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Identification and characterization of two new soluble nuclear antigens reactive with sera of patients with connective tissue diseases.

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803
IDENTIFICATION AND
CHARACTERIZATION OF TWO NEW
SOLUBLE NUCLEAR ANTIGENS
REACTIVE WITH SERA OF PATIENTS
WITH CONNECTIVE TISSUE DISEASES
SHOJI MIYAWAKI, KIICHI KOHMOTO, NORIYUKI KURATA, and TADASHI OFUJI
Two antigens, temporarily referred to as MU and
TM antigens after the original serum, that are closely
related to the nucleolus have been identified in the nuclear
soluble extract. Both antigens reacted with the sera of a
small number of patients with various connective tissue
diseases, and both were different from antigens described
previously in these disorders. MU antigen was sensitive to
the digestion with RNase and trypsin, and was shown to be
equivalent to the nuclear ribosomal components in the
nuclear extract. TM antigen was resistant to the digestion
with RNase and trypsin, and was localized in the nucleolus
and extranucleolar portion of nuclei, but the precise nature of TM has not been established.
Recent studies have identified a variety of antibodies to the macromolecules extracted in the saline
soluble fraction of nuclei in the sera of patients with
connective tissue diseases. These include antibodies to
acidic nucleoprotein (Sm)(l), nuclear ribonucleoprotein
From the Third Department of Internal Medicine, Okayama
University Medical School, Okayama, Japan.
Supported in part by Grant-in-Aid No. 167094 for Scientific
Research from the Japan Ministry- of Education.
Shoji Miyawaki, M.D.: Lecturer of Internal Medicine; Kiichi
Kohmoto, M.D.: Research Fellow; Noriyuki Kurata, M.D.: Lecturer
of Internal Medicine; Tadashi Ofuji, M.D.: Professor of Internal
Medicine.
Address reprint requests to Shoji Miyawaki, M.D., The Third
Department of Internal Medicine, Okayama University Medical
School, 2-5-1 Shikata-cho Okayama 700, Japan.
Submitted for publication December 5, 1977; accepted January 30, 1978.
Arthritis and Rheumatism, Vol. 21, No. 7 (September-October 1978)
(RNP) (2,3), SS-B (4) or H a ( 5 ) antigen, and PM-1
antigen (6). In the course of studying the precipitin
reactions between sera from patients with connective
tissue diseases and the nuclear extract, two new antigens
were observed, provisionally referred t o as MU and
TM antigens after the original serum. These antigens
appear to be related t o the nucleolus but are distinct
from previous nuclear macromolecules. The purpose of
this article is to describe the characteristics of these antigens t o which antibodies were found in a small group of
patients with various connective tissue diseases.
MATERIALS AND METHODS
Sera Specimens. Eighty sera were from patients with
systemic lupus erythematosus (SLE)*whomet the preliminary
criteria of the American Rheumatism Association (ARA) (7)
for this disease, 20 sera were from patients with dermatomyositis (DM) or polymyositis, and 15 sera were from patients
with progressive systemic sclerosis (PSS).Forty sera were from
patients with rheumatoid arthritis (RA) whose symptoms and
laboratory data met the criteria for classic and definite RA as
defined by the ARA (8). Forty-five sera were from patients
with sicca syndrome whose diagnoses were made on the presence of clinical symptoms of dry eye or dry mouth in addition
to one of the three features of keratoconjunctivitis sicca, positive salivary gland biopsy, and abnormal sialographic findings.
There were 20 sera from patients with the diagnosis of mixed
connective tissue disease (MCTD)who had the-clinical overlapping features of connective tissue diseases and high titers of
antibodies to RNP (3,9). All sera were stored at -2OOC until
examined and were decomplemented before use by heating to
56°C for 30 minutes.
804
Preparation of Antigens. Calf thymus nuclei were isolated according to the method of Allfrey et al. (10). Isolated
nuclei were extracted with 5 volumes of 0.15 M NaCl (pH 7.2,
0.01 M phosphate) containing 0.005 M MgCl, by blending in
a Waring blender for 5 minutes at medium speed. The nuclear
homogenate was centrifuged at 20,000 g for 30 minutes. The
extract was processed by centrifugation of 105,000 g for 2
hours. The supernatant from this centrifugation was designated as the nuclear extract and used throughout this study.
To obtain the nuclear ribosomes, the pellets from the
centrifugation of 105,000 g were homogenized in the initial
extractant, and sodium deoxycholate (DOC) was added to a
final concentration of 1%. The DOC suspension was further
stirred for 1 5 minutes and centrifuged at 105,000 g for 2 hours.
