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To find a less-invasive and lung-specific clinical biomarker, we measured serum levels of surfactant proteins A and D (SP-A and SP-D) by sandwich enzyme-linked immunosorbent assays in 42 patients with progressive systemic sclerosis (PSS) to evaluate their significance in relation to the presence of interstitial lung disease (ILD) and to assess their diagnostic merits. The patients were divided into two groups based on findings by chest computed tomography (CT): 30 patients with ILD (CT-positive ILD group), and 12 patients without any lung abnormalities (CT-negative ILD group). The CT-positive ILD group was further divided into two groups: 24 patients with ILD detectable by chest plain radiography (X-ray-positive ILD group) and six patients with ILD showing no abnormality (X-ray-negative ILD group). The levels of SP-A and SP-D in sera were significantly higher in the CT-positive ILD group than in the CT-negative ILD group. They were also significantly higher in the X-ray-positive ILD group than in the CT-negative ILD group. In the X-ray-negative ILD group, their levels were higher than those of the CT-negative ILD group. We next estimated sensitivity and specificity of SP-A, SP-D, and X-ray for detecting ILD on CT. Sensitivity of SP-D was high (77%) as well as that of X-ray (80%), whereas SP-A showed a low sensitivity (33%). Remarkably, five of six patients in the X-ray-negative ILD group showed SP-D concentrations over its cut-off level, thereby demonstrating that an SP-D assay contributes to the detection of ILD overlooked by X-ray. Moreover, a combination of X-ray and SP-D dramatically increases sensitivity to 97%. Specificity of SP-A, SP-D, and X-ray to the CT-negative ILD group was 100%, 83%, and 100%, respectively. In conclusion, this study indicates that elevated levels of serum SP-A and SP-D reflect well the presence of ILD and that the combination of SP-D and X-ray contributes to reduce the risk of clinicians overlooking ILD complicated by PSS, although a repetition in another set of subjects is needed to confirm these indications.
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Progressive systemic sclerosis (PSS) is a systemic collagen vascular disease (CVD) of unknown etiology characterized by vascular and connective tissue abnormalities. Patients with PSS, compared with patients with other types of CVD, frequently complicate with interstitial lung disease (ILD) and changes in lung parenchyma, which are detected by chest plain radiography (X-ray) in 25-65% of patients (1).
ILD is pathohistologically characterized by alveolitis and fibrosis known as fibrosing alveolitis (4). Alveolitis is an inflammatory change observed in an early stage of ILD. Alveolitis gradually leads to fibrosis resulting in a restrictive pulmonary function and an increase of morbidity. Alveolitis is generally presumed to be reversible by successful therapy, whereas fibrosis is not. An epidemiological study of 496 Japanese patients with PSS showed that common causes of death were pulmonary insufficiency and fibrosis, which occurred as frequently as heart failure and renal failure (5). When radiographic abnormalities or severe pulmonary dysfunction is present, the 5-yr mortality rate approaches 50% (6). Steen and coworkers (7) have suggested that PSS patients with an earlier stage of ILD respond better to drugs such as cyclophosphamide. Therefore, the presence of alveolitis needs to be judged precisely so that clinicians can initiate a timely therapy.
Computed tomography (CT) has an effective role in the evaluation of alveolitis. It is more sensitive and more accurate in detecting this intrapulmonary change than X-ray. However, frequent examination by CT is difficult for patients with PSS because of its high cost. For these reasons, X-ray has been commonly used for periodic checkups performed while patients with PSS are being followed up. In addition, there is no serological diagnostic method for detecting ILD, at least in the early stage, although serum lactate dehydrogenase (LDH) activity is elevated in patients with severe interstitial lung disorder.
Surfactant proteins A and D (SP-A and SP-D) belong to the collectin subgroup of the C-type lectin superfamily (8). They are produced by two types of epithelial cells in the peripheral airway, Clara cells and alveolar type II cells (9, 10), and play important roles in the innate immune system of the lung. We developed an enzyme-linked immunosorbent assay (ELISA) to detect SP-A (11) and evaluated the levels of SP-A in sera from patients with idiopathic pulmonary fibrosis (IPF) (12, 13) and ILD complicated by CVD (14). SP-A concentrations increase significantly in patients with these disorders as compared with healthy volunteers. We also previously prepared monoclonal antibodies against human SP-D and developed an ELISA, and then determined serum SP-D concentrations in 16 patients with ILD, of whom three had PSS complicated by CVD (15). The mean SP-D value of these patients was significantly higher than that of healthy volunteers, suggesting that this protein has a value as a clinical marker for CVD.
