Guinea pig myelin basic protein

Experimental autoimmune encephalomyelitis (EAE) was induced as described previously [33 (link)]. Briefly, female Lewis rats (200 g) received subcutaneous injections at both sides of the base of the tail with 0.2 mL of an emulsion containing 1 g/L Guinea pig myelin basic protein and 1 g/L M. tuberculosis in Freund's complete adjuvant (all from Sigma-Aldrich). The injections were performed under full inhalation anesthesia. EAE was induced in 30 animals; 6 healthy, age-matched, unimmunized rats were used as controls. Between day 0 and day 21, the weight and clinical signs of EAE were recorded daily for all animals. Clinical signs were scored as follows: 0, no abnormality; 0.5, weak tail; 1, limp tail; 2, mild palsy of one or both hind legs; 3, severe palsy of one or both hind legs; 4, complete paralysis of one or both hind legs; 5, paralysis of one or both hind legs and beginning paralysis of front legs; 6, moribund. Animals with a clinical score ≥4 were sacrificed immediately.
Starting at day 10 after induction of EAE, animals were treated once/day for 5 consecutive days with Epobis (10 mg/kg, 1 mL/kg, s.c.) or PBS (1.0 mL/kg, s.c.). The control group is identical to the control group of previously published data [33 (link)] performed simultaneously with the Epobis study.

ovalbumin-specific or myelin basic protein-specific rat T cells were resuspended in medium + 5% FBS and seeded in a 96-well plate (5 × 10⁴ per well) in the presence of irradiated rat thymocytes (2 × 10⁶ per well), serving as antigen-presenting cells38 (link)54 55 (link)56 (link). Rat mononuclear splenocytes and human peripheral blood cells were resuspended in medium + 5% FBS and seeded in a 96-well plate (1 × 10⁵ per well)40 (link)57 (link). Cells were incubated with or without antioxidants: trolox (Sigma-Aldrich), vitamin C (Sigma-Aldrich), PEG-HCCs, or HCCs for 30 min at 37 °C and 5% CO₂. ovalbumin-specific and myelin basic protein-specific rat T cells were stimulated with 10 μg/mL ovalbumin or guinea pig myelin basic protein (Sigma-Aldrich), respectively36 (link)38 (link)58 (link). Rat mononuclear splenocytes and human peripheral blood cells were stimulated with 1 μg/mL concanavalin A or phytohemagglutinin, respectively36 (link)59 (link). Cells were cultured for 72 h at 37 °C and 5% CO₂, and 1 μCi [³H] thymidine (MP Biomedicals) was added to each well during the final 16–18 h of incubation. Cells were then lysed by freezing and DNA was harvested on fiberglass filters using a cell harvester (Inotech Biosystems International). [³H] thymidine incorporation was measured on a β-scintillation counter (Beckman Coulter).