Departments of 1 Internal Medicine and 2 Microbiology, 3 Howard Hughes Medical Institute, University of Iowa, Iowa City, Iowa 52242; and 4 Department of Internal Medicine, University of Colorado, Denver, Colorado 80262
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ABSTRACT |
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In this study, we utilized the
reverse transcriptase component of telomerase, hTERT, and human
papillomavirus type 16 (HPV-16) E6 and E7 genes to transform normal and
cystic fibrosis (CF) human airway epithelial (HAE) cells. One cell
line, designated NuLi-1 (normal lung, University of Iowa), was derived
from HAE of normal genotype; three cell lines, designated CuFi (cystic
fibrosis, University of Iowa)-1, CuFi-3, and CuFi-4, were derived from
HAE of various CF genotypes. When grown at the air-liquid interface, the cell lines were capable of forming polarized differentiated epithelia that exhibited transepithelial resistance and maintained the
ion channel physiology expected for the genotypes. The CF transmembrane
conductance regulator defect in the CuFi cell lines could be corrected
by infecting from the basolateral surface using adenoviral vectors.
Using nuclear factor-B promoter reporter constructs, we also
demonstrated that the NuLi and CuFi cell lines retained nuclear
factor-
B responses to lipopolysaccharide. These cell lines should
therefore be useful as models for studying ion physiology, therapeutic
intervention for CF, and innate immunity.
cystic fibrosis transmembrane conductance regulator; human papillomavirus type 16; human airway epithelial cells; telomerase
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INTRODUCTION |
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THE AIRWAY EPITHELIUM IS NOT completely understood. It lines the airway ducts that connect the environment with the gas exchange epithelium of the lung. The surface epithelium lining the large human airways is pseudostratified; that is, all cells extend to the basement membrane, but not all cells extend to the luminal surface. The airway epithelium forms a barrier between the external and internal environments, prepares the air for optimal gas exchange at the alveoli, and purifies the air by removing particles and bacteria. The mucus at its surface helps prevent invasion of microorganisms and viruses. Its ciliated surface allows for an efficient mucociliary escalator that allows clearance of particles. The airway epithelium secretes numerous agents into the airway surface liquid, including immunoglobulins and antimicrobial factors; these form part of the defensive shield that protects the airways and lungs from bacterial infection. By active transepithelial transport of electrolytes, it controls the composition and volume of the airway surface liquid covering the epithelium to ensure proper mucociliary clearance and innate immunity. Finally, the airway epithelium plays an important role in the inflammatory response when challenged with environmental factors or infectious agents. It responds to and produces a number of cytokines and other pro- and anti-inflammatory agents that modulate innate immunity.
Several genetic and acquired diseases of the lungs involve the airway epithelia in their pathogenesis and highlight the importance of the airway epithelium (46). These include cystic fibrosis (CF), immotile cilia syndrome, pseudohypoaldosteronims, chronic bronchitis, lung cancer, and viral infections. Therefore, in vitro models of the epithelium would be helpful in understanding the pathogenesis of disease and in developing new therapies.
In earlier studies, methods for culturing and differentiating primary airway epithelial cells have been described. The differentiation was facilitated by air-liquid interface culture (10, 14, 19, 49). Compared with in vivo studies, such models have the important advantage of flexibility, control of experimental conditions, and greater opportunities for interventions. However, primary cultures have some disadvantages: limited availability of primary cells and significant variability between donors. Therefore, several cell lines have been developed. These cell lines have been derived from carcinomas or have been transformed using viral genes (for review of airway epithelial cell lines see Refs. 11, 15, 51). Although these cell lines have been very valuable, they often have limitations. Primarily, the morphology and some important functions are not always retained.
The present data strongly support the hypothesis that immortalization of human cells requires activation of a mechanism to maintain telomeres (for review see Ref. 40). It has recently been demonstrated that exogenous expression of hTERT, the catalytic component of telomerase, can efficiently immortalize certain human cell types (4) and that these immortal cell lines exhibit few, if any, phenotypic alterations (25). Some cell types, for reasons that are not completely clear, cannot be immortalized by hTERT alone and require abrogation of the retinoblastoma (Rb) and/or p53 pathways, usually by expression of viral oncoproteins (20, 32). The goal of the present work was to develop human airway epithelial (HAE) cell lines that maintain phenotypic qualities that would make them useful to study airway epithelial biology, viral pathogenesis, and airway surface liquid composition and to develop models for gene therapy for CF studies.
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METHODS |
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Primary HAE cell culture model.
Primary epithelial cells were enzymatically isolated from bronchial
epithelium of human donor lungs, as previously described (19). During transformation and subsequent passaging, the
cells were cultured on collagen-coated plastic dishes (type VI, human placental; catalog no. C-7521, Sigma) in serum-free bronchial epithelial cell growth medium with supplements (catalog no. CC3170, Clonetics/BioWhittaker). For the epithelial cultures, cells were seeded
onto collagen-coated, semipermeable membranes (0.6 cm2,
Millicell-PCF; Millipore, Bedford, MA), grown at an air-liquid interface as previously described (39, 49, 57), and
cultured for the 1st day in 50:50 DMEM-Ham's F-12 medium supplemented
with 5% fetal bovine serum. On the day after seeding, the cells were grown and then maintained in 50:50 DMEM-Ham's F-12 medium supplemented with 2% Ultroser G (Biosepra, Cergy-Saint-Christophe, France). Basolateral culture medium was changed every 2-4 days. Viable polarized epithelial cultures are stable for 3-6 mo
(44). Samples were collected with approval from the
University of Iowa Institutional Review Board.