Although the previous studies were performed by analyzing findings on chest X-ray, there was no information available regarding the relationship between serum SP levels and minimal change of ILD that could be evaluated by CT (14, 15). In this study we evaluated ILD on the basis of CT diagnosis and examined whether assays for SP in patients with PSS are diagnostically useful tools.
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Subjects
We studied 42 patients (5 male and 37 female) ranging in age from 30 to 73 yr (mean, 54.9 ± 9.3 yr) who were enrolled from among outpatients at Sapporo Medical University Hospital, Sapporo Yamanoue
Hospital, and Iwamizawa General Hospital between 1995 and 1997, and who met the preliminary criteria of the American Rheumatism
Association for the diagnosis of PSS. These patients with PSS consisted of two current smokers and 40 nonsmokers. The healthy subjects were 108 volunteers (42 male and 66 female, 34 current smokers
and 74 nonsmokers) ranging in age from 20 to 61 yr with no history of
any respiratory diseases. Peripheral venous blood samples were collected from all the patients and healthy subjects at the start of registration for this study. The serum samples were stored at 
80° C until
use. This study was approved by the Sapporo Medical University Ethics Committee, and informed consent was obtained from each subject
prior to radiographic examinations.
Study Design
Chest plain radiography and CT were performed as a routine clinical evaluation less than 4 wk after serological examinations. CT was conventionally performed at the end inspiration using 10-mm collimation with 10-mm intervals from the apex to the diaphragm. Three or more images by high-resolution (HR) CT using 1.5-mm collimation were also obtained between the middle and lower lung field. CT scan is more effective for detection of ILD compared to chest plain X-ray (16, 17). Therefore, on the basis of the presence or absence of ILD judged by using CT, patients were divided into two groups: the CT-positive ILD group and the CT-negative ILD group. To compare cases of mild ILD with severer ones, the patients in the CT-positive ILD group were further divided according to their findings by chest plain X-ray into the X-ray-positive ILD group and the X-ray-negative ILD group. The X-ray-positive ILD group was defined as a group showing reticulonodular and/or honeycomb-like shadow, and the X-ray-negative ILD group was defined as a group showing no abnormal shadow. The evaluations of X-ray and CT were made by three clinicians who are experts in radiographic diagnosis and were all blinded to the serum values.
Immunoblotting Analysis of Serum Proteins
Because SP-A and SP-D are members of the C-type lectin superfamily, we analyzed the serum proteins that bound to the mannose-Sepharose column by immunoblotting. Forty milliliters of normal serum was applied to a mannose-Sepharose 6B column in the presence of 5 mM CaCl2 and the column was washed with 5 mM Tris buffer (pH 7.4) containing 5 mM CaCl2. The serum proteins binding to the affinity matrix were then eluted with 5 mM Tris buffer (pH 7.4) containing 5 mM ethylenediaminetetraacetic acid (EDTA). The eluted fractions were combined, divided into three fractions, and concentrated. Each fraction was electrophoresed on 13% polyacrylamide gel and electrotransferred onto polyvinylidenefluoride (PVDF) membrane. One lane was stained with Coomassie brilliant blue and two lanes were used for immunoblotting analysis, which was performed by the method of Towbin and colleagues (18). The membranes were incubated with phosphate-buffered saline (PBS) containing 3% (wt/vol) skim milk and 0.1% (vol/vol) Triton X-100 (blocking buffer) to block nonspecific binding and further incubated with 10 µg/ml of anti-SP-A monoclonal antibody PE10 or anti-SP-D monoclonal antibody 6B2 at room temperature for 90 min. The membranes were then washed with the blocking buffer, followed by incubation with horseradish peroxidase (HRP)-labeled anti-mouse immunoglobulin G (IgG) (1:1,500) for 30 min. After the incubation, the membranes were washed with PBS containing 0.1% (vol/vol) Triton X-100. It was then incubated with chemiluminescence reagent (NEN) and the proteins were visualized on an X-ray film. Staining with Coomassie blue showed that several serum proteins bound to the mannose-Sepharose column (Figure 1). Anti-SP-A monoclonal antibody PE10 and anti-SP-D monoclonal antibody 6B2 clearly recognized the serum proteins with apparent molecular masses of 35 and 62 kD and 43 kD, respectively, which are identical to lung collectins isolated from broncheoalveolar lavage (BAL) fluids (12, 15). Because monoclonal antibodies do not recognize another collectin that exists in serum, mannose-binding protein (19), the serum proteins detected by these antibodies are likely to be SP-A and SP-D. Collectively, the data demonstrate that our ELISA can detect SP-A and SP-D that exist in serum.
