 1B). While unimmunized group and control (immunized with BSA) mice displayed normal behavior (data not shown). Therefore, Que treatment clearly attenuates the clinical scores in EAE. Additionally, pre-treatment with Que from 1 week before immunization resulted in a significant delay in the onset of EAE and diminishes the severity of symptoms (Figure S1). As demyelination is one of the major histopathologic hallmarks in EAE and MS, histological observation indicate that the spinal cords from the immunized group without Que treatment were weakly stained with MBP and LFB, especially in the white matter tracts (arrows, Fig. 2A, B), while a more intense expression of MBP and LFB staining were observed in the Que treatment group but less intense than unimmunized group or control (Fig. 2A, B) on 30 PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e42746