Mycobacterium tuberculosis h37ra

Ten-week-old female mice were immunized by subcutaneous injection of 300-μg myelin oligodendrocyte glycoprotein-35-55 (MOG_35–55) (Hooke Laboratories, Lawrence, Massachusetts, USA) in 200-μl Freund’s Adjuvant containing 300-μg/mL mycobacterium tuberculosis H37Ra (Hooke Laboratories) in the dorsum. Mice were administered with 500-ng pertussis toxin (PTX) intraperitoneously on day of sensitization and 1 day later (Hooke Laboratories). The emulsion provides antigen, which initiates expansion and differentiation of MOG-specific autoimmune T cells. PTX enhances EAE development by providing additional adjuvant. EAE will develop in mice 7 to 14 days after immunization (Day 0): Animals that develop EAE will become paralyzed. Disease was scored (0, no symptoms; 1, floppy tail; 2, hind limb weakness (paraparesis); 3, hind limb paralysis (paraplegia); 4, fore and hind limb paralysis; 5, death)) [31 (link)] from day 7 post immunization until day 32 post immunization. At Day 32, all the animals were euthanized by pentobarbital (Richter Pharma) overdose. For histological assessment, cervical, lumbar, and dorsal sections of each animal spinal cord, as well as the spleen, were harvested.

To investigate the effects of macrophage CD40 and the CD40–TRAF pathways in EAE, 12‐week‐old female CD40^flflLysM^cre, CD40^flfl, MHCII–CD40–T2/3/5^−/−, MHCII–CD40–T6^−/−, CD40^−/− mice and WT mice had ad libitum access to food and water and were housed under a 12 h light/dark cycle. On day 0, mice were immunized subcutaneously with 200 µg of a MOG peptide (MOG_35–55) emulsified in CFA Complete Freund's Adjuvant supplemented with 4 mg/ml Mycobacterium tuberculosis H37Ra (Hooke Laboratories, Lawrence, MA, USA). Mice were injected i.p. on days 0 and 1 with 300 (CD40^flflLysM^cre and CD40^flfl mice, n = 12/group) or 100 ng (MHCII–CD40–T2/3/5^−/−, MHCII–CD40–T6^−/−, CD40^−/− and WT mice, n = 15/group) pertussis toxin (Hooke Laboratories). A control group was included that received only CFA and pertussis toxin (n = 6). Body weight and neurological symptoms were monitored daily. The symptoms were graded using the following scale: 0 = no neurological abnormalities; 0.5 = partial loss of tail tonus; 1 = complete loss of tail tonus; 2 = hind limb paresis; 3 = partial hind limb paralysis; 4 = complete hind limb paralysis; 4.5 = paralysis up to the diaphragm, 5 = death. Scoring of clinical symptoms was performed by an observer who was blinded for the experimental conditions.

Active EAE was induced in 12-week-old female mice by s.c. immunization at two sites, upper and lower back, with 200 μg MOG_35–55 peptide emulsified in CFA containing 400 μg heat-killed Mycobacterium tuberculosis H37Ra (Hooke Laboratories, Lawrence, MA, USA), on day 0. In addition, mice received 200 ng pertussis toxin (Hooke Laboratories) i.p. in 0.1 ml per mouse on days 0 and 1. Clinical signs of EAE were assessed daily with a 0- to 5-point scoring system defined as follows: 0, no obvious changes in motor function compared with non-immunized mice; 0.5, tip of tail is limp; 1, limp tail; 1.5, limp tail and hind leg inhibition; 2, limp tail and weakness of hind legs; 2.5, limp tail and dragging of hind legs; 3, limp tail and complete paralysis of hind legs or paralysis of one front and one hind leg; 3.5, limp tail and complete paralysis of hind legs that are together on one side of body; 4, limp tail, complete hind leg and partial front leg paralysis; mouse is still minimally moving and appears feeding; 4.5, complete hind leg and partial front leg paralysis; no movement around the cage, mouse is not alert; 5, death or severe paralysis. Mice with score ≥4 for two consecutive days and mice with score 5 were killed.