Retroviral infections. Primary or passage 1 cryopreserved airway epithelial stocks were thawed and expanded for retroviral infections with pLXSN- or pBABE-based retroviral vectors using supernatants generated from stably producing lines (PA317) or from transient transfections of Phoenix amphotrophic packaging lines, as previously described (13, 50). Cells were infected with retroviral vector (LXSN) alone or retrovirus expressing hTERT alone or dually infected with hTERT and human papilloma virus (HPV)-16 E6/E7 retroviruses using protocols that have been previously described (13, 20). NuLi (normal lung, University of Iowa)-1 and CuFi (CF, University of Iowa)-1, CuFi-2, and CuFi-3 cells were generated by dual infection with HPV-16 E6/E7-LXSN (13) and hTERT-LXSN (20); NuLi-2 and CuFi-4 cells were generated by dual infection with HPV-16 E6/E7-LXSN and pBabe-neo-hTERT (12). Cells were selected with appropriate antibiotic [G418 (50 µg/ml) and/or hygromycin (8 µg/ml)], split 1:6 with each passage, collected for various assays, and expanded for cryopreservation.
RT-PCR, Western blot, telomere repeat amplification protocol, and cytogenetic analysis. RT-PCR for HPV-16 E7 was performed using a Retroscript kit (Ambion) according to the manufacturer's protocol and E7 specific primers: 5'-ATG ACA GCT CAG AGG AGG AG-3' (forward) and 5'-TCA TAG TGT GCC CAT TAA CAG-3' (reverse). Hot-start PCR was performed with 5 min of denaturing at 95°C followed by 33 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 30 s, with a final extension for 5 min at 72°C. This yielded an E7-specific band of 176 bp.
Western analyses were performed as previously described (20) using polyvinylidene difluoride membranes with an antibody for p53 (Oncogene Science) and secondary horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) and visualized using a chemiluminescence system (Renaissance, DuPont NEN). Telomerase analysis was performed using a telomere repeat amplification protocol ELISA kit (Roche) with 1 µl of cell lysate consisting of 1,000 cells/µl according to the manufacturer's protocol. An HPV-16 E6/E7 immortalized human keratinocyte line was used as a positive control. Cytogenetic analysis was performed at the University of Iowa Cytogenetics Facility on Giemsa-banded chromosomes onStaining for goblet and ciliated cells. Primary cultures of human airway epithelium were stained for fluorescence microscopy as follows. Cultures were fixed in 4% paraformaldehyde and then washed three times in phosphate-buffered saline (PBS). Jacalin (JAC) lectin directly conjugated to FITC (1:200; Vector Laboratories, Burlingame, CA) was applied to the apical surface at 4°C for 30 min (29, 41). After the cultures were washed in PBS, they were mounted on glass slides, and coverslips were applied with Vectashield mounting medium (Vector Laboratories). To quantitatively estimate the number of ciliated cells, epithelia were fixed in 4% paraformaldehyde and washed three times in PBS. Epithelia were then permeabilized with 0.2% Triton X-100, rinsed, and incubated with Superblock (Pierce). Epithelia were incubated with mouse antikeratan sulfate monoclonal antibody (Chemicon, Temecula, CA) diluted 1:100 in Superblock and incubated again in a 1:200 dilution of FITC-labeled anti-mouse IgG (Biomeda) (44, 58). Epithelia were analyzed using a scanning laser confocal microscope (model MRC-1024; Bio-Rad, Richmond, CA). Images were obtained en face. To quantitate the percentage of JAC- or keratan sulfate-positive cells per epithelial preparation, epithelial cultures were counterstained with 4',6-diamidino-2-phenylindole (Vector Laboratories) to label total number of cells.
For scanning electron microscopy, samples were fixed with 2.5% glutaraldehyde and then with 1% osmium tetroxide (43). Samples were dehydrated through a graded series of ethanol and finally with hexamethyldisilizane (Ted Pella, Redding, CA). Samples were mounted onto grids, sputter coated with gold, and imaged on a Hitachi S-4000 scanning electron microscope.Measurement of transepithelial electrical properties.
For measurement of transepithelial electrical properties, epithelia
were mounted in Ussing chambers and studied as previously described
(38, 54). Epithelia were bathed in symmetrical solutions containing (in mM) 135 NaCl, 2.4 K2HPO4, 0.6 KH2PO4, 1.2 CaCl2, 1.2 MgCl2, 10 dextrose, and 5 HEPES (pH 7.2) at 37°C and
gassed with 100% O2. Isc Amil is
the decrease in short-circuit current (Isc)
after apical addition of 10 µM amiloride. cAMP-stimulated Isc (Isc cAMP) is the
increase in current after basolateral addition of cAMP agonists (10 µM forskolin + 100 µM IBMX). Bumetanide-sensitive Isc (Isc Bumet) is the
decrease in current after basolateral addition of 100 µM bumetanide
to epithelia studied in the presence of apical 10 µM amiloride and
basolateral cAMP agonists (10 µM forskolin + 100 µM IBMX);
Isc Bumet is a measure of the transepithelial
Cl transport pathway that includes CF transmembrane
conductance regulator (CFTR). To investigate the ability to correct the
Cl
transport pathway on CuFi cells, we studied the effect
of adenovirus (Ad)-mediated expression of CFTR (AdCFTR). Briefly, to
disrupt the tight junctions, we pretreated the apical surface of the
epithelium for 30 min with 400 µl of 8 mM EGTA in Eagle's modified
essential medium (EMEM) containing 50 multiplicities of infection (MOI) of AdCFTR at 37°C. Epithelia were studied in Ussing chambers 3 days
later as described above.
Polarity of infection by adenoviruses.
Recombinant adenovirus vectors expressing -galactosidase, Ad
Gal,
were prepared as described previously (28, 42) by the University of Iowa Gene Transfer Vector Core at titers of
~1010 infectious units (IU)/ml. At 14 days after the
epithelia were seeded, 25 µl containing 50 MOI of the recombinant
viruses in PBS were added to the apical or basolateral surface for 30 min (particle-to-IU ratio = 25). After incubation, the viral
suspension was removed and the epithelia were rinsed twice with PBS.
