Open Access

Frequency of disease-associated and other nuclear autoantibodies in patients of the German network for systemic scleroderma: correlation with characteristic clinical features

  • Rudolf Mierau1Email author,
  • Pia Moinzadeh2,
  • Gabriela Riemekasten3,
  • Inga Melchers4,
  • Michael Meurer5,
  • Frank Reichenberger6,
  • Michael Buslau7, 8,
  • Margitta Worm9,
  • Norbert Blank10,
  • Rüdiger Hein11,
  • Ulf Müller-Ladner12,
  • Annegret Kuhn13, 14,
  • Cord Sunderkötter14,
  • Aaron Juche15,
  • Christiane Pfeiffer16,
  • Christoph Fiehn17,
  • Michael Sticherling18, 25,
  • Percy Lehmann19,
  • Rudolf Stadler20,
  • Eckhard Schulze-Lohoff21, 26,
  • Cornelia Seitz22, 27,
  • Ivan Foeldvari23,
  • Thomas Krieg2,
  • Ekkehard Genth24 and
  • Nicolas Hunzelmann2
Arthritis Research & Therapy201113:R172

DOI: 10.1186/ar3495

Received: 21 July 2011

Accepted: 21 October 2011

Published: 21 October 2011

Abstract

Introduction

In the present study, we analysed in detail nuclear autoantibodies and their associations in systemic sclerosis (SSc) patients included in the German Network for Systemic Scleroderma Registry.

Methods

Sera of 863 patients were analysed according to a standardised protocol including immunofluorescence, immunoprecipitation, line immunoassay and immunodiffusion.

Results

Antinuclear antibodies (ANA) were detected in 94.2% of patients. In 81.6%, at least one of the autoantibodies highly associated with SSc or with overlap syndromes with scleroderma features was detected, that is, anti-centromere (35.9%) or anti-topoisomerase I (30.1%), followed in markedly lower frequency by antibodies to PM-Scl (4.9%), U1-ribonucleoprotein (U1-RNP) (4.8%), RNA polymerases (RNAPs) (3.8%), fibrillarin (1.4%), Ku (1.2%), aminoacyl-transfer RNA synthetases (0.5%), To (0.2%) and U11-RNP (0.1%). We found that the simultaneous presence of SSc-associated autoantibodies was rare (1.6%). Furthermore, additional autoantibodies were detected in 55.4% of the patients with SSc, of which anti-Ro/anti-La, anti-mitochondrial and anti-p25/p23 antibodies were most frequent. The coexistence of SSc-associated and other autoantibodies was common (43% of patients). SSc-associated autoantibodies disclosed characteristic associations with clinical features of patients, some of which were previously not acknowledged.

Conclusions

This study shows that five autoantigens (that is, centromere, topoisomerase I, PM-Scl, U1-RNP and RNAP) detected more than 95% of the known SSc-associated antibody responses in ANA-positive SSc patients and characterise around 79% of all SSc patients in a central European cohort. These data confirm and extend previous data underlining the central role of the determination of ANAs in defining the diagnosis, subset allocation and prognosis of SSc patients.

Keywords

systemic sclerosis scleroderma autoantibodies antinuclear antibodies

Introduction

Autoantibodies targeting characteristic nuclear antigens are one of the hallmarks of systemic sclerosis (SSc) [13]. The occurrence of different antinuclear antibodies (ANAs) is associated with distinct disease subtypes and with differences in disease severity, including extent of skin involvement, internal organ manifestation and prognosis. Although the current SSc criteria of the American College of Rheumatology [4] do not include the presence of ANAs, The detection of scleroderma-associated antibodies may be a valuable tool in the diagnosis of patients with very early SSc or only subtle symptoms [5, 6]. For instance, in a recent study of patients with Raynaud's phenomenon, the presence of ANAs (adjusted HR = 5.67) and SSc-associated antibodies (HR = 4.7) was the strongest independent predictor of definite SSc [6]. Some of the autoantibodies in SSc are regarded as disease-specific and can be correlated with genetic, demographic, diagnostic, clinical and prognostic aspects of the disease [1, 3]. Therefore, autoantibodies are pivotal tools in the diagnosis of SSc by helping clinicians make decisions whether to perform further, more detailed and efficient diagnostic procedures, as well as decisions addressing disease management.

For frequently occurring antibodies such as anti-centromere (ACA) and anti-topoisomerase I (ATA), reliable detection systems based on ELISA or other binding tests have been developed. Other antibodies (that is, to fibrillarin, RNA polymerases (RNAPs) and so on) are not identified by common test procedures, but rather by laboratories able to perform sophisticated procedures to confirm the results on the basis of more than one independent method. Even for the most common autoantibodies, the choice of the detection method used is critical to the sensitivity and specificity of the results and hence their diagnostic value [7].

Researchers in numerous studies have examined the presence of antibodies to single predefined antigens in SSc and their clinical associations, whereas many of the investigators who have comprehensively examined large SSc patient cohorts have often restricted their autoantibody analyses to the most common SSc antibodies, ACA and ATA [812], or analysed only a few additional antibodies [1317]. The aim of this study was therefore to characterise all known non-organ-specific, SSc-associated autoantibodies, as well as other, potentially new nuclear autoantibodies by using a standardised protocol in the large SSc patient cohort included in the German Network for Systemic Scleroderma Registry, and to correlate these findings with the clinical characteristics of these patients.

Materials and methods

Patients

Serum samples from 863 consecutive patients included in the German Network for Systemic Scleroderma Registry between 2004 and 2007 from 23 different clinical centres were analysed. Patient data are gathered and registered using a consensual registration form and reference documents with item definitions and recommendations for organ-specific diagnostic procedures as previously described [18, 19]. Of the patients included, 82.9% were female, their mean age ± SD was 58.0 ± 13.4 years (median = 60 years, range 12 to 93). The patients' age at disease onset ranged from 3 to 87 years (median = 49 years, mean = 47.7 ± 14.2).

The registry defines five subsets, that is, limited cutaneous and diffuse cutaneous SSc [20], overlap syndrome [21, 22], systemic sclerosis sine scleroderma [23, 24] and undifferentiated connective tissue disease with features of scleroderma [25, 26], as recently described [18]. The latter subset corresponds largely to the subgroup 'early SSc' as described by LeRoy and Medsger [5] but may also include patients who will never develop definite SSc. The study, including the patients' informed consent regarding data storage, was approved by the lead Ethics committee of the Cologne University Hospital and by the respective ethics committees of the contributing centres.