The pellets from this centrifugation were the isolated nuclear
ribosomes (11). The material was analyzed spectrophotometrically. The ratio of 260 n M to 280 nM was 1.7.
The saline extracts of isolated rat liver nuclei and
nucleoli were obtained as described previously (12). The cytoplasmic ribosomes were prepared from rat liver as described
by Rendi and Hultin (13). The 260/280 ratios of cytoplasmic
ribosomes were 1.9.
Fluorescent Antibody Technique. Frozen young rat
liver sections, 4 r m thick, were placed on slides, fixed for 30
minutes i n a solution of acetone-ethanol (4: 6 ) , air dried, and
reacted with the patients’ serum for 30 minutes at room temperature. Excess serum was removed by two 5 minute’washings in buffered 0.15 M saline. A drop of the fluorescein
conjugated antiserum to human IgG was then applied for 30
minutes at room temperature. The sections were washed as
before, mounted in buffered saline containing 10% glycerol,
and examined with a Nikon ultraviolet microscope. The fluorescein conjugated antiserum to human IgG was prepared by
raising antiserum in rabbits to human IgG obtained by DEAE
column chromatography. The gammaglobulin fraction of the
rabbit antiserum was conjugated to fluorescein isothiocyanate.
Only conjugates with a fluorescein to protein ratio between 1 .O
and 2.0 were used in this study. The cultures of mouse fibroblast cells (L-929; Flow Laboratories, Inc.) monolayered on
the glass slides were grown in petri dishes in Earles’ medium
plus 10%fetal calf thymus. The cells were air dried and processed for immunofluorescence in a manner similar to the rat
liver sections. The use of cultured cells as the second substrate
provided further information concerning the location of nuclear and cytoplasmic macromolecular antigens, as will be
described below.
Precipitin Reactions. Agar gel diffusion was performed
in 0.6% agarose (Seakem Agarose, Marine Colloidal Inc.,
Rockland, Maine) with 0.1% sodium azide added at pH 7.4.
The reference sera of known antibody specificitieswere used to
detect and characterize the precipitating antibodies. These
standard sera characterized as containing anti-RNP antibody,
anti-Sm antibody, and anti-SS-A plus B antibodies (all determined using sera kindly provided by Dr. Eng Tan), and antibody to the cytoplasmic ribsosomes were placed in wells adjacent to the unknown sera being tested in order to identify the
precipitin line that resulted after interaction with the nuclear
extract.
Enzymes. Bovine pancreatic ribonuclease (RNase, 5 X
crystallized), deoxyribonuclease I (DNase, RNase free), and
trypsin (5 X crystallized) were obtained from Worthington
MIYAWAKI ET AL
Biochemical Corporation. Enzyme digestion of antigens was
performed according to the method of Tan (14).
Other Immunological Methods. Sheep tanned red cells
coated with the nuclear extract which were formalinized were
used for detecting the hemagglutinating antibodies to the nuclear extract. Antibody to native DNA was determined by the
method of Carr et al. (15) with tritiated actinomycin D-labeled
calf thymus native DNA and a gammaglobulin binding assay,
the normal range being 0-1570. Total hemolytic complement
activity was detected by a standard sensitized sheep red cell
assay (16).
RESULTS
As shown in Figure 1, serum MU from a patient
with dermatomyositis ( D M ) or serum TM from a patient with systemic lupus erythematosus (SLE) reacted
with t h e nuclear extract to give one line that was nonidentical with each other. It was important to establish
that t h e precipitin systems presented here were different
from precipitating antibodies described in other reports.
This was accomplished by using previously analyzed
sera containing known antibodies. Anti-Sm in Figure
I A was a serum from a patient with SLE that gave a
precipitin line with t h e nuclear extract that has been
identified in previous studies a s the Sm system. It was
clear that t h e Sm system was different from the MU or
T M system. Anti-RNP in Figure 1B was a serum from a
patient with mixed connective tissue disease (MCTD),
and has been identified as having antibody to RNP of
MCTD. This was also not identical immunologically
with the MU or TM system. Anti-SS-A plus B in Figure
IC was t h e serum of a patient with sicca syndrome that
had antibodies t o SS-A a n d B. This reacted with the
nuclear extract t o give a line identified previously as t h e
SS-B system that was not identical to the line of the M U
or TM system. N o precipitating antibody to the SS-A
was observed between this serum and t h e nuclear extract. Studies not illustrated here showed that the MU
or T M system was not related t o precipitating systems
involving soluble nucleoprotein (SNP), native D N A ,
a n d denatured D N A .