After infection, the epithelia were incubated at 37°C for an
additional 48 h. Total
-galactosidase activity was then
measured using a commercially available method (Galacto-Light; Tropix,
Bedford, MA). Briefly, after they were rinsed with PBS, the cells were removed from the filters by incubation with 120 µl of lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM
1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10%
glycerol, and 1% Triton X-100) for 15 min. Light emission was
quantified in a luminometer (Analytical Luminescence Laboratory, San
Diego, CA).
Adenovirus-mediated gene transfer corrects the CF epithelial
Cl transport defect.
To study the ability to correct the Cl
transport pathway
on CuFi cells, we studied the effect of adenovirus-mediated expression of CFTR cDNA on the three CuFi cell lines. Recombinant adenovirus vectors expressing CFTR cDNA under the cytomegaolvirus promoter, AdCFTR, were prepared as described previously (52).
Briefly, to disrupt the tight junctions of the epithelia and allow
access of the recombinant viruses to the basolateral surface, we
pretreated the apical surface of the epithelium for 10 min with 400 µl of 8 mM EGTA in EMEM containing 50 MOI of AdCFTR or adenovirus
expressing green fluorescent protein (AdGFP) as control
(42). The virus was rinsed, and epithelia were studied to
assess Cl
transport in Ussing chambers 3 days later (see
above). We also studied epithelia derived from the primary cells that
were used to develop the CuFi-1, CuFi-2, CuFi-3, and CuFi-4 cell lines.
Lipopolysaccharide-induced activation of nuclear factor-B.
The cDNA for luciferase was introduced into primary and NuLi airway
epithelial cells by treating the epithelia with 400 µl of 8 mM EGTA
in EMEM containing 50 MOI of the adenovirus that expresses nuclear
factor-
B (AdNF-
B) (35) luciferase at 37°C. After 1 day, the epithelia were exposed to Pseudomonas aeruginosa lipopolysaccharide (LPS) at 0.1 ng/ml, and luciferase activity was
analyzed (1). After the cells were rinsed with PBS, they were removed from the filters by incubation with 120 µl of lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10%
glycerol, and 1% Triton X-100) for 15 min. Light emission was
quantified in a luminometer (Analytical Luminescence Laboratory).
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RESULTS |
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Passaging primary HAE cells.
Primary cells generally are good for electrophysiology studies for only
the first couple of passages; then they lose their ability to form
tight junctions and transepithelial resistance (Rt) when grown at the air-liquid interface. In
our experience, the passaging of HAE cells results in increased
Rt and decreased Isc by
passage 1 with a significant drop of
Rt on further passages. Most of the time, cells
passaged two to three times fail to form structures that exhibit
Rt. In Fig. 1, we
show the results of passaging cells from one donor three times.
Rt increased by 40% on passages 1 and 2 and dropped substantially on passage 3 (Fig. 1A). We observed a linear drop in
Isc (Fig. 1B) and
Isc Amil (Fig. 1C) as well as in
Isc cAMP (Fig. 1D). By passage 3, the epithelia exhibited minimal active Na+ and
Cl transport. Data for passages 4 and beyond
are not shown, because epithelia that could be studied in Ussing
chambers fail. These data suggest that it is important to develop
airway epithelial cell lines that retain the ability to differentiate,
form tight junctions, and maintain ion channel physiology when grown at
the air-liquid interface.
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Generation of HAE cell lines.
To develop HAE cell lines, we wanted to first determine whether
immortalization could be achieved by infection with retroviruses expressing hTERT alone. Because it has been shown that some cell types
require other factors for immortalization, we also decided to perform
experiments in parallel in which cells were coinfected with hTERT and
HPV-16 E6/E7 retroviral vectors. The latter retrovirus expresses HPV-16
E6 and E7, which are capable of abrogating the p53 and Rb pathways,
respectively (20). Two normal HAE and four CF HAE cell
strains were used for this study. We found that exogenous expression of
hTERT was inefficient at immortalizing HAE cells, and, in general,
hTERT-expressing cells grown on submerged cultures senesced at
passages 10-15, which was approximately the same time at which vector control cells senesced (data not shown). Expression of
hTERT and HPV-16 E6/E7 together, on the other hand, resulted in
efficient extension of lifespan to beyond passage 30 with no apparent crisis or slow down in growth (as evidenced by the time required to achieve confluency after splitting). The hTERT-
E6/E7-transduced cell lines derived by this method were designated
NuLi-1 and NuLi-2; the cell lines from CF tissue were designated
CuFi-1, CuFi-2, CuFi-3, and CuFi-4 (Table
1). The CF genotypes of the lines were verified by commercially available multiplex PCR (Genzyme Genetics, Framingham, MA) and are shown in Table 1. All the cell lines were
analyzed for telomerase activity, expression of HPV-16 E7, and p53
levels (as a functional assay for expression of HPV-16 E6). RT-PCR
analysis for HPV-16 E7 revealed that all the cell lines expressed E7
transcripts (Fig. 2A). Western
analysis demonstrated downregulated levels of p53 protein in all the
cell lines compared with normal or vector control cells, as would be
expected of cells that expressed HPV-16 E6 (Fig. 2B). The
cell lines were also shown to express high levels of telomerase as
measured by an ELISA-based telomere repeat amplification protocol assay
(Fig. 2C). These data demonstrated that the cell lines
contained and expressed the expected hTERT and E6/E7 genes.
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NuLi-1 cells retain the ability to develop epithelia that are
active in Na+ and
Cl transport.
Primary lung epithelial cells infected with retroviruses expressing
hTERT and HPV-16 E6/E7 (NuLi-1) were cultured on plastic up to
passage 30. Every couple of passages the airway epithelial cells were trypsinized and seeded onto semipermeable filters and grown
at the air-liquid interface. After 3-4 wk, the epithelia were
studied in Ussing chambers, and the Isc,
transepithelial voltage (Vt), and
Rt were recorded. Rt was
slightly higher after passage 7 than in the primary cultures
(685 ± 30.7 vs. 532 ± 146.7
), and
Rt showed a downward trend as the passage number
increased (Fig. 3).
