A Marker of Leakage through the Visceral Pleura |
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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Pleural fluid (PF) proteins either derive from serum by diffusion or are locally secreted within the
pleural space. Another hypothetical origin is a leakage of lung secretory proteins across the visceral
pleura. To test this hypothesis, we investigated the occurrence, sources, and determinants in PF of
CC16, a small-size and readily diffusible protein of 16 kDa secreted by bronchiolar Clara cells. CC16
concentration was determined by a sensitive latex immunoassay in serum and PF of 117 subjects (86 exudates and 31 transudates) and, for purpose of comparison, in ascites samples from another group
of 38 subjects (7 exudates and 31 transudates). CC16 was also studied in serum and PF of normal rats
and in rats with pleural exudate induced by 
-naphthyl-thiourea (ANTU). The levels of CC16 in PF
and ascites were highly correlated with that in serum, suggesting a diffusional exchange across the
pleural/blood and peritoneal/blood barriers. Whereas CC16 occurs at similar levels in ascites and serum, the protein was found to be more concentrated in PF than in serum in both humans (geometric
mean in µg/L, 26.2 versus 14.6, p < 0.0001) and rats (213 versus 16.2, p < 0.001). A local synthesis of
CC16 appeared unlikely in view of the lack of CC16-immunostaining in pleura of both species. The
only plausible explanation for these findings is that CC16 in PF originates from two sources: diffusion
from plasma and a leakage from the lung into the pleural space across the semipermeable visceral
pleura. This interpretation is supported by a markedly increased leakage of CC16 in experimental exudates induced by ANTU and the finding of high CC16 concentrations in human transudates associated with congestive heart failure, two conditions wherein PF has been shown to arise from the interstitial spaces of the lung.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Determination of total protein level is a cornerstone in the diagnosis of pleural disorders. It indeed serves as one of the criteria for separating transudates from exudates (1). Most proteins present in pleural effusion originate from blood by a size-restricted diffusional process, accounting for their lower concentrations in pleural fluid (PF) than in serum (2). Some proteins have been shown to be more concentrated in PF than in serum as a result of a local production by inflammatory or malignant cells (6). A possible although unexplored origin of PF proteins is the lung itself. It can indeed be assumed that proteins secreted in large amounts within the respiratory tract might penetrate into the pleural space as a consequence of their leakage across the visceral pleura.
With the exception of surfactant-associated protein A secreted by alveolar type II cells and serosal mesothelium (7, 8), no lung-specific secretory protein has indeed so far been studied in pleural effusion. Another lung-specific protein is the Clara cell protein, a low-molecular-weight protein (LMWP) of 16 kDa (CC16), mainly secreted by bronchiolar Clara cells (9). As a result of its intense secretion into the respiratory tract, CC16 is present in high concentrations in sputum and bronchoalveolar lavage fluid (BALF) (10). Interestingly, CC16 occurs in small amounts in plasma where it penetrates by diffusion through the broncho-alveolar/blood barrier (10, 11). Like other LMWP, CC16 is eliminated by glomerular filtration and accordingly plasma levels increase in renal insufficiency (12). Another major determinant of the concentration of CC16 in serum identified so far is tobacco smoking, which induces a dose-related decrease of CC16 in BALF and serum (13).