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Measurement of SP-A in Sera
SP-A assay was performed by using ELISA kits provided from the Teijin Institute of Biomedicine. In using the kit a method based on that of Shimizu and coworkers (20) was adapted with minor modifications. Briefly, 50 µl of standard materials containing 0-250 ng/ml human SP-A or 50 µl of serum samples, 200 µl of buffer I (10 mM PBS at pH 7.2 containing 1.5% [wt/vol] sodium dodecyl sulfate and 3% [vol/vol] Triton X-100), and 200 µl of monoclonal antibody PE10 labeled with HRP dissolved in buffer II (10 mM PBS at pH 7.4 containing 0.25% [wt/vol] skim milk) were mixed thoroughly. A plastic bead coated with monoclonal antibody PC6 was added to each tube containing the mixture described above. The assay tube was then incubated at 37° C for 90 min. After the incubation, the beads were washed three times with saline. Four hundred microliters of substrate solution (0.1 M phosphate-citrate buffer at pH 4.0 containing 5 mM H2O2 and 0.06% [wt/vol] tetramethylbenzidine) was added and incubated at 37° C for 30 min. The reaction was finally stopped by the addition of 1 ml of 1 N sulfuric acid, and the absorbance of each tube was measured at 450 nm. This assay system was able to detect SP-A at 2.0-250 ng/ml. All assays were performed in duplicate, and results were given as the mean value.
Measurement of SP-D in Sera
The concentration of SP-D in sera was measured by an ELISA using recombinant SP-D as a standard and two monoclonal antibodies against human SP-D. The use of the HRP-conjugated F(ab')2 fragment enables more accurate detection of SP-D in sera from patients with CVD without interference of the rheumatoid factor (21). Briefly, the microtiter wells (Immunoplate; Maxsorp, Nunc, Denmark) were coated with 100 µl of monoclonal antibody 7C6 (10 µg/ml in PBS) at 4° C overnight. After washing three times with PBS, the wells were incubated with 200 µl of PBS containing 1.0% bovine serum albumin (BSA) at room temperature for 1 h to block nonspecific binding. The wells were then incubated at 4° C overnight with 100 µl of the SP-D standard solution (1.56-100 ng/ml of recombinant SP-D solution) or samples diluted with 10 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 0.5% Triton X-100, and 1.0% BSA (HEPES-TB). The wells were next incubated with 100 µl of the HRP-conjugated F(ab')2-6B2 diluted with HEPES-TB at room temperature for 2 h. After washing, the wells were finally incubated with 100 µl of 0.3 mM 3,3',5,5'-tetramethylbenzidine containing 0.005% H2O2 at room temperature for exactly 15 min. The reaction was terminated by adding 100 µl of 1 N sulfuric acid and the absorbance was measured at 450 nm. This assay system was able to detect SP-D at 1.56-100 ng/ml. All assays were performed in duplicate, and results were given as the mean value.
Laboratory Parameters Compared with SP-A and SP-D
To evaluate the significance of SP-A and SP-D as a clinical tool we also measured various laboratory parameters in inflammatory disorders and collagen vascular diseases: lactate dehydrogenase (LDH), IgG, IgM, IgA, complement (C) 3, C4, white blood cell count (WBC), and C-reactive protein (CRP).
Statistical Analysis
Comparison between the means obtained from the two groups was statistically analyzed by the Mann-Whitney U test. Comparisons of data from the three groups (X-ray-positive ILD, X-ray-negative ILD, and CT-negative ILD) were made by one-factor ANOVA. When there were significant differences among the means, they were analyzed with a post-hoc test using Scheffé F. Correlation analyses were performed using the Spearman rank correlation. A p value of less than 0.05 was considered to be significant. A cut-off level of serum SP-A (43.8 ng/ml) was mean + 2 SD of the values in healthy volunteers, and that of SP-D (110 ng/ml) was set up based on the Receiver Operating Characteristics curve for IPF patients.
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Characteristics of Subjects and Concentrations of SP-A and SP-D
The characteristics and concentrations of SP-A and SP-D in
sera for all subjects are summarized in Table 1. The PSS patients were grouped by chest CT findings: CT-positive ILD
group (n = 30) and CT-negative ILD group (n = 12). In the
CT-positive ILD group, 24 subjects grouped into an X-ray-positive ILD group and six into an X-ray-negative ILD group.