Rt toward passage 29 was only
slightly lower at 389 ± 20.7
. To evaluate ion transport, we
recorded Isc at baseline (Fig.
4A),
Isc Amil (Fig. 4C),
Isc DIDS (Fig. 4D), Isc cAMP (Fig. 4E), and
Isc Bumet (Fig. 4F). We also
recorded baseline Vt under open-circuit
conditions (Fig. 4B). Baseline Isc
was preserved throughout passages 7-29 at 30-40 µA/cm2 and was very similar to that of the primary
untransformed cells (34.9 ± 7.7 µA/cm2). Most of
this Isc could be accounted for by
amiloride-sensitive Na+ transport, and, not surprisingly,
Isc Amil was also preserved throughout
passaging. Isc Amil at passage 29 was similar to that of the untransformed passage 1 (25.7 ± 9.2 vs. 27.3 ± 1.4). DIDS blocks Cl
transport via non-CFTR Cl
channels.
Isc DIDS in primary airway epithelia is <1
µA/cm2, which is much less than
Isc Amil and Isc cAMP and declines as the passage number is increased. CFTR-mediated Cl
transport can be stimulated with the addition of cAMP
agonists (Isc cAMP). In primary cultures,
Isc cAMP was similar to that in epithelia grown
from transformed passage 7 NuLi cells (33.33 ± 2.9 vs.
29.3 ± 2.5). However, we observed a linear decline in
Isc cAMP as the passage number increased.
Passage 29 exhibited a cAMP-stimulated Cl
current that was only 16.3% of the current in untransformed primary cells. Isc cAMP, a reflection of
cAMP-stimulated Cl
current, and
Isc Bumet, a reflection of total
(cAMP-stimulated + baseline) Cl
current, mirror each
other in magnitude and trends with subsequent passages. We also
examined the response of NuLi-1 epithelia to 1 mM ATP in the presence
of amiloride (Isc ATP).
Isc ATP was significantly higher (68%) in
NuLi-1 epithelia than in primary cultures of non-CF epithelia
(15.0 ± 1.35 vs. 8.9 ± 2.0 µA/cm2,
n = 6, P < 0.01). Thus NuLi-1 cells
can form electrically tight airway epithelia that preserve
Na+ transport compared with primary cells. For reasons
unknown, NuLi-2 cells did not exhibit measurable
Rt when grown in a similar fashion (data not
shown). The progressive decline in Cl
transport in the
NuLi-1 cells could imply a decrease in CFTR or a decrease in
basolateral K+ channels. Although less than ideal, the
significant amount of Cl
transport observed in earlier
passages would be useful for studies of CFTR function.
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NuLi-1 airway epithelia are highly reproducible.
A major advantage of using an immortalized airway epithelial model over
primary lung airway epithelial cells would be the ability to decrease
the variability from donor to donor. Thus we asked whether NuLi-1 cells
independently thawed and grown at the air-liquid interface at different
times would exhibit similar electrophysiology. We grew three different
cryostocks of NuLi-1 cells at passage 11 at the air-liquid
interface and examined their Rt. After 3 wk, the
epithelia were studied in Ussing chambers, and we recorded the baseline
Isc and the change in Isc
after sequential administration of 10 µM amiloride, 10 µM DIDS, 10 µM forskolin + 100 µM IBMX, and 100 µM bumetanide. Figure
5 shows that three different
cryostock-derived epithelia resulted in identical
Isc. This suggests that using NuLi-1-derived
epithelia can significantly improve the reproducibility of experimental
results.
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CuFi cells retain the ability to develop epithelia that are active
in Na+ transport and lack
Cl transport.
Primary lung epithelial cells from CF lung transplant recipients were
infected with hTERT and HPV-16 E6/E7 to generate four different
immortalized CF cells: CuFi-1 (
508/
508), CuFi-2 (
508/
508), CuFi-3 (
508/R553X), and CuFi-4 (
508/G551D). Cells were passaged on plastic up to 30 generations. At passage 18, cryovials
were individually thawed and seeded onto semipermeable filters and compared with their primary source. After 3-4 wk, the epithelia were studied in Ussing chambers, and Isc,
Vt, and Rt were recorded. All cell lines except CuFi-2 exhibited good Rt
(Table 1). The ion transport properties of passaged CuFi-1, CuFi-3, and
CuFi-4 epithelia were compared with ion transport properties of
epithelia grown from their corresponding primary cells and passaged
epithelia in which wild-type CFTR was expressed using a recombinant
adenovirus (AdCFTR). We recorded the Isc as we
did on NuLi cells. Baseline Isc values for all
three cell lines were very similar to values for the primary
untransformed cells (Fig. 6,
A-C). Most of this Isc could be accounted for by
amiloride-sensitive Na+ transport and, not surprisingly,
Isc Amil was also preserved throughout
passaging (Fig. 6D). Addition of cAMP agonists failed to
stimulate CFTR-mediated Cl
transport (Fig.
6E). We also examined the response of CuFi-1 epithelia to 1 mM ATP in the presence of amiloride (Isc ATP). Isc ATP in CuFi-1 epithelia was twice as high
as in NuLi-1 epithelia and fivefold higher than in primary cultures of
CF epithelia (28.8 ± 1.7 vs. 5.5 ± 0.5 µA/cm2, n = 6, P < 0.01). Restoration of Isc cAMP and
Isc Bumet in the CuFi epithelia treated with
AdCFTR suggests that these epithelia could be used as an intermediate
model for therapeutic interventions that restore Cl
current (Fig. 6).
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Morphology of NuLi-1-derived airway epithelia.