To test the hypothesis of a passage of lung proteins across the visceral pleura, we investigated the occurrence, sources and determinants of CC16 in both human and experimental PF.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Patients
One hundred seventeen patients from the internal medicine divisions of the Cliniques Universitaires Saint-Luc, Brussels, Belgium and
Sherbrooke University, Québec, Canada who had a diagnostic or
therapeutic thoracentesis were included. The study population involved 47 patients with neoplasia (37 with metastatic carcinoma, eight
with hematologic malignancy, and two with mesothelioma), 49 patients with inflammatory pleural effusion (25 with parapneumonic and
seven with postsurgical effusion, six with empyema, two with subphrenic abscess, two with collagen vascular disease, and seven with
various other causes), 17 patients with congestive heart failure (CHF)
and four patients with cirrhosis and hydrothorax. The inpatient and
outpatient follow-up medical notes were reviewed and the diagnosis
was established using the following criteria: positive cytology and/or
pleural biopsy in malignant effusions. Parapneumonic effusions were
diagnosed when there was an associated infiltrate with signs of infection but with negative Gram stain and culture of the PF. Empyema
was defined as neutrophilic effusion with a positive Gram stain or
growth of bacteria on culture of PF. CHF was diagnosed if the patient
had an appropriate history, physical findings, and objective evidence
of cardiac dysfunction. Other clinical diagnoses such as an inflammatory process or cirrhosis with ascites were also included. Pleural effusions were categorized as transudates or exudates according to Light's
criteria (1). The following measurements were performed on all PF
samples: CC16, total protein, LDH, cell count, Gram stain, bacterial
culture, and cytology. A sample of serum (preferably taken simultaneously or within 24 h after thoracentesis) was obtained in order to
measure CC16, total protein, LDH levels, and creatinine. In a subgroup of 47 patients selected randomly including 12 transudates and 35 exudates, PF and serum CC16 levels were compared with those of
two other LMWP, retinol-binding protein (RBP), a 21 kD liver protein (16), and 
2-microglobulin (2-m), a 11.8 kD ubiquitous protein
that is locally produced in inflammatory pleural or peritoneal disorders (17, 18). Measurements similar to that on PF, including RBP and
2-m, were performed in another group of 38 patients with ascites
(seven exudates and 31 transudates).
Animal Studies
Pleural effusion was induced by systemic administration to rats of
ANTU (kindly provided by Dr. G. Saumon, Institut National de la
Santé et de la Recherche Médicale, Faculté Xavier Bichat, Paris), an
edematogenic agent known to cause pulmonary edema and pleural effusion (19). This experiment was performed on adult female Sprague-
Dawley rats (B&K Universal Limited, N. Humberside, UK) weighing
between 230 and 280 g. The animals were treated in compliance with
the guidelines edicted by the Belgian Ministry of Middle Class and
Agriculture. Throughout the study, the animals were housed in an air-conditioned room (25° C, 50% relative humidity) with a regular 12-h
light-dark cycle and were allowed standard chow and tap water ad libitum. Two experimental groups of 10 animals were used in this study.
ANTU (5 mg/kg) was given in one intraperitoneal injection of a 5 mg/
ml solution prepared in arachis oil. Control animals were given pure
arachis oil intraperitoneal (1 ml/kg) and processed along with treated
rats. Six hours after treatment with ANTU, the animals were terminated by an intraperitoneal injection of sodium pentobarbital (60 mg/
kg). When the respiration had ceased, whole blood was collected following aortic cannulation and stored at 4° C for 3 h. After diaphragm
incision, PF was aspirated from pleural spaces and quantified. Lungs
were removed and weighed. The lung/body weight ratio was calculated
for each rat. Thoracic cage fragments with lining parietal pleura and
lung samples with visceral pleural membrane were fixed in Bouin's
fluid and formaldehyde 4% solutions for histological studies. After centrifugation of the clotted blood at 2,000 g for 10 min, the sera were stored at 
20° C until analysis.
CC16 Assays
The concentration of CC16 in human biological fluids was determined by a sensitive immunoassay relying on the agglutination of latex particles. A detailed description of this immunoassay has been previously published in its application to urinary CC16 (20). The assay uses the rabbit anti-protein 1 antibody from Dakopatts (Glostrup, Denmark) and as standard the protein purified in our laboratory. To avoid possible interferences by the complement, rheumatoid factor or chylomicrons, sera were pretreated by heating at 56° C for 30 min and by the addition of polyethylene glycol (16%, vol/vol, 1 /1) and trichloroacetic acid (10%, vol/vol, 1/40). After overnight precipitation at 4° C, the samples were centrifuged (2,000 g × 10 min), and CC16 was determined in the supernatants. All samples were analyzed in duplicate at two different dilutions. The validity and the analytical performances of the CC16 latex immunoassay (LIA) in different biological media have been reported previously (10, 20). Briefly, applied to serum, this assay has a detection limit of 0.5 µg/L and an average analytical recovery of 95%. The within- and between-run coefficients of variation range from 5 to 10%. When pooled sera samples from healthy subjects are fractionated by fast protein liquid chromatography on Sephacryl S-200 (Pharmacia Biotechnology, Uppsala, Sweden), CC16 elutes as a single component with an apparent molecular size around 16 kD which is indistinguishable from that of the native protein. CC16 levels in serum determined by our LIA are in good agreement with those obtained with a prototype fluorescence enzyme immunoassay using monoclonal antibodies recently developed by Pharmacia and Upjohn, Diagnostics (r = 0.91, p = 0.0001, n = 96).
