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Although destructive airway disease is evident in young children with cystic fibrosis (CF), little is known about the nature of the early CF lung environment triggering the disease. To elucidate early CF pulmonary pathophysiology, we performed mucus, inflammation, metabolomic, and microbiome analyses on bronchoalveolar lavage fluid (BALF) from 46 preschool children with CF enrolled in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) program and 16 non-CF disease controls. Total airway mucins were elevated in CF compared to non-CF BALF irrespective of infection, and higher densities of mucus flakes containing mucin 5B and mucin 5AC were observed in samples from CF patients. Total mucins and mucus flakes correlated with inflammation, hypoxia, and oxidative stress. Many CF BALFs appeared sterile by culture and molecular analyses, whereas other samples exhibiting bacterial taxa associated with the oral cavity. Children without computed tomography–defined structural lung disease exhibited elevated BALF mucus flakes and neutrophils, but little/no bacterial infection. Although CF mucus flakes appeared “permanent” because they did not dissolve in dilute BALF matrix, they could be solubilized by a previously unidentified reducing agent (P2062), but not N -acetylcysteine or deoxyribonuclease. These findings indicate that early CF lung disease is characterized by an increased mucus burden and inflammatory markers without infection or structural lung disease and suggest that mucolytic and anti-inflammatory agents should be explored as preventive therapy.
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Experimental and statistical methods for microrheological characterization of heterogeneity in human respiratory mucus mimics of health and disease progression
Human respiratory mucus (HRM) is extremely soft, compelling passive microrheology for linear viscoelastic characterization. We focus this study on the use of passive microrheology to characterize HRM heterogeneity, a phenomenon in normal HRM that becomes extreme during cystic fibrosis (CF) disease. Specifically, a fraction of the mucin polymers comprising HRM phase-separate into insoluble structures, called flakes, dispersed in mucin-depleted solution. We first reconstitute HRM samples to the MUC5B:MUC5AC mucin ratios consistent with normal and CF clinical samples, which we show recapitulate progressive flake formation and heterogeneity. We then employ passive particle tracking with 200 nm and 1 μm diameter beads in each reconstituted sample. To robustly analyze the tracking data, we introduce statistical denoising methods for low signal-to-noise tracking data within flakes, tested and verified using model-generated synthetic data. These statistical methods provide a fractional Brownian motion classifier of all successfully denoised, tracked beads in flakes and the dilute solution. From the ensemble of classifier data, per bead diameter and mucus sample, we then employ clustering methods to learn and infer multiple levels of heterogeneity: (i) tracked bead data within vs. outside flakes and (ii) within-flake data buried within or distinguishable from the experimental noise floor. Simulated data consistent with experimental data (within and outside flakes) are used to explore form(s) of the generalized Stokes–Einstein relation (GSER) that recover the dynamic moduli of homogeneous and heterogeneous truth sets of purely flakelike, dilute solution, and mixture samples. The appropriate form of GSER is applied to experimental data to show (i) flakes are heterogeneous with gel and sol domains; (ii) dilute solutions are heterogeneous with only sol domains; and (iii) flake and dilute solution properties vary with probe diameter.
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- Award ID(s):
- 1931516
- PAR ID:
- 10572002
- Publisher / Repository:
- AIP Publishing LLC
- Date Published:
- Journal Name:
- Journal of Rheology
- Volume:
- 68
- Issue:
- 6
- ISSN:
- 0148-6055
- Page Range / eLocation ID:
- 995 to 1011
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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