EDITORIAL FOCUS
X-ray microanalysis of airway surface liquid collected in cystic fibrosis mice

Jean-Marie Zahm1,*, Sonia Baconnais2,*, Donald J. Davidson3, Sheila Webb3, Julia Dorin3, Noel Bonnet1, Gérard Balossier2, and Edith Puchelle1

1 Institut National de la Santé et de la Recherche Médicale Unité 514, Institut Federatif de Recherche 53, Reims 51092; 2 Laboratoire de Microscopie Electronique, Unité de Formation et de Recherche Sciences, Reims 51685, France; and 3 Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The airway surface liquid (ASL) that lines the airway surface epithelium plays a major role in airway antibacterial defense and mucociliary transport efficiency, two key factors in cystic fibrosis (CF) disease. A major difficulty is to collect ASL in native conditions without stimulation or alteration of the underlying airway epithelium. Using a cryoprobe specifically adapted to collect native ASL from the tracheal mouse surface, we analyzed by X-ray microanalysis the complete ASL and plasma ion content in Cftrtm1Hgu/Cftrtm1Hgu mice compared with that in control littermates. ASL ion content from eight Cftrtm1Hgu/Cftrtm1Hgu mice and eight control littermates did not appear significantly different. The mean (±SE) concentrations were 2,352 ± 367 and 2,058 ± 401 mmol/kg dry weight for Na, 1,659 ± 272 and 1,448 ± 281 mmol/kg dry weight for Cl, 357 ± 57 and 337 ± 38 mmol/kg dry weight for S, 1,066 ± 220 and 787 ± 182 mmol/kg dry weight for K, 400 ± 82 and 301 ± 58 mmol/kg dry weight for Ca, 105 ± 31 and 105 ± 20 mmol/kg dry weight for Mg, 33 ± 15 and 29 ± 9 mmol/kg dry weight for P in non-CF and CF mice, respectively. This cryotechnique appears to be a promising technique for analyzing the complete elemental composition of native ASL in CF and non-CF tissues.

cryoprobe; ion concentration


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE AIRWAY SURFACE LIQUID (ASL) is a thin layer of fluid covering the airways that contains proteins, glycoproteins, lipids, peptides, ions, and water. The ionic composition of the ASL plays a crucial role in airway host defense by controlling the ciliary activity, mucin release (16), and antimicrobial activity (15) and regulating the volume and/or ionic composition of the ASL by ion transport across the airway epithelium. The major active ion transport process across human airway epithelia is amiloride-sensitive absorption of Na, with Cl acting as the major counteranion (9, 16).

Two hypotheses linking cystic fibrosis (CF) transmembrane conductance regulator (CFTR) dysfunction to lung disease pathogenesis have emerged and have focused investigation on salt and fluid absorption mechanisms across the airway epithelium. The first hypothesis proposes that active transcellular absorption of Na is accompanied by the diffusion of Cl ions through tight junctions between the cells and that water rapidly follows, thus maintaining osmolarity (11). As a consequence, this hypothesis predicts that the NaCl concentration in ASL should be similar to that in plasma. According to this hypothesis, the pathogenesis of lung disease in CF is related to a reduction in ASL volume due to an increased rate of isotonic ion and water absorption. Consequently, ciliary transport of ASL is impaired and airway infection develops.

The second hypothesis considers that Cl ions follow Na, transported transcellularly via Cl channels in the apical membrane (17). This hypothesis predicts that the NaCl concentration in ASL should be lower than that in plasma, with the volume maintained constant by capillary pressure from the cilia, osmotic pressure from nondiffusable osmolytes, or impermeability of the epithelium to water. Among the Cl channels regulating these ion effluxes, the CFTR protein has been heavily implicated. Hence this theory predicts that CFTR dysfunction compromises the ability of the cells to absorb Cl ions from the ASL. This failure and the consequent impairment of Na absorption would result in a raised salt concentration in the ASL of CF individuals. This hypothesis is supported by the observation that ASL displays broad-spectrum antibacterial activity that is impaired by a high-salt environment and is defective in CF (15). More recently, using a noninvasive in vivo fluorescence measurement of salt concentration, Jayaraman et al. (7) have shown that the Cl and Na contents in ASL were similar in wild-type and CFTR-null mice and that the ASL was approximately isotonic.