All patients with indications for ILD on chest plain X-ray
showed a typical pattern of ILD on CT findings
various degrees of ground glass opacity and a honeycombing pattern.
The majority of patients in the X-ray-negative ILD group showed a mild degree of ground glass opacity and/or curve linear sign near their pleura, but not honeycombing. High-resolution CT (HRCT) is a gold standard diagnostic method for
ILD; therefore we performed HRCT using 1.5-mm collimation with 10-mm intervals concurrently with conventional CT
in approximately two-thirds of the patients and then compared the results from these two methods. The rate of detection of ILD by HRCT was the same as by conventional CT
(data not shown).
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Comparison of Parameters in Patients with PSS and Healthy Volunteers
In healthy volunteers, the concentrations of SP-A and SP-D were 26.7 ± 8.5 ng/ml (range 12.0-59.5) and 46.4 ± 32.9 ng/ml (range 7.1-174.5), respectively. There was no significant difference in concentrations of SP-A or SP-D between males and females among the healthy volunteers (SP-A, 27.9 ± 8.6 ng/ml versus 26.0 ± 8.4 ng/ml; SP-D, 51.1 ± 33.2 ng/ml versus 43.5 ± 32.7 ng/ml). The concentrations of serum SP-A were slightly higher in smokers (29.1 ± 10.1 ng/ml) than in nonsmokers (25.1 ± 6.8 ng/ml), although these differences were not significant. SP-D concentrations were not significantly different between smokers and nonsmokers. No correlation between the concentrations of SP-A and SP-D was observed in healthy volunteers and there was no significant correlation between SP-A or SP-D concentrations and aging. As shown in Table 2, SP-A concentrations in the CT-positive ILD group (38.2 ± 17.0 ng/ ml) were significantly higher than those in the CT-negative group (19.2 ± 9.8 ng/ml, p < 0.001) and in healthy volunteers (26.7 ± 8.5 ng/ml, p < 0.001). SP-D concentrations in the CT-positive ILD group (182.3 ± 118.9 ng/ml) were also significantly higher than those in the CT-negative ILD group (76.2 ± 42.5 ng/ml, p < 0.001) and in healthy volunteers (46.4 ± 32.9 ng/ml, p < 0.001). The degrees of increase of SP-A and SP-D were approximately 2.0- and 2.4-fold, respectively. IgG and IgM concentrations in the CT-positive ILD group were also significantly increased as compared with those in the CT-negative ILD group, although their degrees of increases were small (approximately 1.3- and 1.5-fold, respectively). Other parameters showed no significant difference between the two groups.
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We further analyzed the levels of SP-A and SP-D and other parameters in the two CT-positive ILD groups defined by their positive or negative status by X-ray and the other group consisting of CT-negative PSS patients. Among these three groups, a significant difference in SP-A and SP-D was seen (p = 0.0029 and p = 0.0075, respectively). Six patients in the X-ray-negative ILD group as well as 24 patients in the X-ray-positive ILD group showed high concentrations of both SP-A (37.9 ± 18.9 ng/ml) and SP-D (157.8 ± 71.8 ng/ml) when compared with the CT-negative ILD group. These differences were significant when analyzed with the Mann-Whitney U test, whereas they were not significant when analyzed with the Scheffé F test, presumably because the number of X-ray-negative ILD group subjects was small. Five of six subjects in the X-ray negative ILD group showed high SP-D concentrations (111.5- 284.2 ng/ml) that were over the cut-off level (110 ng/ml). In contrast, only two patients in this group showed high SP-A concentrations (59.5 and 63.1 ng/ml) that were over the cut-off level (43.8 ng/ml) (Figure 2). Parameters other than SP-A and SP-D showed no significant differences between the X-ray-negative ILD group and the CT-negative ILD group.
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Sensitivity and Specificity of X-ray and Serum Parameters for Detecting IP
The sensitivity and specificity of plain X-ray were 80% and 100%, respectively. In other words, 20% of patients with ILD showed no abnormalities by X-ray. The sensitivity and specificity of the main parameters are shown in Table 3. Cut-off levels are set at 43.8 ng/ml for SP-A, 110 ng/ml for SP-D, and 440 IU/ml for LDH. The sensitivity of SP-A, SP-D, and LDH to CT-positive ILD patients was 33%, 77%, and 17%, respectively. The specificity of these to the CT-negative ILD group patients was 100%, 83% and 100%, respectively. In 108 healthy subjects, only 7% and 5% showed concentrations of SP-A and SP-D, respectively, over their cut-off levels.
