High Sensitivity Detection of Anti-DFS70 Antibodies by Radioimmunoprecipitation Assay (RIPA).

BACKGROUND: Autoantibodies against the chromosome associated protein DFS70/LEDGF (dense fine speckled 70/ lens epithelial growth factor; anti-DSF70) are increasingly being regarded as biomarkers for the diagnostic exclusion of systemic autoimmune rheumatic diseases (SARD). In routine ANA screening by indirect immunofluores-cence (IIFT) tests the presence of anti-DFS70 may first be presumed because of their characteristic immunofluo-rescence pattern (AC-2 pattern) and then be confirmed by antigen specific assays, a sequential approach, which may underestimate the prevalence of anti-DFS70 because of the inherent shortcomings of the ANA-IIFT. We therefore, for the first time, determined the prevalence of anti-DFS70 in patient sera by means of a sensitive and specific radioimmunoprecipitation assay (RIPA) as compared to a commercial ELISA. METHODS: Blood specimens referred for routine ANA screening (n = 1.100, ANA-Series) or for basic clinical chemistry tests (n = 350, CC-Series) were assayed for the prevalence of anti-DFS70 by RIPA using 35S-methionine labelled full-length DFS70 (FL-DFS70) as well as a C-terminal DFS70 fragment (CT-DFS70) generated by in vitro transcription/translation (ivTT) of the respective cDNAs. ELISA was performed using an anti-DFS70 test-kit (Eu-roimmun) and ANA-IIFT by means of commercial HEp-2 cells (INOVA) and appropriately chessboard titrated conjugates (Dianova). Accessory SARD markers (anti-dsDNA, anti-ENA) were determined in sera positive for anti-DFS70. RESULTS: The detection of anti-DFS70 by RIPA was considerably more sensitive than by ELISA, resulting in an overall detection rate of 9.0% (ANA-Series) and 8.0% (CC-Series) compared to ELISA revealing 4.6% (ANA-Se-ries) and 2.6% (CC-Series) anti-DFS70 positive sera. Of 99 RIPA reactive sera (ANA-Series) 72% were reactive against anti-FL-DFS70, 93% against CT-DFS70, polyspecific antibodies coexisted in 65%, reacting with both antigen specificities, 28% showed monospecific reaction with CT-DFS70 and 7% monospecific with FL-DFS70, indicating also the possible existence of antibodies specific for N-terminal epitopes in DSF70. Similar frequencies were seen in sera of the CC-series. The RIPA measured antibody concentrations (Rratio) obtained with FL-DSF70 antigen and CT-DSF70 antigen showed a correlation. There was also a correlation between the IIFT-ANA titers and Rratio found by RIPA. The consensus of suspected AC-2 pattern in ANA-IIFT and anti-DFS70 measured by RIPA was about 80%. No significant correlation existed between the antibody concentrations measured by RIPA and ELISA. Additional SARD markers were present in 24% of anti-DFS70 positive sera referred for ANA screening. No additional markers were seen in sera of the CC-Series. CONCLUSIONS: RIPA constitutes a highly-sensitive assay for detection of anti-DFS70 in human sera. ANA-IIFT screening performed under consideration of the AC-2 pattern for verification of antibodies to DFS70 under routine conditions may incorrectly estimate a considerable number of not only low but also high titer anti-DSF70 positive sera. The significance of RIPA reactive antibodies, especially of low titer range, in the context of SARD and healthy individuals now has to be scrutinized in further clinical studies.

Autoantibodies Against DFS70/LEDGF Exclusion Markers for Systemic Autoimmune Rheumatic Diseases (SARD).

