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Objectives: Whereas antinuclear antibodies (ANAs) detected by indirect immunofluorescence (IIF) have diagnostic significance, the dense fine speckled (DFS) pattern on HEp-2 cells may be an exclusionary marker for ANA-associated rheumatic disease (AARD). The aim of this study was to evaluate a new algorithm considering anti-DFS70 antibodies for routine ANA testing., Method: From ANA requested sequential 10 528 sera, 181 sera samples showing the DFS pattern were additionally tested for anti-DFS70 antibodies by an enzyme-linked immunosorbent assay (ELISA-DFS70) and for specific-ANAs. Specific-ANAs(+)/IIF-DFS(-) control sera samples (n = 50) were also tested., Results: Of the 181 IIF-DFS-positive sera samples, 82.9% (n = 150) were from non-AARD patients and 112 (61.9%) patients had non-rheumatic diseases (NRD), including the most common clinical feature of dermatitis (18.2%). The ELISA-DFS70 was positive in 109 (60.2%) sera and was negative in all control sera. Specific-ANAs were similarly detected as 25.7% (28/109) and 22.2% (16/72) of ELISA-DFS70(+) and ELISA-DFS70(+) patients, respectively (p > 0.05). The prevalence of non-AARD was 95.1% and 25.1% in the ELISA-DFS70(+)/specific-ANAs(-) and ELISA-DFS70(-)/specific-ANAs (+) groups, respectively., Conclusions: In patients with a HEp-2 DFS pattern, the additional ELISA-DFS70 and specific-ANAs test could improve the efficiency of diagnosing AARD. The detection of anti-DFS70 antibodies should be included in test algorithms for ANA testing.

Objective: To investigate the levels and relationship between IL-10, TNF-alpha, anti-U1snRNP antibodies and disease activity in longitudinally collected serum samples from patients with mixed connective tissue disease (MCTD)., Methods: Six patients followed for 17-138 months were investigated with ELISA for estimation of cytokine levels and antibodies to the different epitopes of the U1snRNP. Disease activity was assessed by systemic lupus activity measure (SLAM)., Results: IL-10 and TNF-alpha levels fluctuated with time in at least half of the patients. Three patients had increased IL-10 levels and two had increased TNF-alpha in all samples. There was no correlation between cytokine levels and disease activity or clinical manifestations. All patients had increased levels of antibodies to the main components of the U1snRNP. Both antibody levels and disease activity decreased with time. A correlation between TNF-alpha and U1snRNP antibody levels were observed in five patients., Conclusions: Increased and fluctuating levels of IL-10 or TNF-alpha without correlation to disease activity were observed in MCTD patients. In some patients increased cytokine levels were observed over several years irrespective of disease activity indicating that they could be constitutively increased in these individuals.

To evaluate a role of myeloperoxidase (MPO) and antibody to myeloperoxidase (anti-MPO) in vasculitis, MPO and anti-MPO were determined by enzyme-linked immunosorbent assays in sera from 43 patients with vasculitis, 40 with rheumatoid arthritis, 36 with systemic lupus erythematosus (SLE), 23 with mixed connective tissue disease, 13 with systemic sclerosis, 22 with polymyositis/dermatomyositis, 18 with Sjögren's syndrome, and 30 normal controls. Kidney and lung sections from patients with vasculitis were stained for MPO. Anti-MPO titers were significantly higher (p<0.005) in the patients with vasculitis (mean+/- SD absorbance at 405 nm: 0.53 +/- 0.37) than in any other groups (0.15 +/- 0.04 to approximately 0.21 +/- 0.11). MPO levels in patients with vasculitis were comparable with those in patients with other diseases except SLE. In two patients with vasculitis, anti-MPO decreased sharply with simultaneous increases in MPO 1-2 weeks after they developed pulmonary hemorrhage. Numerous cells positive for MPO infiltrated the Bowman's spaces. These results indicate that MPO may contribute to the pathogenesis of vasculitis and a sudden fall in anti-MPO may predict a poor prognosis in some cases.

We describe a 60-year-old woman who developed mixed connective tissue disease (MCTD) associated with multicentric Castleman's disease (MCD) and Crow-Fukase syndrome (CFS). She showed HLA DR-4 antigen and an abnormal X chromosome (47,XXX). The serum interleukin-6 (IL-6) level was markedly increased and IL-6 mRNA was detected in enlarged lymph node cells. After prednisolone was administered, her IL-6 level decreased and the symptoms of MCTD, MCD, and CFS all improved. Thus, IL-6 may be involved in the modification of the pathologic condition in this patient.

