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Introduction: Anti "nucleolar" antibodies are estimated to be present in about 5-10% of anti-nuclear antibodies-positive (ANA), but only in about 15% of cases (sometimes in lower percentages) it is possible to assess precisely the specificity of antibodies responsible for this pattern of ANA. Nucleoli are composed of many proteins, e.g., nucleolin, fibrillarin, Pm-Scl (spliceosome component), RNA-polymerases and different sizes rRNA transcripts. Nucleoli also contain annexins (ANX) II and V. Many of these proteins are target molecules for autoantibodies generated in connective tissue diseases (CTD)--especially in scleroderma and overlap syndromes., Objectives: The aim of this study was to work out methods for differentiation of antibody specificities in the case of sera ANA-positive with the nucleolar pattern in indirect immunofluorescence (IIF), with a particular concern directed to anti-Annexin V antibodies (belonging to anticofactor antibody system--characteristic of antiphospholipid syndrome [APS])., Patients and Methods: In the sera of the tested group of 12 (selected from 150 subjects suffering from different kinds of CTD) ANA-positive (Western-blott negative) and showing the nucleolar type of ANA pattern patients, the following autoantibodies were assessed: ANA-IIF, anti-Pm-Scl (overlap syndrome marker), anticardiolipin, antiannexin V, anti-RNA-ase--all using the ELISA method. With the use of the Western-blott method a basic set "marker" autoantibody in CTD was estimated, and the RNA-ase was used for elimination (by enzymatic digestion) of RNA-s and RNA-protein complexes--a possible target for autoantibodies giving the nucleolar pattern., Results: In 5 out of 12 sera (41.7%) of ANA-positive/Western-blott-negative CTD patients antibodies against Annexin V, cardiolipin, Pm-Scl were detected. In 2 out of 12 patient sera antibodies to the RNA-ase were found. None of the tested sera showed the presence of autoantibodies against antigen Scl-70 (scleroderma marker), and SS-A (Sjögren syndrome marker), which can also give the atypical nucleolar ANA-pattern. Only in few sera "my ositis"--blotts showed antibodies to antigen Ku (nuclear protein), Jo-1 and PI-7 (anti-tRNA synthetases--polymyositis markers)--myositis and overlap syndrome markers. In 80% of tested sera the co-appearance of anti-ANX-V and anticardiolipin antibodies was observed. The presence of antibodies against ANX-V together with Pm-Scl was confirmed in 60% of sera The RNA-ase, used as a specific rRNA and RNP-protein blocker, resulted in a partial or total disappearance of the "nucleolar" pattern, but independently of the tested serum specificity (anti-ANX-V, anti-Pm-Scl or both together). The observed RNA-ase treatment of the Hep-2 cells effects are equivocal and they may result from either binding or digestion the antigen by the RNA-ase, but also from forming a sterical hindrance for the autoantigen binding to another "nucleolar" antigens., Conclusions: These preliminary results are interesting, especially as concerns anti-ANX-V autoantigens (associated with APS), but they require further research including bigger groups of CTD patients.