to the anal verge and the oxazolone or FITC was injected with a total volume of 100 μl. The mice were held in a vertical position for 30 s after the injection to ensure distribution of the oxazolone or FITC within the entire colon. The colon was removed 24 h after challenge and the morphological and immunological changes were observed. Seirogan treatment. Sugar-coated and uncoated Seirogan were supplied by Taiko Pharmaceutical Co. Ltd. (Osaka, Japan). Seirogan is coated with white soft sugar (sucrose) which prevents any smell from being noticed during oral ingestion. One group received 30 mg/day of coated Seirogan in solution orally throughout the experiment period. Only distilled water was administered orally to the control animals. The other group was exposed to the uncoated Seirogan for 2 h per day throughout the experimental period. The uncoated Seirogan smell was diluted with ultrapure water to 0.03% (v/w). This concentration of uncoated Seirogan smell has been shown to be effective in reducing the increase in the plasma concentration of adrenocorti- cotropic hormone (ACTH) induced by acute immobilization stress (data not shown). Quantification of cytokines by enzyme-linked immuno- sorbent assay (ELISA). Blood samples were taken from the heart 5 h after sensitization, and then the plasma was fractionated. Skin samples were removed 1 day after the challenge and immediately frozen in liquid nitrogen and stored at −80°C. For extraction, the skin samples were homogenized, then the superna- tant after centrifugation was used for the analysis. The plasma tumor necrosis factor-alpha (TNF-α), cortisol, and ACTH concentration and the colonic level of IgA were determined using a commercial ELISA kit (TNF-α, Pierce Biotechnology, IL; ACTH, Phenix Pharmaceuticals Inc., CA; Cortisol, Oxford Biochemical Research Inc., MI; IgA, BETHYL Laboratories Inc., TX) according to the manufacturer’s instructions. Histological analysis. The colon specimens were fixed in phosphate-buffered paraformaldehyde (4%), embedded in frozen Tissue Tek, OCT compound, and cut into 5 μm thick sections. Thin sections were stained with hematoxylin-eosin and analyzed histologically to evaluate the degree of colon inflammation caused by the FITC and oxazolone solution. Other thin sections, were washed with phosphate-buffered saline (PBS), and then were incubated with goat anti-mouse IgA (1:100) polyclonal antibody (ZYMED, San Francisco, CA) overnight at 4°C. The sections were thereafter washed in PBS, and incubated at room temperature for 2 h with FITC-conjugated anti-goat immunoglobulin made from rabbit (1:30; Dako Cytomation, Denmark). The expression of IgA was evaluated immunohistochemically under fluorescence microscopy. Statistical analysis. All data were expressed as the mean ± SD. The results obtained from the three animal groups were analyzed by either Student’s t test or an ANOVA using a computer software. Differences were considered to be significant when p<0.05. Results Effect of Seirogan on systemic suppression of CHS ear thickness by FITC and oxazolone. CHS induced by FITC or oxazolone was suppressed by 55% and 68%, respectively, following oral administration of coated Seirogan. The group that smelled uncoated Seirogan, showed a greater suppression of CHS by FITC in comparison to the oral administration group (Fig. 1). Seirogan reduced TNF-α, cortisol and ACTH production induced by FITC or oxazolone. The plasma level of TNF-α, cortisol and ACTH increased with either FITC or oxazolone sensitization (Fig. 2). The oral administration of coated Seirogan after FITC or oxazolone sensitization decreased the cytokine and hormone levels this study in comparison to the levels in the mice not treated with Seirogan. In addition, uncoated Seirogan smell 92 Fig. 1. Effects of Seirogan treatment on FITC- or oxazolone-induced CHS. CHS is expressed as the amount of ear swelling 24 h after FITC or oxazolone challenge. The data are presented as the mean ± SD from ten animals. *p<0.05. Fig. 2. Effects of Seirogan treatment on the plasma concentration of TNF-α (A), ACTH (B), or cortisol (C). The plasma concentration of each mediators were determined 24 h after challenge with FITC or oxazolone challenge. The data are presented as the mean ± SD from ten animals. *p<0.05. treatment after FITC sensitization decreased the TNF-α, cortisol and ACTH levels in comparison to the levels in the coated Seirogan oral administration group. Morphological and histopathological changes in Seirogan treated mice. Biopsy specimens were taken from the regions of positive colon reactions. The morphological observations are shown in Fig. 3. There was remarkable edema and congestion due doi: 10.3164/jcbn.11-82 ©2012 JCBN