Abstract
The composition of airway surface liquid (ASL) is partly determined by active ion and water transport through the respiratory epithelium. It is usually stated that in cystic fibrosis (CF), CF transmembrane conductance regulator protein abnormality results in imbalanced ion composition and dehydration of ASL, leading to abnormal rheologic and transport properties. To explore the relationship between ion composition, water content, and viscosity of airway liquid (AL), we used a human xenograft model of fetal airways developed in severe combined immunodeficiency (SCID) mice. Six non-CF and six CF portions of fetal tracheas were engrafted subcutaneously in the flanks of SCID mice raised in pathogen-free conditions. AL accumulated in the closed cylindric grafts was harvested 9 to 17 wk after implantation. At the time of AL sampling, all tracheal grafts displayed well-differentiated pseudostratified surface epithelium and submucosal glands. The viscosity of AL was measured using a controlled-stress rheometer. The ion composition of AL was quantified by X-ray microanalysis. No significant difference was observed for AL viscosity between non-CF (0.6 ± 0.5 Pa · s) and CF (0.2 ± 0.1 Pa · s) samples. In AL from non-CF and CF samples, the ion concentrations were Na: 63.9 ± 7.6, 79.7 ± 11.6; Cl: 64.9 ± 13.2, 82.6 ± 15.7; Mg: 1.9 ± 0.3, 2.2 ± 0.4; S: 4.9 ± 1.3, 4.8 ± 0.5; K: 2.4 ± 0.5, 3.2 ± 1.6; and Ca: 1.2 ± 0.3, 2.6 ± 0.8 mmol/liter, respectively. The ion composition of AL from CF versus non-CF xenografts was not significantly different. These results suggest that prior to inflammation and infection, the viscosity and ion composition of the fetal AL do not differ in CF and non-CF.
Cystic fibrosis (CF) is characterized by defective regulation of chloride (Cl−) and sodium (Na+) transports due to the defect in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. In particular, an increase in active amiloride-sensitive Na+ absorption and a defect in cyclic adenosine monophosphate (cAMP)-activated Cl− secretion are believed to alter the physical properties as well as the ion and water composition of CF airway surface liquid (ASL). The electrolyte transport defects may thus result in an abnormal regulation of fluid secretion (1, 2) and in an impaired mucociliary clearance, leading to the obstruction of airways by an excess of viscous and adhesive secretions associated with recurrent bacterial infection (3). The mechanisms that regulate the ASL hydration in non-CF and in CF respiratory epithelium, as well as the interactions between the ion composition, the water content, and rheologic properties of ASL, are still poorly known.
In the lung, ASL represents the first line of defense against microbial and environmental aggressions. The ion composition and osmolarity of ASL control the ciliary beat frequency and may also regulate mucin exocytosis (4) and the bactericidal capacity of neutrophils (5). Smith and colleagues (6) have shown that ASL from primary cultures of normal respiratory epithelia was capable of killing pathogens such as Pseudomonas aeruginosa, whereas the ASL from CF cells was not. They also suggested that the CFTR defect could lead to elevated NaCl in ASL from CF patients, which could, in turn, inactivate antimicrobial molecules in CF airways. More recently, Goldman and associates (7), using a xenograft model, confirmed that ASL from CF grafts contained abnormally high NaCl and concomitantly failed to kill bacteria. This defective antimicrobial activity could be corrected by CFTR gene transfer and was shown to be salt-dependent and related to human β1-defensin activity in ASL. At present, most of the studies on the ion composition of CF airway secretions have been limited to Na, Cl, K, and Ca composition (8, 9). Moreover, the ASL expectorated by patients or directly collected with absorbent material (such as filter paper) is likely to be contaminated by plasma exudate, inflammatory cells, and exfoliated epithelial cells. To explore the interactions between the ion composition, water content, and rheologic properties of airway liquid (AL), we used an in vivo animal model of fully differentiated CF and non-CF human tracheobronchial respiratory tissue developed from fetal tracheas after implantation into severe combined immunodeficiency (SCID) mice (10, 11). Human non-CF and CF fetal xenografts in SCID mice reach similar end-stage histologic differentiation, including a pseudostratified secretory and ciliated surface epithelium, submucosal glands, mesenchymal cells, and cartilage rings. This model has the unique property of maintaining for long term (more than 50 wk) well-differentiated and sterile non-CF and CF human tracheal mucosae that have never been in contact with air.
