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1 Department of Medicine, Veterans Affairs Medical Center and Duke University Medical Center, and 2 Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27705
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The goal of this study was to compare the functions of Clara and type II cells during alveolar clearance and recycling of surfactant protein (SP) A, a secretory product of both cell types. We examined the incorporation of instilled biotinylated SP-A (bSP-A) into rat lung type II and Clara cells as a measure of clearance and recycling of the protein. Ultrastructural localization of bSP-A was accomplished by an electron-microscopic immunogold technique at 7, 30, and 120 min after intratracheal instillation. Localization of bSP-A was quantitatively evaluated within extracellular surfactant components (lipid-rich forms: myelin figures, vesicles, and tubular myelin; and lipid-poor hypophase) and in compartments of type II and Clara cells. bSP-A was incorporated into myelinic and vesicular forms of extracellular surfactant, but tubular myelin and hypophase had little bSP-A. Lamellar bodies of type II cells demonstrated a significant time-dependent increase in their incorporation of bSP-A. There was a concentration of bSP-A in the secretory granules and mitochondria of Clara cells, but no Clara cell compartment showed a pattern of time-dependent change in immunolabeling. Our immunolabeling data demonstrated a time-dependent movement of exogenous SP-A from extracellular components into type II cells and their secretory granules. Clara cells did not demonstrate a time-dependent incorporation of bSP-A into their secretory granules during the period of this study. If Clara cells recycle SP-A, they must reach a steady state very quickly or very slowly.
surfactant recycling; surfactant subfraction; immunocytochemistry; electron microscopy
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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PULMONARY SURFACTANT is a complex mix of lipids and lung-specific surfactant-associated proteins. Surface tension-lowering functions as well as host defense functions have been attributed to lung surfactant. Surfactant has an extracellular metabolic pathway that is only partially understood and that includes morphological transformations of surfactant forms within the alveolar liquid phase (13, 17). There are several possible pathways of surfactant clearance from the alveolar space: degradation within alveoli or airways, uptake by several cell types, movement up the airways, or removal through the air-blood barrier (25). Internalized surfactant components could undergo either recycling or degradation. Although many details of intracellular metabolism remain to be discovered, recycling within alveolar type II cells may be an important part of surfactant metabolism. Both surfactant phospholipid and surfactant-associated protein are synthesized as well as recycled by alveolar type II cells (6, 25, 28).
Surfactant protein (SP) A is a major protein of the alveolar fluid and is the most extensively characterized of the four known surfactant-associated proteins. It is synthesized, secreted, and recycled by type II alveolar cells. It is found in alveolar macrophages, but it is not synthesized by them and is degraded by macrophages after phagocytosis (25). Nonciliated bronchiolar (Clara) cells are another source of SP-A production in the lung (1, 2, 21). Humans produce SP-A not only in type II and Clara cells but also in the cells of tracheal and bronchial glands (10, 14). Whether Clara cells have the ability of type II cells not only to produce but also to recycle SP-A is a subject of this report on the distribution of biotinylated SP-A (bSP-A) instilled into rat lungs.
The majority of extracellular SP-A is evidently bound with the alveolar lipids, with <1% reported as free in the surfactant hypophase (3). We also report herein on the distribution of exogenously administrated bSP-A within extracellular surfactant compartments.