Growing airway epithelia that can retain electrical properties of the
epithelia will be very useful for the understanding and development of
therapies directed at restoring Cl transport. To further
characterize the morphology of these epithelia, we compared the number
of ciliated and goblet cells as detected by JAC lectin directly
conjugated to FITC (goblet cells) and mouse antikeratan sulfate
monoclonal antibody (ciliated cells) (58). The number of
goblet cells progressively increased as the passage number increased
(Fig. 7). Moreover, the number of
ciliated cells decreased significantly from >50% on primary cells to
~5% by passage 20. Thus most of the cells in the
epithelia derived from NuLi-1 cells do not appear to be ciliated or to
be goblet cell markers. This suggests that although the epithelia
retain significant amounts of Cl
and Na+
transport, the morphology of the epithelia only slightly resembles that
of the human airway in vivo after extensive passaging.
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Polarity of infection by adenoviruses.
To further characterize these transformed epithelia, we asked whether
receptors are polarized appropriately to the basolateral side. The data
from the Ussing chamber experiments clearly suggested that channels
were correctly localized; for example, administration of amiloride
resulted in blocking of the epithelial Na+ channel. The
receptor for adenovirus (CAR) is also localized exclusively to the
basolateral side of the epithelial cells. We and others
(30) have shown that basolateral administration of adenovirus results in a more efficient fiber-dependent infection of the
airway epithelia. Thus we compared the polarity of adenovirus infection
on primary and passaged NuLi-1-derived epithelia. Epithelia were
incubated with 10 MOI of a recombinant adenovirus that expresses -galactosidase. After infection, the epithelia were incubated at
37°C for an additional 48 h. Similar to our previous
observations, infection of NuLi-1-derived epithelia was 2 logarithmic
units more efficient from the basolateral side than from the apical side (Fig. 8). This suggests that
NuLi-1-derived epithelia retain the appropriate polarity of expression
of receptors such as CAR. Moreover, these results indicate that these
cell lines could be a good model for developing vectors targeted to the
apical side.
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LPS-induced activation of NF-B.
One of the properties of the airway epithelia is to offer the
first-line release of proinflammatory cytokines and recruitment of
polymorphonuclear leukocytes to the air space required to maintain a
sterile environment (innate immunity). We tested the ability of
NuLi-derived airway epithelia to respond to apical administration of
LPS. To accomplish this, we utilized a recombinant adenovirus to
introduce the cDNA for luciferase driven by an NF-
B response promoter. Thus activation of NF-
B could be easily detected by measuring luciferase activity in a luminometer. Figure
9 demonstrates that primary cells
expressed little luciferase activity and that exposure to LPS results
in a 10-fold increase in the amount of luciferase activity. Passaged
cells exhibited increased basal levels of luciferase activity but
continue to respond to LPS stimulation. These data suggest that
NuLi-1-derived airway epithelia conserve the TLR-4 NF-
B axis and may
be utilized to further understand innate immunity.
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DISCUSSION |
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The development of methods for growing HAE cells that can reliably
form electrically tight epithelia has been indispensable for the
understanding of the function of epithelial ion transport, composition
of airway surface liquid (23, 56), the CF defect (18, 48), transcytosis of immunoglobulins (7,
9), pathogenesis of viral and bacterial infections (30,
37, 53), and the repair process of the airways and development
of novel vectors for gene therapy (17, 24, 27, 33, 44,
55). The reliance on primary cultures has limited the access to
this resource, and the variability between different donor epithelia
complicates the design and interpretation of the data. Here we show a
series of airway epithelial cells that retain epithelial properties
after multiple passages and will significantly aid in the understanding of epithelial biology. Although these epithelia failed to completely reproduce the morphology and differentiation aspects of distinct airway
epithelial cell types, they may become a useful tool to understand the
cues required for airway epithelia to assume a ciliated or secretory
phenotype. Moreover, these non-CF epithelia exhibited decreased
cAMP-stimulated Cl currents with serial passaging, but
these currents were still significantly different from those seen in CF epithelia.
The generation of CuFi cells might also be useful in the development of
mutation-specific therapies. Several groups have screened compounds
that may affect F508 or G551D CFTR-expressing cells to restore
Cl
channel function (3, 8, 22, 34, 36, 47).
This work has been done mostly in yeast and heterologous cell lines.
Therapeutic candidates could easily be tested in CuFi-4 epithelia, or
novel strategies could be developed to perform high-throughput
screening in epithelia. Moreover, compounds that induce read through of stop codon mutations have been tested in cell lines and humans. CuFi-3
cells that express R553X (2, 5, 16, 47) could be an
excellent candidate for screening those drugs.
Our success at generating HAE cell lines that retain at least some aspects of normal primary cells may be related to exogenous expression of hTERT in conjunction with HPV-16 E6/E7. Most previous attempts to generate HAE cell lines have relied on transfection of the entire HPV-16 or HPV-18 genomes, E6/E7 alone, or infection with the entire genome of simian virus-40 (11, 15). Without maintenance of telomeres by telomerase activation, these strategies have the potential of generating cell lines that are genetically unstable. Cytogenetic analysis of the NuLi and CuFi cell lines that we have developed indicates that they do not contain numerous chromosome rearrangements and are fairly stable in chromosome number. Interestingly, all the cell lines contained extra copies of chromosome 20, and most contained extra copies of chromosome 5. The relevance of these changes is unknown, although 20q amplifications have been observed in uroepithelial cells immortalized by HPV-16 E7 (6). It is possible that exogenous expression of hTERT confers some measure of stability on the cell lines, both genotypically and phenotypically. This needs to be studied in more detail by comparing cells immortalized by E6/E7 alone and cells immortalized by hTERT and E6/E7 together. Even with the addition of hTERT, however, the transduced cell lines still lost some of their phenotypic properties with time in culture, and not all the transduced cell lines retained the ability to form structures that exhibited Rt, even at early passage (i.e., NuLi-2 and CuFi-2). The reason for this is unknown. Immortalization of cells by hTERT alone, without the addition of viral oncogenes, might have resulted in more stable cell lines. We were, however, unsuccessful at efficiently and consistently generating immortal cell lines by expression of hTERT alone. In another study (unpublished observations), we were able to obtain one immortal HAE cell line by hTERT expression alone, but this cell line failed to form an electrically tight epithelium when grown at the air-liquid interface. The observation of poor immortalization of cells by hTERT is similar to a study recently reported by others in which immortalization of HAE cells required expression of hTERT and the early region of simian virus-40 (21). It is unknown why HAE cells are resistant to immortalization by hTERT alone, but it may be related to growth conditions. For example, there is evidence that immortalization of human keratinocytes grown on plastic requires telomerase activation and abrogation of the Rb pathway, but when the cells are grown with irradiated feeders, only activation of telomerase is required (31). One way to potentially overcome the phenotypic changes that may be associated with expression of viral oncogenes would be use of a system in which the viral genes could be turned off or removed by a recombinase after initial expansion of the cells. Such a strategy has recently been used to generate reversibly immortal endothelial cells (26).