To question whether AL was altered in such well-differentiated and sterile CF xenografts as compared with non-CF xenografts, microsamples of AL were collected in the tracheal lumen of these xenografts. The ion composition of AL, including Na, Cl, K, Ca, P, S, and Mg, was further analyzed by X-ray microanalysis, and the viscosity and the water content of AL were simultaneously measured.
Portions of fetal tracheas were obtained from medical abortions in compliance with the current French legislation. Experiments on human tissues and live animals were approved by Centre National de la Recherche Scientifique Ethics Committee for Life Sciences. CF fetal tracheal portions were obtained from three CF fetuses (gestational age: 12.6 ± 0.9 wk). Among the CF fetuses, two were homozygous for the ΔF508 mutation and one carried the ΔF508 and R347L mutations. Non-CF fetal tracheal portions originated from four non-CF fetuses (gestational age: 20.0 ± 1.0 wk) with no history of genetic or infectious disease known to affect the respiratory tract. Prior to implantation, the tracheal samples were kept in phosphate-buffered saline (PBS) (140 mM NaCl, 4 mM KCl, 0.5 mM Na2HPO4, 0.15 mM KH2PO4, pH 7.4) (Sigma France, St. Quentin Fallavier, France) supplemented with antibiotics (streptomycin–penicillin 1%; GIBCO, Eragny, France). Each fetal trachea was dissected into two to five cylindrical pieces, 3 to 5 mm in length. These tracheal pieces were then blotted on absorbent paper to remove intraluminal PBS and surgically implanted under the skin of the flanks of anesthetized 6- to 8-wk-old C.B.17 scid/scid mice. Each mouse received at most two grafts of the same origin (one per flank). A total of six CF and six non-CF xenografts were used in this study. These grafts were maintained for at least 10 wk in the host mice before AL sampling to ensure complete histologic differentiation of the airway mucosa.
All manipulations (e.g., dissection, implantation, collection of AL, and biopsy) were performed under a laminar flow hood using sterile instruments. Host mice were bred and maintained in germ-free conditions. During the xenograft implantation and the subsequent AL collection and biopsy, the mice were anesthetized by intraperitoneal injection of 0.4 ml of Hypnomidate (ethomidate; Janssen-Cilag, Boulogne-Billancourt, France).
Control histology was performed on the fetal tracheas before engraftment and after the final experimental procedure for each xenograft. A portion of the fetal trachea and of the xenograft was dissected, fixed in 4% formaldehyde in PBS, rinsed three times in PBS, and immersed overnight at 4°C in 15% sucrose in PBS. The tissue was then embedded in Cryo-M-Bed medium (BRIGHT, Huntingdon, UK) and frozen in liquid nitrogen. Histologic observation was performed on 5-μm cryostat sections stained with periodic acid Schiff–alcian blue.
For transmission electron microscopic observation, the xenografts were fixed for 1 h with 2.5% glutaraldehyde in 0.15 M PBS and postfixed in 2% OsO4. After dehydration in ethanol, the grafts were embedded in Agar 100 resin (Agar Scientific, Stansted, UK), cut as ultrathin sections (Ultracut E; Leica, Rueil Malmaison, France), and observed using a transmission electron microscope (Hitachi H300; Elexience, Verrières le Buisson, France) at 75 kV.
To allow the collection of AL, xenografts were exposed by a limited incision of the host's skin. The membrane occluding the terminal part of the xenograft was incised in a nonvascularized area. AL that had accumulated in the lumen was collected with a positive displacement micropipette (Hirschmann Laborgeräte, Mannheim, Germany) through the incised membrane. Immediatly after AL collection, the tip of the filled micropipette was sealed with Teflon ribbon (OSI, Elancourt, France) while the piston was kept at the proximal end of the micropipette, thereby avoiding any contamination and limiting the dehydration of the AL sample. Samples were kept at 4°C and analyzed within 14 h. We verified that the evaporation rate of control samples stored under identical conditions was less than 1.5%.
