Mesenchymal determination of mechanical strain-induced fetal
lung cell proliferation
Jing
Xu1,
Mingyao
Liu2,
A. Keith
Tanswell1, and
Martin
Post1
1 Lung Biology Research
Program, Department of Pediatrics, Hospital for Sick Children
Research Institute, and 2 Thoracic
Surgery Research Division, Toronto Hospital, University of Toronto,
Toronto, Canada M5G 1X8
 |
ABSTRACT |
Fetal breathing
movements play an important role in normal fetal lung growth. We have
previously shown that an intermittent mechanical strain regimen (60 cycles/min, 15 min/h), simulating normal fetal breathing movements,
stimulated growth of mixed fetal rat lung cells in organotypic culture.
In the present study, we examined the individual responses of the two
major fetal lung cell types, fibroblasts and epithelial cells, to
mechanical strain. Also, we investigated the effect of
mesenchymal-epithelial interactions on strain-induced cell
proliferation during fetal lung development. Fibroblasts and epithelial
cells from day 18 to day
21 fetal rat lung (term = 22 days),
cultured alone or as various recombinants, were subjected to either a
48-h static culture or to strain, and DNA synthesis was measured. Both
cell types responded individually to strain with enhanced DNA synthesis
throughout late fetal lung development. Independent of the
recombination ratio, there was no additive response to strain when
fibroblasts and epithelial cells from the same gestation were
recombined. In contrast, strain-induced DNA synthesis was suppressed
when cells from different gestations were recombined. The ontogenic
response pattern of recombinants to mechanical strain was similar to
that of fibroblasts but not of epithelial cells. Strain-induced
proliferation increased and peaked at the early canalicular stage of
lung development at 19 days of gestation and declined thereafter. We
conclude that strain-enhanced growth of the fetal lung is gestation
dependent and that the gestational response to mechanical force is
regulated by the mesenchyme.
fetal lung cell growth; physical forces; mesenchymal-epithelial
interaction
 |
INTRODUCTION |
PULMONARY HYPOPLASIA is a major cause of neonatal
respiratory diseases (15). Although fetal lung growth has been shown to depend on a proper lung fluid balance and normal fetal breathing movements (9), the precise mechanism(s) by which these physical factors
influence lung cell proliferation remains unknown. To investigate the
effect of phasic strain generated by fetal breathing movements on lung
cell growth, we used a strain system for organotypic cultures of mixed
fetal rat lung cells (17). DNA synthesis and cell division of mixed
lung cells isolated from the canalicular stage of lung development were
increased by an optimized intermittent strain regimen (11), which is
very similar to the reported frequency, amplitude, and periodicity of
normal human fetal breathing movements in vivo (7). Strain-induced cell
proliferation as a function of lung development and lung cell type has
not been previously investigated.
Fetal rat lung development can be divided into four periods:
1) embryonic period
(days
11-13),
development of major airways; 2)
pseudoglandular period (days
14-18),
development of airways to terminal bronchioles;
3) canalicular period
(days
19-20),
development of the acinus and vascularization; and
4) terminal sac period (days
21-22,
term = 22 days), subdivision of saccules by secondary crests. The fifth
and final stage, formation of true alveoli, occurs postnatally in the
rat. Fetal lung cells change their phenotype (e.g., morphology, growth
rate, cellular function) during each of these periods, and it is likely
that the response of cells to mechanical stimulation also changes with
gestation.
As assessed by autoradiography, both major cell types in organotypic
cultures, epithelial cells and fibroblasts, respond to strain with
increased DNA synthesis (11). This observation that both cell types can
respond to strain when combined in a mixed-cell organotypic culture
(11) does not mean that each cell type when cultured in isolation can
respond to mechanical stimulation. Epithelial-mesenchymal interactions
play a critical role in lung development (1, 18), and tissue
interactions may also influence cellular responses to mechanical
strain.
