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Investigation of type i and type iii collagens of the lung in progressive systemic sclerosis.

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The interstitial collagens, type I and type 111,
were investigated in lung tissue from patients with progressive systemic sclerosis (PSS) with pulmonary involvement. By use of CNBr digestion of whole tissue,
the relative content of type I versus type I11 collagen
was unchanged. This contrasts with idiopathic pulmonary fibrosis. Limited pepsin digestion released greater
amounts of collagen (55%) than normal (16%), but the
individual collagen chains were chemically indistinguishable. A reduced amount of the more stable collagen crosslink, hydroxylysinonorleucine,was observed
which was consistent with the relatively greater degree
of solubilization.
Progressive systemic sclerosis (PSS) or systemic
scleroderma is characterized by extensive fibrotic destruction of several organs associated with increased accumulation of collagen and other extracellular macromolecules in the involved tissues. Most commonly
involved in the process is skin, but certain internal organs such as the lung, heart, kidney, and gastrointestinal tract are also frequently affected. The etiology and
pathogenesis of this disorder are not completely understood.
Theoretically, the increased accumulation of col~~
From the Veterans Administration Medical Center and the
Departments of Biochemistry and Medicine, University of Tennessee
Center for the Health Sciences, Memphis, Tennessee 38104; and the
Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.
Supported in part by USPHS grants HL-19731 and AM16506, and by the Veterans Administration.
Address reprint requests to J.M. Seyer, PhD, Research Service (151). VA Medical Center, 1030 Jefferson Avenue, Memphis, Tennessee 38104.
Submitted for publication August 26, 1980; accepted in revised form October 28, 1980.
Arthritis and Rheumatism, Vol. 24, No. 4 (April 1981)
lagen may be due to its increased rate of biosynthesis, or
a decreased breakdown rate, which in turn may be due
to a diminished activity of collagenase or possible alterations in the structure of collagens. The preponderance
of available evidence suggests that the rate of collagen
synthesis by dermal fibroblasts derived from the affected area of PSS patients is increased, although the issue still remains controversial (1-5). There is also no definitive evidence that the rate of its breakdown or the
activity of specific collagenase is diminished in PSS (68).
The questions concerning possible alterations in
the structure of collagen or in the relative distribution of
different types of genetically distinct collagens in PSS
lungs have not been definitively resolved. Extensive histologic and ultrastructural studies have shown abnormalities in the organization and arrangement of collagen fibrils in the involved tissues (9). Yet the molecular
and biochemical bases for the observed alterations have
not been elucidated. Several groups of investigators
have reported abnormalities in the ratio of type I to type
111 collagen in several other fibrotic disorders including
liver cirrhosis, idiopathic pulmonary fibrosis, hypertrophic scars, granulomatous colitis, and inflamed gingival tissue (10-15). In these fibrotic tissues, type I collagen was increased out of proportion to type 111 collagen.
Immunofluorescent studies of PSS skin using type-specific antibodies of type I and type I11 collagen have suggested increased quantities of type I11 collagen in the
deeper levels of the dermis (16), but biochemical studies
with CNBr digests of whole dermal tissue revealed no
alterations in the relative content of type I and type 111
collagen in PSS (17).
In the present study, we have isolated and characterized type I and type 111 collagen obtained from the
involved lungs from 3 patients with PSS. The constituent collagen a chains were found to be identical with
those from normal individuals with respect to amino
acid and carbohydrate composition, chromatographic
behavior, and C N B r peptide maps. W e have also determined the relative distribution of the two collagens by
directly determining the type-specific peptides, a l(1)CB8, al(III)-CB8, al(I)-CB3, a n d al(II1)-CB4, in the
CNBr digests of whole lung tissues. Unlike other fibrotic tissues investigated up to this time, the ratio of
type I to type 111 collagen was not altered. In addition,
PSS collagen in lung tissue contained a decreased
amount of t h e more stable hydroxylysino-hydroxynorleucine ( H L H N L ) as compared to normal lung collagen after reduction with 'H-borohydride.
Lung samples. Diseased lungs were obtained during
autopsy from 1 female and 2 male subjects who had thickening of the skin, Raynaud's phenomenon, esophageal dysfunction, and pulmonary fibrosis at the time of death. (Gross
and microscopic findings of PSS were documented by Dr. J.