Rat CC16 concentrations in both serum and pleural effusion were determined by the same automated LIA using a rabbit polyclonal antibody raised against rat CC16 purified by us. PF and serum samples were treated according to the same protocol described for the human CC16 assay. The analytical performances of this LIA applied to rat CC16 were similar to that reported in humans (10).
Other Assays
Creatinine was determined in serum by the Jaffé's method. Total protein was determined by the Coomassie blue method (Bayer Diagnostics, Belgium). LDH dehydrogenase activity was assayed by monitoring the reduction of NAD+ at 340 nm in the presence of lactate. 2-m
and RBP were measured in PF, ascites, and serum by LIA as previously described (21, 22).
CC16 Immunohistochemistry
In humans, parietal pleura specimens were obtained from 10 patients involved in the study who had a pleural biopsy. Visceral pleura specimens were from 10 patients who had a lung resection for lung carcinoma. In human and rat, lung samples and pieces of thoracic cages were fixed by immersion in 4% formaldehyde or in Bouin's fluid for at least 48 h, paraffin-embedded and cut to 6-µm thick sections. CC16-immunoreactive cells were detected by using the rabbit polyclonal antibody against human or rat CC16 and the immunoperxidase technique, as previously described (23).
Statistical Analysis
All statistical analyses were performed using the Statview SE software. Non-normally distributed parameters (CC16 and other LMWP in different biological fluids and creatinine in serum) were log-transformed before the application of parametric tests.
CC16 levels in serum and in corresponding pleural or ascites fluid were compared by the Student's paired t test. Correlations between CC16 concentrations in serum and those in PF or ascites were evaluated by Pearson's correlation coefficient. The determinants significantly affecting the concentrations of CC16 in PF and serum were identified in two models of stepwise regression analysis testing as independent variables sex, serum creatinine (as estimator of the renal function), the smoking status (categorized as smokers and never-/ ex-smokers), and the etiological (model 1) or the pathophysiological (model 2) characteristics of pleural effusion. In this analysis, etiology was categorized as neoplasia, inflammatory disease, heart failure or cirrhosis, and pathophysiology as exudate or transudate according to Light's criteria.
Changes of CC16 in PF were adjusted to that in serum by calculating either the CC16 concentration ratio between PF and serum (PF/S
CC16 ratio) or the difference between the concentrations of the protein between the two media (PF S CC16 
). The same indices were
applied to CC16 in ascites and to the other LMWP tested in the study
(RBP and 2-m). Differences between etiological groups were assessed by one-way analysis of variance followed by the Fisher's least
significant difference multiple comparison test. Comparison of the different parameters between transudates and exudates was made using
the Student's unpaired t test. Values are reported as the geometric
mean with range or as the arithmetic mean ± SD. The level of statistical significance was set at p < 0.05.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Human Studies
The characteristics of the study population are given in Table 1. The four etiological categories were, on the whole, well matched with respect to sex, age, smoking status, and the renal function. When applying Light's criteria, 86 effusions were classified as exudates and 31 as transudates. As expected, most exudates were associated with neoplastic and inflammatory diseases, and almost all patients with heart failure and cirrhosis had transudates. Although pleural effusions due to neoplastic and inflammatory disorders are usually exudates, 11 patients with such disease were still classified as having transudates on the basis of Light's criteria.