To be certain of the absence of bacteria in AL, 10 μl of each AL sample was mixed with 10 ml of trypticase soy broth, incubated for 1 wk at 37°C, and then plated on blood agar. Bacteria were never seen to develop after 1-wk culture of AL samples, which confirms that the xenografts were kept sterile throughout the experimental procedure.
To ensure that the collected AL was not contaminated with blood or epithelial cells, 10 μl of each AL sample was diluted in 90 μl PBS, deposited on a glass slide, air-dried, and finally observed after Gill's hematoxylin staining. Microscopic observation of AL sample smears revealed no contamination with blood cells, whereas rare epithelial cells were seen in some AL samples.
The collected AL was deposited on an electron microscope copper grid (Maxtaform, 200 mesh; Touzart et Matignon, Vitry sur Seine, France) coated with a collodion membrane and covered with a 10-nm-thick carbon film. The AL was further cryofixed by plunging the grid into a liquid ethane bath and was freeze-dried at low pressure (10−6 mm Hg). The dehydrated AL samples were finally transferred on a specimen holder (GATAN, Pleansanton, CA) into a scanning transmission electron microscope (CM30, Philips, Limeil-Brévannes, France) and analyzed at low temperature (−172°C) by X-ray microanalysis using an energy-dispersive detector with a beryllium window (Edax, Praire View, IL).
We checked that the thickness of the dehydrated AL layer deposited on the grid was less than 1 μm. The layer thickness was measured by electron energy-loss spectroscopy by determining the t/λ ratio (t = sample thickness, and λ = total inelastic mean free path). Under such conditions, the assumption was made that both the fluorescence and the absorption of X rays within the thin specimen were negligible.
The ion composition of dehydrated AL samples was determined using the continuum or Hall method (12, 13). This technique permitted the simultaneous, nondestructive, quantitative analysis of the elements of interest (Na, Mg, P, S, Cl, K, and Ca).
The dehydrated AL was analyzed in the scanning transmission mode, at 100 keV (tilt angle: 30 degrees; sample temperature: −172°C, spectrum acquisition time: 200 s). A mean concentration value for each of the ions in the sample was obtained by scanning the electron probe over a 40-nm2 surface and recording 15 to 20 spectra in different areas of each AL sample.
We used calcium standard solutions embedded in resin at concentrations of 50 and 125 mmol/kg of resin (Agar Scientific) to calibrate the spectrometer using the Hall method. We measured calcium concentrations (48 ± 12 and 110 ± 21 mmol/kg), which were not significantly different from the expected values.
In addition, drops of 99 mM NaCl, 9.9 mM MgCl2, 80 mM CaCl2, and 10 mM KCl solutions were deposited on copper grids, dehydrated overnight at 40°C and analyzed by X-ray microanalysis. The mean ion concentrations that we measured differed by 2.5% from the expected values.
The viscosity of AL was analyzed using a controlled-stress rheometer (Carri-Med Rheometer; TA Instruments, Voisins le Bretonneux, France) equipped with a cone-plate geometry (14). The angle between the cone and the plate was 0.017 radians and the sample volume required was 20 μl. The measurements were carried out at 25°C using the creep-test technique. A constant stress of 10 Pa was applied to the sample, and the resultant strain was recorded versus time. When a steady flow was achieved, the applied stress was recorded. The slope of the strain-versus-time curve, representative of the shear rate applied to the AL sample, was calculated. The shear stress–to–shear rate ratio was used to calculate the AL viscosity. According to the sample volume, one to three measurements were performed.
A drop of AL (10 to 30 μl) was deposited on a preweighed glass slide, weighed using an analytical balance (A210P; Sartorius, Göttingen, Germany), and dehydrated for 12 h at 60°C. After the dehydration period, the AL dry weight was immediately determined and expressed as percentage of AL wet weight.
Data are expressed as means ± standard error. The Student's t test was used to compare the data obtained from non-CF versus CF xenografts. A linear regression between viscosity and ion concentration was performed to determine the specific roles of the different ions in the viscous properties of AL.
The age of CF and non-CF human fetal tracheas before engraftment ranged between 11 to 16 and 17 to 23 wk of development, respectively. The histologic aspect of the fetal tracheal mucosa before implantation varied according to the gestational age of the fetuses. At 11 to 13 wk of development, the surface epithelium exhibited more secretory cells than ciliated cells and no submucosal glands were present. Late fetal tracheas at 20 to 23 wk of development were characterized by a mature pseudostratified surface epithelium composed mainly of ciliated cells with fewer secretory cells, along with submucosal glands.