Several in vitro studies identified important functions of SP-A in the regulation of extracellular surfactant metabolism. SP-A inhibits lipid secretion by type II cells (5) and enhances lipid uptake by type II cells (27). SP-A participates in the formation of tubular myelin (TM) (15, 20), increases the rate of surface adsorption of surfactant lipids (18), enhances the resistance of surfactant to inactivation by pulmonary edema fluid (4), and plays a role in lung host defense (26). Because of these properties, SP-A may be a potential component of surfactant replacement therapy. Information about the handling of instilled SP-A, therefore, may have practical value.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Purification of SP-A. SP-A was purified from human alveolar proteinosis fluid by a butanol extraction method as previously described (27). Briefly, lavage fluid from alveolar proteinosis patients was allowed to settle overnight at 4°C, and the pellet was resuspended in a minimal amount of water and frozen in aliquots. The thawed pellet was injected into butanol to extract hydrophobic proteins. The butanol extract was centrifuged, and the resulting pellet was reextracted, recentrifuged, and dried under a stream of nitrogen gas. The dried pellet was extracted twice with octylglucoside and sodium chloride to remove serum proteins; the resulting pellet was treated with octylglucoside and polymyxin B-agarose to remove bacterial endotoxin and dialyzed against sterile 5 mM Tris-buffered water, pH 7.4. After dialysis, residual polymyxin, lipid, and insoluble proteins were removed by centrifugation.
Labeling of SP-A with biotin. SP-A was biotinylated at low pH according to the procedure described by Ryan et al. (16). Sulfo-NHS-biotin (Pierce, Rockford, IL) was added to SP-A in a 2:1 weight ratio, and the mixture, pH 6.3, was incubated 20 min at room temperature with rotation. The reaction was stopped by the addition of glutamine to a final concentration of 20 mM (to react with free NHS-biotin), and then the resulting mixture was placed on ice for 10 min and dialyzed against Tris-buffered water (2 liters × 3 changes) to remove excess biotin and glutamine. bSP-A purity was checked by SDS-PAGE and Western blot with streptavidin-labeled horseradish peroxidase as a probe.
Dose preparation. We mixed 1 µl of 10% bovine serum albumin (BSA), 2 µl of 1% Evans blue dye, both in phosphate-buffered saline (PBS), and 22 µl of additional PBS. The dye was used to determine the lung location of the instillate; the ratio of dye to albumin was chosen to minimize any free dye that might bind to bSP-A. Then bSP-A was added to the mixture to obtain 58.5 µg of SP-A in each 50-µl dose. The instillate did not contain lipids. The instilled dose of protein was subsequently localized in a part of only one lobe, usually in the right lung.
Animals. Eight specific pathogen-free male Sprague-Dawley rats (Charles River, Raleigh, NC) were used. The animals were lightly anesthetized with halothane (Fluothane) vaporized in a large glass vessel and intratracheally intubated by the transoral insertion of a 16-gauge blunt steel needle. A polyethylene tube (Clay Adams PE-10, Intramedic) was passed through the steel needle until slight positive resistance was felt, and then 50 µl of the mixture described in Dose preparation were instilled and the animal was extubated. A period of hyperpnea occurred during the next 2-4 min, after which the animal appeared recovered.
The animals were killed at times from 1 min to 2 h after instillation of the bSP-A. After anesthesia with 100 mg/kg of intraperitoneal pentobarbital sodium, the trachea was cannulated, the rats were exsanguinated, and the pulmonary artery was cannulated with PE-250 tubing. Vascular perfusion of the lungs was begun with a solution of 0.9 NaCl containing 0.3% NaNO3 and 100 U/ml of heparin. Fixation proceeded immediately with a mixture of 2% glutaraldehyde and 1% paraformaldehyde buffered at pH 7.4 with 85 mM sodium cacodylate buffer. After 30 min, areas of interest were selected by identifying the region colored by Evans blue, and small tissue blocks were cut. Specimens were fixed by immersion for 30 min in the same fixative mixture.Tissue processing. After primary fixation, the samples were washed in 85 mM sodium cacodylate buffer 3 times for 10 min and then postfixed in 2% osmium tetroxide in the same buffer at pH 7.4 for 1 h. Then tissues were washed (3 × 10 min) with distilled H2O and stained with 2% uranyl acetate in distilled H2O for 90 min at room temperature. Finally, the tissues were washed again with 85 mM sodium cacodylate buffer and dehydrated through graded ethanols. An unselected sample of eight tissue blocks was infiltrated in an ethanol-LR White resin (Polysciences, Warrington, PA) mixture and embedded and polymerized in LR White at 50-55°C for 72 h. Three of the eight blocks were prepared for thin sectioning by sequential cutting on a Reichert-Jung ultratome with a glass knife. The surface of these blocks was examined for the presence of small airways and the absence of large airways and vessels.