In view of these emergent but contradictory hypotheses, it is critical that the precise ASL composition be determined. However, the depth of this layer is so small in normal airways that it is difficult to sample ASL in vivo without disturbing the underlying epithelium. In addition, ASL is a complex mixture produced by the secretory cells of the surface epithelium and by glands in the trachea and bronchi and may also include contributions from the bronchiolar and alveolar liquids. Thus physiological studies on isolated or cultured epithelium do not appear to be the most appropriate methods for analyzing ASL composition.

Baconnais et al. (1a) have recently developed methods to collect and analyze the elemental composition of tracheal ASL in mice. The collection is carried out under conditions that do not induce any stimulatory, morphological, or functional alterations of the airway cells that produce ASL. After collection, the complete ionic composition is determined by X-ray microanalysis. The aim of this study was to use these techniques to compare the ionic composition of ASL collected from wild-type and transgenic CF mutant mice (6).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CF mouse model. The Cftrtm1Hgu/Cftrtm1Hgu transgenic mouse model used in this study was generated after targeted insertional mutagenesis into exon 10 of the murine Cftr gene in embryonic stem cells (6). The mutation is slightly "leaky," resulting in the production of a low level of wild-type CFTR as a result of exon skipping and aberrant splicing. Nevertheless, these mutant mice display the electrophysiological defect in the gastrointestinal and respiratory tracts that is characteristic of CF and can be unequivocally distinguished from their non-CF littermates on this basis (6). The mutant mice also display significantly reduced pulmonary clearance of Staphylococcus aureus and Burkholderia cepacia and significantly more severe lung pathology after repeated challenge with these same pathogens (5).

ASL was collected from pathogen-free mice: eight Cftrtm1Hgu/Cftrtm1Hgu on an outbred MF1/129 strain background (mean age and weight, 87.3 ± 22.9 days and 27.8 ± 1.5 g, respectively) and eight non-CF littermate controls (mean age and weight, 108.9 ± 43.1 days and 28.5 ± 1.8 g, respectively). The mice were housed in germ-free conditions in an isolator unit under positive pressure and maintained with sterilized food and bedding. The diet was the same for the CF mice and the littermate controls. The mice were anesthetized with an intraperitoneal injection of 100 µl of pentobarbital sodium. The trachea of each mouse was incised longitudinally along 5 mm, and the ASL was collected within 2 min with a specially designed cryoprobe.

ASL collection. A special steel probe was designed to collect the native ASL at the tracheal surface. The tip of the probe had a curvature less important (radius = 0.6 mm) compared with the internal curvature of the mouse trachea (radius = 1-1.5 mm) so only the extreme bottom part of the probe came in contact with the tracheal mucosa. In addition, the use of mice of consistent weight (25-30 g) led to a similar surface contact between the probe and the tracheal mucosa from one mouse to the other. Before the collection of ASL, the probe was washed with ultrapure water (Fluka, St. Quentin Fallavier, France) and pure methanol (Sigma, St. Quentin Fallavier, France), dried at room temperature, and cooled by plunging it into a liquid nitrogen bath (-180°C) until the thermal balance was reached. The top of the probe consisted of a small liquid nitrogen tank, allowing the probe tip to be maintained at a low temperature during the experiment. The cryoprobe was then attached to a transducer that controlled the pressure at which the cryoprobe was applied on the tracheal mucosa during ASL collection (Fig. 1). The mouse, placed in the supine position, was then moved upward at a constant speed (1 cm/min) until a contact pressure of <2,000 Pa between the cryoprobe and the tracheal mucosa was reached. Under such conditions, the ASL making contact with the cryoprobe was immediately frozen, resulting in a small amount (<10 pl) of ASL adhering to the cryoprobe tip. With warming to room temperature, the ASL adhering to the cryoprobe was thawed and deposited on an electron microscope copper grid (Maxtaform 200 mesh, Touzard et Matignon, Vitry sur Seine, France) that had successively been coated with a collodion membrane and a 10-nm carbon film.