BACKGROUND: Antinuclear antibodies (ANA) are considered as a key serological feature of systemic autoimmune rheumatic diseases (SARD) which include syndromes like systemic lupus erythematodes (SLE), systemic sclerosis (SSc), mixed connective tissue disease (MCTD), Sjögren's syndrome (SS) or dermatomyositis/polymyositis (DM/PM). ANA, commonly detected by indirect immunofluorescence assays on HEp-2 cells (IF-ANA), recommended as the screening test of choice (ACR), comprise a plethora of antibody specificities, a part of which are important serological markers of the diagnostic armamentarium in SARD. However, the applicability of IF-ANA as global screening test is hampered by its limited diagnostic specificity for resulting positive in up to 20% of apparently healthy individuals. About half of IF-ANA in healthy individuals target the chromosome associated protein DFS70/LEDGF, which tethers transcriptional coactivators or lentiviral integrases to transcriptionally active chromatin moieties and induces pro-survival stress factor transcriptions. Because of their rare prevalence in SARD patients, isolated anti-DFS70 antibodies are being increasingly considered as important biomarker to exclude the diagnosis of SARD. METHODS: Scrutinizing the relevant articles cited in NCBI concerning the DFS70/LEDGF protein, the diverse methods of anti-DFS70 determination in human sera supplemented by own experiences and critical review of the complete literature relevant to anti-DFS70 and SARD. RESULTS: Antibodies to DFS70/LEDGF (anti-DFS70), disclosed by IF-ANA, are characterized by a dense fine speckled (DFS) nucleoplasmic fluorescence pattern (DFS-ANA) accompanied by a striking staining of the condensed chromosomes in mitotic cells. By means of various methods anti-DFS70 may be found in 7.8 ± 6.2% (MD 7.6%) of apparently healthy individuals, may sometimes display rather high antibody titers and antibody carriers do not seem to manifest SARD symptoms within a five year interval. Their prevalence in non-selected cohorts originating from routine IF-ANA screenings (38643 tested individuals) fluctuates between 0.8 and 8.4% (MD 1.7%), depending on patient selection criteria and test performance. The proper appreciation of these data is hampered partially because of missing verification of antibody specificities partially by lack of specifications of associated disease or accompanying SARD specific marker antibodies. A metaanalysis of five studies including 1243 SARD patients confirms the rare mean prevalence of solitary anti-DFS70 (0.7 ± 0.9%, MD 0.45%) in SARD patients. The mean prevalence of anti-DFS70 accompanied by SARD specific markers is 3.8 ± 2.9% (MD 2.9%). In patients exclusively harbouring anti-DFS70 the likelihood ratio (LR+) for the absence of SARD approaches a significant value of 10.9. CONCLUSIONS: Since anti-DFS70 according to the available data may being regarded as a possible biomarker for ruling out the diagnosis of a systemic autoimmune rheumatic disease, it seems to be indispensable to identify properly DFS-ANA patterns in the routine IF-ANA screening, to confirm the anti-DFS70 specificity by appropriate confirmation assays and to communicate the results with annotating comments to the clinician, in order to ameliorate the proper assessment of the pathological significance of serological results and the selection of adequate follow-up investigations.

Anti-cytoplasmic Autoantibodies in Hodgkin's Lymphoma.

BACKGROUND: A 42-year old male patient with anamnesis of bronchial asthma presented himself with exertional dyspnea. High titer antibodies of cytoplasmic pattern in the indirect immunofluorescence test (IIFT) on HEp-2cells, not attributable to common specificities like anti-tRNA-synthetases, anti-signal recognition particle (SRP) or anti-ribosomal proteins (RPs) prompted the molecular biological approach for determination of antigen specificity. In-depth investigation of the patient's clinical settings revealed a lymphocyte predominant Hodgkin's lymphoma, which could be brought to complete remission by chemotherapy. METHODS: Immunoprecipitation of 35S-methionine labelled cell proteins, cDNA-library screening, sequencing of clones reactive with the patient serum, and expression of recombinant candidate proteins in E. coli, their purification, and western blotting were performed to verify and confirm the antibody specificities. RESULTS: The patient's autoantibodies reacted with the three insulin-growth-factor-2-RNA binding proteins (IGF2BP1-3) and a rare centromere protein (CNP27). The antibody titer determined by IIFT declined from initially 1:3000 to 1:800 five years after successful therapy of the Hodgkin's lymphoma. CONCLUSIONS: Anti-cytoplasmic antibodies, detectable by conventional IIFT, mainly belonging to the category of anti-tRNA-synthetases (e.g. anti-Jo1), SRP or RPs are in the first instance suspicious for systemic autoimmune rheumatic diseases (myopathies, interstitial lung disease). Their association with Hodgkin's lymphoma has been described only once in association with anti-Jo 1-syndrome [1]. The existence of anti-cytoplasmic antibodies of different specificities, demonstrated in this patient, may alert the laboratory's attention to provide the clinician with diagnostically relevant information beyond the perspective of systemic autoimmune rheumatic diseases.

Hypokalemic periodic paralysis induced by thymic hyperplasia and relieved by thymectomy.