T h e antigenic uniqueness in the M U system was
further established relative to t h e ribosomal antigen.
This is illustrated in Figure 2 where serum MU reacted
with the nuclear ribosomes to form a sharp line and with
t h e cytoplasmic ribosomes to form a weak line that was
completely identical to the MU precipitin line. T h e sera
containing precipitating antibody to t h e cytoplasmic
ribosomes, however, did not always react equally with
t h e nuclear extract. These data provide strong evidence
that the antigenic material of the MU system was related
NEW SOLUBLE NUCLEAR ANTIGENS
A
805
C
B
Figure 1. Precipitin reaction showing the independence of the MU and TM systems from A the Sm system.
system. MU = patient MU serum; TM = patient TM serum; NE = calf thymus nuclear extracf.
to the nuclear ribosomal components, partially sharing
antigenecity with the cytoplasmic ribosomes.
Serum MU reacted well with the extract of calf
thymus nuclei but reacted weakly with the extracts of rat
liver nuclei and nucleoli where it existed in low concentration. Serum TM reacted equally well with all extracts
from these sources.
Cellular Localization of Antigens. The antigens
were localized by the technique of studying the indirect
immunofluorescence patterns obtained by reacting
serum M U or T M with human white blood cells, rat
liver sections, or cultured L-929 cells. When serum MU
was applied to substrates, intense immunofluorescence
was seen in nucleoli and cytoplasm, and the extranucleolar portion of the nuclei was scattered sparsely
with fine speckles. These findings were observed most
conspicuously in L-929 cells (Figure 3A) and in lymphocytes (Figure 3B), less intensely in rat liver sections, but
were not observed in polymorphonuclear blood cells.
Different staining patterns were obtained by reacting
serum TM with substrates. Serum TM produced intense
nucleolar patterns finely mixed with coarse speckles in
the extranucleolar area of nuclei (Figure 3C). No cytoplasmic stainings were observed with serum TM.
Nucleoli mixed with fine speckles and intense
cytoplasmic stainings produced by MU serum were inhibited by adsorption of the serum either with the nuclear extract and ribosomes, or with a large amount of
cytoplasmic ribosomes, whereas nucleoli mixed with
B the RNP system, and C the SS-A and B
speckled stainings produced by T M serum were inhibited
only with the nuclear extract.
In order t o evaluate the relationship between
M U antigen and ribosomes, a specific immune precipitate was formed in vitro with serum MU and the nuclear
ribosomes, and then specifically purified antibody was
derived from this precipitate using glycine-HCI buffer
(pH 2.7). After neutralization, purified antibody was
applied to substrates and an indirect fluorescent antibody technique was again performed. This is illustrated
in Figure 3D where it can be seen that purified anti-
Figure 2. Precipitin reaction of M U serum (MU) with the calf
thymus nucleac extract (NE),the nuclear ribosomes (n-rib). and
the cytoplasmic ribosomes (cyt-rib).
MIYAWAKI ET AL
806
Figure 3. Indirectjuorescent antibody technique was employed to identijy M U and TM antigens in cultured L-929 cells (A, C , and D) and
lymphocyte (B). A and B show pattern of MU antigen distribution and C TM antigen distribution. D illustrates the same immunojuorescence pattern as with MU serum when specific antibody purifiedfrom precipitate with M U serum and the nuclear ribosomes was
applied to the cultured cells.
body gave fluorescent stainings completely identical to
the stainings produced by original MU serum.
Physicochemical Properties of Antigens. The antigen which produced an MU precipitin line was completely destroyed by RNase or trypsin treatment (Figure
4A). The antigen relative to the TM system was not
sensitive to the digestion with RNase and trypsin (Figure 4B). DNase had no effect on both antigens.
Exposure of the nuclear extract to 56°C for 30
minutes completely destroyed the antigenic activities of
the MU and TM systems.
Both antigens were soluble in buffered 0.15 M
NaCl saline as the activities were recovered in the supernatants of the nuclear extracts separated by the centrifugation at 105,000 g for 2 hours. Parts of MU antigen,
however, were precipitated by this centrifugation and
were recovered as the nuclear ribosomes after the DOC
treatment (see Materials and Methods).