M. Young, Chief, Pathology Service, Memphis Veterans Administration Medical Center.) The pertinent clinical and pathologic features of the 3 patients are summarized in Table 1.
Normal human lungs were obtained from subjects who had
died of causes unrelated to lung.
Bronchial tissues, blood vessels, and pleura were re-
moved as much as possible by dissection and the remaining
peripheral lung cut into pieces, ground in a mechanical meat
grinder and homogenized briefly in a Waring blender with
chips of ice (Waring Products Division, Dynamics Corp. of
America, New Hartford, CT). Multiple aliquots of 10 ml each
of the homogenate were removed for quantitation of total hydroxyproline and protein content. The remaining homogenate
was washed with large volumes of cold 0.05M Tris, pH 7.4,
which removed large quantities of noncollagenous substances,
and finally with cold distilled H,O. No detectable collagen
was removed by the above procedures as determined by hydroxyproline analysis of the extracts (18). The remaining insoluble residue was used directly for CNBr cleavage or limited
pepsin digestion ( 10,12,17).
Cleavage with cyanogen bromide. Samples of either
washed lung homogenates or purified collagen chains were digested with CNBr (Pierce Chemical Co., Rockford, IL) in 70%
formic acid as previously described (10). The liberated peptides were separated from insoluble material by centrifugation
for 10 minutes at 40,OOOg and lyophilized after a 10-fold dilution with cold distilled H20. The degree of solubilization of
collagenous protein was determined by hydroxyproline analysis of portions of the supernate and the residue (18).
Solubilization of collagen by pepsin digestion and isolation of individual chains. The washed lung residues were suspended in 0.5Macetic acid (pH adjusted to 2.8 with 70%0 formic acid) and digested with pepsin for 72 hours 3 times as
previously described (12). Collagen in the three pepsin extracts was precipitated by dialysis against 0.01M Na2HP0,
and collected by centrifugation. The precipitate was resolubilized in 0.5M acetic acid, precipitated with NaCl (5%), and
resolubilized in 0.05MTris/lM NaCI, pH 7.4. Type I11 colla-
Table 1. Clinical characteristics of the scleroderma patients studied'
Age (years)
Disease duration (years)
Organ systems
Cause of death
Chest x-ray
Pulmonary function tests
Total lung capacity, I
C O diffusing capacity,
ml/minutes/mm Hg
Histology of lung
Skin, heart, lung, kidney
Malignant hypertension
leading to renal failure
Bibasilar fibrosis
Skin, heart, lung.
esophagus, kidney
Acute right lower lobe
Bilateral diffuse
granular infiltration;
right lower lobe
Skin, lung, heart,
Gangrene of left foot
and sepsis
Bilateral basilar
interstitial marlungs
3.71 (74)t
2.93 (60)
13.3 (70)
Severe interstitial
12.3 (62)
Marked interstitial
Patches of marked
interstitial fibrosis.
thickening of
alveolar septa, and
obliteration of
air spaces
Both lungs of subject I. left lung of subject 2, and right lower lobe of the lung of subject 3 were analyzed for the present studies.
t Numbers in parentheses indicate the percent of the predicted values.
gen was selectively precipitated by the careful addition of 5M
NaCl to a final concentration of 1.7M NaCl. Type I collagen
was precipitated by the addition of NaCl to a final concentration of 2.5M NaC1. Each collagen type was dissolved in 0.5M
acetic acid, dialyzed against the same solution, and lyophilized.
Ion exchange chromatography. Pepsin-solubilized collagen was separated into constituent a chains by chromatography on columns of carboxymethylcellulose (CM-cellulose)
(Whatman Inc., Clifton, NJ) as previously described (19).
Samples were solubilized in 0.02M sodium acetate/ 1M urea,
pH 4.8, denatured at 43°C for 20 minutes, and separated on
columns (2.5 X 10 cm) equilibrated with the above buffer.
Elution was achieved with a linear gradient from 0 to 0.12M
of NaCl.
Peptides generated by CNBr digestion of either whole
lung homogenate or individual a chains were chromatographed on a column (0.9 x 15 cm) of CM-cellulose at 43OC.
Chromatographic conditions were identical to those previously used (10).