IMPORTANCE: Hypokalemic periodic paralysis is a muscle channelopathy based on mutations or predisposing variants or secondary to potassium wasting. In contrast to myasthenia gravis, an association with thymic hyperplasia has not yet been reported, to our knowledge. OBSERVATIONS: We report a male patient in his mid-20s with progressive episodes of flaccid muscle weakness, associated low serum potassium levels, and a pathologic decrement in the long exercise test. Because the familial inheritance in the family was initially unknown, thorough diagnostic tests were performed including contrast-enhanced computed tomography scan, which displayed a mass in the anterior mediastinum. The test results for autoantibodies against myasthenia gravis (acetylcholine receptor, muscle-specific tyrosine kinase, and low-density lipoprotein receptor-related protein 4) and other end plate channelopathies were negative, and test results for hypokalemia-inducing hormones (thyroid, corticotropin, and cortisol) were negative. Surgery identified a thymus of 13 × 8 × 3 cm(3). Histologic analysis was consistent with thymic hyperplasia of the follicular subtype and immunohistologic analysis showed cytokeratin 5/6 in hyperplastic epithelial cells. A 2-year follow-up revealed the postoperative absence of weakness episodes. As in 30% of familial cases, molecular genetics testing failed to identify a mutation in periodic paralysis genes. CONCLUSIONS AND RELEVANCE: Thymic hyperplasia can clinically manifest susceptibility to hypokalemic periodic paralysis. For patients with late onset or increasing weakness episodes, we recommend imaging to assess for thymic enlargement and thymectomy at thymic hyperplasia.

Antibody to aquaporin 4 in the diagnosis of neuromyelitis optica.

BACKGROUND: Neuromyelitis optica (NMO) is a demyelinating disease of the central nervous system (CNS) of putative autoimmune aetiology. Early discrimination between multiple sclerosis (MS) and NMO is important, as optimum treatment for both diseases may differ considerably. Recently, using indirect immunofluorescence analysis, a new serum autoantibody (NMO-IgG) has been detected in NMO patients. The binding sites of this autoantibody were reported to colocalize with aquaporin 4 (AQP4) water channels. Thus we hypothesized that AQP4 antibodies in fact characterize NMO patients. METHODS AND FINDINGS: Based on these observations we cloned human water channel AQP4, expressed the protein in a eukaryotic transcription/translation system, and employed the recombinant AQP4 to establish a new radioimmunoprecipitation assay (RIPA). Indeed, application of this RIPA showed that antibodies against AQP4 exist in the majority of patients with NMO (n = 37; 21 positive) as well as in patients with isolated longitudinally extensive transverse myelitis (n = 6; six positive), corresponding to a sensitivity of 62.8% and a specificity of 98.3%. By contrast, AQP4 antibodies were virtually absent in 291 other participants, which included patients with MS (n = 144; four positive), patients with other inflammatory and noninflammatory neurological diseases (n = 73; one positive), patients with systemic autoimmune diseases (n = 45; 0 positive), and healthy participants (n = 29; 0 positive). CONCLUSIONS: In the largest series reported so far to our knowledge, we quantified AQP4 antibodies in patients with NMO versus various other diseases, and showed that the aquaporin 4 water channel is a target antigen in a majority of patients with NMO. The newly developed assay represents a highly specific, observer-independent, and easily reproducible detection method facilitating clinically relevant discrimination between NMO, MS, and other inflammatory diseases.

PM-Scl-75 is the main autoantigen in patients with the polymyositis/scleroderma overlap syndrome.

OBJECTIVE: To compare the autoantigenicity of the recently described N-terminally elongated PM-Scl-75 protein with that of PM-Scl-100 and the originally defined PM-Scl-75 polypeptide, and to determine its value for analyzing sera from patients with the polymyositis (PM)/scleroderma overlap syndrome. METHODS: Serum samples obtained from patients with the PM/scleroderma overlap syndrome and from patients with several other diseases were analyzed for the presence of autoantibodies reactive with recombinant PM-Scl-100 and PM-Scl-75 (both the original and the longer form) proteins, in an enzyme-linked immunosorbent assay (ELISA). RESULTS: Autoantibodies recognizing the longer PM-Scl-75 protein isoform were detected in 28% of the patients with PM/scleroderma. This percentage is slightly higher than that for PM-Scl-100 (25%) and is significantly higher than that for the previously defined PM-Scl-75 protein (11%). In addition, we identified a significant number of patients who had anti-PM-Scl-75 but not anti-PM-Scl-100 antibodies. This finding contrasts with what has been previously reported for the shorter version of the PM-Scl-75 protein. CONCLUSION: Our data indicate that use of the long PM-Scl-75 isoform in addition to PM-Scl-100 in ELISAs significantly increases the number of patients in whom anti-PM-Scl autoantibodies can be detected.

Autoantibodies directed to novel components of the PM/Scl complex, the human exosome.

The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex. Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex. The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10). The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins. Our results show that each human exosome component is recognized by autoantibodies. The hRrp4p and hRrp42p components were most frequently targeted. The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody. Other clear associations between autoantibody activities were not found. These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components.