In the present study, epithelial cells and fibroblasts were isolated
from the pseudoglandular, canalicular, and saccular stages of rat lung
development, incubated in Gelfoam sponges alone or recombined in
various ratios, and subjected to intermittent strain. We report that
mechanical strain enhanced proliferation of epithelial cells and
fibroblasts when cultured alone or when recombined. However, the
proliferative response of mixed cells to strain was found to be both
gestation dependent and modulated by mesenchymal-epithelial interactions.
 |
METHODS |
Materials. Female (200-250 g) and
male (250-300 g) Wistar rats were purchased from Charles River and
bred in our animal facilities. Cell culture media, antibiotics, and
trypsin were obtained from GIBCO
Canada (Burlington, ON). Fetal bovine serum (FBS) was from Flow
Laboratories (McLean, VA), and collagenase and DNase were from
Worthington (Freehold, NJ). Cell culture flasks were from Falcon
(Becton Dickinson, Lincoln Park, NJ), and multiwell plates were from
Costar (Johns Scientific, Toronto, ON). Gelfoam sponges were from
Upjohn (Toronto, ON), and
[3H]thymidine was from
Amersham Canada (Oakville, ON). All other chemicals were from Sigma
(St. Louis, MO).
Cell culture. The isolation of
epithelial cells and fibroblasts has been described previously (5, 8,
14). Briefly, fetuses were removed from the dam at
day
18 to
day
21 of gestation (term = 22 days), and the fetal lungs were dissected out into cold Hanks'
balanced salt solution without calcium or magnesium [HBSS(
)] and cleared of major airways and vessels.
The lungs were washed twice in HBSS(
), minced, and suspended in
HBSS(
). The lung tissue was digested with trypsin solution
[0.125% (wt/vol) trypsin and 0.4 mg/ml of DNase] for 20 min. After the lung tissue was filtered through a 100-µm mesh nylon
bolting cloth, minimal essential medium (MEM) with 10% (vol/vol) FBS
was added, and the mixture was centrifuged. The pellet was resuspended
in MEM containing 0.1% (wt /vol) collagenase. After a 15-min
incubation, the collagenase activity was neutralized by adding MEM + 10% (vol/vol) FBS. Two differential adhesion periods of 1 h in tissue
culture flasks allowed attachment of fibroblasts. The nonadherent cells
were removed, transferred to new culture flasks, and incubated
overnight for attachment of epithelial cells. Nonadherent cells were
removed from cell cultures after overnight incubation. Viability and
purity of the cultures were comparable to previously published data
(5). Fibroblasts stained positively (>95%) with anti-vimentin, but staining was negative for cytokeratin and smooth muscle cell actin, indicating that the fibroblast cultures were not contaminated with
epithelial and smooth muscle cells. Epithelial cell cultures were
cytokeratin positive (>95%) and vimentin negative. Although we
previously reported fibroblast heterogeneity in fetal rat lung (5),
herein we did not attempt to isolate the different fibroblast populations. After a 48-h culture, fibroblasts and epithelial cells
were floated with 0.25% (wt /vol) trypsin + 0.4 mM EDTA, as
previously described (14). Fibroblasts and epithelial cells were
inoculated alone or as various recombinations (fibroblast-to-epithelial cell ratio in percentages: 100:0, 80:20, 50:50, 20:80, and 0:100) onto
2 × 2 × 0.2-cm Gelfoam sponges at a density of 1.6 × 106 cells per sponge. After
inoculation, cells were incubated at 37°C for 1 h before the
addition of MEM + 10% (vol/vol) FBS.
After overnight incubation, sponges were washed twice with MEM to
remove nonadhered cells. Attached cells were harvested from sponges by
collagenase and trypsin digestion (11) and counted electronically
to determine inoculation efficiency. In separate experiments, sponges
were changed to MEM + 1% (vol/vol) FBS supplemented with 1 µCi/ml of
[3H]thymidine and
subjected to strain or static culture for 24 h.
Strain of fetal rat lung cells and assessment of cell
proliferation. The mechanical strain device used in
these studies has been described in detail elsewhere (11, 17). It
consists of a programmable burst timer, a control unit, a
direct-current power supply, and a set of solenoids. A culture dish
with a Gelfoam sponge was placed in front of each solenoid. One end of
each sponge was glued to the bottom of the dish, and the other end was
attached to a movable metal bar, which was wrapped and sealed in
plastic tubing. A magnetic force, generated through the solenoids,
acted on the metal bar to apply strain to the organotypic cultures. Sponges were subjected for 48 h to a 5% elongation from their original
length at 60 cycles/min for 15 min/h. We previously reported (11) that
such a strain regimen optimally stimulated mixed fetal lung cell growth
without cell injury, and the increase in proliferation, as assessed by
thymidine incorporation, was not due to enhanced mixing of nutrients.