Molecular sieve chromatography. The separated collagen chains were further purified and their molecular weight
estimated on calibrated columns (2 X 120 cm) of agarose
beads, A-15M or A-1.5M (200-400 mesh, Bio-Rad Laboratories) in 0.05M Tris/lM CaCI,, pH 7.4 (20). A drop of tritiated
water was added to each sample to mark the column volumes
(20). Type I11 collagen ( y component) was reduced with 0.1%
mercaptoethanol and carboxymethylated (21) for isolation of
type I11 monomeric a chains.
The relative distribution of type I and type I11 collagen was determined by quantitating the relative amount of
two sets of peptides, aI(I)-CB8 and aI(III)-CB8, and al(1)CB3 and al(III)-CB4 in the CNBr digests of whole lung tissue. These two sets of peptides coelute with each other from
CM-cellulose and were quantitated by amino acid analysis after gel filtration on agarose A- 1.5M (22,23).
Amino acid and carbohydrate analysis. Samples for
amino acid analysis were hydrolyzed in doubly distilled, constantly boiling HC1 under an atmosphere of nitrogen for 24
hours at 108°C. Analyses were performed on an automatic
analyzer (Model 121, Beckman Instruments Inc., Spinco Division, Palo Alto, CA) by using a four buffer elution system
Samples for analysis of hydroxylysine glycosides were
hydrolyzed in 2N NaOH in borosilicate-free test tubes at
108°C for 24 hours. The hydrolyzates were neutralized with
6N HCl and applied directly to a Beckman 121 analyzer as
described by Askenasi and Kefalides (25).
Reduction and labeling of crosslinks. Washed lung
residue (200 mg wet weight) was suspended in 10 ml of 0.1M
sodium phosphate, pH 7.4, and stirred briefly. NaB[3H], (New
England Nuclear: specific activity 200 pCi/mM, 10 mg/ml in
0.01M NaOH) at 3 mg/gm of protein was added (26). Reduction was allowed to proceed for 30 minutes at 22OC; the solution was then acidified with acetic acid to pH 3.5 in order to
stop the reaction. Excess reagents were removed by centrifugation and multiple washings with deionized water. The residue was lyophilized prior to acid hydrolysis.
Crosslink analysis. The reduced radioactive labeled
lung tissue was hydrolyzed in 3N HCl under N2 for 48 hours
at 105°C. Analysis was performed on a column (0.9 X 60 cm)
of Beckman PA-35 resin maintained at 60°C using 0.35M sodium citrate, pH 5.25, on an automatic amino acid analyzer
(Technicon Institute, Ardsley, NY) equipped with a split
stream device. A portion of the effluent was continuously analyzed for ninhydrin reactivity; the remainder was collected in
fractions of 2.6 ml. An aliquot of 0.5 ml of each fraction was
used for the assay of radioactivity. The crosslink compounds
were identified on the basis of elution positions of authentic
compounds (27).
A significantly greater proportion of collagen
(Table 2) was solubilized with cold, dilute acetic acid
and limited pepsin digestion in PSS lungs (55%) as compared with either normal (16%) or fibrotic lungs (idiopathic pulmonary fibrosis) (35%). The pepsin-extracted
collagen was purified by dialysis against 0.02M
Na,HPO,, NaCl precipitation from acetic acid solution,
and sequential precipitation at 1.7 and 2.5M NaCl at
pH 7.4 for type I11 and type I collagens, respectively. Final purification of type I11 collagen (1.0-1.7M NaCl
Table 2. Relative amounts of collagen and protein extracted from normal, fibrotic, and PSS human
0.05MTris HC1, pH 7.4
First pepsin digestion
Second pepsin digestion
Third pepsin digestion
Idiopathic pulmonary
Progressive systemic
* The results are expressed as percentage of collagen or protein solubilized by each extractant.
t From Seyer et a1 (12).
$ Collagen content based on hydroxyproline analysis.
8 Total protein content was determined from amino acid analysis of a portion of each extract and converted to protein, assuming a mean residue weight of 100.
precipitate) was achieved by CM-cellulose and molecular sieve chromatography. The elution peaks were identified by agarose chromatography (not shown) and
amino acid analysis. Type I11 collagen eluted from agarose A-15M primarily at the position corresponding to a
y component, molecular weight (mol wt), 280,000 with
smaller amounts of higher mol wt components. This
material was identified as type 111 collagen by reduction
with mercaptoethanol followed by carboxymethylation
to the corresponding 95,000 mol wt a chains. The amino
acid composition of al(I11) was, within experimental error, identical with type 111 collagen of human lung previously reported (12).
