[9] Stimulation indices (SI) were calculated

as prolifera

[9] Stimulation indices (SI) were calculated

as proliferative response in the presence of antigen divided by response in the absence of antigen. Brains and spinal cords were fixed in 5% formalin saline and processed for routine histology. Sections, 5 μm thick, were cut and stained with haematoxylin & eosin to evaluate inflammatory infiltrates or Luxol fast blue/cresyl fast violet (LFB/CFV) to assess the degree of demyelination. Data were analysed using Graphpad prism and expressed as mean ± standard error of the mean (SEM). The EAE clinical scores were assessed by Mann–Whitney U-test and day of onset and disease incidence were analysed by Kaplan–Meier using sigmastat software (SPSS Inc., Chicago, IL). Group EAE score represents the maximum neurological deficit in all animals within the group and mean EAE score represents the maximum neurological deficit developed by mice, which exhibited EAE, as

previously described EPZ-6438 supplier and the mean day of onset of signs.[3, 16] P-values < 0·05 were considered significant. To identify the immunodominant B-cell epitopes C57BL/6 WT (MOG+/+) and MOG-deficient (MOG−/−) mice, which will lack any immune tolerance and deficits in their immune repertoire to MOG, were immunized with rmMOG corresponding to MOG sequence 1–116. On day 20, plasma was collected and examined using ELISA to identify responses to 23 mer overlapping peptides (Table S3). No differences were observed between the responses of MOG+/+ and MOG−/− mice to rmMOG on day 20 (Fig. 1). Similarly, antibody responses to peptides in both Celecoxib selleck WT and MOG−/− knockout mice were restricted to sequences below residues 82 and dominant responses to epitopes within residues MOG45–67 and MOG50–72 (Fig. 1a).

Similar to responses to MOG35–55 (see ref. [9]) antibody responses to the 23 mer peptide MOG35–57, encompassing the encephalitogenic peptide MOG35–55, were not dominant. As expected, no responses were found in peptides above residues 116 (Fig. 1a). To examine antibody responses in more detail, C57BL/6 WT (MOG+/+) and MOG-deficient (MOG−/−) mice (n = 5) were immunized with a pool of 15 mer peptides and recall responses on day 20 to individual peptides were examined using ELISA. We identified immunodominant epitopes with residues MOG113–127 and MOG148–162 (Fig. 1b) in C57BL/6 WT (MOG+/+) and MOG-deficient (MOG−/−) mice. No responses were observed to any other peptide or in mice immunized with complete Freund’s adjuvant only. No differences were observed between responses in C57BL/6 WT (MOG+/+) and MOG-deficient (MOG−/−) mice (Fig. 1). Next, to identify the immunogenic T-cell epitopes within mouse MOG, mice were immunized with the overlapping peptide spanning the mouse MOG sequences. On day 10 responses were examined using a thymine incorporation assay as described previously.[9] This study revealed that while a T-cell response to MOG36–50 (SI = 3·90) was detectable (Fig. 2) a stronger response to peptide MOG183–197 (SI = 5·2) was also induced.

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