Figures
- FIGURE 1
In vivo mucociliary clearance (MMC) response to hypertonic saline (HS) inhalation correlates with in vitro measures of fluorescence recovery after photobleaching (FRAP) diffusion time measured in human nasal epithelial (HNE) cell cultures from the cystic fibrosis (CF) participants (R2=0.55, p=0.04). Only a portion of the CF group had FRAP measurements available (n=10).
- FIGURE 2
Relationship between fluorescence recovery after photobleaching (FRAP) diffusion time and airway surface liquid absorption rate in cystic fibrosis (CF) human nasal epithelial (HNE) cell cultures (R2=0.34, p=0.07). Only a portion of the CF group had FRAP measurements available (n=10).
- FIGURE 3
Mucociliary clearance (MCC) rate decreases with age in single cystic fibrosis transmembrane conductance regulator mutation carriers (CR group) (R2=0.44, p=0.002; n=16).
Tables
- TABLE 1
Demographics of study participants along with pulmonary function, Lung Clearance Index (LCI) and sweat chloride data
CF (n=26) CR (n=16) HC (n=14) p-value p-value
(CF vs HC)p-value
(CF vs CR)p-value
(CR vs HC)Age (years) 26.5 (19–39) 48.0 (44–63.5) 22.5 (20–23) 0.0001 0.09 <0.0001 <0.0001 Female/male (n) 14/12 10/6 6/8 0.56 0.51 0.29 0.43 FEV1 % pred 70 (50–93) 97 (93–109) 102 (95.5–113) (n=12) 0.0003 0.0008 0.0014 0.30 FVC % pred 94.5 (76–104) 104.0 (96–109) 107.5 (100–116) (n=12) 0.02 0.01 0.05 0.23 FEF25–75% % pred 40.5 (22–66) 100.5 (74–119.5) 83.0 (68–99.5) (n=12) 0.0001 0.004 0.0002 0.29 LCI 9.0 (7.6–13) (n=23) 7.8 (7.1–8.3) 7.2 (6.7–7.5) (n=11) 0.0007 0.0005 0.01 0.10 Sweat chloride (mmol·L−1) 101 (91–110) 36 (21–53) (n=14) 22 (10–29) (n=12) 0.0001 <0.0001 0.0002 0.11 Data are presented as median (interquartile range), unless otherwise stated. CF: cystic fibrosis; CR: single CF transmembrane conductance regulator mutation carrier; HC: healthy control; FEV1: forced expiratory volume in 1 s; FVC: forced vital capacity; FEF25–75%: forced expiratory flow at 25–75% of FVC; LCI: Lung Clearance Index (from multiple-breath washout testing). p-values comparing all groups by Kruskal–Wallis (nonparametric) except sex which is Chi-squared. Group comparisons by Dunn's test with Holm adjustment (nonparametric, multiple comparisons).
- TABLE 2
In vitro measures of human nasal epithelial (HNE) cell physiology across the groups
CF (n=26) CR (n=16) HC (n=14) p-value p-value
(CF vs HC)p-value
(CF vs CR)p-value
(CR vs HC)Culture success/failed (n) 23/3 9/7 14/0 0.004 0.18 0.004 0.001 Cl− current (µA·cm−2) 0.21 (−0.2–0.5) (n=16) 4.0 (1.5–5.2) (n=7) 5.4 (4.9–10.2) (n=13) 0.0001 <0.0001 0.003 0.13 Na+ current (µA·cm−2) 22.1 (7.8–41.3) (n=16) 9.3 (2.5–18.4) (n=7) 37.2 (26.0–46.9) (n=13) 0.012 0.10 0.04 0.004 i-ratio (Na+ current/
Cl− current)29.4 (−3.9–112.3) (n=16) 1.2 (0.7–3.9) (n=7) 6.0 (4.2–8.0) (n=13) 0.03 0.06 0.02 0.18 TER (ohm·cm2) 651 (453–807) (n=16) 717 (205–1066) (n=7) 556 (499–673) (n=13) 0.63 0.50 0.37 0.66 Cell ABS (% cleared per 24 h) 49.7 (38.2–54.1) (n=23) 42.4 (35.6–54.3) (n=9) 36.8 (27.1–45.2) (n=14) 0.03 0.01 0.19 0.22 Normalised liquid absorption rate (% per 24 h) 68.4 (61.2–80.2) (n=17) 53.7 (39.5–72.5) (n=7) 59.4 (37.2–66.5) (n=13) 0.03 0.02 0.11 0.31 HNE ASL FRAP diffusion time (τ/τsaline) 3.5 (2.8–4.0) (n=10) 2.7 (1.2–3.0) (n=5) 2.1 (1.3–3.1) (n=13) 0.03 0.02 0.08 0.40 Data are presented as median (interquartile range), unless otherwise stated. CF: cystic fibrosis; CR: single CF transmembrane conductance regulator mutation carrier; HC: healthy control; TER: transepithelial resistance; ABS: technetium-99m-DTPA absorption rate; ASL: airway surface liquid; FRAP: fluorescence recovery after photobleaching. Cl− and Na+ currents and TER were measured using Ussing chamber assessments. Cell ABS is the absorption rate of technetium-99m-DTPA from the apical surface of the cultures after addition in a 10 µL volume. Liquid absorption is measured via an optical technique [6] based on changes in ASL volume after 10 µL volume addition. Not all sampled cultures were viable and available for all measurements. Data presented graphically in supplementary figure S3. p-values comparing all groups by Kruskal–Wallis (nonparametric) except for culture success/failure which is Chi-squared. Group comparisons by Dunn's test with Holm adjustment (nonparametric, multiple comparisons). The number of individual cell donors is indicated. A minimum of three cultures is included in each measurement.