The collagen precipitating between 1.7 and 2.5M
NaCl eluted from CM-cellulose as two components (not
shown). The peak containing al(1) was identified as
such by its elution position from agarose A-15M gel filtration (mol wt 95,000) and amino acid analysis. Gel filtration of a2 (mol wt 95,000) provided final purification
of this a chain from contaminating [al(IlI)], (mol wt
280,000). No peaks corresponding to PI, or j3,2were observed by gel filtration.
The amino acid and carbohydrate composition
of PSS lung collagens al(I), al(lII), and a2 were identical, within experimental error, to that previously found
for the corresponding chains in lung collagens from
idiopathic pulmonary fibrosis (12). Compared with normal human lung collagens, the present data indicate a
L L l J l l O N VOLLlhlL
possible slight decrease in hydroxylysine content for
al(1)and al(II1) collagen chains (10.0 and 11.1 in normal to 8.1 and 7.8 in PSS, respectively). In all cases, the
aI(1) and al(II1) collagen chains contained no significant difference in the amount of glucosylgalactosylhydroxylysine or galactosylhydroxylysine per collagen
Further characterization of the covalent structure of al(1) and al(II1) was achieved by CM-cellulose
chromatography of peptides derived after CNBr digestion of the purified collagen chains. No differences were
apparent from the elution pattern of CNBr digests of
human normal and fibrotic lung al(1) and al(II1) reported elsewhere (10). Although the peptides were not
further investigated, the peptide elution patterns indicate that they are indistinguishable from the corresponding normal lung collagens.
The question of collagen polymorphism was also
examined with PSS lung. Due to only a limited and variable solubilization of normal versus PSS lung collagen
by pepsin extraction, CNBr digestion of whole lung tissues was used to quantitate the relative distribution of
type I and type 111 collagen. Briefly, the total lung homogenates were subjected to CNBr digestion directly
after preliminary washing with 0.05M Tris, pH 7.4, and
distilled water. Under the conditions used, 88% or more
of the total hydroxyproline was solubilized by CNBr digestion. The chromatographic pattern obtained by sepa-
:, .
Figure 1. A, CM-cellulose chromatography of peptides solubilized by CNBr digestion of human PSS lung. The tissue was washed exhaustively with
O.OSM Tris, pH 7.4, then in distilled water and digested with CNBr at 4OoC for 6 hours. CM-cellulose chromatography of a 150 mg sample was
performed at 43°C with 0.02M sodium citrate, pH 3.8, as the starting buffer and a linear NaCl gradient from 0.02 to 0.16M with a total volume of
500 ml. The peaks designated al(lII)-CB4, al(l)-CB3 and al(lll)-CBS + al(I)-CR8 were pooled as indicated by bars. B, Peak CB8 was further
separated and used for quantitative analysis of a l ( l ) and al(1ll) ratios. Agarose A-1.5M gel filtration of the CNBr peptides eluting in the CB8
region from CM-cellulose. Material eluting at a volume corresponding to 24,000 and 12,000 was identified as al(l)-CB8 and al(lll)-CB8, and
hydroxyproline analysis of each peak was used to calculate molar concentrations of each peptide based on their known molecular weights. Further
calculations provided relative quantities of [a l(l)]2a2:[al(lll)]3.
Table 3. Percentage of type 111 collagen in human lung. The values
are expressed as percent type I11 collagen with respect to type I*
Idiopathic pulmonary
Each value represents an analysis of an individual lung. Additional
values in series represent actual values obtained from repeated CNBr
digestion of different aliquots of the same lung. The values presented
were obtained by quantitating the two peptides al(I)-CBB plus al(1)CB8-3 versus al(III)-CB8 as described in the text.
f From Seyer et a1 (12). A total of 5 lungs were assayed; 2 were assayed 3 times and 3 were assayed once.
ration on CM-cellulose was complex due to the presence of both type I and type I11 collagens (Figure 1A).
The characteristic pattern of type I was more evident
and, therefore, was used for orientation. The peptide
a 1(111)-CB4 was immediately followed by a1(I)-CB3,
and the fractions containing both were pooled together.
