Unstabilized DNA breaks in lymphocytes of patients with systemic sclerosis.

The clastogenic effects on DNA, proven by the presence of micronuclei (MN), and the protective cellular mechanisms normally used to stabilize DNA breaks were investigated in patients with systemic sclerosis (SSc). The frequency of micronucleated cells found in cultures of peripheral lymphocytes in patients was significantly higher than in the control group. The patient group with anti-centromere antibodies showed a significantly higher frequency of micronucleated cells than that observed in the patients with anti-topoisomerase I antibodies (4.22% versus 2.34%, p < 0.001). Moreover, we attempted to characterize MN for the presence or absence of DNA fragments with free 3'-OH ends by digoxigenin-dUTP (DIG-dUTP) using terminal deoxynucleotidil transferase. It was found that the frequency of MN containing DNA fragments with 3'-OH free ends (unstable fragments) increased in SSc patients compared to that observed in the control group. Moreover, this increase was significantly higher in lymphocytes of the patients with anti-centromere antibodies than in those with anti-topoisomerase I antibodies (35% versus 20.08%, p < 0.001). Our results indicate that in SSc patients there is an interference in the protective cellular mechanisms, normally stabilizing DNA breaks.

Use of a new silica clotting time for diagnosing lupus anticoagulant in patients who meet the clinical criteria for antiphospholipid syndrome.

The silica clotting time (SCT) is a phospholipid-dependent coagulation assay used for the laboratory diagnosis of lupus anticoagulant (LA) antibodies. The sensitivity and specificity of a new commercial SCT for identifying LA in patients who meet the clinical criteria for antiphospholipid syndrome (APS), and its association with thrombotic events were evaluated here. Forty-five patients who met the clinical criteria for APS according to the Sapporo International Consensus Statement were examined. Sixty-nine patients who did not meet the clinical criteria for APS, and 20 blood donors were used as controls. Plasma samples from the patients and controls were tested for LA using a new commercial SCT with low and high synthetic phospholipid concentrations. The results were compared with those obtained by diluted Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT). SCT's sensitivity for identifying LA in patients who met the clinical criteria of APS was higher compared to APTT and dRVVT (53.3% vs. 31.1% and 31.1%), and the specificities of these assays were 96.6%, 100%, and 98.9%, respectively. When dRVVT was combined with SCT, and dRVVT was combined with APTT their sensitivities were 57.7% and 48.8%, and their specificities were 96.6% and 98.9%, respectively. A stepwise logistic regression analysis indicated that the combination of dRVVT with SCT was associated with total thrombotic events (odds ratio (OR)=11.5, 95% confidence interval (CI)=1.25-106.3, P=0.031) as well as with venous thrombosis (OR=4.09, 95% CI=1.16-14.43, P=0.028). According to our results, SCT is the most sensitive assay for identifying LA in patients who meet the clinical criteria for APS. Moreover, the highest sensitivity was reached with a combination of SCT and dRVVT. The method's association with total thrombotic events and venous thrombosis was in fact significant.

Sensitivity and specificity for primary Sjögren's syndrome of IgA and IgG anti-alpha-fodrin antibodies detected by ELISA.

OBJECTIVE: To investigate the sensitivity and specificity of anti-alpha-fodrin antibodies in patients with primary Sjögren's syndrome (pSS). METHODS: IgA and IgG anti-alpha-fodrin antibodies were measured in the sera of 80 patients with pSS, 60 blood donors matched for age and sex, 50 patients with systemic lupus erythematosus (SLE), 30 with rheumatoid arthritis (RA), 20 with systemic sclerosis (SSc), and 10 with polymyositis or dermatomyositis (PM/DM) by an ELISA method employing recombinant human alpha-fodrin as antigen. RESULTS: The sensitivity of IgA and IgG anti-alpha-fodrin antibodies for pSS was 32.50% and 21.25%, respectively. When the prevalence of these antibodies in patients with SLE, RA, SSc, and PM/DM was evaluated, we observed specificity of these antibodies of 68.18% and 79.09%, respectively. The sensitivity and specificity for pSS of the combined determination of IgA and IgG anti-alpha-fodrin antibodies were 40% and 58.18%, respectively. CONCLUSION: The prevalences of IgA and IgG anti-alpha-fodrin antibodies in our patients with pSS and other chronic autoimmune diseases have induced us to doubt their use as diagnostic markers of pSS.

Autoantibodies to proteinase 3 and myeloperoxidase in systemic sclerosis.

OBJECTIVE: We evaluated the prevalence and clinical significance of proteinase 3 (PR3-) and myeloperoxidase (MPO-) antineutrophil cytoplasmic antibodies (ANCA) in 115 patients with systemic sclerosis (SSc, scleroderma). METHODS: Sera were assayed by 2 independent centers, which used indirect immunofluorescence (IIF) and direct ELISA as screening tests. Inhibition-ELISA for PR3- and MPO-ANCA and PR3 capture-ELISA experiments were also performed. The clinical features of the ANCA positive were compared with those of the ANCA negative scleroderma patients. RESULTS: The IIF test revealed 5 P-ANCA positive sera (4.34%). Surprisingly, by ELISA 2 of these were PR3-ANCA positive, one was MPO-ANCA positive, and 2 were both PR3- and MPO-ANCA positive. In addition, 3 IIF negative sera were ELISA positive, 2 for PR3- and one for MPO-ANCA. ELISA results were confirmed by fluid phase inhibition experiments. Only 2 out of the 6 PR3-direct ELISA positive sera were positive by PR3-capture ELISA at low titers. Neither PR3- nor MPO-ANCA were significantly associated to any clinical feature of patients with SSc. CONCLUSION: As well as the previously described MPO-ANCA, even PR3-ANCA may be detected in some sera from patients with SSc. The IIF pattern and the negative results obtained with PR3-capture ELISA suggest that different epitopes from those recognized by vasculitis sera might be involved with PR3-ANCA in SSc, and show the importance of combining IIF and ELISA tests for ANCA detection.