View larger version (58K):
[in this window]
[in a new window]
 
Fig. 1.   Schematic representation of the device used to collect airway surface liquid (ASL) from the mouse trachea. The cryoprobe was cooled in liquid nitrogen and then attached to a transducer that controlled the pressure at which the cryoprobe was applied to the tracheal mucosa during ASL collection. The mouse, placed in supine position, was then moved upward at constant speed (1 cm/min) until a contact pressure of <2,000 Pa between the cryoprobe and the tracheal mucosa was reached.

The ASL deposited on the coated copper grid was cryofixed by plunging it into a liquid nitrogen bath and storing it in liquid nitrogen until analyzed. For analysis, the sample was dehydrated by freeze-drying. The freeze-drying process of the sample was done in a vacuum chamber at a residual pressure of 10-6 Torr. To increase the sublimation of the solidified ASL, the sample was warmed to the yield point of the degassing of molecular water as determined by pressure variation. Under such conditions, the degassing of water did not perturb the molecular organization of the sample.

This process had several advantages. First, the sample could be dehydrated without introducing any artifactual contamination while keeping the diffusible ions restricted to their original site. Second, the process provided a 16-fold increase in the elemental concentration of the sample in relation to the inverse of the dry mass fraction of the hydrated mucus sample. The process was completed by transferring the samples to an electron microscope fitted with a specimen cryoholder (Gatant, Pleasanton, Ca) after which they were analyzed at low temperature (-172°C) by energy-dispersive X-ray microanalysis.

X-ray microanalysis. The samples were analyzed in a scanning transmission electron microscope (CM30, Philips, Limeil-Brevannes, France) equipped with a 30-mm2 (0.13-steradian subtending solid angle, 172-eV resolution) Edax Si(Li) detector with a beryllium window.

The elemental composition of dehydrated ASL samples was determined with the continuum method (1), which implies the measurement of the specimen background intensity. The experimental background may be due to the specimen itself but may also be related to the grid fluorescence. In our experiments, we used pure copper grids. The X-ray spectra were therefore characterized by the presence of the Kalpha (8,044-eV), Kbeta , and Lalpha (940-eV) copper peaks that were constant while the X-ray spectrum was recorded at a distance higher than 10 µm from the grid bar. The method used to detect and quantify the elements from an experimental spectrum consisted of filtering and fitting the experimental spectrum with reference spectra. This method allowed us to separate the peaks from the continuous background and to differentiate overlapping peaks and permitted the simultaneous, nondestructive quantitative analysis of the biological elements of interest (Na, Cl, Mg, P, S, K, and Ca).

The dehydrated ASL was analyzed in the scanning transmission mode at 100 keV (tilt angle, 30°; sample temperature, -172°C, spectrum acquisition time, 200 s). A mean value for each of the elemental concentrations of the sample was obtained by scanning the electron probe over a 40-nm2 surface and recording 15-20 spectra in different areas of the sample. The elemental concentration was calculated in millimoles per kilogram of dry weight.

The accuracy of the methods has been checked with standards. Drops of NaCl (99 mM), MgCl2 (9.9 mM), CaCl2 (80 mM), and KCl (10 mM) dextran solutions were deposited on the pure copper grid and dehydrated overnight at 40°C. The salt crystals were then analyzed by X-ray microanalysis.

Statistical analysis. Means ± SE were calculated for ionic concentrations for the non-CF and CF groups. The two groups of mice were compared by the nonparametric Mann-Whitney test. The values were considered significantly different at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Standard solutions. The results obtained from the analysis of the standard solutions are presented in Table 1. The measured concentrations of elements in the NaCl, MgCl2, CaCl2, and KCl solutions were not significantly different compared with the expected values.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Ion concentrations in standard salt solutions

