DAO isolated from Fusarium spp. (fDAO) was a kind gift from Ikeda Food Research Co., Ltd, Minami- ku Kumamoto, Japan. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Molecular Probes (Eugene, OR, USA), respectively. H2O2 and tert-butyl hydroperoxide (t-BuOOH) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). D-Phenylalanine, D-proline, and rhodamine B isothiocyanate were purchased from Wako Pure Chemical Industries, Ltd.
Succinimidyl-activated PEG (SUNBRIGHT MEGC-50HS-PEG), with an average molecular size of 5250 Da, was purchased from NOF Corp., Tokyo, Japan. All other reagents were of reagent grade and used without further purification.
CGD mice (gp91-phox knockout) obtained from Dr. Mary C. Dinauer  were backcrossed at least 12 times with C57BL/6 mice to ensure similar genetic backgrounds. Experiments on 8–12-week-old CGD C57BL/6 mice were performed according to the guidelines of the Laboratory Protocol of Animal Handling, Sojo University Faculty of Pharmaceutical Sciences. All animals were housed under specific pathogen-free conditions.
Preparation of nCA
nCA was kindly gifted by Dr. Ohno . Briefly, a C-limiting medium was used to grow Candida albicans and cultured at 27 °C with aeration. Viable cells were collected by centrifugation, killed with ethanol, and dried with acetone. The resulting nCA was suspended in phosphate-buffered saline (PBS).
Preparation of PEG-fDAO
PEGylation of fDAO was conducted as described previously . In brief, to the fDAO solution (2.0 mg/mL protein in 0.1 M sodium bicarbonate), succinimide-activated PEG was added at a 3-fold molar excess of PEG to free the amino groups in fDAO and allowed to react for 1 hour at 4 °C. The reaction mixture containing PEG-fDAO was purified to remove free PEG and other low-molecular-weight reactants by ultrafiltration with a YM-10 membrane (Merck, Darmstadt, Germany) using 0.1 M sodium bicarbonate aqueous solution. PEG-fDAO was stored at -80 °C until further use.
Evaluation of the enzymatic activities of fDAO and PEG-fDAO
The enzymatic activity of fDAO and PEG-fDAO were determined by a horseradish peroxidase-coupled colorimetric assay with o-dianisidine as the substrate. In this assay, the substrate was reduced, and a color developed with a maximal absorption of 460 nm. D-alanine was used as the substrate for fDAO at a final concentration of 10 mM. The enzymatic reaction was performed at 25 °C in 0.1 M Tris-HCl buffer (pH 8.2), where 1 U of fDAO activity is defined as the rate of formation of 1 µmol of H2O2 per minute. The maximal rate of activity (Vmax) and Km for each D-amino acid were calculated by curve fitting of the non- linear plot of reaction rate versus substrate concentration using the gnu plot software.
Analysis of in vivo pharmacokinetics of fDAO and PEG-fDAO using the plasma
For the in vivo pharmacokinetics analysis, fDAO or PEG-fDAO (20 U/mL, 0.1 mL/mouse, n = 3) was injected intravenously into C57BL/6 (BALB/cAjcl) mice. Blood was withdrawn from the medial canthus of the eye using a microhematocrit at 0.5, 4, 24, 48, and 72 hour after fDAO or PEG-fDAO administration. Each blood sample was centrifuged and the plasma was obtained in an ice-cold buffer (100 mM Tris-HCl, pH 8.0) containing a mixture of protease inhibitors (1mM PMSF, 10 µg/mL leupeptin, and 2.5 mM EDTA). The DAO activity in the plasma was determined based on the formation of pyruvic acid during the reaction between D-Alanine and DAO, as described previously .
Induction of lung inflammation by nCA aspiration
Mice were anesthetized by intraperitoneal injection with 200 mg/kg of 2,2,2-tribromoethanol (Sigma- Aldrich), as described by Dr. Aratani. CGD mice were challenged with intranasal administration of 107 nCA cells in a volume of 30 μL of PBS, and control mice were administered 30 μL of PBS alone . Since we had no information on the time course of in vivo production of H2O2 induced by PEG- fDAO with D-amino acids in the nCA-induced lung inflammation model, a short, medium, and long treatment schedule was explored.
Mice were divided into two groups. The first group was administered nCA intranasally as the positive control (n = 5). The second group was administered nCA intranasally on day 0 of experiment and PEG- fDAO was injected through the tail vein on the second day, followed by intraperitoneal administration of D-phenylalanine (0.1 M, 0.5 mL/mouse) on the third, fourth, and fifth days (n = 5). Mice were sacrificed on the eighth day.
Mice were divided into three groups. The first group was administered PBS intranasally as the negative control (n = 3). The second group was administered nCA intranasally as the positive control (n = 3). The third group was administered nCA on day 0 and injected with PEG-fDAO (10 U/mL, 0.1 mL/mouse) (n = 3) through the tail vein on the fourth day, followed by intraperitoneal injection of D-phenylalanine (0.1 M, 0.5 mL/mouse) on the fifth, sixth, and seventh days instead of D-proline. Rhodamine-labeled bovine serum albumin (rhodamine-BSA) (10 mg/kg) was injected one day before sacrifice on the 14th day. Fluorescent imaging of the excised lungs was conducted using the IVIS Lumina XR (excitation: 555–585 emission: 695–770 nm; PerkinElmer Japan Co., Ltd., kanagawa, Japan).
Mice were divided into three groups. The first group was administered PBS intranasally as the negative control (n = 3). The second group was administered nCA intranasally as the positive control (n = 3). The third group was administered nCA on day 0 and injected with PEG-fDAO (10 U/mL, 0.1 mL/mouse) (n = 3) on the ninth day, followed by intraperitoneal injection of D-proline (1 M, 0.5 mL/mouse) on the 10th, 11th, and 12th days. Rhodamine-BSA (10 mg/kg) was injected one day before sacrifice on the 21st day. Fluorescent imaging of the excised lungs was performed using the IVIS Lumina XR (PerkinElmer Japan Co., Ltd.).
Analysis of the lung pathology
Mice were sacrificed on the eighth day in Exp-1, 14th day in Exp-2, or 21st day in Exp-3 after nCA administration. The lungs were removed and fixed in 10% buffered formalin. For light microscopy, tissues were fixed overnight, dehydrated in graded ethanol solutions, embedded in paraffin, sectioned at 2-μm thickness, and stained with hematoxylin and eosin using standard protocols.
Statistical analysis of the obtained data was performed using a two-tailed unpaired Student’s t-test. Differences were considered statistically significant at p < 0.05.