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Table 2 compares the CC16 levels between PF and serum,
by considering either the whole population or each etiological
category separately. For the whole population, the mean CC16
level was significantly higher in PF than in serum with an average PF/S CC16 ratio of 1.8 and a mean PF S CC16 of 14.2 µg/L. When the comparison is made for each disease category,
a statistically significant excess of CC16 in PF compared with
serum was found in patients with inflammatory disease, neoplasia, and heart failure. However, patients with cirrhosis associated with hydrothorax had similar CC16 levels in PF and
serum. An important point to note is that CC16 in PF was nearly
systematically in excess to that in serum on an individual basis
also since on a total of 117 patients only 11 had a CC16 level in
PF lower than that in serum. With respect to other LMWP, on
average, 2-m concentration was 20% higher in PF than in serum whereas RBP concentration was 60% lower (Table 4).
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When a similar comparison is made on patients divided between transudates and exudates according to Light's criteria,
CC16 concentrations were found to be significantly higher in
both PF and serum of transudates compared with exudates. This
difference did not emerge from the ratio but well from the
PF S CC16 which was twice higher in transudates than in
exudates (Table 3). Such a difference was not found with 2-m
and RBP which showed similar levels in PF and serum for
both transudates and exudates (Table ). These CC16 differences appear PF-specific since concentrations of CC16 in another effusion fluid, such as ascites, did not differ from that in
serum nor between exudates and transudates (Table 5). By
contrast, the two other LMWP studied showed in ascites the
same pattern of concentrations as in PF (2-m being more and RBP less concentrated in these two fluids than in serum).
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As illustrated in Figure 1, in both fluids, CC16 concentrations were well correlated with that in serum, the best correlation being found between serum and ascites. Regarding pleural effusions, the correlation was better for exudates than
for transudates. Interestingly, with increasing CC16 in serum
(mainly due to renal insufficiency), the three regression lines
tended to converge, which probably reflects the overwhelming
contribution of serum CC16. Significant correlations were also
found between effusion fluids and serum levels for both 2-m
(PF: r2 = 0.56, p = 0.0001, n = 47 and ascites r2 = 0.57, p = 0.0001, n = 38) and for RBP (PF: r2 = 0.23, p = 0.0006, n = 47 and ascites r2 = 0.57, p = 0.0001, n = 38).
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Among the variables tested by stepwise regression analysis
(sex, serum creatinine, smoking status, etiology, or pathophysiology of pleural effusion), the renal function estimated by the
measurement of creatinine and the smoking status were found
to have a major influence on the concentrations of CC16 not
only in serum but also in PF (Table 6). In these two fluids,
CC16 was correlated positively with serum creatinine and negatively with the smoking status. The pathophysiology as well
as the etiology of pleural effusion, which were tested separately in two different regression models, had both an influence on the CC16 level in PF. When the analysis was carried
out by expressing the results as ratio or difference, the influence of renal function and smoking status disappeared. The
ratio was only influenced by the etiology whereas the difference was dependent upon both the etiology and the pathophysiology. Using similar regression models, 2-m and RBP in
PF were only influenced by their respective levels in serum,
the smoking status and the etiology as well as the pathophysiology of PF being not retained as significant determinants.
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By immunohistochemistry, CC16-positive cells were identified in terminal and respiratory bronchioles. By contrast, no CC16 immunoreactivity was detected on both visceral and parietal mesothelial and sub-mesothelial cells (Figure 2).