Ionic composition of airway liquid in Cftrtm1Hgu/Cftrtm1Hgu mice. A typical spectrum obtained from ASL X-ray microanalysis is presented in Fig. 2. This spectrum clearly identifies the major ions in ASL, i.e., Na, Cl, S, and Ca. The copper peak observed at a high-energy level corresponds to the copper grid on which the ASL sample was deposited. Table 2 shows the concentration of the different ions analyzed in ASL samples collected in Cftrtm1Hgu/Cftrtm1Hgu and wild-type mice. The data show that in ASL collected from mice tracheae, Na and Cl represent the two major unbound ions. The mean concentrations of Na, Cl, Mg, S, P, K, and Ca were not significantly different in Cftrtm1Hgu/Cftrtm1Hgu mice compared with wild-type mice. A huge interindividual variability was observed for the different ion concentrations in both groups of mice.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2.   Typical spectrum obtained from a mouse ASL sample after background subtraction.


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Ionic composition of airway surface liquid collected in the trachea of Cftrtm1Hgu/Cftrtm1Hgu and wild-type mice


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we report an original noninvasive method for analyzing the ionic composition of the in situ ASL collected with a specially designed cryoprobe from non-CF and CF mice. The complete ionic composition of the ASL collected from the tracheal surface of pathogen-free mice was determined by X-ray microanalysis. The main advantage of the X-ray microanalysis technique is that it requires a very low sample volume (10-50 pl), which can be easily collected with the cryoprobe described in the present paper. This small sample volume is therefore easily collectable without any mechanical stimulation, even in healthy animals. The technique also allows the simultaneous, nondestructive quantitative analysis of the different ions of interest, whereas other methods like radiotracer dilution or selective microelectrode methods (2, 12) are restricted to the measurement of one or two ions, generally Cl and Na. Moreover, the radiotracer dilution technique can only be applied to culture models. A novel noninvasive fluorescence microscopy technique has been recently described (7) to measure ASL thickness, salt concentration, and pH either in cell culture models, in vivo in the mouse trachea, or in freshly excised human bronchi. The main advantage of this technique is that it permits continuous quantitative measurements of ASL properties, although it is at present only applied to Na and Cl measurement. The cryoprobe technique that we used, associated with the X-ray microanalysis technique, allows the sampling of native ASL, but there is a limitation in the expression mode of the data that are expressed in millimoles per kilogram of dry mass.

The sampling technique may be a key factor in the ionic composition determination of ASL. The cryotechnique used in the present study allowed the rapid collection of ASL under conditions that did not induce any stimulatory, morphological, or functional alterations of the airways cells that produce ASL (1a). We carefully checked the pressure under which the probe was applied to the tracheal mucosa. Having previously shown that a pressure of <2,000 Pa did not damage the surface epithelium, we paid particular attention to limit the pressure to this level during ASL collection (1a). When analyzing with scanning electron microscopy the tracheal area where the cryoprobe had been applied, we did not observe any damage to the Clara cells and ciliated cells that cover the mouse tracheal epithelium. Moreover, we did not observe any change in the ciliary beating frequency of the ciliated cells present in the area where ASL had been collected (1a). This suggests that the ASL is mainly collected from the upper gel layer and is instantaneously frozen, thus preventing any metabolic or histological alterations of the underlying epithelial cells. In contrast, most of the techniques described in the literature (3, 8) require that the collection probe be in contact with the airway mucosa for a prolonged period, thus potentially stimulating ASL secretion during the sampling process. Such variables may well be the explanation for the apparent incompatibility of many of the published results in this field to date and suggest that the two most prominent models describing the ASL composition may not be mutually exclusive but may reflect key differences in the experimental model systems used.

We demonstrate that when housed in germ-free conditions, i.e., in the absence of any previous bacterial infection, CF mice exhibit a NaCl content in their ASL that is not significantly different compared with that in their littermate controls. Our results are consistent with the recent data reported by Cowley et al. (3), who demonstrated no difference in NaCl composition in ASL from CF and non-CF mice except when the mice were infected with Pseudomonas aeruginosa. These results are emphasized by previous data demonstrating that ASL collected from CF and non-CF xenografts of human fetal airways did not show a significant difference in the ion composition (1). Here, again, the lack of difference could be related to the perfect sterility of the mature fetal tracheal xenografts. The huge variability in the ion concentrations that we observed in both groups of mice could be related to any genes capable of modifying the genotype present in the inbred 129 or MF1 outbred genetic background. The fact that the Cftrtm1Hgu/Cftrtm1Hgu mice are not genetically identical may well account for the variation that we observed in the measured phenotypes. Genetic modifiers may influence the degree of wild-type CFTR transcription from the Cftrtm1Hgu allele and/or the level of non-CFTR-mediated Cl ion transport (14).