Autoantibody analysis

To detect SSc-associated autoantibodies in a comprehensive way, we used the search strategy commonly performed in diagnostic procedures for connective tissue diseases based on a HEp-2 cell immunofluorescence assay followed by tests using cellular extracts and/or recombinant antigens. This strategy is focused on circulating antibodies against non-organ-specific cellular autoantigens. Cell- or tissue-specific autoantibodies, which have also been described in scleroderma patients [3], were not included in the analytical protocol. At least one serum draw from each patient (N = 863) was analysed for circulating autoantibodies by a predefined protocol (Figure 1) with at least four assay systems performed in a single laboratory by a single group of technologists.
https://static-content.springer.com/image/art%3A10.1186%2Far3495/MediaObjects/13075_2011_Article_3451_Fig1_HTML.jpg
Figure 1

Protocol for serological analysis of systemic sclerosis patient sera.

Indirect immunofluorescence using fluorescein isothiocyanate-conjugated goat anti-human immunoglobulin G was performed as a screening method for the detection of ANAs on HEp-2 cells (HEp-20-10; Euroimmun, Lübeck, Germany) seeded onto a microscope slide [27]. Titres of at least 1:80 dilution were regarded as positive. Different nuclear and cytoplasmic fluorescence patterns were documented.

A line immunoassay (EUROLINE ANA Profile 3; Euroimmun) was performed according to the manufacturer's instructions. This assay is able to detect, by binding to recombinant or purified antigens, the following autoantibodies: U1-ribonucleoprotein (U1-RNP), Sm, Ro60, Ro52, La (SS-B), Scl-70, PM-Scl, centromere protein B (CENP-B), proliferating cell nuclear antigen (PCNA), double-stranded DNA (dsDNA), nucleosomes, histones, ribosomal P proteins and the mitochondrial M2 antigen.

Immunoprecipitation (IP) of radiolabelled HeLa cell extract was performed as described [28, 29] with slight modifications. In brief, HeLa S3 cells in suspension culture in methionine-deficient RPMI medium with 10% dialyzed foetal bovine serum were incubated with 35S methionine (to a final activity of 0.3 MBq/ml) overnight. They were (1) washed in Tris-buffered saline and (2) lysed by resuspension in IP buffer (10 mM Tris·HCl, pH 8.0, with 500 mM NaCl, 0.1% Igepal (Sigma, Munich, Germany) and 2 mM phenylmethylsulfonyl fluoride) and sonication on ice. IP was performed by incubation of patient sera (10 μl) with protein A Sepharose beads (2 mg in 500 μl; Sigma) for two hours, three short washing steps, end-over-end rotation with the radiolabelled cell extract overnight, five washing steps, separation of the precipitates on 5% to 20% gradient SDS-PAGE gels and subsequent autoradiography for six to ten days. Bands typical of autoantibodies were identified according to their apparent molecular weight and comigration with bands produced by reference sera with known autoantibody specificity. After we completed this procedure, the autoantibodies to the following antigens were routinely detectable: topoisomerase I (Scl-70), RNAPs (I and/or III), Ku, fibrillarin, To, NOR-90, Pl-7, Pl-12, EJ, OJ, KS, Mi-2, signal recognition particle, ribonucleoprotein (usually U1-RNP), SL, PCNA, ribosomal P proteins and p25/p23, also known as 'anti-chromo' [30, 31]. In our hands, the detection of antibodies to Ro, La, PM-Scl, Jo-1, centromere antigens and the mitochondrial M2 antigen was unreliable by this method. Bands of unknown nature were registered and entered into the database of autoantibody results.

Immunodiffusion (ID) was performed in Agarose gels with rabbit and/or calf thymus extract (Pel-Freez Biologicals, Rogers, AR, USA) as described previously [32]. Autoantibodies were identified by the identity of precipitation lines with patient sera compared with monospecific prototype sera with known autoantibody specificity. The use of prototype serum was guided by results of the immunofluorescence pattern on HEp-2 cells. The following autoantibodies were detectable: topoisomerase 1 (Scl-70), PM-Scl, Ku, SL, Jo-1, Pl-7, U1-RNP, Sm and La (SS-B). Precipitation bands that did not merge with any of the prototype sera were registered as unknown autoantibodies and entered into the database of autoantibody results.

In selected sera, confirmatory assays using recombinant or synthetic antigens (see Table 1 Antibody detection criteria) were performed. More than one serum sample was available from 213 patients (from 2 to 25 samples). At least one serum sample per patient was tested with the whole protocol described in Figure 1, whereas in most cases the follow-up sera drawn were at least partially characterised with, for example, the HEp-2 cell test. The criteria for classifying sera as positive for autoantibodies are listed in Table 1 together with additional serological results commonly found and helpful to identify the antibodies.
Table 1

Methodological criteria for assignment of autoantibodies

Autoantibody against

Findings classifying patients as antibody-positive

Usual additional findings

Centromere

Centromeric immunofluorescence pattern on HEp-2 cells (308 of 310 were positive) or a CENP-B band in line assay (309 of 310 positive)

 

Topoisomerase I

At least two of three findings: Scl-70-positive signal in line assay (258 of 260 positive), a band comigrating with a prototype band in IP (all positive), a line of identity in ID with the Scl-70 prototype serum (244 of 260 positive)

Typical HEp-2 cell immunofluorescence pattern: fine granular karyoplasmic, weakly nucleolar, metaphase chromosome-positive

RNA polymerases

Characteristic IP pattern comigrating with the pattern of a prototype serum mainly consisting of four bands: Ia, Ib, IIIa and IIIb [58]

Confirmation by ELISA in 32 of 32 cases with the immunodominant epitope of RNA polymerase III subunit RPC155 according to Kuwana et al. [71], provided by Matritec, Freiburg, Germany. ANA immunofluorescence on HEp-2 cells was predominantly fine granular only sometimes (five cases) in addition nucleolar [72].