The mean duration of engraftment in SCID mice was 11.6 ± 1.4 wk for CF xenografts and 13.8 ± 0.5 wk for non-CF xenografts. At the time of AL collection, all xenografts were surrounded by connective tissue expansions and contained AL. A newly formed membrane occluded and protruded at both ends of the xenografts. Whatever their gestational age before implantation, all non-CF and CF tracheal xenografts included in this study were completely lined by a typical well-differentiated pseudostratified respiratory epithelium with basal cells and ciliated cells. Periodic acid Schiff–alcian blue–positive mucous and serous cells were present in both the surface epithelium and submucosal glands (Figures 1a and 1b). The liquid filling the xenograft lumen was also stained by periodic acid Schiff–alcian blue. The ultrastructural observation of the epithelium lining the xenografts confirmed the presence of numerous ciliated cells with cilia uniformly distributed at the apex and interspersed with microvilli. Mucous cells filled with secretory granules were also visible (Figure 2). As previously reported (11), the newly formed membranes that occluded both ends of the xenografts were also lined with a pseudostratified ciliated and secretory epithelium. No difference in the histologic aspect of the mucosa lining tracheal xenografts could be observed whether the tracheas originated from CF or non-CF fetuses.
Fig. 1. Microscopic observation at low magnification of a section of fetal tracheal xenograft at the time of AL collection. The xenograft was lined by a mature pseudostratified respiratory epithelium with periodic acid Schiff–alcian blue–positive secretory cells present in both the surface epithelium (a) and the submucosal glands (b). Note the lumen completely filled with AL, which was also periodic acid Schiff–alcian blue–positive.
[More] [Minimize]
Fig. 2. Transmission electron microscopic micrograph of the surface epithelium from a fetal tracheal xenograft. The surface epithelium is well differentiated and pseudostratified, covered by numerous cilia uniformly distributed at the apex of the ciliated cells (Cc), and interspersed with microvilli. Mucous cells filled with secretory granules (Sg) are also visible.
[More] [Minimize]All the tracheal xenografts were filled with AL that could be collected directly after incision of the occluding membrane.
As shown in Figure 3, individual AL viscosity values were low in both non-CF (range: 0.08 to 4.1 Pa · s) and CF xenografts (range: 0.04 to 0.57 Pa · s). Each individual value corresponded to between one and three measurements, with an intraindividual variation coefficient of 15.5 ± 6.7%. No significant difference was observed between the mean viscosity of AL collected in non-CF (0.6 ± 0.5 Pa · s) versus CF (0.2 ± 0.1 Pa · s) xenografts.
Fig. 3. Individual values of viscosity of AL collected from non-CF and CF xenografts (in Pa · s). No significant difference was observed between non-CF and CF.
[More] [Minimize]The water content in AL from non-CF xenografts ranged between 97.5 and 98.5%, and between 98.0 and 98.5% in AL from CF xenografts. No significant difference could be observed between the water content of AL in non-CF and CF tracheal xenografts.
Table 1 shows the concentrations of the different ions analyzed in AL samples collected in non-CF and CF xenografts. Each individual value was obtained from 15 to 20 spectra from different areas of the sample. The mean intraindividual coefficient of variation varied from 22.1 to 31.3% for all of the elements except Mg (80.3%). The data show that in AL collected from tracheal xenografts, Na and Cl represent the two major unbound ions. Although the mean concentrations of Na and Cl were higher in CF samples than in non-CF, the difference was not significant. Furthermore, no significant difference could be observed for Mg, S, K, and Ca between CF and non-CF AL samples.
| Concentration of Ions (mmol/liter) | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Xenografts | Na | Cl | Mg | S | K | Ca | ||||||
| Non-CF | 63.9 ± 7.5 | 64.9 ± 13.2 | 1.9 ± 0.3 | 4.9 ± 1.3 | 2.4 ± 0.5 | 1.2 ± 0.3 | ||||||
| CF | 78.9 ± 14.1 | 79.3 ± 18.8 | 2.3 ± 0.4 | 4.8 ± 0.6 | 3.7 ± 1.9 | 2.6 ± 0.9 | ||||||
The calculation of the index of osmolarity (i.e., twice the sum of Na plus K, which is about 285 mOsm/liter for isotonic body fluids) (9), showed that AL samples from both non-CF and CF xenografts were markedly hypotonic (132.6 and 165.2 mOsm/liter, respectively) compared with plasma (290 mOsm/liter).