Cell proliferation was assessed by
[3H]thymidine
incorporation into DNA, as described previously (11). Results were
determined as disintegrations per minute per sponge and are expressed
as percentage of static control groups. In contrast to adult lung cells, especially adult type II pneumocytes, fetal lung cells undergo
continuous proliferation both in vivo and in vitro. We have recently
demonstrated that
[3H]thymidine
incorporation into DNA correlates directly with the change in (
)
cell number in three-dimensional (3-D) organotypic cultures (12) of
fetal lung cells. The correlation between disintegrations per minute
per sponge and
cell number per sponge was highly significant (r = 0.777, P < 0.001 for the unstrained control
group; r = 0.940, P < 0.001 for the strained group)
(12), implying that under the 3-D organotypic culture conditions
thymidine incorporation into DNA mainly reflects DNA synthesis by
proliferating fetal lung cells. Therefore, DNA synthesis, as measured
by [3H]thymidine
incorporation into DNA, was used as an index of cell proliferation.
Effect of conditioned media on DNA
synthesis. Conditioned media were collected after a
24-h strain period (S-CM) and from static control cultures (C-CM). The
conditioned media were centrifuged at 420 g to remove cell debris and then split in aliquots for storage at
20°C until mitogenic activities were measured.
The mitogenic activity of conditioned media was assessed by measuring [3H]thymidine
incorporation into DNA of monolayer cell cultures. One milliliter of a
cell suspension (104 cells/ml) in
MEM + 10% (vol/vol) FBS, containing either
day
19 or
day
21 fetal rat lung fibroblasts or
epithelial cells, was seeded in wells of 24-multiwell culture plates
and incubated overnight. The wells were rinsed twice with fresh MEM.
Cells were incubated for another 24 h in MEM + 1% (vol/vol) FBS for
suboptimal cell growth. Cells were then cultured in the presence of
either C-CM or S-CM supplemented with 1 µCi/ml of
[3H]thymidine. Based
on our previous findings (14), conditioned media were diluted 10 times
with MEM. Because the conditioned media originally contained 1%
(vol/vol) FBS, MEM supplemented with 0.1% (vol/vol) FBS was used as a
control medium. After a 24-h incubation, media were aspirated, and
cells were rinsed twice with ice-cold PBS. The amount of radioactive
thymidine incorporated into DNA was then measured (14). Results were
determined as disintegrations per minute per well and are expressed as
percentage of C-CM values. It is worthwhile mentioning that we have
also demonstrated that
[3H]thymidine
incorporation into DNA paralleled the increase in cell number
(
cells) in two-dimensional (2-D) monolayer cultures (13) of fetal
lung cells. The correlation coefficient between disintegrations per
minute per well and
cells per well was highly significant (
= 0.918, P < 0.001). Thus
[3H]thymidine
incorporation into DNA is a reliable index of cell proliferation in 2-D
cultures.
Statistical analysis. All values are
shown as means ± SE. Statistical analysis was by Student's
t-test or, for comparison of more than
two groups, by one-way ANOVA, followed by Duncan's multiple range
comparison test, with significance defined as
P < 0.05 (19).
 |
RESULTS |
Cell attachment to Gelfoam sponges. To
determine the inoculation efficiency of cells on sponges, epithelial
cells and fibroblasts isolated from fetal lungs at
day
18-21
of gestation were seeded on sponges separately or as various
recombinations. After 24 h of incubation, nonadherent cells were
removed and attached cells were collected and counted to determine the
inoculation efficiency, which is expressed as percentage of number of
inoculated cells (1.6 × 106
cells/sponge). The average inoculation efficiency was 73.8 ± 1.95%. The efficiency did not change significantly as a function of
development, cell type, or epithelial-mesenchymal recombination (Table
1).
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Table 1.