Ultrathin sections were cut on the ultratome. The sections were picked up on Formvar-coated 200-mesh nickel grids and air-dried for 30 min.Immunogold labeling. Tissue sections on grids were stained at room temperature by immersion (section side down) in 30-µl drops on dental wax in the following sequence: 1) PBS plus 1% BSA for 10 min; 2) 5% normal rabbit serum for 30 min; 3) primary antibody (goat anti-biotin; Calbiochem, La Jolla, CA) in 1:120 PBS-Tween for 6 h at room temperature in a moisture chamber; 4) PBS for three washes of 10 min each; 5) secondary antibody (10-nm gold-conjugated rabbit anti-goat IgG; Sigma, St. Louis, MO) in 1:120 PBS-Tween for 4 h also at room temperature in a moisture chamber; 6) PBS for three washes of 10 min each; and 7) three washes with double-distilled H2O for 10 min each. Grids were air-dried overnight. Then they were stained with lead citrate for 4 min and viewed in a JOEL 1200 electron microscope.
Several controls were performed: lung tissue without bSP-A taken from the contralateral lung, omission of primary antibody, or its substitution with nonspecific serum or irrelevant goat IgG.Morphometry (quantitative immunolabeling analysis). All extracellular surfactant compartments as well as all type II and Clara cell profiles that were in contact with immunoreactive bSP-A (the instilled dose) were photographed at ×6,000 magnification. Four hundred twenty-six micrographs were printed on 11 × 14-inch paper at ×21,000 magnification. Two hundred sixty micrographs of Clara cells and type II cells were made; this resulted in ~16 micrographs per cell type per animal or between 60 and 100 micrographs per cell type per time point. Point counting volumetry was done by using a plastic grid overlay of 464 1-cm lines in a pattern described by Weibel (22). The end of each line was considered to be a point, giving a total of 928 points/micrograph. Point counting was employed to calculate the volume density of different compartments at high magnification (×21,000).
Three different extracellular surfactant compartments were quantified. Two of them represented a lipid-rich part of the surfactant layer: TM plus two vesicular forms [unilamellar and multilamellar, also called myelin figures (MFs)]. The third compartment quantified was the lipid-poor liquid hypophase. Large vesicle-like structures found within the surfactant layer and having any diameter of 0.5 µm or more were omitted, and their area was not included in the calculations. Such structures are of uncertain origin, are uncommon, and are not present in undisturbed lungs fixed in a similar manner. Inclusion of these would have a disproportionate effect on the calculated areas of the typical small vesicular pool. The volume density of the cytoplasm, nuclei, and mitochondria of all type II and Clara cell profiles was also measured. For type II cells, lamellar bodies (LBs) were also quantified. Clara cell granules were distinguished from the cytoplasm and quantified. Enough total points falling over the profiles of interest were counted to reduce to <5% the estimated error of the smallest compartment measured (23). Because gold particles presumably demonstrate biotin-immunoreactive sites (gold identifies location of bSP-A), the gold particles overlying each of the above-mentioned extra- and intracellular compartments were counted. The density of labeling is reported as the number of gold particles divided by the number of points overlying each extracellular form and cellular organelle. These ratios were calculated for every type II or Clara cell profile as well as for each extracellular surfactant compartment found on the same electron-microscopic micrograph. Average values for every animal were calculated, and then the mean results for each experimental group were determined and are presented as the number of gold particles per square micrometer of measured area.Statistics. ANOVA followed by pairwise comparisons with Scheffé's correction procedures was done with commercial software (SAS, Cary, NC) (19).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The human bSP-A migrated on SDS gels as expected for the protein
(Fig. 1). bSP-A instilled into rat lungs
was found within the bronchiolar mucous layer and was also incorporated
into alveolar surfactant forms. Because we examined only those areas
where we found the instilled bSP-A in close contact with alveolar type II or bronchiolar Clara cells, bSP-A should have had an opportunity to
be recognized and internalized by the epithelial cells studied.