Fibrillarin

An IP band (approximately 34 kDa) comigrating with a prototype serum band, plus a nucleolar immunofluorescence pattern on HEp-2 cells

Confirmation by investigational ELISA kindly provided by Euroimmun, Lübeck, Germany, positive in 11 and borderline in 1 of the 12 cases

To

An IP band of approximately 40 kDa plus a nucleolar immunofluorescence pattern on HEp-2 cells; confirmation by immunoblot analysis with recombinant To antigen kindly supplied by Dr M Blüthner, Labor Seelig, Karlsruhe, Germany

 

PM-Scl

A line of identity in ID with a PM-Scl prototype serum (41 of 42 cases) and/or positive result of ELISA with the synthetic peptide PM-1α [73] (Dr Fooke Laboratorien GmbH, Neuss, Germany) (12 of 13 cases)

Positive reaction in 37 of 41 cases for PM-Scl by line assay. ANA immunofluorescence on HEp-2 cells usually was nucleolar plus fine granular karyoplasmic.

Ku

Two prominent IP bands at about 70 and 80 kDa comigrating with prototype bands

In 3 of 10 cases, a line identical to a Ku prototype band in ID. ANA immunofluorescence was finely granular, usually at a high titre.

U1-RNP

A positive signal for RNP/Sm in line assay, with or without a positive signal for Sm, plus a typical IP pattern consisting of at least antigen A (about 33 kDa), antigen B/B' (about 28/29 kDa) and antigen C (about 22 kDa)

In 37 of 41 cases, a line of identity with a U1-RNP prototype in ID. ANA pattern on HEp-2 cells usually was coarsely speckled.

Sm

A positive signal for RNP/Sm as well as for Sm in line assay

In two of four cases, a band identical to a Sm prototype in ID with ribonuclease-digested calf thymus extract. IP and immunofluorescence patterns were similar to those found for anti-U1-RNP.

Jo-1

A positive signal for Jo-1 in line assay plus a band identical to a Jo-1 prototype band in ID

Immunofluorescence on HEp-2 cells was inconsistent.

Pl-7

IP band of about 80 kDa comigrating with prototype band plus a band identical to a Pl-7 prototype band in ID

Cytoplasmic immunofluorescence on HEp-2 cells

OJ

Typical triplet band in IP comigrating with prototype bands

Cytoplasmic immunofluorescence on HEp-2 cells

U11-RNP [74, 75]

An RNP-like IP pattern and coarsely speckled ANA immunofluorescence, without any U1-RNP signals in line assay and ID; U11-RNP specificity detected by C Will and R Lührmann, Marburg, Germany

 

p25/p23 [76, 77]

Doublet IP bands of about 25 and 23 kDa, with the 25 kDa band comigrating with the precipitate of rabbit anti-p25 kindly provided by E Chan, Gainesville, FL, USA

HEp-2 cell immunofluorescence pattern was always centromeric because anti-p25/p23 was exclusively found together with anticentromere.

SL

A band identical to the SL prototype band in ID plus an IP band at about 31 kDa comigrating with the precipitate of the SL reference serum

HEp-2 cell immunofluorescence pattern was fine granular, but in this study often was masked because of other coexisting antibodies

NOR-90

Doublet IP bands at about 90 kDa comigrating with the precipitate of a NOR-90 reference serum [78]

The nucleolar immunofluorescence pattern expected on HEp-2 cells was hard to detect in the sera examined in this study, because NOR-90 antibodies in all cases coincided with other autoantibodies visible on HEp-2 cells.

Mitochondrial M2

AMA M2-positive signal by line assay (40 of 41 positive) and/or AMA typical cytoplasmic immunofluorescence on HEp-2 cells and/or rat kidney sections (27 of 41 positive)

In IP, a band of around 70 kDa was present in 36 of 41 cases.

Sp100

Multiple nuclear dot pattern on HEp-2 cells [79] plus Sp100 signal in the line assay HUMAN IMTEC-Liver Line Immunoassay (HUMAN Diagnostics GmbH, Wiesbaden, Germany)

 

Ro52

Ro52-positive signal by line assay

 

Ro60

Ro60-positive signal by line assay

 

La

La-positive signal by line assay

 

AMA = antimitochondrial antibodies; ANA = antinuclear antibodies; CENP-B = centromere protein B; ID = immunodiffusion; IP = immunoprecipitation; PM-Scl = polymyositis and scleroderma; RNAP = RNA polymerase; RNP = ribonucleoprotein;.

In addition to the autoantibodies defined in Table 1 other circulating autoantibodies detected by at least one of the above-mentioned procedures, either known (for example, anti-histone, anti-dsDNA) or unknown (for example, either unidentified bands in IP or ID or antinuclear or anticytoplasmic antibodies on HEp-2 cells without subsequent identification), were registered. Sera which were negative for ANAs in immunofluorescence on HEp-2 cells but exhibited cytoplasmic fluorescence in that assay and/or a positive signal in any of the other assays were grouped together as ANA-negative. Sera without any positive signal, neither defined nor undefined, in all four assay systems described above were listed as autoantibody-negative.

Statistics

The data were analysed using Microsoft Excel (Microsoft Corp, Redmond, WA, USA) and SPSS version 14.0 software (SPSS, Inc, Chicago, IL, USA) for tabular and graphic representation. Statistical evaluation was performed using contingency table tests with the help of GraphPad Prism version 3.02 software (GraphPad, La Jolla, CA, USA). We calculated OR and 95% CI data. P-values were calculated using Fisher's exact test. When multiple tests were performed, P-values below 0.005 were recorded without performing strict Bonferroni correction. For most variables, less than 5% of data were missing. Quantitative data (erythrocyte sedimentation rate (ESR), age at disease onset and Rodnan skin score), depending on the presence or absence of different autoantibodies, were analysed using the Mann-Whitney rank-sum test.

Results and discussion

Of the 863 SSc patients studied, 513 (59.4%) were classified as having limited disease and 173 (20.1%) we classified as having diffuse cutaneous disease. Another 108 patients (12.5%) had a scleroderma overlap syndrome, 64 (7.4%) had undifferentiated connective tissue disease with scleroderma features and 5 (0.6%) had systemic sclerosis sine scleroderma.