In summary, we have described a method to efficiently extend the lifespan of CF and non-CF HAE cells while still allowing, in most cases, the retention of normal phenotypic qualities such as the ability to form an electrically tight epithelium. These cell lines should be useful for studies of ion physiology of airway cells, therapeutic intervention for CF, and innate immunity of epithelial cells.
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ACKNOWLEDGEMENTS |
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We thank Jan Launspach, Pary Weber, Tamara Nesselhauf, Lisa Einwalter, David Welsh, Daniel Vermeer, Theresa Mayhew, and Rosanna Smith for excellent assistance; Joel Kline and Dwight Look for insightful discussion; Geron for the hTERT cDNA used in construction of pLXSN-hTERT; Robert Weinberg for the hTERT-neo-pBABE retroviral construct; and Denise Galloway for the HPV-16 E6/E7 retrovirus.
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FOOTNOTES |
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This study was supported by the University of Iowa Central Microscopy Research Facility, the Gene Transfer Morphology Core (supported by the National Institute of Diabetes and Digestive and Kidney Diseases), the In Vitro Cell Models Core (supported by the National Institutes of Health and the Cystic Fibrosis Foundation), and Iowa Statewide Organ Procurement and National Institutes of Health Grants HL-61234, DK-60113, and P30 DK-54759 and Cystic Fibrosis Foundation Grant ENGELH98S0.
Address for reprint requests and other correspondence: J. Zabner, Dept. of Internal Medicine, University of Iowa, Iowa City, IA 52242 (E-mail: joseph-zabner{at}uiowa.edu).
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.
First published January 10, 2003;10.1152/ajplung.00355.2002
Received 23 October 2002; accepted in final form 14 December 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arbour, NC,
Lorenz E,
Schutte BC,
Zabner J,
Kline JN,
Jones M,
Frees K,
Watt JL,
and
Schwartz DA.
TLR4 mutations are associated with endotoxin hyporesponsiveness in humans.
Nat Genet
25:
187-191,
2000[ISI][Medline].
2.
Bal, J,
Stuhrmann M,
Schloesser M,
Schmidtke J,
and
Reiss J.
A cystic fibrosis patient homozygous for the nonsense mutation R553X.
J Med Genet
28:
715-717,
1991[Abstract].
3.
Bebok, Z,
Venglarik CJ,
Panczel Z,
Jilling T,
Kirk KL,
and
Sorscher EJ.
Activation of F508 CFTR in an epithelial monolayer.
Am J Physiol Cell Physiol
275:
C599-C607,
1998
4.
Bodnar, AG,
Ouellette M,
Frolkis M,
Holt SE,
Chiu CP,
Morin GB,
Harley CB,
Shay JW,
Lichtsteiner S,
and
Wright WE.
Extension of life-span by introduction of telomerase into normal human cells.
Science
279:
349-352,
1998
5.
Clancy, JP,
Bebok Z,
Ruiz F,
King C,
Jones J,
Walker L,
Greer H,
Hong J,
Wing L,
Macaluso M,
Lyrene R,
Sorscher EJ,
and
Bedwell DM.
Evidence that systemic gentamicin suppresses premature stop mutations in patients with cystic fibrosis.
Am J Respir Crit Care Med
163:
1683-1692,
2001
6.
Cuthill, S,
Agarwal P,
Sarkar S,
Savelieva E,
and
Reznikoff CA.
Dominant genetic alterations in immortalization: role for 20q gain.
Genes Chromosomes Cancer
26:
304-311,
1999[ISI][Medline].
7.
Deffebach, ME,
Bryan CJ,
and
Hoy CM.
Protein movement across cultured guinea pig trachea: specificity and effect of transcytosis inhibitors.
Am J Physiol Lung Cell Mol Physiol
271:
L744-L752,
1996
8.
Devor, DC,
Bridges RJ,
and
Pilewski JM.
Pharmacological modulation of ion transport across wild-type and F508 CFTR-expressing human bronchial epithelia.
Am J Physiol Cell Physiol
279:
C461-C479,
2000
9.
Ferkol, T,
Eckman E,
Swaidani S,
Silski C,
and
Davis P.
Transport of bifunctional proteins across respiratory epithelial cells via the polymeric immunoglobulin receptor.
Am J Respir Crit Care Med
161:
944-951,
2000
10.
Gruenert, DC,
Basbaum CB,
and
Widdicombe JH.
Long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions.
In Vitro Cell Dev Biol
26:
411-418,
1990[ISI][Medline].
11.
Gruenert, DC,
Finkbeiner WE,
and
Widdicombe JH.
Culture and transformation of human airway epithelial cells.
Am J Physiol Lung Cell Mol Physiol
268:
L347-L360,
1995
12.