Filtration of the nuclear extract through Sephadex G 200 (Pharmacia, Uppsala, Sweden) gave two
major peaks. MU and TM antigen activities detected by
immunodiffusion were eluated in the descending portion
of the first peak and coincided with the elution volumes
for Sm and R N P antigens (Figure 5).
Clinical and Serological Features. The diagnosis
and relevant serological findings for the MU or TM
precipitin line positive patients are listed in Tables 1 and
2, respectively.
From Table 1 it can be seen that 10 patients had
antibodies to MU antigen in their sera. There was one
patient with the clinical diagnosis of DM, 5 with SLE
(including one associated with sicca syndrome), 2 with
MCTD sharing clinical features of SLE and DM, and 2
with sicca syndrome. When rat liver sections were used
as substrates, all patients had significant titers of antinuclear antibodies (ANA) and cytoplasmic stainings
NEW SOLUBLE NUCLEAR ANTIGENS
807
B
A
Figure 4. Eflect of enzyme on the precipitating activities of (A) the M U system and (B)the TM system. MU
serum: NE = calf thymus nuclear extract.
consistent with findings of antiribosomal antibodies in
their sera. Four of 10 had nucleolar patterns in addition
to a variety of other nuclear stainings. In the rest of the
patients the staining patterns could not be interpreted as
being predominantly of the nucleolus because of the
interfering fluorescence of surrounding nuclear structures. Slightly raised native DNA-binding activities were
found in 2 patients with SLE, and slight to moderate
decrease of serum complement levels was observed in 8.
No patient had serious renal disease.
Antibodies to TM antigen were seen in 2 patients
with SLE whose sera showed moderate titers of ANA
with nucleolar staining patterns, but did not have cytoplasmic stainings and antibodies to cytoplasmic riboI
1
1
1
1
I
1
,-.
I-\
0.9
~..
Q8
1
-260nm
---280nm
--__-----____
m 0.2
M U serum; TM
= patient
TM
somes (Table 1). Both had moderate titers of anti-native
DNA antibodies. One showed marked decrease of
serum complement level because of renal involvement,
whereas the other had normal levels of serum complement without renal involvement.
Five of 10 sera containing antibodies to M U
antigen and 1 of 2 sera with antibodies to TM antigen
also contained anti-RNP antibodies, one of which was
from an SLE patient and also had anti-Sm antibody
demonstrable as precipitins (Table 2). All sera had the
hemagglutinating antibodies to the nuclear extract. The
hemagglutinating titers, however, in the range of I : 1280
to 1 : 1,048,576 in patients having anti-RNP (Sm) antibodies, seemed to distinguish them from patients with
only anti-MU or TM antibodies in whom the hemagglutinating titers were in the range of 1 : 16 to 1 :256.
RNase treatment of the red cells sensitized with the
nuclear extract slightly or markedly reduced the hemagglutinating reaction with the sera of patients with positive MU line or of those with both MU and R N P lines
positive, but this reaction was not reduced with TM
serum.
DISCUSSION
0.1
'
-
= patient
1
0
9
S m + t H i t t I t ) + - - - - $ + * - - - MU
TM
I
1
1
t
1
I
-
-,
2
3
4
5
6
7
* * - - - - - I
-
It has been pointed out that the nuclear extract
contains several unidentified antigens that are distinct
from either R N P or Sm antigen, to which antibodies are
detected in some of the sera from patients with connective tissue diseases (2,9). The present study confirmed two of these antigens, provisionally called MU
and TM antigens, that were not related to the precipitating systems involving Sm, RNP, SS-A and B, SNP,
MIYAWAKI ET A L
808
Table 1. Clinical Diagnosis and Other Serological Findings in Patients with Precipitating Antibodies to M U and
TM Antigens
ANA Titer and Staining Pattern*
Patients
Diagnosis
Titer
Low Serum
Dilution
Anti
RiboCyto- somal Native
High Serum plasmic AntiDNA
Dilution Staining body? Binding$ C H 5 q
~~
M U Line
Positive
MU
TN
MI
UY
KD
SY
KO
KI
DM
SLE
SLE
SLE
SLE
SLE + Sicca
MCTD
MCTD
Sicca
RA + Sicca
SM
MO
TM Line
Positive
TM
SLE
YN
SLE
1:128
1:128
1: I28
I : I6
1:128
1:64
1:32
1:512
Noll
H#
H
Sp** No
H +PH
H+P
SP
H+P
No
H+P
1:512
1:128
Sp No
H+P
1:64
1 : 128
+
+
* Indirect fluorescent antibody technique
t Precipitin line to cytoplasm ribosomes.