The frequency of autoantibodies detected in these patients is shown in Table 2. Overall, ANAs were detected in 94.2% of patients. This frequency was similar to data previously published [12, 14, 15, 33, 34] in which ANA frequencies reported were between 85% and 99%. Among our patients with ANAs, 86.6% (704 of 813) had autoantibodies known to be highly associated with SSc, and among these latter patients, 96.4% (679 of 704) had antibodies that detected five antigens: centromere, topoisomerase I, PM-Scl, U1-RNP and RNAPs.
Table 2

Prevalence of autoantibodies in 863 scleroderma patients

Autoantibodies

Patients, n(%)

Positive for antinuclear antibodies

813 (94.2)

Antibodies highly associated with SSc or scleroderma overlap syndromes

704 (81.6)

   Anti-centromere

310 (35.9)

   Anti-topoisomerase I

260 (30.1)

   Anti-PM-Scl

42 (4.9)

   Anti-U1-RNP

41 (4.8)

   Anti-RNA polymerase

33 (3.8)

   Anti-fibrillarin

12 (1.4)

   Anti-To

2 (0.2)

   Anti-Ku

10 (1.2)

   Anti-Jo-1/-Pl-7/-OJ

4 (0.5)

   Anti-U11-RNP

1 (0.1)

Other autoantibodies

 

   Anti-Ro and/or anti-La

206 (23.9)

Anti-Ro52

187 (21.7)

Anti-Ro60

59 (6.8)

Anti-La

16 (1.9)

   Anti-mitochondrial M2

41 (4.8)

   Anti-p25/p23

28 (3.2)

   Anti-NOR-90

6 (0.7)

   Anti-SL

9 (1.0)

   Anti-Sm

4 (0.5)

   Anti-Sp100

4 (0.5)

   Other (known or unknown)

363 (42.1)

Negative for all highly SSc-associated antibodies

159 (18.4)

Negative for antinuclear antibodies

50 (5.8)

Autoantibody-negative by all criteria used

38 (4.4)

ANA = antinuclear antibodies; SSc = systemic sclerosis.

A coincidence of SSc-associated autoantibodies (Table 3) was rare, being detected in only 1.6% patients (11 of 704). The presence of a SSc-associated antibody without any other autoantibody detectable by the methods used was found at varying frequencies, being highest for anti-PM-Scl (73.8%) and lower for, for example, anti-centromere (33.9%) and anti-fibrillarin (33.3%) (see Table 3). That SSc-associated autoantibodies are largely mutually exclusive is well-known [14, 15, 35]. Coincidences in individual patients do occur but are rare [33, 36, 37]. Our study shows that this statement holds true even if all known non-organ-specific, SSc-associated autoantibodies are sought using a rigorous protocol in all patients. On the other hand, additional (mainly not SSc-specific) autoantibodies are common and were detected in about 53% in our patient cohort with SSc-associated antibodies (and in 55.4% of all of our patients). In 14.0% of patients (n = 121), none of the above-mentioned SSc-associated but other (defined or undefined) autoantibodies were found, whereas in 4.4% no autoantibodies at all were detected by the methods used. Defined autoantibodies not regarded as SSc-specific, such as anti-Ro/La, anti-NOR-90 or AMAs rarely occurred without the evidence of SSc-associated autoantibodies (Table 3). Antibodies to p25/p23 were detected exclusively in conjunction with ACA.
Table 3

Coincidence* of autoantibodies in 863 individual systemic sclerosis patients

Antibodies

ACA

ATA

Anti-PM-Scl

Anti-U1-RNP

Anti-RNAP

Anti-fibrilla-rin

Anti-To

Anti-Ku

Anti-Jo-1/Pl-7/OJ

Anti-U11-RNP

Anti-Ro52

Anti-Ro60

Anti-La

AMA

Anti-p25/p23

Anti-NOR-90

Anti-SL

Anti-Sm

Anti-Sp100

Other

ACA

 

1

0

1

0

0

0

0

0

0

92

11

1

31

28

4

2

0

3

144

ATA

  

1

2

0

0

0

4

0

0

36

26

6

4

0

1

4

1

0

77

Anti-PM-Scl

   

0

0

0

0

1

0

0

6

2

0

0

0

0

0

0

0

4

Anti-U1-RNP

    

1

0

0

0

0

0

11

6

1

2

0

1

0

4

0

11

Anti-RNAP

     

0

0

0

0

0

5

1

1

1

0

0

1

0

0

5

Anti-fibrillarin

      

0

0

0

0

0

0

0

1

0

0

0

0

0

8

Anti-To

       

0

0

0

1

1

0

0

0

0

0

0

0

2

Anti-Ku

        

0

0

2

2

0

0

0

0

1

0

0

2

Anti-Jo-1/Pl-7/OJ

         

0

3

0

0

0

0

0

0

0

0

1

Anti-U11-RNP

          

0

0

0

0

0

0

0

0

0

0

Anti-Ro52

           

41

15

14

11

2

5

2

2

97

Anti-Ro60

            

14

6

0

0

0

1

1

33

Anti-La

             

1

0

0

0

1

0

8

AMA

              

2

0

1

1

2

29

Anti-p25/p23

               

1

1

0

1

18

Anti-NOR-90

                

0

1

0

4

Anti-SL

                 

0

0

3

Anti-Sm

                  

0

0

Anti-Sp100

                   

2

Other

                    

Isolateda

104

153

31

19

23

4

0

4

1

1

2

1

0

0

0

0

1

0

0

79

Totalb

310

260

42

41

33

12

2

10

4

1

187

59

16

41

28

6

9

4

4

363

*Number of patients with co-occurrences of autoantibodies. aIsolated: presence without coincidence of any other autoantibody by all detection methods used. bTotal number of individuals with the respective autoantibody; these numbers mostly are smaller than the sum of all co-occurrences plus the 'isolated' individuals listed because of some triple, quadruple and higher-order coincidences. ACA = anti-centromere antibodies; AMA = antimitochondrial antibodies; ATA = anti-topoisomerase I antibodies;; RNAP = RNA polymerase; RNP = ribonucleoprotein.

From 213 patients, more than one serum sample was available (from 2 to 25 samples). In the majority (86.4%) of cases, the results of follow-up testing remained essentially the same and differed in ANA titre by up to only two titre steps. In 11.3% (24 patients), ANA titre changes exceeded two steps (up to eight steps), in two cases ANAs turned negative, in one case the ANA pattern changed from finely granular to nucleolar and in only two cases new, additional typical SSc autoantibodies emerged.