Having previously observed no alteration in beat frequency of ciliated cells from Cftrtm1Hgu/Cftrtm1Hgu mice but a decreased mucociliary transport rate (18), we proposed that this latter could be related to a decreased ASL water content. Data reported by Zhang and Engelhardt (19), using a human bronchial xenograft model, suggest that the hydration in CF airways may be impaired. Loss of water from the ASL is likely responsible for an increase in the mucin content of ASL, thus reducing the transport capacity of ASL (13). The "isotonic volume transport/mucus clearance" hypothesis proposed by Matsui and colleagues (10, 11) supports the proposal that in CF, the primary defect is characterized by an increased water absorption with an excessive isotonic volume absorption, leading to alterations in ASL properties. Widdicombe and Widdicombe (16), using primary cultures of human tracheal epithelium in unstimulated and uninfected conditions, obtained a hypotonic ASL (120 meq/l), and Cowley and colleagues (3, 4) reported hypotonic ASL collected by capillary electrophoresis in both mice and rats. McCray et al. (12), using a new radiotracer technique to measure the NaCl content of the ASL produced by primary cultures of murine tracheal epithelium from Cftrtm1Delta F508Uta/Cftrtm1Delta F508Uta and wild-type control mice, also reported that murine ASL is hypotonic. However, neither Cowley et al. (3) nor McCray et al. (12) found any difference between the ASL composition of CF mice and control littermates. These results are emphasized by Jayaraman et al. (7), who reported no difference in ASL salt concentrations in CF and non-CF mice.

In summary, we report an original technique to collect in vivo murine ASL and to analyze its ionic composition. We found no differences in the salinity of ASL from CF mice compared with wild-type littermates. Our results suggest that it would be of major interest to develop a technique allowing to quantify the water content in parallel with the ion content of the murine ASL.


    ACKNOWLEDGEMENTS

We thank Laurence Killian and Alain Perchet for help and contribution to this work and Prof. Thierry Chinet for critically reading the manuscript.


    FOOTNOTES

* Jean-Marie Zahm and Sonia Baconnais contributed equally to this work.

This work was supported by the Association Française de Lutte contre la Mucoviscidose and Région Champagne Ardenne (France), the Cystic Fibrosis Research Trust, and the Medical Research Council (United Kingdom).

Address for reprint requests and other correspondence: E. Puchelle, INSERM U514, 45 rue Cognacq-Jay, 51092 Reims Cedex, France (E-mail: epuche{at}worldnet.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 August 2000; accepted in final form 28 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Baconnais, S, Tirouvanziam R, Zahm JM, de Bentzmann S, Péault B, Balossier G, and Puchelle E. Ion composition and rheology of airway liquid from cystic fibrosis fetal tracheal xenografts. Am J Respir Cell Mol Biol 20: 605-611, 1999[Abstract/Free Full Text].

1a.   Baconnais, S, Zahm JM, Killian L, Bonhomme P, Gobillard D, Perchet A, Puchelle E, and Balossier G. X-ray microanalysis of native airway surface liquid collected by cryotechnique. J Microsc 191: 311-319, 1998[ISI].

2.   Boucher, RC, Stutts MJ, Bronberg PA, and Gatzy JT. Regional differences in airway surface liquid composition. J Appl Physiol 50: 613-620, 1981[Abstract/Free Full Text].

3.   Cowley, EA, Govindaraju K, Guilbault C, Radzioch D, and Eidelman DH. Airway surface liquid composition in mice. Am J Physiol Lung Cell Mol Physiol 278: L1213-L1220, 2000[Abstract/Free Full Text].

