Animals and Experimental protocol
Six groups of mice were prepared in the present study (Fig. 1). Four-week-old female BALB/c mice (Charles River Japan, Inc, Yokohama, Japan) were immunized twice intraperitoneally on days 1 and 14 with 0.5 mg per mouse of Dermatophagoides farinae (Df) (LG-5339; Cosmo Bio, Tokyo, Japan) precipitated in aluminum hydroxide. These mice were then challenged intranasally (i.n.) with 50 µg/50 µL Df allergen (crude extract of the mite) on days 14, 16 and 18. Following that, 5 × 106 of Af conidia were administered i.n. on days 19, 21 and 23. Various drugs were administered from on day21 to 25 and ten groups of mice were prepared as follows. DfAf, no drug was administered; DfAf/Dex, 0.02mg of Dexamethasone (Sigma, St Louis, Mo) were injected subcutaneously (SC); DfAf/PRN, 0.5mg of pranlukast hydrate (ONO Pharmaceutical Co., Osaka, Japan) were injected subcutaneously (SC); DfAf/CAM, 4mg of clarithromycin (Taisho Toyama, Tokyo, Japan) were orally administered; DfAf/Dex/PRN, combination therapy with Dexamethasone and pranlukast hydrate; DfAf/Dex/CAM, combination therapy with Dexamethasone and clarithromycin. On day 26, all mice were sacrificed. Bronchoalveolar lavage fluid (BALF) and lung tissues were obtained from each group. The procedures were reviewed and approved by Nagasaki University School of Medicine Committee on Animal Research. All experiments were repeated at least 3 times.
Preparation of Af conidia
A. fumigatus MF-13, which was isolated from the sputum of a patient with pulmonary aspergilloma, was subcultured on Sabouraud dextrose agar (Becton Dickinson, Cockeysville, MD) at 30°C for 7 days. The conidia were then harvested with sterile saline containing 0.02% Tween 80 (Wako Pure Chemical Industries, Tokyo, Japan), counted in a hemocytometer, and diluted with sterile saline for intranasal infection.
Bronchoalveolar lavage and lung pathology
On day 26, mice were sacrificed and BAL was conducted utilizing 1 ml of PBS in the immediate postmortem period. The obtained BALF was centrifuged. Differential cell counts were performed using cytocentrifuged BALF stained with May-Grünwald-Gìemsa. Formaldehyde fixative was gently infused through the lavage catheter set in the trachea. Resected lungs were fixed for an additional 24 hours and embedded in paraffin. Sections (4 µm) were stained with hematoxylin and eosin (HE).
Analysis of cytokines concentrations in homogenized lung
Lung homogenates were prepared by homogenizing a freshly excised lung. Concentrations of IL-5, IL-13 and MIP-2 in homogenized lung samples were measured using enzyme-linked immunosorbent assay using the methods described by the manufacturer. Detection limits for IL-5, IL-13 and MIP-2 were 5 pg/ml, 1.5 pg/ml and 5 pg/ml respectively.
Statistical analysis
Results are expressed as mean (standard error of mean). Differences between groups were examined for statistical significance using repeated-measures ANOVA with a Bonferroni multiple comparison test. p values of < 0.05 were considered significant.