No
H
SP No
SP
H
SP
SP
SP
No
+
+
+
+
+
+
H
+
+
+
+
+
Sp + No
Sp + No
-
+
+
+
+
+
+
+
+
t
+
-
12.4
11.5
12.1
7.1
21.1
18.0
7.5
13.2
9.3
7.8
13
34
16
16
15
16
22
10
44
24
21.3
23.0
0
31
using rat liver sections.
$Normal range less than 15%.
(j Normal range 30-40.
1 1 No = Nucleolar.
# H = Homogeneous.
** Sp = Speckled.
$$ P = Peripheral.
and DNA. Since the antibodies to MU and TM antigens
possessed the same hemagglutinating properties against
the nuclear extract, though in low titer, as those of
antibodies to R N P and/or Sm, the precipitin reaction
using reference sera made it possible to differentiate
these antibodies.
The indirect fluorescent antibody technique with
TM serum disclosed that TM antigen was localized in
nucleoli and the extranucleolar portion of nuclei, but
was not present in cytoplasm. It had essentially the same
physiocochemical properties as those of Sm antigen but
was distinguishable from it in the nonidentical precipitin
pattern. We have no further knowledge of TM antigen
at present.
The sera with the MU precipitating antibody
showed predominantly nucleolar and cytoplasmic staining patterns by immunofluorescence. However, some
could not be interpreted as being predominantly of the
nucleolus because of the interference by the antibodies
to other nuclear antigens that produced peripheral, homogenous, and speckled stainings.
It is suggested that M U antigen is equivalent to
Table 2. Hemagglutinating Reaction with Nuclear Extract. Anti-RNP.
and Anti-Sm Antibodies in Patients with Positive M U or T M Lines
Titer of Hemagglutinating
Reaction with Nuclear Extract
Patients
M U Line
Positive
MU
TN
MI
UY
KD
SY
KO
KI
SM
MO
TM Line
Positive
TM
YN
Buffer
After Treatment
with RNase
Precipitating
Antibody to
RNP
Sm
-
-
I :2,560
1 : 10.240
1: I6
I :65,536
1 : 1,048,576
I :256
I : I6
1:80
1 : 640
1:640
1:40
1 : 1,024
-
1:64
1 :40,960
1 : 64
1 :2,560
-
1:64
I : 320
I : 1,280
+
+
+
+
+
-
+
-
+
-
-
-
-
NEW SOLUBLE NUCLEAR ANTIGENS
the nuclear ribosomes and partially shares antigenecity
with the cytoplasmic ribosomes. This conclusion was
based on 5 major findings: 1) The antibody to MU
antigen reacted with the nuclear ribosomes to give an
identical line, but it reacted occasionally and less intensely with cytoplasmic ribosomes. 2 ) The antigen was
sensitive to the digestion with RNase and trypsin. 3) The
antigen was localized in nucleoli, extranucleolar portion
of nuclei, and cytoplasm identified by indirect fluorescent antibody technique. 4 ) Immunofluorescent stainings produced by MU serum were inhibited by adsorption of MU serum with the nuclear extract and the
nuclear ribosomes, but was weakly inhibited with the
cytoplasmic ribosomes. 5 ) Purified antibody obtained
from a specific immune precipitate with MU serum and
the nuclear ribosomes gave immunofluorescent stainings
completely identical to the stainings produced by MU
serum.
Genetic, cytochemical, and biochemical studies
show that the nucleolus is a most active site of synthesis
of ribosomal RNA. The present day concepts are that
either whole ribosomes, larger complex of polysomes, or
interchromatinic granules move through the nuclear
pores into the cytoplasm (17). Electron microscope
studies have revealed the presence of a number of types
of particulate bodies in nucleoli and nuclear ribonucleoprotein network which are believed to be ribosomes
(17). Wang isolated the nuclear ribosomes from calf
thymus nuclei by treating the crude nuclear fraction
with DOC, which showed that the nuclear ribosomes
contain 50% RNA ( 1 I ) . Nuclear ribosomal protein
shows basic and similar amino acid composition to the
protein of the cytoplasmic ribosomes (18). Thus, our
findings identifying the antibody as reacting with the
nuclear ribosomes may have the distinct implication
that such an antibody provides a means of identifying
the presence of the ribosomes in the nucleus by immunochemical means.