A linear regression analysis allowed us to determine which ions were specifically related to the viscous properties of AL. The concentration of S in non-CF AL was significantly correlated (r = 0.95, P < 0.01) to the viscosity. Although not significant, a similar relationship was observed for the CF samples (r = 0.49).
In the present study, we used the model system of human CF and non-CF fetal tracheal xenografts in the SCID mouse. An important characteristic of this model is that CF and non-CF fetal grafts, regardless of their initial gestational age, are shown to reach similar end-stage histologic differentiation a few weeks after implantation in the host. In addition, we have previously demonstrated (11) that in such well-differentiated xenografts the bioelectric properties are independent of both the gestational age and the duration of engraftment. This previous work also showed the absence of forskolin-induced chloride secretion in CF xenografts, whereas the responses to both amiloride and extracellular adenosine triphosphate were increased compared with non-CF, as previously described in CF patients (15, 16). Histologic and bioelectric analyses thus validate the use of this in vivo model of well-differentiated human airways which, importantly, have never been exposed to air and pathogens.
The present work demonstrated that AL collected from CF and non-CF human xenografts of fetal airways exhibited very low viscosity values and a high water content compared with the values reported for adult patients. Furthermore, no significant difference could be observed regarding the viscosity and ion composition of CF versus non-CF AL.
Previous studies in the literature report high values of AL viscosity in CF, ranging between 1 Pa · s and 2 kPa · s (17-19). In contrast, associated with low values of viscosity, we also described in the present study low values of solid content in the AL from CF xenografts, suggesting that dehydration does not occur in the human fetal xenografts, despite an increased number of amiloride-sensitive sodium channels and an inefficient cAMP-dependent chloride channel.
Several factors are likely to account for the absence of AL dehydration in CF xenografts. The presence of numerous glands in the human tracheal xenografts may modulate ion and water transport. The very low viscosity of AL associated with high water content may also be related to the maturational regulation of human tracheal xenografts in SCID mice. Although the histology of human fetal airway mucosa in SCID mice clearly shows full differentiation, the xenograft remains a closed, nonventilated system that resembles the fetal lung more than the adult lung. We can therefore hypothesize that volume-activated chloride channels, such as ClC-2, contribute to the fluid secretion as was previously reported in the late stages of lung development (20).
Our data suggest that the viscosity of airway fluid is not increased at the end-stage of fetal development and that the hyperviscosity and dehydration characteristic of adult CF airway secretions are dependent on environmental factors such as infection and associated inflammation. Furthermore, AL collected in human fetal xenografts contained neither desquamated epithelial cells nor inflammatory cells, the DNA from which is known to enhance markedly the mucus viscosity (21).
Although mucus hydration is considered the main determinant of mucus rheology, the concentration of free and bound ions itself might also alter viscosity by modifying the degree of folding of the mucin peptide core (17). The regression between viscosity and ion composition showed that sulfur, which bound to mucins such as sulfomucins, is positively correlated with AL viscosity. In the present study, as already reported in the literature (8, 9, 22-24), sodium and chloride are the major ions identified in terms of relative concentrations in AL. Although these two major ions tend to be higher in CF than in non-CF airway xenografts, the difference was not statistically significant. These results differ from those of Joris and colleagues (9), who reported an increased concentration of Na and Cl in AL collected by fiberoptic bronchoscopy in CF patients. Nevertheless, regarding the CF patients included in this latter study, the potential influence of infection and inflammation cannot be ruled out, even if the AL collection was carried out after a full course of antibiotics. One very important advantage of the in vivo model that we used was that the AL collected in mature human fetal tracheal xenografts is perfectly sterile.