The peptides al(I)-CB8 and al(I)-CB(8-3) characteristically elute together from CM-cellulose with the
smaller molecular weight al(III)-CB8 (22).
Quantitation of the al(I):al(III) ratio was obtained from the above al(1)- and al(II1)-specific CNBr
peptides. Specific details and quantitative methods have
been documented elsewhere (10,12,17,22,28). Briefly,
al(1)-CB8, al(I)-CB(8-3), and al(II1)-CB8 coelute
from CM-cellulose as a clearly identifiable peak (Figure
1A). These peptides were collected as indicated by the
bar and separated from each other by molecular sieve
chromatography on agarose A- 1.5M (Figure 1B). The
three peaks eluted at positions corresponding to 37,000,
24,000, and 12,000 mol wt for al(1)-CB(8-3), al(1)CB8 and al(II1)-CB8, respectively. Identity of these
peptides was established by amino acid analysis, and
quantitation was achieved by hydroxyproline analysis.
Generally, the uncleaved a 1(I)-CB(8-3) represented
10-15% of the total al(I)-CB8 on a molar basis and was
included in the calculation of the amount of al(1). The
al(III)-CB8 peak, which eluted at a position corresponding to 12,000 mol wt, was used to determine the
quantity of type I11 collagen present. The values obtained for PSS lungs averaged 33 to 36% type I11 collagen with the remainder being type I (Table 3).
The two previously mentioned peptides, al(II1)CB4 and al(I)-CB3 (Figure lA), were used to verify the
above quantitation. These two peptides are homologous
within their respective collagen chains, each having 149
amino acid residues and equal molecular weight. Their
amino acid composition varies significantly in the
amount of threonine and valine (23). The peptide from
type I11 collagen, al(III)-CB4, has 5 threonine residues
but no valine per peptide, whereas al(1)-CB3 contains
no threonine but 5 valine residues. The peptide mixture
was collected and separated from contaminating peptides by agarose A-1.5M gel filtration (not shown). Because of their identical molecular weight (13,500), they
coelute from the molecular sieve column. Therefore, the
amino acid composition of this mixture yielded an average of 42 m o l e s of threonine and 56 nmoles of valine.
These values correspond to 33% type I11 collagen with
respect to type I (12).
The amount and type of aldamine crosslinkages
in PSS lung tissue were assayed after Na[’H],B reduction and separation by ion exchange chromatography.
The ’H-labeled peaks were identified by comparison of
their elution times with those of authentic compounds
as previously established (27). Only the difunctional
(HLHNL and HLNL) crosslinks were quantitated. Separate aliquots were used for hydroxyproline analysis
and the amount of radioactivity was expressed as cpm/
pg hydroxyproline (Table 4). All the reducible crosslink
compounds are present in markedly diminished
amounts in PSS lungs as compared with lung of normal
or idiopathic pulmonary fibrosis. However, the content
of the more stable crosslink, HLHLN, is diminished to a
much greater extent than HLNL, so that the ratio of
HLHNL to HLNL is much smaller in PSS. These data
are consistent with the greater solubility of PSS lung
collagen as compared with normal or idiopathic fibrotic
lungs (Table 2). No attempts were made to quantitate
glycosidically-linked lysine-derived crosslinks, since
they were present in much smaller quantities after 2N
NaOH hydrolysis.
The chemical nature of collagen in PSS lung has
never been fully investigated. Analysis of this protein
Table 4. Reducible crosslinks in human lung*
Idiopathic pulmonary fibrosis
Progressive systemic sclerosis (TM)
Progressive systemic sclerosis (AV)
17 1
30 1
* Values expressed as cpm 3H per pmole of hydroxyproline.
t Lung samples (TM) and (AV) represent 2 individual cases.
$: HLHNL = hydroxylysinohydroxynorleucine;HLNL = hydroxy1ysinonorleucine.
was the purpose of this investigation. Type I and type
I11 collagens were made soluble by limited pepsin digestion using conditions which preserve the large helical
portion of the molecule (19). Solubilization occurs when
the lysine-derived, small interchain nonhelical crosslinking regions of each end of the collagens are cleaved
with pepsin, leaving the remainder of the collagen molecules intact with physical-chemical properties similar to
the complete molecule.