Although highly purified calf thymus nuclei were
used in this study, the possibility cannot be completely
excluded that the calf thymus nuclear extract is contaminated by a small amount of the cytoplasmic ribosomes.
However, if MU antigen is simply thought to be equivalent to the cytoplasmic ribosomes contaminated in the
nuclear extract, the nuclear immunofluorescence produced by the MU antibody and the results of inhibition
study mentioned above cannot be explained. Since the
ribosomes settle in the precipitate by centrifugation at
105,000 g , the present finding that the nuclear extract
obtained as the 105,000 g supernatant contained an
antigen identical to the nuclear ribosomes may be attributable to the presence of a precursor of ribosomes.
809
The reason that the serum MU reacted well with
the calf thymus nuclear extract, but reacted weakly with
the extracts of rat liver nuclei and nucleoli, may be the
difference o f concentration of antigen. Similar phenomena are frequently seen in other antigens, such as R N P
and Sm antigens which are usually extracted in high
concentration from calf thymus nuclei, but in low concentration from rat liver nuclei. While some sera having
antibody activity to MU antigen reacted equally with
the cytoplasmic ribosomes, others produced a less intense precipitin line with the cytoplasmic ribosomes.
A most informative explanation on this subject is given
in Wang’s report (18), in which the nuclear ribosomes
differ from their cytoplasmic counterpart by containing
more antigenically active protein components.
The MU system bears some similarities to two
previous systems. One is the system described by Watanabe et al. (19) who reported 2 patients with SLE whose
sera contained antibodies reacting with nucleoli and
cytoplasm of mononuclear cells that were absorbed with
the cytoplasmic ribosomes. However, they have not defined the antigenicity relative to this system. The other
one to be differentiated from the MU system is the
cytoplasmic ribosomal system reported by Sturgill and
Carpenter (20) and Schur et al. (21). They found antibodies to cytoplasmic ribosomes in the sera of patients
with SLE. Although some of their sera might contain
antibodies to the MU antigen, they did not give any
attention to the relationship between nucleus and cytoplasm. In our observations the sera with antibodies to
cytoplasmic ribosomes always contained antibodies to
several known and/or unknown nuclear antigens which
were not restricted to the sera of patients with SLE. The
MU antigen has been identified as one of those nuclear
antigens in connection with the cytoplasmic ribosomes
and described as new in this report.
Although MU and T M antigens are closely related to the nucleolus, both are distinct from the previously reported nucleolar antigen ( 1 2,22). Because this
nucleolar antigen, which is a nucleolar specific RNA
with slow sedimentation coefficients of 4S-7S, is sensitive to the digestion only with RNase (12) and the antibody to this antigen does not react with the cytoplasmic
ribosomes, it is clear that MU and/or TM antigen is
different from the nucleolar specific antigen. The soluble
cytoplasmic antigens, Ro described by Clark ef al. (23)
and La by Mattioli and Reichlin (24), are distinguishable
from MU and TM antigens by a different cellular localization. Moreover, the relationship to the PM-1 antigen
described by Wolfe et al. ( 6 )is uncertain because we lack
the reference serum of the PM-I system. However, it can
be said only that our antigens are distinguishable from
MIYAWAKI ET AL
810
PM-1, firstly, by t h e different sensitivity t o the enzymes
a n d , secondly, by a different disease distribution.
Antibodies to M U antigen were positive in sera
of patients with D M , SLE, M C T D , a n d sicca syndrome,
and antibodies to T M antigen were present in SLE and
MCTD. There were no such o b v i o u s correlations between t h e presence of antibodies t o TM or M U antigen
and t h e clinical status of t h e patients, as exist between
t h e antibodies to Sm antigen and SLE ( l ) , or antibodies
t o R N P antigen a n d MCTD (3,9) as well as SLE (2,9).
Furthermore, the frequency of t h o s e antibodies in connective tissue diseases w a s by f a r lower t h a n t h a t of t h e
antibodies to RNP or Sm antigen. A t t h e present time
w e h a v e no information t h a t suggests t h a t t h e serological characteristics described here reflect different expressions of t h e s a m e basic disease process, nor whether
they reflect t h e expression of different pathogenic process. However, t h e differentiation and identification o f
uncharacterized individual antibodies t h a t react with t h e
extractable nuclear macromolecules h a v e been t h o u g h t
in t h e p a s t t o be of clinically characteristic v a l u e a n d
p e r h a p s t o offer a clue t o t h e pathogenesis of these
diseases.
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