The detection of antibodies in different disease subsets is shown in Table 4. It is obvious that ACA and ATA are not exclusive to either the limited or the diffuse subset. In patients with overlap syndrome, anti-U1-RNP, anti-PM-Scl and anti-synthetase antibodies are characteristic.
Table 4

Autoantibodies in different disease subsets in 863 individual systemic sclerosis patients

 

Limited (N= 513)

Diffuse (N= 173)

Overlap (N= 108)

Undifferentiated (N= 64)

Antibodies

n(%)

ORa (P-value)

n(%)

OR (P-value)

n(%)

OR (P-value)

n(%)

ACA

253 (49.3)

5.00 ( P < 0.0001)

12 (6.9)

 

16 (14.8)

 

28 (43.8)

ATA

141 (27.5)

 

98 (56.6)

4.26 ( P < 0.0001)

11 (10.2)

 

9 (14.1)

Anti-RNAP

14 (2.7)

 

14 (8.1)

3.11 ( P = 0.0029)

2 (1.9)

 

2 (3.1)

Anti-U1-RNP

7 (1.4)

 

0 (0.0)

 

31 (28.7)

30.00 ( P < 0.0001)

2 (3.1)

Anti-PM-Scl

16 (3.1)

 

2 (1.2)

 

22 (20.4)

9.40 ( P < 0.0001)

2 (3.1)

Anti-fibrillarin

3 (0.6)

 

8 (4.6)

8.32 ( P = 0.0005)

1 (0.9)

 

0 (0.0)

Anti-To

1 (0.2)

 

0 (0)

 

1 (0.9)

 

0 (0.0)

Anti-Jo-1/Pl-7/OJ

0 (0.0)

 

0 (0)

 

4 (3.7)

65.07 ( P = 0.0002)

0 (0.0)

Anti-U11-RNP

0 (0.0)

 

1 (0.6)

 

0 (0.0)

 

0 (0.0)

Anti-Ku

5 (1.0)

 

1 (0.6)

 

3 (2.8)

 

1 (1.6)

Anti-SL

5 (1.0)

 

3 (1.7)

 

1 (0.9)

 

0 (0.0)

Anti-Sm

0 (0.0)

 

0 (0.0)

 

3 (2.8)

21.54 (P = 0.0069)

0 (0.0)

Anti-NOR-90

5 (1.0)

 

0 (0.0)

 

1 (0.9)

 

0 (0.0)

AMA

28 (5.5)

 

4 (2.3)

 

4 (3.7)

 

5 (7.8)

Anti-Sp100

3 (0.6)

 

0 (0.0)

 

0 (0.0)

 

1 (1.6)

Anti-Ro52

125 (24.4)

1.50 (P = 0.023)

20 (11.6)

 

27 (25.0)

 

15 (23.4)

Anti-Ro60

28 (5.4)

 

14 (8.1)

 

13 (12.0)

2.11 (P = 0.0382)

4 (6.3)

Anti-La

9 (1.8)

 

3 (1.7)

 

1 (0.9)

 

3 (4.7)

Anti-p25/p23

24 (4.5)

4.25 ( P = 0.0031)

0 (0.0)

 

2 (1.9)

 

2 (3.1)

Other

209 (40.7)

 

74 (42.8)

 

44 (40.7)

 

34 (53.1)

ANA-negative

32 (6.2)

 

6 (3.5)

 

5 (4.6)

 

7 (10.9)

ACA, anti-centromere antibodies; AMA, antimitochondrial antibodies; ANA, antinuclear antibodies; ATA, anti-topoisomerase I antibodies; RNAP, RNA polymerase;. aOR for antibody in that subset compared with all other subsets. Only significant positive associations are documented by OR, and those with P-values < 0.005 are printed in bold.

The correlation of demographic features or signs and symptoms of SSc with the presence or absence of defined autoantibodies was investigated by contingency table analysis. The frequencies of these features and their positive or negative correlations with specific autoantibodies are listed in Table 5. For the purpose of clarity, only those comparisons that led to P-values below 0.05 derived by Fisher's exact test are shown. Significance was calculated without correction of P-values for multiple comparisons, because not all variables used were independent; however, we are aware of the fact that some of the weak associations listed in Table 5 might have arisen by chance due to the high number of comparisons made. Therefore, we focused on those differences calculated that were highly significant (P < 0.005; OR printed in bold in Table 5).
Table 5

Correlations of clinical features with SSc associated autoantibodies

 

ACA

(310)

ATA

(260)

Anti-PM-Scl

(42)

Anti-U1-RNP

(41)

Anti-RNA -P

(33)

Anti-Fibrillarin

(12)

Anti-Ku

(10)

Anti-Ro52

(187)

Anti-Ro60

(59)

Anti-La

(16)

AMA

(41)

Anti-p25/23

(28)

ANA-negative

(50)

no SSc associated ab's

(161)

male sex

148 (17.1%)

17

0.19 (0.11 to 0.32)

P < 0.0001

64

2.02 (1.4 to 2.91)

p = 0.0002

6

9

8

4

2

23

0.62 (0.38 to 0.997

p = 0.049

12

2

1

1

10

41

1.90 (1.261 to 2.863)

p = 0,0035

age at disease onset < 50 y

399/781 (51.1%)

115

0.57 (0.43 to 0.77)

p = 0.0003

135

1.39 (1.02 to 1.88)

p = 0.0431

22

29

2.92 (1.40 to 6.07)

p = 0.0029

13

8

4

77

31

10

20

8

18

73

Rodnan skin score > 10

294/750 (39.2%)

58

0.27 (0.19 to 0.38)

P < 0.0001

132

3.10 (2.24 to 4.27)

P < 0.0001

14

9

18

3.24 (1.44 to 7.31)

p = 0.0042

7

1

58

23

6

10

11

14

56

Raynaud's phenomenon

819 (94.9%)

301

2.26 (1.07 to 4.77)

p = 0.0349

252

37

41

32

12

10

184

3.96 (1.2 to 12.94

p = 0.0133

58

16

41

28

43

0.29 (0.12 to 0.70)

p = 0.0104

140

0.23 (0.12 to 0.42)

P < 0.0001

Digital ulcers

216/840 (25.7%)

55

0.50 (0.36 to 0.71)

P < 0.0001

106

3.18 (2.30 to 4.41)

P < 0.0001

9

11

7

4

1

48

19

6

13

6

4

0.26 (0.09 to 0.76)

p = 0.0076

28

0.60 (0.39 to 0.94)

p = 0.024

Pulmonary hypertension

126 (14.6%)

57

1.58 (1.08 to 2.32)

p = 0.0208

36

2

9

4

1

0

31

14

2

2

7

2

0.23 (0.06 to 0.97)

p = 0.0232

17

Pulmonary fibrosis

287 (33.3%)

39

0.18 (0.12 to 0.26)