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The concentrations of SP-A and SP-D in patients with PSS are plotted in Figure 2. Eighteen of the 30 CT-positive ILD patients were positive for ILD in both plain X-ray and in SP-D concentration. Six and five patients were positive for ILD in either plain X-ray or in SP-D, respectively. Only one of the CT-positive ILD patients was negative for ILD in both X-ray and in SP-D, and collectively, of the CT-positive ILD patients who were negative in X-ray, 83% (five of six) were positive in SP-D. Of the CT-positive ILD patients who were negative in SP-D 86% (six of seven) were positive in X-ray. When X-ray and SP-D measurements are combined, a high sensitivity (97% [29 of 30]) is obtained for detection of ILD by CT scan.
Correlation between SP-A and SP-D Levels and Other Laboratory Parameters Measured in Patients with PSS
As shown in Figure 3, serum levels of SP-A and SP-D in patients with PSS were correlated (r = 0.641, p < 0.001), whereas neither SP-A nor SP-D was correlated with LDH, IgG, IgM, IgA, C3, C4, WBC, and CRP. In healthy volunteers, unlike patients with PSS, there was no significant correlation between SP-A and SP-D (data not shown).
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Open lung biopsy and BAL are useful in identifying the presence of alveolitis (22). However, it is difficult for patients being monitored to repeat these invasive examinations. Clinicians therefore generally speculate on the presence of those pathohistological changes by imaging diagnosis using chest X-ray and/or CT scan. CT is more sensitive than X-ray in the detection of lung abnormalities so that it is now used in an assessment of patients with various diffuse lung diseases (16, 17). In patients with ILD complicated with PSS, CT also showed a high efficiency (1). It is likely to be the best practical examination for this disease. However, CT also has some problems, such as high cost. Some serum biomarkers seem to have overcome these problems (26, 27), but they lack organ specificity.
Alveolar type II cells are the major sources of the SP secreted in alveoli, although Clara cells express SP-A, -B, and -D but not SP-C (28). Analysis by immunoblot and reverse transcriptase-polymerase chain reaction (RT-PCR) has revealed that SP-A is expressed in rat small intestine and colon (29) and that SP-D is expressed in rat (30) and human (31) gastric mucosa, whereas secretion of these two proteins by gastrointestinal mucosal cells has not been demonstrated. Expression of SP-A and SP-D in the gastrointestinal tract appears very weak compared with expression in the lung. A previous study revealed that the serum protein binding to the mannose-affinity matrix exhibits SP-A with an apparent molecular mass identical to that of SP-A isolated from BAL fluids and is recognized by anti-SP-A monoclonal antibody, indicating the presence of SP-A in serum (32). Thus, serum SP-A and SP-D are likely to be specific to lung.
This study showed that the concentrations of both SP-A and SP-D in the CT-positive ILD group were significantly higher than those in the CT-negative ILD group, suggesting that their elevations in sera closely reflect the appearance of abnormalities in the lung. Moreover, in most of the patients who were negative for X-ray but positive for CT (the X-ray-negative ILD group), both SP-A and SP-D were also higher than those in the CT-negative ILD group. This may indicate that these proteins increase even in patients with mild interstitial lung abnormalities that can be detected by CT but not by X-ray. However, their sensitivities are quite different. Unlike SP-A, SP-D clearly showed high sensitivity (77%) and high specificity (83%). Even in the X-ray-negative ILD group, serum SP-D exhibited a high sensitivity (83%: five of six). These data clearly demonstrate that the assay of serum SP-D is a clinically useful tool for detecting ILD complicated with PSS. In CT-positive ILD patients showing low SP-D concentration, X-ray also showed a high sensitivity (86%: six of seven). These results suggest there is a qualitative difference between the use of X-ray and SP-D as diagnostic tools for ILD; the elevated SP-D level could be closely associated with alveolitis but not fibrosis, whereas detecting alveolitis (imaged as a ground glass shadow by plain X-ray) seems to be more difficult than detecting fibrosis (imaged as a honeycombing pattern). The conclusion reached by a combination of SP-D and X-ray provides extremely high sensitivity (97%) and specificity (83%). This implies that this combination assay is almost equivalent to CT in the detection of ILD. However, repetition in another set of subjects is needed before cut-off levels of SP-A and SP-D can be proposed in a clinical setting as the number of patients in this study was small.