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Animal Studies
All rats treated by ANTU developed large amounts (9.5 ± 1.76 ml in treated animals versus 0.04 ± 0.012 ml in control animals, p = 0.0001) of protein-rich pleural effusion (PF/S total
protein ratio, 0.84 ± 0.07) with concomitant pulmonary edema
(lung/body weight ratio [g/g], 0.008 ± 0.001 in treated animals
versus 0.005 ± 0.001 in controls, p = 0.0001). As shown in Figure 3, CC16 was significantly increased in serum of the ANTU-treated group as compared with the control group. In PF,
CC16 level was significantly higher than in serum but did not
differ between treated and control animals. By contrast, the
total amount of CC16 in recovered PF was markedly increased
in treated animals compared with controls. No alteration of
the renal function estimated by serum creatinine was present
in ANTU-treated animals. The PF/S CC16 ratio was lower in
animals treated by ANTU than in control animals (3.12 [1.0- 13.4] versus 11.52 [8.0-17.8], p = 0.005) whereas the PF S
CC16 was not significantly different (84.4 [8.0-244.0] versus
186.7 [95.9-334.8] µg/L, p = 0.161). As in humans, there was
no immunohistochemical labeling for CC16 in both visceral
and parietal pleura but well in bronchioles (Figure 4).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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To test the hypothesis that lung secretory proteins might leak
into the pleural space, we investigated the occurrence, source and determinants of CC16, a major lung secretory protein, in
human PF of various etiologies. For purpose of comparison,
the protein was also measured in ascites from an additional
group of patients. In both effusion fluids, CC16 was found to
occur at concentrations that were significantly correlated with
those found in serum, most likely as a result of a diffusional
exchange between intravascular, pleural and peritoneal spaces.
This interpretation is supported by the fact that significant
correlations between effusion fluids and serum were also found
for 2-m and RBP, two proteins of similar size as CC16 known
to be readily exchangeable. It is further supported by the fact
that the main factors influencing serum CC16 levels, i.e., the
renal function and the smoking status, emerge also as significant determinants of CC16 in PF.
The most interesting finding in our study is that the concentration of CC16 in PF is in excess to that in serum as opposed to ascites. To study the sources and the determinants of this excess of CC16 in PF, an accurate mathematical expression
was required. A first possibility was to calculate the ratio of
CC16 levels in PF to that in serum, as for the Light's criteria.
The value of this ratio was significantly greater in PF than in
ascites, pointing to an additional source than exudation from
serum. Although this mode of calculation allows an adjustment for the changes in serum concentrations, it cannot provide a quantitative estimation of the amount of CC16 that
might originate from other sources than serum. Indeed for a
similar absolute amount of CC16 entering the PF, ratio values
may greatly differ depending on the baseline level. This is especially true for serum CC16, which is subjected to wide variations reaching values more than 10 times than normal in patients with advanced renal failure. That this ratio should be
used with caution clearly emerges from the fact that it is negatively correlated with CC16 level in serum, which would not be expected in the case of an accurate adjustment (such a negative correlation is not found with Light's criteria, results not
shown). For a better mathematical representation of the transfer of CC16 in PF, we also calculated the difference between
the concentrations of CC16 in PF and that in serum (PF S
CC16 ). This second mode of expression presents indeed the
advantage of being independent of serum CC16 levels (results
not shown) and therefore appears as a more reliable index to
investigate the contribution to PF CC16 from other sources
than serum.
Our study clearly shows an excess of CC16 in PF of most
etiologies (inflammatory, neoplastic, and heart failure) at the
exception of those associated with cirrhosis. This excess appears specific of PF since it was not found in ascites. Moreover, it seems also specific of CC16 since the liver protein,
RBP, showed the opposite pattern, being less concentrated in
both PF and ascites than in serum. 2-m was slightly increased
in these two fluids but this excess was found mostly in exudates (in contrast to CC16) and is known to result from a local
production (17). The excess of CC16 in PF compared to serum
was confirmed in rat PF of physiological origin and induced by
ANTU.
The finding of higher CC16 levels in PF than in serum in
both human and rat can be explained only by assuming a pleural or a pulmonary source of CC16. Local production has been
reported for some proteins such as LDH, orosomucoid and
2-m in inflammatory or neoplastic pleural disorders (17, 24,
25) but this appears very unlikely for CC16 in view of the lack
of CC16 immunoreactivity on both pleura in human and rat.
As to a production by inflammatory cells recruited into the
pleural space, this hypothesis is refuted by the absence of correlation between CC16 and the number of white cells in the
PF (data not shown). The only remaining plausible explanation is that CC16 can reach the pleural space not only from serum but also from the lung by moving across the visceral pleura.
This passage is probably facilitated by the concentration gradient for CC16 across the pleural semipermeable membrane.