Inoculation efficiency of fetal rat lung epithelial cells,
fibroblasts, and their recombinations on Gelfoam sponges
|
|
Effect of mechanical strain on cell
proliferation. To investigate the responses of fetal
lung cells to mechanical stimulation, fibroblasts, epithelial cells,
and various recombinations were subjected to static culture or
intermittent strain. Cell proliferation was assessed by measuring DNA
synthesis. To compare the responses of fibroblasts, epithelial cells,
and their recombinants with strain, strain-induced DNA synthesis was
plotted as a function of epithelial cell-to-fibroblast ratio of the
recombinations. Table 2 shows the DNA
synthesis of fetal lung cells during static culture. Independent of
gestation, DNA synthesis decreased with an increasing amount of
epithelial cells. Figure 1 shows that, except for day
18 fetal lung fibroblasts, cells from
different gestations, individually or recombined, responded to strain
with a significant increase in DNA synthesis compared with static
control groups (P < 0.05). These
data suggest that increased DNA synthesis is a common response to
strain of both fetal lung epithelial cells and fibroblasts throughout
late gestation. Regression analysis and ANOVA revealed that there was
no significant additive effect of fibroblasts on strain-induced DNA
synthesis of epithelial cells or vice versa at any day of gestation
(Fig. 1). Because the regression lines did not change significantly at
any day of gestation, we combined all the data of the 1:1
recombinations of each day of gestation to determine at which day of
gestation cells were most responsive to mechanical strain.
Strain-induced stimulation of DNA synthesis of the recombinations was
maximal at the early canalicular stage of lung development at 19 days
of gestation, after which there was a decline in strain-induced DNA
synthesis (Fig. 2). The developmental
pattern of strain-induced DNA synthesis of fibroblasts was similar to
that of the recombinants. Strain-induced DNA synthesis of epithelial
cells increased slightly but not significantly with advancing gestation
of cells.
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Table 2.
DNA synthesis of fetal rat lung epithelial cells, fibroblasts, and
their recombinations on Gelfoam sponges during static cell culture
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Fig. 1.
Mechanical strain stimulates DNA synthesis of fetal rat lung
cells. Epithelial cells and fibroblasts were isolated
from pseudoglandular stage (day
18), canalicular stage
(days
19 and
20), and saccular stage
(day
21) of lung development, cultured on
sponges alone or recombined in various ratios, and subjected to an
intermittent strain or static culture for 24 h.
[3H]thymidine
incorporation into DNA was measured, and strain-induced DNA synthesis
is expressed as percentage of control groups. Values are means ± SE. Data from 3-4 separate experiments carried out in duplicate
were combined and plotted as a function of recombination ratios.
Independent of gestation, except day
18 fibroblasts
(# P > 0.05), and
recombination ratio, DNA synthesis of strained cells was significantly
greater compared with that of static control groups
(P < 0.05). Fib, fibroblasts; Epi,
epithelial cells.
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Fig. 2.
Gestation-dependent proliferative response of mixed cells to mechanical
strain originates in the mesenchyme. Epithelial cells
(A) and fibroblasts
(B) were isolated from
pseudoglandular stage (day
18), canalicular stage
(days
19 and
20), and saccular stage
(day
21) of lung development, cultured on
sponges alone or recombined in 1:1 ratios
(C), and subjected to an
intermittent strain or static culture for 24 h.
[3H]thymidine
incorporation into DNA was measured, and strain-induced DNA synthesis
was expressed as percentage of static control groups. Values are means ± SE. Data from 3-4 separate experiments carried out in
duplicate were combined and plotted as a function of gestation.
Epithelial cells showed a slight but not significant increased
proliferative response to mechanical strain with advancing gestation.
Strain-induced DNA synthesis increased and peaked in fibroblasts at 19 days of gestation and declined thereafter. The gestational
proliferative response of 1:1 recombinants to strain was identical to
that of fibroblasts alone. * P < 0.05 compared with day
18 cells.
