Hans-Peter Seelig

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.

We have previously described a SWI/SNF-related protein complex (PYR complex) that is restricted to definitive (adult-type) hematopoietic cells and that specifically binds DNA sequences containing long stretches of pyrimidines. Deletion of an intergenic DNA-binding site for this complex from a human beta-globin locus construct results in delayed human gamma- to beta-globin switching in transgenic mice, suggesting that the PYR complex acts to facilitate the switch. We now show that PYR complex DNA-binding activity also copurifies with subunits of a second type of chromatin-remodeling complex, nucleosome-remodeling deacetylase (NuRD), that has been shown to have both nucleosome-remodeling and histone deacetylase activities. Gel supershift assays using antibodies to the ATPase-helicase subunit of the NuRD complex, Mi-2 (CHD4), confirm that Mi-2 is a component of the PYR complex. In addition, we show that the hematopoietic cell-restricted zinc finger protein Ikaros copurifies with PYR complex DNA-binding activity and that antibodies to Ikaros also supershift the complex. We also show that NuRD and SWI/SNF components coimmunopurify with each other as well as with Ikaros. Competition gel shift experiments using partially purified PYR complex and recombinant Ikaros protein indicate that Ikaros functions as a DNA-binding subunit of the PYR complex. Our results suggest that Ikaros targets two types of chromatin-remodeling factors-activators (SWI/SNF) and repressors (NuRD)-in a single complex (PYR complex) to the beta-globin locus in adult erythroid cells. At the time of the switch from fetal to adult globin production, the PYR complex is assembled and may function to repress gamma-globin gene expression and facilitate gamma- to beta-globin switching.

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.