Hydrocortisone acetate

Mice were injected subcutaneously with hydrocortisone acetate (Sigma-Aldrich) (112 mg/kg of body weight) alone (C group) or together with cyclophosphamide (Sigma-Aldrich) (150 mg/kg) intraperitoneally on days –3 and –1 prior to infection (CC group). Mice were myeloablatively irradiated (8 Gy) by the use of an electron linear accelerator (Mevatron Primus; Siemens, Germany) 3 h before infection (Irr group).
On day 0, mice were intranasally infected with 2 × 10⁵ freshly harvested conidia (suspended in 0.9% NaCl plus 0.005% Tween 20) of the A. fumigatus clinical wild-type isolate ATCC 46645. Infection was left to progress for 16 h or 3 days, and mice were then processed for lung harvesting.

In the cyclophosphamide and corticosteroid treated (CCT) model, mice were intraperitoneally injected with 150 mg kg^-1 cyclophosphamide (Sigma–Aldrich, Munich, Germany) and subcutaneously (s.c.) with 112 mg kg^-1 hydrocortisone acetate (Sigma–Aldrich) on days -3 and -1 before A. fumigatus infection. In the corticosteroid treated (CT) model, mice were s.c. injected with 112 mg kg^-1 hydrocortisone acetate on days -3 and -1 before infection.

Fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) was centrifuged for 15 hours at 120,000 g, 4 °C to remove exosomes and was used to make exosome free cell culture media. Human neonatal dermal lymphatic endothelial cells (LECs) were originally harvested as described previously39 (link). LECs were expanded in flasks coated for 1 h with 50 μg/mL type I rat tail collagen (BD Biosciences, Bedford, MA) in 0.1% acetic acid and were cultured in EBM (Lonza, Walkersville, MD) supplemented with 20% exosome free FBS, 1% penicillin-streptomycin-amphotericin, 1% Glutamax (both from Life Technologies, Grand Island, NY), 25 mg/mL cyclic-AMP, and 1 mg/mL hydrocortisone acetate (both from Sigma, St. Louis, MO). Media was changed every 2–3 days and LECs were used for experiments at passages 9 and 10. Human ovarian adenocarcinoma cell line, HEY cells (Cedarlane Labs, Ontario, Canada) were cultured in RPMI 1640 (Mediatech, Manassas, VA) supplemented with 10% exosome free fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES buffer (both from Mediatech), penicillin (100 U/ml), and streptomycin (100 μg/mL) (Life Technologies) for 48 hours and the culture media was used for isolation of exosomes by ultracentrifugation. SV-LECs were cultured as previously described30 (link) and were used as a source of mouse exosomes.

The following solvents: ethanol (96%), chloroform, ammonia (25%), toluene, and ethyl acetate were purchased from POCh (Gliwice, Poland). Acetone was procured from Chempur (Piekary Śląskie, Poland). All solvents used for the TLC-densitometric analysis were of analytical grade. hydrocortisone acetate (Ph. Eur. Grade) produced by Fluka Chemicals (Milwaukee, USA), lidocaine hydrochloride (>99%, Sigma-Aldrich, St. Louis, MO, USA), and the substances related to hydrocortisone acetate such as prednisolone (>99%, Sigma-Aldrich, St. Louis, MO, USA), cortisone acetate (>99%, Sigma-Aldrich, St. Louis, MO, USA), and also hydrocortisone (>99%, Sigma-Aldrich, St. Louis, MO, USA) were used as reference substances in the preparation of standard solutions.

All chemicals and solvents were of analytical grade. Water for chromatography was obtained from a reverse osmosis system (Merck Millipore, Darmstadt, Germany). Hydrocortisone (target substance, purity > 99.0%) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Impurities of Hydrocortisone were procured from Sigma-Aldrich (Tokyo, Japan) as follows: prednisolone (impurity A), cortisone (impurity B), Hydrocortisone acetate (impurity C), 6β-hydroxy-Hydrocortisone (impurity D), 6-dehydrocortisol (impurity E), Reichstein substance S (impurity F), Hydrocortisone-21-aldehyde (impurity G), 7α-hydroxy-Hydrocortisone (impurity H), 14α-hydroxy-Hydrocortisone (impurity I), and Hydrocortisone dimer (impurity N) [16 ,17 (link),18 (link),19 (link)] (Figure 1). Lactose monohydrate (extra-fine crystal lactose hydrate “Hoei”, Pfizer, Tokyo, Japan) was used as a diluting agent. Standard solutions for Hydrocortisone (100 μg/mL) and its impurities (100 μg/mL each) were prepared by dissolving 1 mg of the respective substances in 10 mL of 50% (v/v) methanol/water. Hydrocortisone test solutions were then prepared by dissolving 10 mg of stored compounded powders in 10 mL of 50% (v/v) methanol/water and then diluting with the solvent mixture to obtain final concentrations of 100 μg/mL. Test solutions were then prepared in triplicate.