Hahn, WC,
Counter CM,
Lundberg AS,
Beijersbergen RL,
Brooks MW,
and
Weinberg RA.
Creation of human tumour cells with defined genetic elements.
Nature
400:
464-468,
1999[ISI][Medline].
13.
Halbert, CL,
Demers GW,
and
Galloway DA.
The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells.
J Virol
66:
2125-2134,
1992[Abstract].
14.
Harris, CC,
Lechner JF,
Yoakum GH,
Amstad P,
Korba BE,
Gabrielson E,
Grafstrom R,
Shamsuddin A,
and
Trump BF.
In vitro studies of human lung carcinogenesis.
Carcinog Compr Surv
9:
257-269,
1985[Medline].
15.
Hopfer, U,
Jacobberger JW,
Gruenert DC,
Eckert RL,
Jat PS,
and
Whitsett JA.
Immortalization of epithelial cells.
Am J Physiol Cell Physiol
270:
C1-C11,
1996
16.
Howard, M,
Frizzell RA,
and
Bedwell DM.
Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations.
Nat Med
2:
467-469,
1996[ISI][Medline].
17.
Johnson, LG,
Boyles SE,
Wilson J,
and
Boucher RC.
Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells.
J Clin Invest
95:
1377-1382,
1995[ISI][Medline].
18.
Johnson, LG,
Dickman KG,
Moore KL,
Mandel LJ,
and
Boucher RC.
Enhanced Na+ transport in an air-liquid interface culture system.
Am J Physiol Lung Cell Mol Physiol
264:
L560-L565,
1993
19.
Karp, PH,
Moninger TO,
Weber SP,
Nesselhauf TS,
Launspach J,
Zabner J,
and
Welsh M.
Developing an in vitro model of differentiated human airway epithelia: methods for establishing primary cultures.
In: Epithelial Cell Culture Protocols, edited by Wise C.. Totowa, NJ: Humana, 2002, p. 115-137.
20.
Kiyono, T,
Foster SA,
Koop JI,
McDougall JK,
Galloway DA,
and
Klingelhutz AJ.
Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells.
Nature
396:
84-88,
1998[ISI][Medline].
21.
Lundberg, AS,
Randell SH,
Stewart SA,
Elenbaas B,
Hartwell KA,
Brooks MW,
Fleming MD,
Olsen JC,
Miller SW,
Weinberg RA,
and
Hahn WC.
Immortalization and transformation of primary human airway epithelial cells by gene transfer.
Oncogene
21:
4577-4586,
2002[ISI][Medline].
22.
Ma, T,
Vetrivel L,
Yang H,
Pedemonte N,
Zegarra-Moran O,
Galietta LJ,
and
Verkman AS.
High-affinity activators of CFTR chloride conductance identified by high-throughput screening of 60,000 diverse compounds.
J Biol Chem
277:
37235-37241,
2002
23.
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].
24.
Matsui, H,
Johnson LG,
Randell SH,
and
Boucher RC.
Loss of binding and entry of liposome-DNA complexes decrease transfection efficiency in differentiated airway epithelial cells.
J Biol Chem
272:
1117-1126,
1997
25.
Morales, CP,
Holt SE,
Ouellette M,
Kaur KJ,
Yan Y,
Wilson KS,
White MA,
Wright WE,
and
Shay JW.
Absence of cancer-associated changes in human fibroblasts immortalized with telomerase.
Nat Genet
21:
115-118,
1999[ISI][Medline].
26.
Noguchi, H,
Kobayashi N,
Westerman KA,
Sakaguchi M,
Okitsu T,
Totsugawa T,
Watanabe T,
Matsumura T,
Fujiwara T,
Ueda T,
Miyazaki M,
Tanaka N,
and
Leboulch P.
Controlled expansion of human endothelial cell populations by Cre-loxP-based reversible immortalization.
Hum Gene Ther
13:
321-334,
2002[ISI][Medline].
27.
Olsen, JC,
Johnson LG,
Stutts MJ,
Sarkadi B,
Yankaskas JR,
Swanstrom R,
and
Boucher RC.
Correction of the apical membrane chloride permeability defect in polarized cystic fibrosis airway epithelia following retroviral-mediated gene transfer.
Hum Gene Ther
3:
253-266,
1992[ISI][Medline].
28.
Ostedgaard, LS,
Zabner J,
Vermeer DW,
Rokhlina T,
Karp PH,
Stecenko AA,
Randak C,
and
Welsh MJ.
CFTR with a partially deleted R domain corrects the cystic fibrosis chloride transport defect in human airway epithelia in vitro and in mouse nasal mucosa in vivo.
Proc Natl Acad Sci USA
99:
3093-3098,
2002
29.
Paulsen, FP,
Tschernig T,
Debertin AS,
Kleemann WJ,
Pabst R,
and
Tillmann BN.
Similarities and differences in lectin cytochemistry of laryngeal and tracheal epithelium and subepithelial seromucous glands in cases of sudden infant death and controls.
Thorax
56:
223-227,
2001
30.
Pickles, RJ,
Barker PM,
Ye H,
and
Boucher RC.
Efficient adenovirus-mediated gene transfer to basal but not columnar cells of cartilaginous airway epithelia.
Hum Gene Ther
7:
921-931,
1996[ISI][Medline].
31.
Ramirez, RD,
Morales CP,
Herbert BS,
Rohde JM,
Passons C,
Shay JW,
and
Wright WE.
Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions.
Genes Dev
15:
398-403,
2001
32.
Rheinwald, JG,
Hahn WC,
Ramsey MR,
Wu JY,
Guo Z,
Tsao H,
De Luca M,
Catricala C,
and
O'Toole KM.
A two-stage, p16INK4A- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status.
Mol Cell Biol
22:
5157-5172,
2002
33.
