Deferoxamine

NHBE cells were cultured in 12 well plates in BEGM with supplements and exposed for 24 h to media alone, 200 μM FAC, and 50 μM deferoxamine. Total RNA was isolated using a Qiagen kit (Qiagen, Valencia, CA) and reverse transcribed to generate cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Oligonucleotide primer pairs and fluorescent probes for MUC5B, MUC5AC, and GAPDH were designed using a primer design program (Primer Express; Applied Biosystems) and obtained from Integrated DNA Technologies (Coralville, IA). Quantitative fluorogenic amplification of cDNA was performed using the ABI Prism 7500 Sequence Detection System (Applied Biosystems), primer/probe sets of interest, and TaqMan Universal PCR Master Mix (Applied Biosystems). The relative abundance of GAPDH mRNA was used to normalize mRNA levels.

${\text{O}}_2^{•-}$ concentration in whole blood was determined immediately after blood removal. 25 µL of whole blood were mixed with an aliquot of 25 µL freshly thawed CMH (1-Hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) spin probe solution. The CMH solution contained 400 µM CMH spin probe, 25 µM deferoxamine, and 5 µM diethyldithiocarbamate to chelate transition metal ions in Krebs-HEPES-Buffer (KHB) (Noxygen, Elzach, Germany). After mixing whole blood with CMH spin probe solution, it was transferred to a 50 µL glass capillary, sealed, and measured with an EMXnano electron spin resonance (ESR) spectrometer (Bruker, Billerica, MA, USA) after 5 min incubation at 37°C (Bio-III, Noxygen, Elzach, Germany). The device settings are detailed in the Supplements. Radical concentration was quantified by comparison with a series of CP° (3-Carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy) radical standards solved in KHB. As a blank sample, KHB added to the respective amount of CMH spin probe solution was measured and subtracted from the sample value.
For determination of radical production by immune cells, 25 µL of a cell suspension containing 2.5 × 10⁶ cells/mL RPMI 1640 medium (Glucose 1.8 mg/mL, Glutamine 0.6 mg/mL, NaHCO₃ 100 µg/mL) were mixed with 25 µL of CMH spin probe solution. In contrast to whole blood, cell samples were measured over a 30 min interval to calculate the radical production rate. A sample of RPMI 1640 medium mixed 1:1 with CMH spin probe solution was used as a blank value for measuring cell suspensions and subtracted from sample values. Data were evaluated with the Xenon_nano software (version 1.3; Bruker BioSpin GmbH, Rheinstetten, Germany) and Microsoft Excel. Results regarding ROS determination by ESR were included in a dissertation by one of our co-authors (51 ). Additionally, the extracellular H₂O₂ concentration was determined in a suspension of 1 × 10⁶ PBMCs/granulocytes in 100 µL PBS after 30 min at RT. A three-electrode setup that has been previously thoroughly described was used for this purpose (52 (link)). The determination of the H₂O₂ concentration was not performed for each animal due to limited availability of the measurement device.

Sample size estimation was calculated based on an assumption of a baseline NTBI of 2.95 μmol/L, an SD of 0.6, and an alpha of 0.05 [34 (link)], resulting in an estimate that ten dogs in each arm would provide 90% power to detect a conservative 30% decrease in NTBI in the deferoxamine arm (treatment group) compared with placebo (control group) [35 (link)].
Normality was assessed through visualization of histograms and Q–Q plots. Data are summarized as the mean (95% confidence interval) for normal data, geometric mean (95% confidence interval) for log-transformed data that approximated a normal distribution, and median (Q1–Q3) for non-normal data. Baseline characteristics were compared between groups using either Student’s t-test or Fisher’s exact test. Linear mixed-effects models were used to compare differences in outcome variables between groups, with time and treatment as fixed effects and dog as a random effect. Post hoc pairwise comparisons with p value correction for multiple comparisons were planned if there was significant interaction between time and treatment (p < 0.05). Given the lack of data in the literature on the kinetics of most of the measured variables in dogs, the effect of time alone (treatment and placebo group) within the model was also reported. For highly skewed data that could not be transformed to approximate a normal distribution, pairwise comparisons using Wilcoxon rank-sum tests between groups at each time point were performed. Data were analyzed using a commercial statistical software package (SAS 9.4, SAS Institute, Cary, NC, USA).