The majority of patients with advanced PSS have a long clinical course during which ILD becomes complicated. Therefore, ILD should be monitored carefully from the onset and its monitoring marker must be not only less invasive but also lung specific. With this in mind, the measurement of serum LDH activity, which is a simple test that appears to reflect changes of disease activity in patients with IPF, has been proposed (26, 27). However, LDH activity in this study showed no significant increase, and moreover it is released from not only the lung but from many other organs. Therefore, serum LDH does not appear to be suitable as a marker of lung complication in systemic diseases such as PSS. In contrast, because SP-D is lung specific, serum levels of SP-D could be such a marker.
The mechanisms by which SP-A and SP-D appear in the bloodstream remain to be determined. Hypothetical mechanisms include alteration of the surfactant protein production by alveolar type II cells, an increased amount of their concentrations in alveoli, the increased permeability of lung vessels, the destruction of the barrier between alveolar epithelium and endothelium caused by injury to the basement membrane, and their decreased clearance rates from the circulation. In patients with PSS with ILD, epithelial abnormalities that consisted of patchy epithelial cell swelling, focal loss of bare epithelial basement membranes, and focal alveolar type II cell proliferation were observed ultrastructurally even in biopsy specimens that appeared normal by light microscopy (33). Therefore, the accelerated production of SP-A and SP-D by alveolar type II cells and the destruction of the epithelium- endothelium barrier are likely to be the main causes of the appearance of SP-A and SP-D in the bloodstream. Harrison and co-workers (33) demonstrated that pulmonary endothelial and/or epithelial injury precedes inflammation and fibrosis. We speculate that SP-A and SP-D may enter the circulation at an early stage of the pathohistological changes seen in the lungs of patients with PSS when epithelium are injured. This study supports this idea since serum SP-D concentrations increased even in patients with minimal changes on imaging diagnosis by CT.
When serum levels of SP-A and SP-D in patients with PSS are compared, they are statistically correlated (see Figure 3). However, only nine patients had prominent elevations of both SP-A and SP-D. Most of patients exhibit SP-A levels below the cut-off value but SP-D levels above the cut-off value. This difference may imply that SP-D leaks more easily than SP-A because of its solubility. Since most of SP-A tightly bind to surfactant lipid aggregates in the alveoli but SP-D appears to be lipid free, the distinct properties of the collectins may cause different solubilities.
The studies, using clearance of inhaled technetium-labeled diethylenetriamine pentacetate (99mTc-DTPA), have revealed that permeability measured is accelerated in patients with PSS with ILD and patients with IPF as compared with healthy subjects and, moreover, that it is more prominent in progressive disease than in stable disease (34, 35). These studies suggest that the increased permeability results in an increase of alveolar-to-vascular leakage of SP-A and SP-D. The amount of SP-A and SP-D in BAL fluid from patients with IPF showed decreased levels of SP-A and little change in SP-D when compared with amounts from healthy subjects (36, 15), suggesting that the increased amount in alveoli is not involved. The clearance system of SP-A and SP-D from the circulation is unknown at present.
In conclusion, there were obvious increases in serum SP-D concentrations in patients with PSS with ILD as well as in patients with IPF. Serum SP-D increased dramatically in patients with ILD when it could not be detected by chest plain X-ray. Although a large number of patients must be examined to set cut-off levels, these results suggest that measurement of serum SP-D concentration is of diagnostic value in patients with PSS followed through a long clinical period. Moreover, a combination of SP-D determination and chest plain X-ray may provide a high level of diagnostic evaluation, preventing clinicians from overlooking ILD complicated by PSS.
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Correspondence and requests for reprints should be addressed to Hiroki Takahashi, M.D., Third Department of Internal Medicine, Sapporo Medical University School of Medicine, South-1 West-16, Chuo-ku, Sapporo 060-8543, Japan. E-mail: htaka@sapmed ac.jp
(Received in original form March 1, 1999 and in revised form December 15, 1999).
Acknowledgments: The authors wish to thank Drs. H. Takahashi (the First Department of Internal Medicine, Sapporo Medical University), M. Shinohara (Sapporo Yamanoue Hospital), and H. Yamamoto and N. Sukoh (Iwamizawa Municipal Hospital) for assistance in identifying study patients. They thank Dr. I. Kaneko (Department of Biochemistry, Sapporo Medical University) for detection of SP-D by Western blotting analysis. They also thank Dr. T. Akino (Sapporo Medical University) and Dr. M. Mori (Department of Public Hygiene, Sapporo Medical University) for valuable suggestions and encouragement.
Supported by a grant-in-aid for scientific research from the Ministry of Education, Japan.
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References |
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1.
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