CC16 concentration in the pulmonary interstitium, which is in
continuity with the connective tissue of the visceral pleura
(26), is indeed likely to be much higher than that in serum. In
addition, the visceral pleura, with its sieving properties (2),
should offer no or little hindrance to this passage of CC16. The
amount of CC16 present in PF and arising from pleural leakage might reasonably be well estimated by the PF S CC16 ,
which takes into account the total CC16 level in PF and the
amount originating from plasma by diffusion and very close to
the S CC16 level. As expected, the PF S CC16 was positive for most pleural effusions but was close to zero and even
negative among the small group of subjects with hepatic hydrothorax, a condition wherein pleural effusion arises from a
liquid flow from the peritoneal space across holes in the diaphragm rather than a leakage through the visceral pleura.
This hypothesis of a leakage of CC16 into the PF is further
supported by the observations that in rat CC16 level was
markedly higher in PF than in serum for both physiological or
ANTU-induced PF. In addition to endothelial cell damage
leading to pulmonary edema and elevation of CC16 in serum,
this agent is known to induce a marked increase in pleural permeability leading to the development of large amounts of protein-rich pleural effusion (19). The observation that the PF
CC16 level remained unchanged despite a 2,000-fold increase
of the PF volume induced by ANTU suggests that this agent
causes the movement of CC16-rich fluid into the pleural
space. The fact that the P/S CC16 ratio and PF S CC16 are
higher in rat than in human effusions can be explained either by a more severe degree of disruption of the barrier following toxic insult or by inter-species anatomical differences. Compared with humans, rats have indeed a thinner visceral pleura
with little connective tissue which probably offers less hindrance to the flux of fluid and proteins (26).
This hypothesis of a CC16 leakage across the visceral pleura is in keeping with a recent theory regarding the formation of PF. Classically it is believed that pleural effusions arise from the pleural capillaries, either because of an imbalance between hydrostatic and osmotic forces, as occurs in transudates, or because of an increased permeability of the pleural capillaries, as present in exudates. Some authors have proposed modifying this classic theory by including the interstitial space of the lungs as another source in both exudates and transudates (27). This view is supported by different experimental and clinical studies showing that transudates associated with hydrostatic pulmonary edema and exudates accompanying increased-permeability pulmonary edema induced by different agents arise from the interstitial space of the lung (28). Our findings are in agreement with this theory, since the highest CC16 ratios and differences were found in transudates associated with CHF and a drastically increased leakage of CC16 was observed in exudates induced by ANTU. One might assume that the flow of interstitial fluid driven by hydrostatic forces in these conditions promotes the movement of CC16 through the visceral pleura into the pleural space. This most likely occurs by convection, although a diffusive component is probably also involved. Whether the measurement of CC16 in PF would be complementary to Light's criteria by providing an estimate of the contribution of the lung interstitial fluid to the formation of pleural effusion remains to be investigated.
In conclusion, this study suggests that C16, which is present in pleural effusions, derives not only from the serum but also from the lung following its passage across the visceral pleura. This is supported by the higher concentrations of CC16 compared to serum in PF of different origins and by the finding of a markedly increased leakage of CC16 accompanying experimental exudates induced by ANTU and high CC16 concentrations in human transudates associated with CHF, two conditions wherein pleural liquid has been shown to arise from the interstitial spaces of the lung.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Professor A. Bernard, Unit of Industrial Toxicology and Occupational Medicine, Faculty of Medicine, Catholic University of Louvain, 30.54 Clos Chapelle-aux-Champs, B-1200 Brussels, Belgium. E-mail: bernard{at}toxi.ucl.ac.be
(Received in original form July 24, 1997 and in revised form October 23, 1997).
This work was partially presented in abstract form (A151) at the American Thoracic Society Meeting, San Francisco, 1997.Acknowledgments: This study was supported by the European Union (EV4-CT96-0171) and the National Fund for Scientific Research (Belgium). C. Hermans is Research Fellow and A. Bernard Research Director of the National Fund for Scientific Research. O. Lesur is Scholar of the Fonds de la Recherche en Santé du Québec.
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References |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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