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Controls.
Sections made from blocks free of dose (contralateral lung) and those
prepared from blocks containing the instilled bSP-A but incubated
without primary antibody did not react with the secondary antibody. The
extracellular surfactant on those sections was nearly free of
gold particles or had very low levels (Table 1). Very little labeling of control
sections was observed over type II or Clara cell profiles (Tables
2 and 3).
Regions of sections free of tissue (including the air spaces and empty
capillaries) were not labeled. Lung from the contralateral side not
instilled with bSP-A had no recognizable labeling even with primary and secondary antibodies.
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Extracellular surfactant compartments.
Lipid-rich components of alveolar surfactant were composed of
unilamellar vesicles, multilamellar (myelin) figures, and other lipid
lamellae including the unique TM figures. Viewed at low power, the
air-liquid interface of dye-stained lung was densely labeled with gold
particles. Lipid-rich vesicles and MFs were strongly gold labeled (Fig.
2a), with an average gold
grain density significantly higher than the labeling of hypophase and
TM profiles at the earliest time period (Fig. 2, b and
c). These differences remained throughout the study period
because there was no significant change in gold density over the
surfactant compartment within the 2-h period. Occasional large
vesicle-like structures were not quantified due to their large size,
uncertain nature, unusual occurrence, and lack of any internal
structure.
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3 particles lying within 30 nm of
each other) over lipid lamellae or close to the lipid vesicles and
speculated that clustered particles might indicate clustering of
antigen. With the two-step immunogold method that we were using, an
ideal geometry would be realized only when a gold particle projected
directly over the antigen and therefore accurately marked the actual
location of the antigen (bSP-A). In principle, there is always a
possibility that a gold particle might project away from the antigen
(maximum distance with this assay would be two immunoglobulin lengths,
~18-20 nm) (8). We did not assume that gold
clusters represented the exact location of clustered antigen molecules
in the underlying structure.
TM consisted of intersecting lipid layers with 50 nm spacing. TM
profiles had a gold grain density three- to fourfold lower than other
lipid-rich compartments. Single gold particles were found over the TM
lattice in no specific order, and no clusters were observed. Regions
from the air-liquid interface that contained TM profiles situated
immediately underneath the interface monolayer were free of gold (Fig.
2c). No significant time-dependent change of gold grain
density over TM was apparent at the 2-h point compared with the earlier periods.
The lipid-poor liquid hypophase also had a very low gold label density
compared with the lipid-rich vesicular components. Especially low were
the number of particles found at the earliest time point. Gold
particles were scattered randomly, and no clusters were observed. There
was a threefold increase in the average number of gold particles after
30 min and 2 h compared with that after the 7-min period. This was
not significant. The total number of gold grains over the hypophase
represented ~2% of all gold grains found over surfactant lipid
profiles in the same set of micrographs. Table 1 shows the average
density of gold labeling (number of gold particles/volume of the
compartment) of extracellular surfactant compartments.
Type II cells.
We studied type II cells only from alveolar spaces that contained
extracellular lipid forms immunostaining for bSP-A. At the early time
periods (7 and 30 min), gold particles were associated with cellular
organelles (nucleus, cytoplasm, mitochondria, and LBs), but no
significant difference in subcellular distribution of the
immunolabeling was found (Fig. 3).
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Clara cells.
Clara cell profiles were studied from bronchioles that contained
recognizable instilled dose and stained positive for bSP-A. At all
three time periods, Clara cell nuclear and cytoplasmic compartments had
lower average labeling densities compared with Clara cell secretory
granules and mitochondria. Clara cell secretory granules were not more
densely labeled than Clara cell mitochondria (Fig.
