Methylated bovine serum albumin (mbsa)

Mice were immunized by subcutaneous administration of 100 μl methylated bovine serum albumin (mBSA; 1 mg/ml, Sigma-Aldrich) emulsified in complete Freund’s adjuvant (CFA, Sigma-Aldrich). Mice were injected i.p. with 200 ng of heat-inactivated Bordetella pertussis toxin (Sigma-Aldrich). The immune response was boosted after 7 days with identical administration of mBSA/CFA. Inflammatory arthritis was induced 21 days after the initial immunizations by intra-articular administration of 10 μl mBSA (10 mg/ml) into the right hind knee joint. For the WT-PBS group, 10 μl phosphate buffered saline (PBS) was injected into the knee joint. Swelling was monitored by measuring knee joint diameter using a POCO 2T micrometer (Kroeplin), and recorded as the difference between right (inflamed) and left (contralateral) diameters. Knee joints were harvested at day 15 post-AIA for histopathological assessment of disease severity (arthritic index).

The following materials were obtained from the indicated sources: recombinant murine IL-22 (rmIL-22; R&D Systems, Minneapolis, MN, USA), anti-IL-22 antibody (α-IL-22) (16-7222-85; eBioscience, San Diego, CA, USA), zymosan, fucoidin, methylated bovine serum albumin (mBSA) and complete Freund adjuvant (CFA; Sigma-Aldrich, St. Louis, MO, USA). The drugs were diluted in sterile saline.

This study was approved by the Ethics in Committee Research for animal studies of University of São Paulo School of Medicine (São Paulo, Brazil) N° 295/12.
Thirty male Lewis rats with an average weight of 220–240 g and an average age of 3 months were used. Food and water were given ad libitum. The animals were kept in a room with a controlled environment and a circadian night–day rhythm of 12 h. All the animals received human care in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health [26 ].
The arthritis group (IA, n = 20) was induced by intraarticular injection (10 μL) of 500 μg of mBSA (Sigma Chemical Co.) diluted in saline (10 μl) and emulsified in an equal volume of complete Freund’s adjuvant, followed by an intraarticular booster of mBSA (50 μg) in saline (50 μl) administered at 7 and 14 days [27 (link)]. After the first intraarticular injection, 10 animals were further randomized to be supplemented via gavage with Col V (500 μg/300 μl) diluted in acetic acid (0.01 N) and administered daily for 30 days (IA/Suppl). The control group (n = 10) received saline (50 μl) using the same protocol and was Col V supplemented in the same way (Suppl) (Fig 1).

Rabbits were sensitized by intradermal injection of methylated bovine serum albumin (mBSA, Sigma; 0.5 ml containing 2 mg of mBSA) suspended in Complete Freund's adjuvant (FCA, Sigma) at 3-5 sites on day 0 and day 14. The controls were injected with saline solution. On day 28, the rabbits were anesthetized with methohexital (10 mg/kg, iv), and the right knee joints were intra-articularly injected with 1 mg of mBSA in 0.5 ml of 5% glucose solution, while the left knee joints were injected with 0.5 ml of saline solution as the sham-operation controls. The perimeter of the joints was measured with a caliper at regular intervals.