P < 0.0001

151

4.76 (3.48 to 6.50)

P < 0.0001

16

11

7

2

6

69

30

2.20 (1.29 to 3.75

p = 0.0040

9

6

0.33 (0.14 to 0.79)

p = 0.01

4

0.33 (0.11 to 0.95)

p = 0.0393

11

55

Lung restrictive defect

218/833 (26.2%)

41

0.31 (0.21 to 0.45)

P < 0.0001

104

2.96 (2.14 to 4.09)

P < 0.0001

9

7

8

3

4

45

19

4

8

4

10

34

Esophageal involvement

535 (62.0%)

198

175

1.39 (1.02 to 1.89)

p = 0.039

14

0.29 (0.152 to 0.56)

p = 0.0001

29

20

7

6

120

39

13

24

21

27

87

0.67 (0.47 to 0.94)

p = 0.0243

Proteinuria

90/830 (10.8%)

23

0.56 (0.34 to 0.92)

p = 0.0207

34

4

4

4

1

1

15

9

2

3

1

5

19

Cardiac involvement

114 (13.2%)

27

0.51 (0.32 to 0.81)

p = 0.0033

45

1.62 (1.08 to 2.44)

p = 0.0216

5

7

5

1

2

20

9

1

4

1

5

22

Musculoskeletal involvement

421/852 (49.4%)

131

0.64 (0.48 to 0.85)

p = 0.0022

130

20

28

2.49 (1.25 to 4.96)

p = 0.009

17

10

5.22 (1.14 to 23.97)

p = 0.0202

6

80

0.71 (0.51 to 0.99)

p = 0.0468

32

12

3.13 (1.002 to 9.79)

p = 0.0447

17

9

25

79

Synovitis

157/837 (18.8%)

35

0.43 (0.29 to 0.65)

P < 0.0001

62

1.71 (1.19 to 2.45)

p = 0.0049

8

13

2.27 (1.14 to 4.53)

p = 0.0326

8

2

2

32

15

5

2

0.21 (0.05 to 0.89)

p = 0.0216

2

10

29

Joint contractures

253/840 (30.1%)

54

0.36 (0.25 to 0.50)

P < 0.0001

107

2.26 (1.65 to 3.08)

P < 0.0001

9

9

10

6

2

49

23

6

9

9

20

1.93 (1.05 to 3.54)

p = 0.0436

56

Tendon friction rubs

88/840 (10.5%)

14

0.30 (0.16 to 0.53)

P < 0.0001

32

2

5

6

3

1

21

5

2

2

0

6

25

1.95 (1.18 to 3.22)

p = 0.0122

CK elevation

74/835 (8.9%)

14

0.37 (0.21 to 0.68)

p = 0.0009

20

19

3.56 (1.67 to 7.57)

p = 0.0023

5

6

2.60 (1.03 to 6.55)

p = 0.0485

0

3

5.32 (1.30 to 21.72)

p = 0.038

21

8

2

1

0

6

16

Sicca syndrome

366/858 (42.7%)

150

1.44 (1.19 to 6.61)

p = 0.0119

98

14

18

12

5

4

96

1.57 (1.13 to 2.17)

p = 0.0075

33

1.85 (1.08 to 3.17)

p = 0.0275

12

4.14 (1.32 to 12.93)

p = 0.0102

19

20

3.50 (1.52 to 8.03)

p = 0.0029

23

66

Mouth involvement

223/829 (26.9%)

64

0.61 (0.44 to 0.85)

p = 0.0034

93

2.1 (1.53 to 2.93)

P < 0.0001

10

7

10

3

4

49

18

6

11

6

12

34

ESR > 25 mm/h

199/741 (26.9%)

58

0.70 (0.49 to 0.99)

p = 0.046

75

1.55 (1.10 to 2.19)

p = 0.015

4

0.33 (0.11 to 0.94)

p = 0.0325

14

5

0

0

60

1.76 (1.21 to 2.54)

p = 0.0039

28

3.39 (1.92 to 5.97)

P < 0.0001

9

3.62 (1.33 to 9.86)

p = 0.0447

10

5

15

42

ACA, anti-centromere antibodies; AMA, antimitochondrial antibodies; ANA, antinuclear antibodies; ATA, anti-topoisomerase I antibodies; CK, creatine kinase; ESR, erythrocyte sedimentation rate; RNAP = RNA polymerase; RNP = ribonucleoprotein; SSc, systemic sclerosis. Dichotomous variables are expressed as raw numbers, OR (95% CI) and P values.

Patients with ACA represented 35.9% of all SSc patients and 38.1% of ANA-positive SSc patients. In accordance with previous reports [8, 9, 12, 15, 33, 36, 38, 39], these patients were less often male, were older at disease onset and had a more limited extension of cutaneous involvement, as documented by a much lower OR for a Rodnan skin score (RSS) above 10 (Table 5) and by a very significantly lower mean RSS (Table 6). They had less involvement of internal organs (pulmonary fibrosis, cardiac, musculoskeletal and oral involvement), with the exception of pulmonary hypertension. An association of ACA with pulmonary hypertension has been observed in several previous reports [2, 12, 33, 40], but not all of them [13, 36, 38, 39]. Digital ulcers in our patients with ACA were less common compared to American patients [2] and more similar to European [9, 12, 38] and Japanese patients [34]. Nevertheless, the presence of ACA does not by any means preclude digital ulcers. To date no marker constellation allows the identification of patients prone to this complication [4143]. ACAs are frequently associated with other antibodies, such as anti-Ro [4446], anti-mitochondrial (M2) [15, 44, 47, 48] and anti-p25/p23 [31, 49, 50] antibodies. These associations were confirmed by this study. The reasons for this frequent co-occurrence are unknown. There is no known antigenic relationship between the individual targets of the antibodies. Probably the (unknown) aetiopathogenetic pathways marked by these antibodies have common components, including common genetic predispositions.
Table 6

Correlations of clinical features with systemic sclerosis-associated autoantibodies

Quantitative traits

Clinical data

Age at disease onset

n

Mean ± SD (years)

P*

   Total

781

47.7 (14.2)

 

   Anti-centromere

273

51.3 (12.5)

< 0.0001

   Anti-topoisomerase I

238

46.0 (14.0)

0.0076

   Anti-fibrillarin

12

38.8 (16.0)

0.0404

   Anti-U1-RNP

39

38.2 (15.0)

< 0.0001

   Anti-La

15

37.9 (18.1)