A comparison of clinical, immunological and HLA-D region antigen features was made between 22 patients with mixed connective tissue disease (MCTD) and 118 patients with systemic lupus erythematosus (SLE), scleroderma or primary Sjögren's syndrome. The MCTD patients had hypergammaglobulinemia more often than did those with SLE and scleroderma, but had less skin ulceration, serositis, nephritis, central nervous system disease and hypocomplementemia than the SLE patients. The frequencies of HLA-DR4 and its Dw4 subtype were significantly increased in MCTD as compared with both the other patient groups and healthy controls. Anti-RNP antibodies and the clinical characteristics together seem to illustrate a disease syndrome which is clinically and genetically distinct and fits with the prevailing concept of MCTD.

Antibodies to three major antigens of the non-histone or saline-extractable nuclear antigen (ENA) complex were sought by counterimmunoelectrophoresis (CEP) in three groups of sera which gave different patterns in the immunofluorescence test for antinuclear antibodies (ANA). Precipitins, mainly anti RNP and anti SS-B, were found most commonly (61%) in 70 sera with a speckled ANA pattern but were less frequent (8%) in 61 sera with a homogeneous ANA pattern and exceptional (1%) in 72 sera which showed fibrillar ANA staining. Rim staining was an insensitive indicator of nDNA antibody. An enzyme immunoassay (EIA), specific for anti-SS-B was more sensitive than CEP and identified this antibody in 28 sera, compared with 18 for CEP.

Human fibroblast monolayers were applied as a nuclear substrate in indirect immunofluorescence for the detection of anti-nuclear antibodies (ANA). Comparison of the results obtained with this substrate and with conventional rat liver sections led to the conclusion that the application of human fibroblast monolayers as a substrate has the following advantages: 1) better recognition of the distinct nuclear staining patterns; 2) more convenient serum titrations, since a cryostat is not needed and twelve tests can be performed on one slide; 3) detection of significantly higher ANA titres in sera from patients with SLE and MCTD; 4) recognition of a type of ANA that was not detected on rat liver sections. These latter antibodies produced a discrete speckled pattern of fluorescence, which was completely lost after acid elution of the fibroblasts. Using HEp-2 cells in metaphase, it was shown that the antibodies were directed against the centromere regions of the chromosomes. These anti-centromere antibodies were detected in sera from patients with severe Raynaud's phenomenon. Underlying disorders were scleroderma (8 patients, 5 of them with CREST syndrome), Sjögren's syndrome (2 patients), and Raynaud's phenomenon in combination with a few symptoms of connective tissue diseases without fulfilling the criteria for a specific disease (5 patients).

Antibodies to soluble ribonucleoprotein (sRNP) are by definition a prerequisite for the diagnosis mixed connective tissue disease (MCTD). They are also found in 30--40% of other rheumatic conditions with a high titred speckled IFL reaction. The same is true for the most characteristic clinical features of MCTD, viz. Raynaud's syndrome and polymyositis--polymyalgia. The serological and clinical symptoms mentioned are closely associated. An association between anti-Sm and the SLE symptoms butterfly erythema, nephropathia, other visceropathias and cerebrovascular disease is confirmed by the present study. Although anti-Sm has less diagnostic specificity for SLE than has anti-ds-DNA, it is useful as a diagnostic aid in SLE cases lacking the latter antibody. The results of the present study tend to support the hypothesis that anti-sRNP may be associated with a prophylactive effect against serious vasculitis and nephropathia. When anti-sRNP and anti-Sm occur together they seem to act competitively so that the symptom associated with the antibody having the highest titre tends to dominate the clinical picture. Both anti-sRNP and anti-Sm produce speckled nuclear patterns in the indirect immunofluorescence test. Sera with immunofluorescence titres below 1/25 were not found to react against either sRNP or Sm. On the other hand all sera tested that had a speckled IFL reaction greater than or equal to 1/800 also had anti-sRNP.

Of 472 consecutive patients with inflammatory rheumatic diseases attending a unit responsible for the treatment of rheumatoid diseases, only 12 patients were found to have anti-ribonucleoprotein (RNP) antibodies. Of these, 5 patients fulfilled the criteria for the diagnosis of systemic lupus erythematosus (SLE) and 7 those of rheumatoid arthritis. Joint symptoms were the predominant clinical features and caused severe deformities requiring operations in most cases. One anti-RNP-positive SLE patient died of chronic active hepatitis.