# P < 0.05 compared with day 18 cells.
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Effect of heterologous recombinations on
strain-induced DNA synthesis. To examine whether
fibroblasts determine the stimulatory effect of strain on DNA
synthesis, we recombined in a 1:1 ratio either
day
19 epithelial cells and
day
21 fibroblasts or
day
21 epithelial cells and
day
19 fibroblasts. Although DNA synthesis of all recombinations was enhanced by mechanical strain
(P < 0.05 compared with static
control groups), this effect was significantly reduced (>50%) in
heterologous recombinations compared with homologous recombinations of
day
19 fetal lung cells
(P < 0.001; Fig.
3). In contrast, DNA synthesis of
unstrained heterologous recombinants was increased compared with
unstrained homologous recombinants (data not shown). To investigate
whether this decreased mitogenic response of heterologous recombinants
to strain is regulated by soluble factors, we collected conditioned
media from day
19 and 21 fetal lung epithelial cells or
fibroblasts after a 24-h static culture (C-CM) or intermittent strain
(S-CM). Mitogenic activities of conditioned media were tested on
day
19 and
21 fetal lung epithelial cells and
fibroblasts. S-CM of day
19 fetal lung fibroblasts
significantly increased DNA synthesis of
day
19 and
21 fetal lung epithelial cells
(P < 0.05), although the stimulatory
effect decreased with advancing gestation of epithelial cells (Fig.
4A).
S-CM of day 21 fetal lung fibroblasts had no
mitogenic effect on day
19 or 21 fetal lung epithelial cells. S-CM
from day
19 and
21 fetal lung epithelial cells
slightly but not significantly inhibited [3H]thymidine
incorporation into DNA of day
19 and
21 fibroblasts.

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Fig. 3.
Mechanical strain-induced DNA synthesis is repressed in heterologous
recombinations. Fibroblasts and epithelial cells isolated from
day
19 and
day
21 fetal rat lungs were cultured as
gestational homologous (19F/19E) or heterologous (19F/21E or 21F/19E)
recombinations and subjected to an intermittent strain or static
culture for 24 h.
[3H]thymidine
incorporation into DNA was measured. Results are expressed as
percentage of control groups. Values are means ± SE of 3 separate
experiments in triplicate. The stimulatory effect of strain on DNA
synthesis was attenuated when cells from different gestational ages
were recombined. F, fibroblasts; E, epithelial cells; 19 or 21, day 19 or
day 21.
* P < 0.05 compared with
19F/19E recombinant.
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Fig. 4.
Mechanical strain-induced mitogenic activities alter with gestation.
Mitogenic activity of conditioned media from strained (S-CM)
fibroblasts (A) and epithelial cells
(B) was assessed with epithelial
cells (A) and fibroblasts
(B) from
day
19 and
21 fetal lung. Conditioned media were
diluted 10 times with MEM and supplemented with 1 µCi/ml of
[3H]thymidine for DNA
synthesis measurements. S-CM results are expressed as percent change of
static control medium (C-CM). Values are means ± SE of 3 separate
experiments in quadruplicate.
* P < 0.05 compared with
C-CM.
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 |
DISCUSSION |
The physical force experienced by fetal lung cells during fetal
breathing movements is potentially an important mediator of normal
fetal lung growth. We previously demonstrated that an intermittent strain regimen, simulating fetal breathing movements, enhanced mixed
fetal lung cell proliferation in vitro (10-13). Uptake of [3H]thymidine was
increased in both epithelial cells and fibroblasts when organotypic
cultures of mixed cells were subjected to mechanical strain (11).
Herein we show that mechanical strain also increased DNA synthesis of
fetal lung epithelial cells and fibroblasts when both cell types were
separately subjected to strain.
Recently, we have shown that the incorporation of
[3H]thymidine into DNA
by fetal lung cells correlates directly with changes in cell number,
indicating that fetal lung cells in 3-D organotypic cultures (12) and
2-D monolayer cultures (13) progress through the cell cycle without
interruption. Thus measurement of DNA synthesis is a reliable indicator
of fetal lung cell proliferation in organotypic and monolayer culture.
Because of the tight correlation between DNA synthesis and
cell
number, increases in DNA synthesis observed in the present study appear
small, but it should be noted that a 25% increase of
[3H]thymidine
incorporation into DNA reflects a similar net increase in cells.