7). No significant changes in
labeling of Clara cell cytoplasm, nucleus, secretory granules, or
mitochondria were found within the 2-h time period. Table 3 and Fig.
8 show the average gold grain density
data of Clara cell compartments obtained at different time periods
after instillation of bSP-A.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, we used human SP-A bound to biotin and a polyclonal antibody (Calbiochem) against biotin to reveal the immunoreactive sites that presumably corresponded to those extra- and intracellular compartments that contained the instilled bSP-A. Postembedding immunogold staining is an assay performed on sections of embedded tissue, and the immunolabeling is restricted to the surface of the sections, resulting in a relatively low signal density because only those binding sites exposed can interact with the specific antibodies. We believe that the observed signal distribution closely reflects the true protein distribution based on antibody control sites and the lack of nonspecific background labeling on the resin. Lung tissue cells were found to have low levels of immunoreactive endogenous biotin, a vitamin that serves as a coenzyme of certain enzymes. Therefore, antibody binding to endogenous biotin during immunocytochemistry did not appear to be a problem (12).
Extracellular surfactant compartments. Extracellular surfactant forms have a very similar phospholipid composition to each other, and TM has been formed from pure lipids plus purified SP-A and SP-B (20). It is not clear how lipid molecules and endogenous surfactant proteins interact in vivo to achieve the different patterns of three-dimensional organization in MFs, TM, vesicles, or monolayers or where exactly exogenously added SP-A might be localized. It is unknown to what extent lipid molecules might interfere with antigen-antibody interactions and therefore might have an impact on our morphological demonstration of the antigens.
Walker et al. (21) detected endogenous SP-A in frozen sections of rat lung with a polyclonal antibody. They found that LBs had a low level of immunostaining for endogenous SP-A, whereas TM was heavily labeled. Haller et al. (7) also described high-density immunogold labeling of TM for endogenous SP-A compared with the labeling over LBs. They reported enhancement of SP-A labeling over the peripheral membranes of multilamellar structures during presumptive TM formation. Inhibition of the antigen-antibody reaction in the tightly spaced lipid-rich environment of the LBs may have accounted for some of this differential immunostaining. Our results for exogenously added bSP-A immunostaining of TM were different. The exogenous bSP-A was present in the extracellular surfactant compartments at all three times we studied. This protein was found associated with lipid-rich vesicular components almost immediately after the instillation and was detectable throughout the period studied. Nevertheless, incorporation of bSP-A into TM was very modest at the early time period and was still low 2 h after the instillation. There are several possible explanations for our findings of the distribution and time course of bSP-A labeling of alveolar surfactant compartments. The human alveolar proteinosis source of the SP-A and our processing of it for biotinylation could affect its distribution, although we think that unlikely because the type II cells incorporated bSP-A into LBs in a time-dependent manner as expected of native SP-A (6, 9, 16, 25, 28). Our method of biotinylation of SP-A was identical to that of Ryan et al. (16). Those authors found bSP-A inhibited phospholipid secretion from isolated (rat) type II cells and that specific bSP-A binding to type II cells was present. We do not know whether TM structures inhibited penetration of the antibody and, therefore, whether the availability of the antigen to the antibody was the same in the lattices as it was over other lipid-rich components. TM is a pool of highly organized lipid and protein molecules usually showing boundary lipid lamellae facing other extracellular surfactant forms nearby (15, 24). A transitional zone apparently exists only between MFs and TM. The tubes of TM that are contiguous to MFs in those pools could be closed at both their ends, possibly inhibiting access to exogenous protein (11). Lattices of TM may be saturated with native SP-A, and thus few sites for binding of exogenous SP-A might be available. Labeling of the hypophase could represent an estimate of the amount of lipid-free SP-A (or lipid-poor SP-A) that might be important for regulatory functions of the protein. Our estimate of ~2% of the total bSP-A accounted for in the hypophase is consistent with the estimate of free SP-A made by Baritussio et al. (3).Type II cells. Prior reports by several investigators (9, 16) have indicated that type II cells recycle surfactant lipid and SP-A. Young et al. (28) previously used 125I-labeled human alveolar proteinosis SP-A and found a time-dependent uptake of the radiolabel from the alveolar space into rat lung type II cell LBs. As a positive control for the present use of bSP-A, we analyzed type II cells and found the expected time-dependent concentration of bSP-A into the LBs. Our electron micrographs showed exogenous SP-A possibly attached to the apical plasma membrane of type II cells and subsequently internalized, but we could not determine whether the transporting organelles were coated pits or coated vesicles because our processing of lung tissue for immunoelectron microscopy resulted in poor preservation of specialized membrane structural detail. Our immunolabeling data were consistent with trafficking of SP-A from the apical plasma membrane via cytoplasmic vacuoles to the LBs of type II cells. Quantitative analysis demonstrated a significant increase in antigen density within LBs after 2 h compared with that at the earlier periods.