We found values for K lower than those reported in previous studies across a variety of species (9, 25). One possibility is that previous reports of AL composition cannot exclude either the presence of surface cells damaged during the sample collection or the presence of inflammatory cells with a consequent release of intracellular K. The K content observed in our study is in agreement with the values recently measured by Cowley and associates (25) in ASL collected in the rat trachea.
Joris and coworkers (9) did not report any significant difference between non-CF and CF AL for concentrations of the potentially bound ions (Ca, P, and S), which is in agreement with our present results. By calculating the index of osmolarity, we confirmed that AL was hypotonic in both CF and non-CF airway xenografts (165.2 and 132.6 mOsm/liter, respectively). The apparently conflicting results reported in the literature suggest that likely there is not a unique ion composition pattern of the AL. The sampling level and epithelial cell phenotype (nasal, tracheal, surface, or submucosal gland cells), as well as the method of sampling, may directly affect the ion composition of the AL. According to Knowles and associates (26), the AL could shift from isotonic values under basal unperturbed conditions to hypotonic after stimulation. Consistent with our results, Widdicombe and coworkers (27), using primary cultures of human tracheal epithelium in unstimulated and uninfected conditions, obtained a hypotonic ASL (120 mOsm/liter).
In conclusion, using a xenograft model of well-differentiated human fetal trachea, we have shown that in CF, prior to inflammation and infection, the viscosity, water content, and ion composition of AL did not differ from non-CF AL. The opening of the human fetal xenografts to the air environment could help in gaining further insight into the role of environmental factors in the regulation of ion and fluid transport across human airway epithelium.
This work was funded in part by the Centre National de la Recherche Scientifique, by Institut National de la Santé et de la Recherche Médicale and by grants from SyStemix, the Association Française de Lutte contre la Mucoviscidose (AFLM) and the Association Française de Lutte contre les Myopathies (AFM). S. Baconnais and R. Tirouvanziam were doctoral students supported by the Region Champagne-Ardenne and by an AFLM fellowship, respectively. The authors thank Dr. Ibrahim Khazaal for participation in mouse surgery. They are also grateful to Drs. Ferec, Narcy, Catala, Delezoide, Chabaud, Marcaurelles, and Levaillant for providing fetal tissues.
| 1. | Yamaya, M., W. E. Finkbeiner, and J. H. Widdicombe. 1991. Altered ion transport by tracheal glands in cystic fibrosis. Am. J. Physiol. 261(Lung Cell. Mol. Physiol.):L491–L494. |
| 2. | Smith J. J., Karp P. H., Welsh M. J.Defective fluid transport by cystic fibrosis airway epithelia. J. Clin. Invest.93199413071311 |
| 3. | Puchelle E., Jacquot J., Beck G., Zahm J. M., Galabert C.Rheological and transport properties of airway secretions in cystic fibrosis: relationship with the degree of infection and severity of the disease. Eur. J. Clin. Invest.151985389394 |
| 4. | Widdicombe J. H., Widdicombe J. G.Regulation of human airway liquid. Respir. Physiol.991995312 |
| 5. | Mizgerd, J. P., L. Kobzik, A. E. Warner, and J. D. Brain. 1995. Effects of sodium concentration on human neutrophil bactericidal functions. Am. J. Physiol. 269(Lung Cell. Mol. Physiol.):L388–L393. |
| 6. | Smith J. J., Travis S. M., Greenberg E. P., Welsh M. J.Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell851996229236 |
| 7. | Goldman M. J., Anderson G. M., Stolzenberg E. D., Kari U. P., Zasloff M., Wilson J. M.Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell881997553560 |
| 8. | Joris, L., and P. M. Quinton. 1992. Filter paper equilibration as a novel technique for in vitro studies of the composition of airway surface fluid. Am. J. Physiol. 263(Lung Cell. Mol. Physiol.):L243–L248. |