The collagens were purified by low salt precipitation, acid precipitation, and selective precipitation at
1.7 and 2.5M NaCl (0.05M Tris, pH 7.4) for type I11
and type I collagen, respectively. Separation into individual a chains was obtained by CM-cellulose chromatography and subsequent agarose A- 15M gel filtration.
The molecular weights of al(1) and a2 were estimated
at 95,000, while [a1(11I)l3eluted from the agarose column in a position of 280,000. After reduction, 2 smaller
fractions were obtained corresponding to molecular
weights of 180,000 and 95,000. These results are identical with collagens of normal human skin (21) and lung
(12), including the occurrence of disulfide bonds in
The amino acid compositions of each PSS lung
collagen chain were found, within experimental error,
to be the same as collagens of idiopathic pulmonary fibrotic lung (12). This contrasts somewhat with normal
lung collagens, which contained slightly greater levels of
hydroxylysine (2-3 residues per chain) than those of
PSS and fibrotic al(1) and al(II1). Proline hydroxylation was not altered in PSS lung. Glycosylation, the final posttranslational event prior to cell secretion, was
measured by isolation and quantitation of glucosylgalactosyl- and galactosyl hydroxylysine. No significant
differences were noted again in either the total content
or ratio of the mono- and disaccharide-linked hydroxylysine. Further characterization of the covalent structure of PSS al(1) and al(II1) was demonstrated by
CNBr digestion. No qualitative differences in the CMcellulose elution pattern of CNBr-derived peptides of
PSS al(1) or PSS al(II1) collagens were noted when
compared with normal or idiopathic pulmonary fibrosis
al(1) and al(II1) CNBr peptide maps. The results indicate no apparent genetic abnormalities in the collagen
primary structure of PSS lung collagen.
The final known posttranslational event in collagen metabolism is oxidative deamination of the &-amino
groups of specific lysine and hydroxylysine residues.
These reactive aldehydic compounds subsequently undergo Schiff base formation to intermolecular crosslinks
necessary for normal collagen maturation. Since these
crosslinks or their ketimine forms are unstable to strong
acid or base hydrolysis, prior reduction with borohydride is necessary to form the more stable, reduced
HLHNL and HLNL. The relative distribution of these
Schiff base compounds were, therefore, analyzed directly in lung tissue and related to the total collagen
content determined by hydroxyproline analysis. The
content of each of the crosslink compounds was significantly diminished in PSS lungs, but the diminution was
more pronounced for HLHNL. This decreased content
of HLHNL in PSS lung tissue compares favorably with
the similar finding noted in PSS skin (28). The reduced
levels of HLHNL may help explain the greater ease
with which PSS lung collagen was extracted from the
tissue (Table 2). The more insoluble tissue collagens,
such as in bone and cartilage, predominantly have
HLHNL. Young rat skin, by contrast, has primarily the
less stable HLNL and is more readily extracted (29).
Finally, the question of the relative accumulation of type I and type I11 collagen in PSS lungs was addressed. By using CNBr digestion of whole lung tissue
which consistently solubilized 88% or more of total lung
collagens, type I and type I11 collagens were shown to
be present in proportions identical to normal lung. Similar results were obtained from analyses of involved PSS
skin by chemical and immunohistologic studies (J. M.
Seyer and A. H. Kang, unpublished data; 4,16,17,3032). This is rather surprising since other fibrotic tissues
examined, including involved tissues of idiopathic pulmonary fibrosis, alcoholic cirrhosis, hypertrophic scar,
and granulomatous colitis (10, 12-14), all showed a
markedly disproportionate increase in type I collagen,
giving rise to the hypothesis that such a response might
represent a final common pathway of fibrotic processes.
Obviously, such an idea is no longer tenable. One might
now postulate that the expression of the particular gene
product in fibrosis may depend on the particular stimuli
operative at the fibrotic process. In the absence of a definitive knowledge of the etiology or pathogenesis of
any of these conditions, it is not possible to speculate on
the nature and mechanism of action of various stimuli.
Nevertheless, these observations may provide a handle
via which the involved components could be further investigated.
The authors wish to express thanks to E. Ryan and D.
Blanton for their expert technical assistance.
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investigation, progressive, lung, systemic, typed, sclerosis, iii, collagen
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