Rich, DP,
Couture LA,
Cardoza LM,
Guiggio VM,
Armentano D,
Espino PC,
Hehir K,
Welsh MJ,
Smith AE,
and
Gregory RJ.
Development and analysis of recombinant adenoviruses for gene therapy of cystic fibrosis.
Hum Gene Ther
4:
461-476,
1993[ISI][Medline].
34.
Rubenstein, RC,
and
Zeitlin PL.
Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of F508-CFTR.
Am J Physiol Cell Physiol
278:
C259-C267,
2000
35.
Sanlioglu, S,
Williams CM,
Samavati L,
Butler NS,
Wang G,
McCray PB, Jr,
Ritchie TC,
Hunninghake GW,
Zandi E,
and
Engelhardt JF.
Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor- secretion through I
K regulation of NF-
B.
J Biol Chem
276:
30188-30198,
2001
36.
Shrimpton, AE,
McIntosh I,
and
Brock DJ.
The incidence of different cystic fibrosis mutations in the Scottish population: effects on prenatal diagnosis and genetic counselling.
J Med Genet
28:
317-321,
1991[Abstract].
37.
Slepushkin, VA,
Staber PD,
Wang G,
McCray PB,
and
Davidson BL.
Infection of human airway epithelia with H1N1, H2N2, and H3N2 influenza A virus strains.
Mol Ther
3:
395-402,
2001[ISI][Medline].
38.
Smith, JJ,
Karp PH,
and
Welsh MJ.
Defective fluid transport by cystic fibrosis airway epithelia.
J Clin Invest
93:
1307-1311,
1994[ISI][Medline].
39.
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].
40.
Stewart, SA,
and
Weinberg RA.
Senescence: does it all happen at the ends?
Oncogene
21:
627-630,
2002[ISI][Medline].
41.
Vermeer, PD,
Harson R,
Einwalter LA,
Moninger TO,
and
Zabner J.
Interleukin-9 induces goblet cell hyperplasia during repair of human airway epithelia.
Am J Respir Cell Mol Biol
28:
286-295,
2003
42.
Walters, RW,
Grunst T,
Bergelson JM,
Finberg RW,
Welsh MJ,
and
Zabner J.
Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia.
J Biol Chem
274:
10219-10226,
1999
43.
Walters, RW,
Pilewski JM,
Chiorini JA,
and
Zabner J.
Secreted and transmembrane mucins inhibit gene transfer with AAV4 more efficiently than AAV5.
J Biol Chem
277:
23709-23713,
2002
44.
Wang, G,
Slepushkin V,
Zabner J,
Keshavjee S,
Johnston JC,
Sauter SL,
Jolly DJ,
Dubensky TW, Jr,
Davidson BL,
and
McCray PB, Jr.
Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect.
J Clin Invest
104:
R55-R62,
1999[ISI][Medline].
45.
Wassink, TH,
Piven J,
and
Patil SR.
Chromosomal abnormalities in a clinic sample of individuals with autistic disorder.
Psychiatr Genet
11:
57-63,
2001[ISI][Medline].
46.
Welsh, MJ,
Ramsey BW,
Accurso F,
and
Cutting GR.
Cystic fibrosis.
In: The Metabolic and Molecular Basis of Inherited Disease (8th ed.), edited by Scriver CR,
Beaudet AL,
Sly WS,
Valle D,
Childs B,
and Vogelstein B.. New York: McGraw-Hill, 2001.
47.
Welsh, MJ,
and
Smith AE.
Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis.
Cell
73:
1251-1254,
1993[ISI][Medline].
48.
Widdicombe, JH,
Welsh MJ,
and
Finkbeiner WE.
Cystic fibrosis decreases the apical membrane chloride permeability of monolayers cultured from cells of tracheal epithelium.
Proc Natl Acad Sci USA
82:
6167-6171,
1985[Abstract].
49.
Yamaya, M,
Finkbeiner WE,
Chun SY,
and
Widdicombe JH.
Differentiated structure and function of cultures from human tracheal epithelium.
Am J Physiol Lung Cell Mol Physiol
262:
L713-L724,
1992
50.
Yang, IC,
Cheng TH,
Wang F,
Price EM,
and
Hwang TC.
Modulation of CFTR chloride channels by calyculin A and genistein.
Am J Physiol Cell Physiol
272:
C142-C155,
1997
51.
Yankaskas, JR,
Haizlip JE,
Conrad M,
Koval D,
Lazarowski E,
Paradiso AM,
Rinehart CA, Jr,
Sarkadi B,
Schlegel R,
and
Boucher RC.
Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype.
Am J Physiol Cell Physiol
264:
C1219-C1230,
1993
52.
Zabner, J,
Couture LA,
Gregory RJ,
Graham SM,
Smith AE,
and
Welsh MJ.
Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis.
Cell
75:
207-216,
1993[ISI][Medline].
53.
Zabner, J,
Freimuth P,
Puga A,
Fabrega A,
and
Welsh MJ.
Lack of high-affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection.
J Clin Invest
100:
1144-1149,
1997
54.
Zabner, J,
Ramsey BW,
Meeker DP,
Aitken MI,
Balfour RP,
Gibson RL,
Launspach J,
Moscicki RA,
Richards SM,
Standaert TA,
Williams-Warren J,
Wadsworth SC,
Smith AE,
and
Welsh MJ.
Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis.
J Clin Invest
97:
1504-1511,
1996
55.
Zabner, J,
Seiler M,
Walters R,
Kotin RM,
Fulgeras W,
Davidson BL,
and
Chiorini JA.
Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer.
J Virol
74:
3852-3858,
2000
56.
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].
57.
Zabner, J,
Zeiher BG,
Friedman E,
and
Welsh MJ.
Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time.
J Virol
70:
6994-7003,
1996[Abstract].
58.
Zhang, L,
Peeples ME,
Boucher RC,
Collins PL,
and
Pickles RJ.
Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology.
J Virol
76:
5654-5666,
2002