Four-week-old female NOD/Lt mice, with average weight of 18.8 g, were raised and maintained under pathogen-free conditions at the Animal Center of this institute purchased from Slaccas Experimental Animal Limited http://www.selleckchem.com/products/byl719.html Company, Shanghai, PR China (SCXK 2003-0003).

The onset of clinical insulitis begins at about 3 months of age and reaches a cumulative incidence of 80% or greater by 8 months of age in this colony for female. The mice were divided into four groups of ten animals each (n = 10 per group). Three groups, respectively, received three i.n. inoculations of 100 μg of purified HSP65-6 × P277, HSP65 and peptide P277 solubilized in sterilized phosphate-buffered saline (PBS, pH 7.4) at 4, 7, and 10 weeks of the age; the control mice received three i.n. inoculations of PBS (pH this website 7.4) at the same time as above. The serum samples were collected before every inoculation, after the third administration, serum samples were collected at monthly interval for 5 months and stored at −20 °C for use in antibody assays. For detection of P277-specific antibodies, a standard ELISA technique was applied as previously described [19]. Briefly, 10 μg/ml of purified VEGF-P277 was applied to ELISA plates (Costar, USA)

overnight at 4 °C. After saturation with 5% BSA for 60 min, the plates were washed and serum samples were added. The binding of antibodies were detected using horseradish peroxidase-conjugated goat anti-rat IgG or isotype-specific anti-mouse IgG1, IgG2a, or IgG2b (Promega, USA). Substrate was added and color development was assayed in an ELISA plate reader (Thermo, USA). Each serum was tested in duplicate. Results were expressed as OD at 450 nm. After until the final administration, serum samples were collected at monthly interval. The concentration

of blood glucose was measured by Hitachi automatic analyzer (model-7150, Tokyo, Japan). A mouse was considered to be diabetic if the blood glucose level was >11 mM on two consecutive examinations. Mice from each treatment group were killed at the age of 8 months, when almost all the control NOD mice were sick. The pancreata were fixed with 10% formalin solution. Formalin-fixed paraffin blocks of pancreas tissue were sectioned with a microtome, stained with hematoxylin (Sangon Company, Shanghai, China) and eosin (Sangon Company, Shanghai, China). We invited a pathologist (Southeast University, Nanjing, China) helping us to evaluate the degree of insulitis in a blinded fashion. The average degree of insulitis was assessed over 20 islets scored per pancreas. Each islet was classified as: clear, if no infiltrate was detected; mildly infiltrated, if peri-insulitis or an intra-islet infiltrate occupied <25% of the islet; infiltrated or heavily infiltrated, if 25–50% or >50% of the islet was occupied by inflammatory cells. Four weeks after the last dose the spleens were removed, and the T-cell proliferative responses were assayed in vitro.