Antalarmin

Streptococcus pneumoniae strain #6301 (ATCC, Manassas, VA) was grown for 16 h to obtain mid‐log phase cultures on blood agar plates (Thermo Fisher Scientific, Lenexa, KS). Mice were infected with 1 × 10⁵ colony‐forming units (CFUs) (LD₅₀) of S. pneumoniae strain #6301 (ATCC, Manassas, VA) by intranasal route in a volume of 40 μL of brain–heart infusion broth (EMD Chemicals, Inc., Gibbtown, NJ) or broth (e.g., sham infection) after anesthesia (100–150 μL ketamine/xylazine intraperitoneally).
Human corticotropin‐releasing hormone, antalarmin, and dexamethasone were from Sigma‐Aldrich, St. Louis, MO. Optimal doses of CRH (1 mg/kg), antalarmin (1 mg/kg), and dexamethasone (1 mg/kg) were administered by intranasal route based on previous published results (Kim et al. 2011).

Three individual patients originated cells were used for the studies. Human DPC culture was established according to the method of Warren [44 (link)] with modifications. Cells were cultured in CnT Basal medium 2 containing two supplements (CnT-05.A and CnT-05.B) (CELLnTEC, Huissen, The Netherlands) with 1× antibiotic-antimycotic and incubated at 37 °C under 5% CO₂.
For human ORSC culture, HFs were isolated from the dermis and attached to dishes coated with collagen type I. Outgrowing ORSCs were cultured in Keratinocyte SFM (Invitrogen-Gibco-BRL) at 37 °C under 5% CO₂.
Cells were exposed to varying concentrations of CRF for up to 72 h at 37 °C. Materials including receptor antagonists were added 1 h before hormone treatments. Antalarmin (Sigma-Aldrich) and astressin 2-B (Sigma-Aldrich) were used as CRFR1- and CRFR2-specific antagonists, respectively.