4.   Cowley, EA, Govindaraju K, Lloyd DK, and Eidelman DH. Airway surface fluid composition in the rat determined by capillary electrophoresis. Am J Physiol Lung Cell Mol Physiol 273: L895-L899, 1997[Abstract/Free Full Text].

5.   Davidson, DJ, Dorin JR, McLachlan G, Ranaldi V, Lamb D, Doherty C, Govan J, and Porteous DJ. Lung disease in the cystic fibrosis mouse exposed to bacterial pathogens. Nat Genet 9: 351-357, 1995[ISI][Medline].

6.   Dorin, JR, Dickinson P, Alton EW, Smith SN, Geddes DM, Stevenson BJ, Kimber WL, Fleming S, Clarke AR, Hooper ML, Anderson L, Beddington RSP, and Porteous DJ. Cystic-fibrosis in the mouse by targeted insertional mutagenesis. Nature 359: 211-215, 1992[ISI][Medline].

7.   Jayaraman, S, Song Y, Vetrivel L, Shankar L, and Verkman AS. Non-invasive in vivo fluorescence measurement of airway surface liquid depth, salt concentration and pH. J Clin Invest 107: 317-324, 2001[Abstract/Free Full Text].

8.   Joris, L, and Quinton PM. Filter paper equilibration as a novel technique for in vitro studies of the composition of airway surface fluid. Am J Physiol Lung Cell Mol Physiol 263: L243-L248, 1992[Abstract/Free Full Text].

9.   Knowles, MR, Murray GF, Shallal JA, Askin F, Ranga V, Gatzy JT, and Boucher RC. Bioelectric properties and ion flow across excised human bronchi. J Appl Physiol 56: 868-877, 1984[Abstract/Free Full Text].

10.   Matsui, H, Davis CW, Tarran R, and Boucher RC. Osmotic water permeabilities of cultured, well-differentiated normal and cystic fibrosis airway epithelia. J Clin Invest 105: 1419-1427, 2000[Abstract/Free Full Text].

11.   Matsui, H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, and Boucher RC. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95: 1005-1015, 1998[ISI][Medline].

12.   McCray, PJ, Zabner J, Jia HP, Welsh MJ, and Thorne PS. Efficient killing of inhaled bacteria in Delta F508 mice: role of airway surface liquid composition. Am J Physiol Lung Cell Mol Physiol 277: L183-L190, 1999[Abstract/Free Full Text].

13.   Puchelle, E, Tournier JM, Petit A, Zahm JM, Lauque D, Vidailhet M, and Sadoul P. The frog palate for studying mucus transport velocity and mucociliary frequency. Eur J Respir Dis 64: 293-303, 1983[ISI].

14.   Rozmahel, R, Wilschanski M, Matin A, Plyte S, Oliver M, Auerbach W, Moore A, Forstner J, Durie P, Nadeau J, Bear C, and Tsui LC. Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor. Nat Genet 12: 280-287, 1996[ISI][Medline].

15.   Smith, JJ, Travis SM, Greenberg EP, and Welsh MJ. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85: 229-236, 1996[ISI][Medline].

16.   Widdicombe, JH, and Widdicombe JG. Regulation of human airway surface liquid. Respir Physiol 99: 3-12, 1995[ISI][Medline].

17.   Zabner, J, Smith JJ, Karp PH, Widdicombe JH, and Welsh MJ. Loss of CFTR chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro. Mol Cell 2: 397-403, 1998[ISI][Medline].

18.   Zahm, JM, Gaillard D, Dupuit F, Hinnrasky J, Porteous DJ, Dorin JR, and Puchelle E. Early alterations in airway mucociliary clearance and inflammation of the lamina propria in CF mice. Am J Physiol Cell Physiol 272: C853-C859, 1997[Abstract/Free Full Text].

19.   Zhang, Y, and Engelhardt JF. Airway surface fluid volume and Cl content in cystic fibrosis and normal bronchial xenografts. Am J Physiol Cell Physiol 276: C469-C476, 1999[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 281(2):L309-L313
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society