Although fetal lung cells responded mitogenically to strain throughout
late gestation, there was a gestation-dependent effect. The
proliferative response of mixed (1:1 recombinants) fetal cells to
strain was low at the pseudoglandular stage on
day
18, peaked during the early
canalicular stage on day
19, and fell again during the late
canalicular and saccular stages at
days
20 and
21 of gestation. A similar ontogenic
profile of strain-induced DNA synthesis was observed for fibroblasts
but not epithelial cells, suggesting that the gestation-dependent
component of the proliferative response of mixed fetal lung cells to
strain originates in the mesenchyme. The developmental growth pattern
of mixed fetal lung cells subjected to intermittent strain was
identical to that of whole fetal lung (4), i.e., ontogenic pattern of
DNA synthesis of the mixed fetal lung cells mimicked developmental
expression pattern of growth-related genes
(c-myc,
c-fos, histone 3) in whole fetal lung.
In contrast, developmental DNA synthesis profiles of intermittently strained epithelial cells and fibroblasts differed from those of static
cultured cells (3, 6). Despite the usual caution regarding cell culture
findings, these results suggest that fetal lung cells subjected to an
intermittent strain regimen, simulating fetal breathing movements, more
closely reflect the in vivo situation than static cell cultures.
Our initial findings suggested that mesenchymal-epithelial interactions
did not influence the mitogenic response of fetal lung to strain in
that all homologous recombinants, independent of tissue ratio and
gestational age, responded to intermittent strain similarly to
individual cells. However, the reduced growth-promoting response of
heterologous recombinants to strain did suggest a role for tissue
interactions. It is plausible that tissue interactions in homologous
recombinants maintain the cells at a proper growth and differentiation
state to sense mechanical signals. Interference with these interactions
by changing the developmental phenotype of one of the cell types in the
heterologous recombinants leads to a diminished biological response to
mechanical strain.
Studies with static cell cultures have suggested that fetal lung cell
growth may be controlled by mesenchymal-epithelial interactions (1, 5,
20). We previously demonstrated that S-CM of
day 19 mixed fetal rat lung cells
stimulated proliferation of day 19 epithelial cells but not that of
day
19 fibroblasts (14). Herein we found
that S-CM collected from day
19 fibroblasts enhanced day
19 epithelial cell proliferation. S-CM
of day 21 fetal lung fibroblasts was
not mitogenic for day
19 fetal lung epithelial cells. Thus
the reduced mitogenic response of the heterologous (day
19/day
21) recombinants to strain compared
with homologous (day
19/day
19) recombinants may be partially
due to a reduced release of mitogenic activities by fibroblasts.
Enhanced growth factor expression in response to mechanical strain may
be a common reaction shared by various cell types. Bishop et al. (2)
have reported that the mitogenic activity of conditioned media from an
embryonic lung fibroblast cell line is increased after mechanical strain. Sadoshima and Izumo (16) found that conditioned media from
strained neonatal cardiac myocytes can induce hypertrophy of
nonstrained cells. We have recently observed that mechanical strain
rapidly upregulates platelet-derived growth factor-B mRNA expression
and protein production in mixed fetal lung cells (10).
In response to mechanical strain, cells may release not only
growth-promoting factors but also inhibitory factors. Studies with
static fetal lung cell cultures have shown that epithelial cells
release inhibitory growth factors for fetal lung fibroblasts (6).
Herein we found that S-CM of epithelial cells inhibited fibroblast
growth independently of gestation. The S-CM of mixed fetal rat lung
cells appeared to be more inhibitory than that of fetal lung epithelial
cells alone (14), again indicating that mesenchymal-epithelial
interaction may influence the production of soluble factors.
Taken together, these data are consistent with a gestation-dependent
proliferative response of fetal lung cells to mechanical strain that
originates in the mesenchyme.
 |
ACKNOWLEDGEMENTS |
This work was supported by a Group Grant from the Medical Research
Council of Canada, Grant R01-HL-43416 from the National Heart, Lung,
and Blood Institute, and an equipment grant from the Ontario Thoracic
Society. M. Liu is supported by a scholarship from the Medical Research
Council of Canada.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: M. Post, Lung Biology Research Program,
Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada
M5G 1X8.
Received 11 February 1998; accepted in final form 13 May 1998.
 |
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