- TABLE 3
Imaging-based measurements across the groups
CF IS (n=26) CR (n=16) HC (n=12) CF HS (n=26) p-value p-value
(CF vs HC)p-value
(CF vs CR)p-value
(CR vs HC)p-value
(CF HS vs CF IS)MCC Whole lung 38 (26–49) 36 (30–43) 36 (26–47) 55 (35–70) 0.97 0.49 1.00 0.83 0.0001 Peripheral lung 36 (16–43) 35 (31–39) 35 (28–43) 54 (35–62) 0.81 0.84 0.63 0.44 <0.0001 ABS Whole lung 21 (8–26) 13 (7–24) 6 (0–13) 18 (6–25) 0.03 0.01 0.14 0.14 0.21 Peripheral lung 20 (11–32) 17 (5–22) 7 (0–18) 14 (8–25) 0.05 0.03 0.15 0.16 0.03 Cen% 51 (47–57) 52 (48–57) 49 (46–51) 52 (48–56) 0.22 0.14 0.43 0.16 0.83 Data are presented as median (interquartile range), unless otherwise stated. CF: cystic fibrosis; IS: isotonic saline; CR: single CF transmembrane conductance regulator mutation carrier; HC: healthy control; HS: hypertonic saline; MCC: mucociliary clearance rate; ABS: technetium-99m-DTPA absorption rate; Cen%: percentage of radioactive counts deposited in the central lung zone (see supplementary material). All groups inhaled IS during the MCC/ABS scan. CF subjects performed an additional study day where they inhaled 7% HS during the scan. Data are presented graphically in supplementary figures S4 and S5 (CF HS versus CF IS). p-values comparing all groups by Kruskal–Wallis (nonparametric). Group comparisons by Dunn's test with Holm adjustment (nonparametric, multiple comparisons). HS versus IS comparison for CF group by Wilcoxon matched-pairs signed-ranks test (nonparametric, paired).
- TABLE 4
Variables that correlated with therapeutic response to hypertonic saline (HS) in the cystic fibrosis group
β0 β1 R2 p-value Baseline MCCadjusted 31.62 −0.44 0.20 0.01 HNE ASL FRAP diffusion time −23.52 11.23 0.55 0.04 MCCadjusted: mucociliary clearance adjusted based on aerosol distribution; HNE: human nasal epithelial; ASL: airway surface liquid; FRAP: fluorescence recovery after photobleaching. Therapeutic response (=β1 (listed variable)+β0) is the improvement in MCCadjusted after inhaling HS compared with a baseline measurement made after isotonic saline inhalation.
- TABLE 5
Multivariable regression models of forced expiratory volume in 1 s (FEV1) % pred in the cystic fibrosis (CF) group
Model Group Model of Age (years) Chronic Pseudomonas aeruginosa# Sex (female) Sweat chloride (mmol·L−1) HNE ASL FRAP Model β1 p-value β2 p-value β3 p-value β4 p-value β5 p-value R2 p-value β0 1 CF FEV1 % pred (n=25) −1.1 0.001 −22.7 0.007 −10.9 0.12 −0.26 0.05 0.66 <0.0001 145 2 CF FEV1 % pred (n=10)¶ −0.85 0.11 −30.0 0.09 −27.8 0.03 −0.21 0.45 −13.8 0.02 0.91 0.01 187 3 CF FEV1 % pred (n=10)¶ −1.1 0.09 −7.95 0.70 −24.8 0.07 −0.25 0.57 0.76 0.03 142 MCC: mucociliary clearance; P. aeruginosa: Pseudomonas aeruginosa; HNE: human nasal epithelial; ASL: airway surface liquid; FRAP: fluorescence recovery after photobleaching. FEV1 % pred=β1(age)+β2(P. aeruginosa)+β3(sex)+β4(sweat chloride)+β5(FRAP)+β0. #: chronic P. aeruginosa is defined as two or more positive throat or sputum cultures in the previous year; ¶: only a portion of the CF group had FRAP measurements available (n=10).
Supplementary Material
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Supplementary material 00382-2022.SUPPLEMENT