0.0431

   Autoantibody-negative

36

52.9 (14.7)

0.0205

Rodnan skin score

n

Mean score ± SD

P

   Total

750

10.2 (9.4)

 

   Anti-centromere

275

6.4 (6.0)

< 0.0001

   Anti-topoisomerase I

227

14.1 (9.7)

< 0.0001

   Anti-RNA polymerase

27

15.7 (11.7)

0.0091

   Anti-fibrillarin

10

21.2 (15.0)

0.0108

   Anti-U1-RNP

35

6.9 (9.2)

0.0053

Erythrocyte sedimentation rate

n

Mean ± SD (mm/hour)

P

   Total

741

19.46 (16.7)

 

   Anti-topoisomerase I

227

22.95 (19.1)

0.0002

   Anti-PM-Scl

36

12.19 (9.4)

0.0014

   Anti-Ro52

167

22.05 (18.8)

0.0374

   Anti-Ro60

53

28.47 (19.6)

< 0.0001

   Anti-La

16

28.56 (20.1)

0.0447

PM-Scl = polymyositis and scleroderma; RNP = ribonucleoprotein; SSc, systemic sclerosis. *P-value calculated by Mann-Whitney rank-sum test for comparison of antibody-positive vs antibody-negative patients.

The prevalence of ATA in our cohort (30.1%) is in line with the numbers published by others, which have varied between 13% and 36% [2, 8, 9, 12, 14, 15, 17, 33, 34, 36, 51]. The patients with ATA in our study were more likely to be male and had higher RSSs (Tables 5 and 6). More common in this patient group were digital ulcers, pulmonary fibrosis, dyspnoea, lung restrictive defect and joint involvement (synovitis, contractures) as well as mouth involvement. However, renal involvement, as measured by proteinuria or renal insufficiency, was not more prevalent in this subgroup, a finding reported by most other researchers [8, 9, 14, 15, 33, 36, 51, 52] but not all of them [2, 34].

The frequency of RNAP antibodies in our cohort was 3.8%, which is at the lower end of the frequency range reported by others, namely, 10% to 25% in North America [33, 5255], 4% to 31.5% in Europe [14, 16, 33, 35, 5659] and 5% to 11% in Japan [34, 36, 60]. This may have several reasons: (1) Our cohort is composed of a broad spectrum of SSc patients, including patients with milder forms and with overlap or undifferentiated subtypes of the disease, (2) regional differences due to genetic background and/or environmental influences and (3) different techniques used to ascertain the presence of RNAP antibodies. A high mean RSS, reflecting diffuse skin involvement, was evident for patients with anti-RNAP antibodies (Tables 5 and 6) as previously observed [2, 14, 33, 34, 36, 40, 5460]. In addition, we found creatine kinase (CK) elevation to be more frequently associated with the presence of anti-RNAP antibodies. This has not been noted before; association with muscular involvement has been found to be nonsignificant [14, 34, 40, 53, 54] or even inverse [2, 36, 55] in previous publications. Our result in this study therefore might be a chance finding due to multiple comparisons. We did not find any significant positive association of RNAP antibodies, or any autoantibody evaluated in this study, with renal involvement. In the German Network for Systemic Scleroderma Registry, 'renal involvement' is defined as renal insufficiency in the form of decreased creatinine clearance and/or proteinuria, as well as a consequence of acute renal crisis. The registry did not include renal crisis as a separate item at that time, which may underestimate the possible correlation of antibodies with renal crisis [18]. Within our network, the frequency of renal crisis currently does not exceed 2% to 3% per year (Hunzelmann N, unpublished observation). The prevalence of renal crisis among patients with RNAP antibodies reported in the literature (for review, see Meyer et al. [57]) varies considerably, between 0% and 43%.

Antibodies to fibrillarin (U3-RNP) were most prominent in patients with the diffuse subtype (table 4), which is in accordance with the findings published in previous reports [2, 13, 34, 40, 61]. In fact, patients with anti-fibrillarin antibodies, on average, had the highest RSS of all patients in our cohort (Tables 5 and 6). The significance of this finding, however, is less pronounced because of the lower number of patients. The detected anti-fibrillarin antibody frequency of 1.4% was considerably lower than that reported in previous cohorts (2.5% to 19%) [2, 13, 34, 36, 40, 6163], which may reflect our broad spectrum of SSc patients that included patients with overlap syndrome and undifferentiated forms, as well as methodological differences and/or the central European background of the patients. Ethnic heterogeneity with a higher frequency of anti-fibrillarin antibodies in black patients has been described previously [2, 13, 40, 6163]. Our findings of a lower age at disease onset and a higher prevalence of musculoskeletal involvement (Tables 5 and 6) are in line with most previous results [2, 36, 61, 63].

The frequency of PM-Scl antibodies (4.9%) detected in our cohort is in accord with previously published studies in which frequencies between 2% and 6% were noted for SSc patients [13, 17, 33]. These antibodies are most well-known as being typical in patients with dermatomyositis-scleroderma overlap syndrome [1, 3]. Accordingly, CK elevation was highly associated with anti-PM-Scl in our cohort. On the other hand, these patients were markedly less likely to have oesophageal involvement (Table 5) and had a low mean ESR (Tables 5 and 6). We found elevated ESR levels in an earlier series of SSc patients with anti-PM-Scl antibodies [32], but others, to the best of our knowledge, did not. (Patients with anti-PM-Scl have rarely been analysed in the large SSc series reported previously.) Therefore, this finding has to be reproduced by independent work in the future. That these patients have a relatively benign prognosis has been mentioned several times before [6466]. Accordingly, 31% of our patients with anti-PM-Scl antibodies were devoid of any internal organ involvement, compared with 13% of the patients without anti-PM-Scl (P = 0.0023, data not shown).

Antibodies to Ku, when found in SSc patients, are often associated with scleroderma overlap syndrome and with muscular involvement [17, 36]. Accordingly, we registered a high OR, but with low significance because of the relatively low patient number, for CK elevation associated with anti-Ku antibody (Table 5). The complete absence of ACA and the occasional presence of ATA described in a large previous study that focused on anti-Ku in patients with SSc [17] were nicely reproduced in our cohort (Table 3). Most of our anti-Ku sera (7 of 10) were positive only by IP and negative in a 'classical' precipitation test with native antigen. In a previous study, on the contrary, a similar test (counterimmunoelectrophoresis) was even more sensitive than a line assay to anti-Ku. A possible explanation for this discrepancy might be the source of the antigen, which was of rabbit origin, in our ID assay. Ku autoantibodies are known to tend to be nonreactive with nonhuman antigens [67].