| 9. | Joris L., Dab I., Quinton P. M.Elemental composition of human airway surface fluid on healthy and diseased airways. Am. Rev. Respir. Dis.148199316331637 |
| 10. | Péault B., Tirouvanziam R., Sombardier M. N., Chen S., Perricaudet M., Gaillard D.Gene transfer to human fetal pulmonary tissue developed in immunodeficient SCID mice. Hum. Gene Ther.5199411311137 |
| 11. | Tirouvanziam, R., M. Desternes, A. Saari, E. Puchelle, B. Péault, and T. Chinet. 1998. Bioelectric properties of human cystic fibrosis and non-cystic fibrosis fetal tracheal xenografts in SCID mice. Am. J. Physiol. 274(Cell. Physiol.):C875–C882. |
| 12. | Hall T. A.Biological X-ray microanalysis. J. Microsc.1171979145163 |
| 13. | Hall T. A., Gupta B. L.Quantification for the X-ray microanalysis of cryosections. J. Microsc.1261982333345 |
| 14. | Puchelle E., Zahm J. M., Duvivier C., Didelon J., Jacquot J., Quemada D.Elastothixotropic properties of bronchial mucus and polymers analogs. Biorheology221985415423 |
| 15. | Krochmal-Mokrzan E. M., Barker P. M., Gatzy J. T.Effects of hormones on potential differences and liquid balance across explants from proximal and distal fetal rat lung. J. Physiol. (Lond.)4631993647665 |
| 16. | O'Brodovich, H. 1991. Epithelial ion transport in the fetal and perinatal lung. Am. J. Physiol. (Cell. Physiol. 30):C555–C564. |
| 17. | Tomkiewicz R. P., App E. M., Zayas J. G., Ramirez O., Church N., Boucher R. C., Knowles M. R., King M.Amiloride inhalation therapy in cystic fibrosis. Am. Rev. Respir. Dis.148199310021007 |
| 18. | Quinton P. M.Viscosity versus composition in airway pathology. Am. J. Respir. Crit. Care Med.149199467 |
| 19. | Deneuville E., Perrot-Minnot C., Pennaforte F., Roussey M., Zahm J. M., Clavel C., Puchelle E., de Bentzmann S.Revisited physicochemical and transport properties of respiratory mucus in genotyped cystic fibrosis patients. Am. J. Respir. Crit. Care Med.156199717 |
| 20. | Murray C. B., Morales M. M., Flotte T. R., McGrath-Morrow S. A., Guggino W. B., Zeitlin P. L.ClC2: a developmentally dependent chloride channel expressed in the fetal lung and downregulated after birth. Am. J. Respir. Cell Mol. Biol.121995597604 |
| 21. | Zahm J. M., Girod de Bentzmann S., Deneuville E., Perrot-Minnot C., Dabadie A., Pennaforte F., Roussey M., Shak S., Puchelle E.Dose-dependent in vitro effect of recombinant human DNase on rheological and transport properties of cystic fibrosis respiratory mucus. Eur. Respir. J.81995381386 |
| 22. | Boucher R. C.Human airway ion transport: part 2. Am. J. Respir. Crit. Care Med.1501994581593 |
| 23. | Potters J. L., Matthews L. W., Spector S., Lemm J.Studies on pulmonary secretions: II. Osmolarity and ionic environment of pulmonary secretions from patients with cystic fibrosis, bronchiectasis, and laryngectomy. Am. Rev. Respir. Dis.9619678387 |
| 24. | Robinson N. P., Kyle H., Webber S. E., Widdicombe J. G.Electrolyte and other chemical concentrations in tracheal airway liquid and mucus. J. Appl. Physiol.66198921292135 |
| 25. | Cowley, E. A., K. Govindaraju, D. K. Lloyd, and D. H. Eidelman. 1997. Airway surface fluid composition in the rat determined by capillary electrophoresis. Am. J. Physiol. 273(Lung Cell. Mol. Physiol.):L895–L899. |
| 26. | Knowles M. R., Robinson J. M., Wood R. E., Pue C. A., Mentz W. M., Wager G. C., Gatzy J. T., Boucher R. C.Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects. J. Clin. Invest.100199725882595 |
| 27. | Widdicombe J. H., Fisher H., Lee C. Y.-C., Uyekubo S. N., Miller S. S.Elemental composition of airway surface liquid. Pediatr. Pulmonol.S1419977475 |
S. Baconnais and R. Tirouvanziam participated equally in the work.
Abbreviations: airway liquid, AL; airway surface liquid, ASL; cyclic adenosine monophosphate, cAMP; cystic fibrosis, CF; cystic fibrosis transmembrane conductance regulator, CFTR; phosphate-buffered saline, PBS; severe combined immunodeficiency, SCID.