Clara cells. Clara cells have been the object of extensive studies, but their role in surfactant biology is not yet defined. The Clara cell of rat lung is a site of production of SP-A mRNA and protein, but little is known about the processing of the protein by Clara cells (21). In particular, a possible recycling of SP-A by Clara cells has not been previously directly addressed.
The cytoplasm and nuclei of Clara cells in this study showed about the same intensity of immunolabeling as the analogous compartments of type II cells, and both were unchanged during the 120-min period studied. The mitochondria of Clara cells contained more immunoreactive biotin labeling than the mitochondria in the type II cells. Kuhn (12) showed that immunoreactive biotin stores in the mitochondria of Clara cells were uniquely greater than the immunoreactive biotin in the mitochondria of type II or ciliated cells. Other organelles of Clara cells did not have high biotin levels according to Kuhn. Clara cell granules showed no time-dependent increase in immunolabeling for bSP-A. We expected that a recycling of exogenous SP-A would be signified by a time-dependent increase in labeled SP-A within those cellular compartments participating in recycling. Because Clara cell granules were maximally labeled by 7 min (the earliest we were able to instill the bSP-A and then perfusion fix the lungs), it is possible that a very fast recycling process would be missed if the recycling were complete in a few minutes. Likewise, our longest measurements at 2 h would not detect a recycling process that reached a steady state over an even longer period. In conclusion, our immunoelectron-microscopic observation of exogenous bSP-A instilled into adult rat lung suggests that specific surfactant lipid forms as well as two different epithelial cells uniquely incorporate this protein during the 2 h after instillation. There was a strong compartmentalization of bSP-A extracellularly. Lipid-rich vesicular components had the greater portion of immunoreactive protein, whereas TM and hypophase were relatively poorly immunolabeled. Although both type II and Clara cells synthesize and apparently secrete SP-A, they do not internalize SP-A in the same way. The uptake of bSP-A by type II cells was confirmed, but Clara cells did not demonstrate a time-dependent uptake. We interpret these results as evidence against recycling of SP-A by the Clara cell, at least within the 7-min to 2-h time frame of our experiments.| |
ACKNOWLEDGEMENTS |
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We were assisted in the statistical analysis of the data by Dr. Steven Grambow (Durham Veterans Affairs Medical Center, Durham, NC). We are grateful to Dr. C. A. Piantadosi for providing the alveolar proteinosis lavage fluid samples.
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FOOTNOTES |
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This work was supported by Veterans Affairs Merit Review and National Heart, Lung, and Blood Institute Grants HL-32188 and HL-30923.
Address for reprint requests and other correspondence: J. Savov, Durham VAMC (151), 508 Fulton St., Durham, NC 27705 (E-mail: jsavov{at}acpub.duke.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. §1734 solely to indicate this fact.
Received 24 May 1999; accepted in final form 10 February 2000.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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