Antibodies to p25/p23 ('anti-chromo') characterise a patient subset within the group of ACA-positive SSc patients. The clinical findings among these patients were heterogeneous in previous reports. Soriano et al. [49] found an elevated prevalence of erosive arthritis and Furuta et al. [50] reported more interstitial lung disease and liver involvement, whereas Japanese groups [30, 31, 68] discovered cytopenias, Sjögren's syndrome, overlap with systemic lupus erythematosus and higher ESR levels. We confirmed the relatively strong association with Sjögren's syndrome on the basis of our finding that 20 (71.4%) of 28 patients with p25/p23 antibodies had sicca symptoms, compared to only 41.7% of SSc patients who were negative for these autoantibodies (Table 5). In fact, the weak association of ACA with sicca symptoms (OR = 1.44) (Table 5) lost significance when those patients with co-occurring anti-p25/p23 were eliminated. Likewise, the low mean RSS calculated for ACA-positive patients (6.4) (see Table 6) turned even lower (6.2) after exclusion of patients with anti-p25/p23.

Antibodies to Ro and/or La, as expected and as previously reported [44, 45, 69, 70], were associated with sicca syndrome. This association was only marginally significant; in fact, the antibodies with the most prominent association with the sicca complex were, as mentioned above, anti-p25/p23 antibodies. Anti-Ro and/or anti-La antibodies showed a particularly high correlation with elevated ESR (Tables 5 and 6). An unexpectedly strong association of anti-Ro60 with pulmonary fibrosis (Table 5) was mainly secondary to the relatively high co-occurrence of this antibody with ATA (see Table 3).

No highly significant differences for any of the autoantibody-defined subgroups could be found for gastric, intestinal or renal involvement, including hypertension and reduced renal function. Furthermore, no significant differences dependent on antibodies against aminoacyl-transfer RNA synthetases, To, Sm, SL or NOR-90 were detected, probably because of the low numbers of patients positive for these antibodies. Patients without highly SSc-associated antibodies were more often male and less frequently had Raynaud's phenomenon.

Conclusions

The occurrence of SSc-related autoantibodies has never been analysed in such detail in a cohort as large as this one. We have shown that five antigens appear to be sufficient to detect more than 95% of the known SSc-associated autoantibody responses in ANA-positive SSc patients. In more than half of patients with a SSc-associated antibody, other nuclear autoantibodies were detected and a considerable patient group (around 40%) still displayed uncharacterised ANAs of as yet unknown significance.

To the best of our knowledge, this is the largest comprehensive analysis of the presence of SSc-associated as well as other non-organ-specific autoantibodies in SSc patients that was performed in a single central laboratory and demonstrates the complexity and heterogeneity of the autoimmune response underlying the pathogenesis of this still enigmatic disease.

Abbreviations

ACA: 

anti-centromere antibody

AMA: 

antimitochondrial antibody

ANA: 

antinuclear antibody

ATA: 

anti-topoisomerase I antibody

CK: 

creatine kinase

ELISA: 

enzyme-linked immunosorbent assay

ESR: 

erythrocyte sedimentation rate

HR: 

hazard ratio

ID: 

immunodiffusion

IP: 

immunoprecipitation

OR: 

odds ratio

RNAP: 

RNA polymerase

RSS: 

Rodnan skin score

SSc: 

systemic sclerosis.

Declarations

Acknowledgements

The expert technical assistance of Marie-Claire Vondegracht and Rita Bernstein is gratefully acknowledged. Furthermore, we are indebted to Dr M Blüthner, Laboratory Prof Seelig, Karlsruhe, Germany, for confirming the anti-To antibodies; to Dr C Will and Professor R Lührmann, Marburg, Germany, for identification of the anti-U11 RNP antibodies; and to Dr EK Chan, Gainesville, FL, USA; Prof B Liedvogel, Diarect, Freiburg, Germany; and Dr W Schlumberger, Euroimmun, Lübeck, Germany, for supplying rabbit anti-sera to p25/p23, recombinant PM-Scl antigens, and investigational anti-fibrillarin ELISA kits, respectively. We also thank A Fehr and B Damm for data acquisition and management and Hildegard Christ and PD Dr Martin Hellmich for statistical advice. This study was supported by the German Federal Ministry of Education and Research (BMBF) (grants 01GM0310 and 01GM0630).

Authors’ Affiliations

(1)
Laboratory at Rheumaklinik Aachen
(2)
Department of Dermatology and Venerology, University of Cologne
(3)
Department of Rheumatology and Clinical Immunology, Charité Universitätsmedizin, Humboldt University
(4)
Clinical Research Unit for Rheumatology, University Medical Center Freiburg
(5)
Department of Dermatology, Dresden University Hospital
(6)
Department of Internal Medicine II, University of Giessen
(7)
Clinic for Rheumatology
(8)
Reha-Rheinfelden
(9)
Department of Dermatology, Venerology and Allergology, Charité Universitätsmedizin, Humboldt University
(10)
Department of Internal Medicine V, University of Heidelberg
(11)
Department of Dermatology and Allergology, Technical University of Munich
(12)
Department of Rheumatology and Clinical Immunology, Kerckhoff Clinic, Justus-Liebig University
(13)
Department of Dermatology, Heinrich-Heine-University
(14)
Department of Dermatology, University of Münster
(15)
Center of Rheumatology of Brandenburg, Johanniter Hospital in Fläming
(16)
Department of Dermatology and Allergology, University of Ulm
(17)
Center for Rheumatology, Acura Hospital
(18)
Department of Dermatology, Venerology and Allergology, University of Leipzig
(19)
Department of Dermatology and Allergology, Helios Klinikum
(20)
Department of Dermatology, Johannes-Wesling-Klinik
(21)
Medical Clinic I, Hospital Cologne-Merheim
(22)
Department of Dermatology, Venerology and Allergology, University of Würzburg
(23)
Hamburg Centre for Pediatric and Adolescence Rheumatology
(24)
Rheumaklinik Aachen
(25)
Department of Dermatology, University Hospital Erlangen
(26)
Medical Clinic 6, Marien-Hospital
(27)
Department of Dermatology, Venerology and Allergology, Georg-August-University

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