Subjects
Sera of anti-Ku-positive patients were collected from six European centers (Ljubljana, Brescia, Pécs, Prague, Nis, and Vienna) and were all secondarily tested; 73 were confirmed as positive in Ljubljana. Among them were 61 female and 12 male subjects. Their mean age was 57.8 years, ranging from 16 to 86 years. Anti-Ku positivity was determined with counter-immunoelectrophoresis (CIE)/line immunoassay (LIA) in the Immunological Laboratory of the Department of Rheumatology, University Medical Center, Ljubljana. Among these, only one patient was LIA but not CIE positive. The diagnoses of SSc, SLE, rheumatoid arthritis, polymyositis, and Sjögren syndrome were defined in the participating centers according to well-established criteria [13–17]. The criteria for UCTD were used, as suggested by Distler et al. [18] because this disease has no uniformly accepted diagnostic criteria. The overlaps were gathered under the diagnosis, which represented the leading clinical symptoms and signs of the disease.
Because of the large amount of clinical and laboratory data, besides some single clinical and laboratory features, a more simplified version of presenting the compilation of data was used.
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SLE skin" is defined by the presence of one or more of the following: malar rash, discoid rash, photosensitivity
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Joint and bone" is defined by the presence of one or more of the following: Joint features were evaluated clinically (synovitis and joint contractions) and by radiographic examination (erosive arthritis, acro-osteolysis)
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Muscle" is defined by the presence of one or more of the following: weakness or atrophy of muscles, serum CK elevation, EMG, and muscle biopsy in line with the appropriate criteria for inflammatory myopathy
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Neurology" is defined by the presence of one or more of the following: seizures, psychosis, or peripheral neuropathy
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Gastrointestinal" is defined by the presence of one or more of the following: dysphagia, gastroesophageal reflux, or early satiety
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Pulmonary" is defined by the presence of one or more of the following: dyspnea, NYHA I/II, fibrosis (plain radiograph), restrictive defect, or pleuritis
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Heart" is defined by the presence of one or more of the following: palpitations, conduction blocks, or abnormal diastolic function
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Renal" is defined by the presence of one or more of the following: proteinuria (more than 0.5 g/day), renal insufficiency, or renal crisis
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Antiphospholipid antibodies" are defined by the presence of one or more of the following: anticardiolipin antibodies, or anti-β2-glycoprotein antibodies, lupus anticoagulant, detected at least on two occasions, as defined by APS criteria [19]
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Cytopenia" means a low number of leukocytes and/or erythrocytes and/or thrombocytes
All clinical and laboratory data are follow-up cumulative data, except for anti-Ku antibodies, as determined with CIE/LIA and anti-p70/anti-p80 immunoblot detection. Informed consent was obtained, and the study was approved by the Ethics Committee of the Slovenian Ministry of Health.
Methods
Stored sera were sent on dry ice to the reference center (Department of Rheumatology, Ljubljana), where they were tested. Antibodies against soluble nuclear antigens (anti-ENA): Ro/SS-A, La/SS-B, Sm, U1RNP, PCNA, SL, Scl-70, PM/Scl, Jo-1, and Ku were screened with CIE, as described [20], which show conformational epitopes and are not able to discriminate between anti-Ku p70 and/or anti-Ku p80.
Sera from the aforementioned centers were additionally tested in the same reference center for antinuclear antibodies (ANAs) with indirect immunofluorescence on HEp-2 cell-line substrate (Immunoconcepts, Sacramento, CA, USA) and with LIA for autoantibodies present in myositis, which detects, besides anti-Ku, also antibodies against Jo-1, Mi-2, PM/Scl, and U1-snRNP (Imtec-Myositis-LIA; Imtec, Immunodiagnostika, Berlin, Germany). The Ku antigen on Imtec-Myositis-LIA is a human recombinant Ku p70/p80, which also does not distinguish between the two subunits. Hep-2 patterns were detected with indirect immunofluorescence, rheumatoid factor, with latex fixation test and Waaler Rose reaction, anti-cyclic citrullinated protein antibodies with enzyme-linked immunosorbent assay (ELISA; Immunoscan CCPlus, Euro-Diagnostica AB, Malmö, Sweden), aPL with an in-house ELISA (for anti-aCL IgG and IgM isotypes and for anti-β2-GPI, IgG, IgM, and IgA have been measured) and anti-dsDNA antibodies with an in-house FARR-RIA assay.
To detect anti-p70 and anti-p80 antibodies, immunoblotting was used. Nuclear extracts were prepared from THP-1 (human acute monocytic leukemia cell line). Centrifuged THP-1s were washed with PBS, and protease inhibitors (Halt Protease Inhibitor Cocktail Kit; Pierce, Rockford, IL, USA) were added. Extraction was done according to instructions (NE-PER Nuclear and Cytoplasmic Extraction Reagents; Pierce), and protein concentration in nuclear extract was measured with the Bio-Rad Protein Assay (Bio Rad, Munich, Germany). The immunoblot method included an initial immunoprecipitation step, which was performed with Protein A/G PLUS-Agarose Immunoprecipitation Reagent; Santa Cruz, Santa Cruz, CA, USA): 500 μg of proteins from the nuclear extract was mixed with 2 μg mouse monoclonal antibody Ku-3 (clone 162) (NeoMarkers, Fremont, CA) and incubated 2 hours at 4°C. Then 20 μl of resuspended A/G Agarose was added, and the mixture was incubated another 2 hours at 4°C. Washing of pellet was done with PBS for 4 times, each time followed by centrifugation. After the final wash, pellets were resuspended in PBS and electrophoresis SDS-sample buffer and boiled for 3 minutes for protein elution. Sample volume corresponding to 50 μg of primary nuclear extract/cm of gel length was analyzed on 10% SDS-polyacrylamide gels in Tris/glycine buffer in BioRad Mini Protean apparatus and transferred to nitrocellulose (BA 85; Schleicher & Schuell, Munich, Germany) at 100 V, 250 mA for 45 minutes. The dry membrane was blocked with TBS/0.05% Tween buffer with 5% milk. Patient sera were diluted 1:50 and incubated 2 hours, followed by washing and 40 minutes of goat anti-human IgG-HRP (1:1,000) (BioRad) incubation. Detection was done with luminol (Western Blotting Luminol Reagent, Santa Cruz, CA, USA) in G:box (Syngene, Cambridge, UK) with chemiluminescence. As positive controls, monoclonal goat antibodies (Ku-86 (C-20), Santa Cruz; Ku-70 (C-19), Santa Cruz, diluted 1:500) and donkey anti-goat IgG-HRP conjugate (diluted 1:1,000, Santa Cruz) were used. All samples were tested also for colorimetric determination, and the secondary antibodies used were rabbit anti-human alkaline phosphatase (Bio Rad) followed by the substrate NBT/BCIP (Pierce).
Statistical analyses
Statistical analyses were performed by using R (V 2.12.1) [21]. The Fisher Exact test was used to evaluate the association between anti-Ku antibodies and diagnosis, gender, clinical signs, and other observed antibodies. The P values were adjusted for multiple testing by using the approach of Benjamini and Hochberg [22]. An adjusted P value below or equal to 0.05 was considered statistically significant. Separation of disease populations based on the presence of antibodies and clinical signs were investigated with PCA. PCA was performed by using the R prcomp function with standard parameters. PCA allows the identification of latent variables (principal components) in the data based on observed variables (in our case, 29 parameters, the presence of those autoantibodies and clinical signs that show five or more observations).
