Double-stranded RNA antibodies in systemic lupus erythematosus.

Antibodies to viral double-stranded RNA (ds RNA) have been found in 40% of patients with systemic lupus erythematosus (SLE) and 14-5% of patients with rheumatoid arthritis. These antibodies were diagnostically more specific SLE than those directed against artificial polynucleotides, poly I:C and poly A:U. Although not disease specific, high titres of antibody to ds viral RNA were found almost exclusively in SLE. Serial studies failed to show that RNA antibody levels correlated with disease activity. Although of considerable interest in experimental studies on the pathogenesis of SLE, ds viral RNA antibodies are of little clinical significance in the management of SLE.     

CLINICAL SIGNIFICANCE OF ANTI–DOUBLE-STRANDED DNA ANTIBODIES DETECTED BY A SOLID PHASE ENZYME IMMUNOASSAY

A solid phase enzyme immunoassay (EIA) detected anti-double-stranded (ds) DNA antibodies in 88% of sera from patients classified clinically as having active systemic lupus erythematosus (SLE) without renal symptoms and 93% with renal disease. Fifty-six percent of sera from patients with inactive SLE were EIA positive for anti-dsDNA antibodies. The EIA had a sensitivity and specificity comparable to radioimmunoassay (RIA) and hemagglutination for patients with active SLE with or without renal disease, but it detected anti-dsDNA antibodies more frequently in patients with inactive SLE than the latter procedures. Precipitating antibodies detected by counterimmunoelectrophoresis (CIE) were less common in patients with renal disease (23% incidence) than clinically active patients without renal disease (79% incidence). Twenty-four SLE sera with elevated levels of CIq binding showed a 96% concordance for a positive EIA for anti-dsDNA antibodies in contrast to 66% concordance by RIA or hemagglutination. These findings suggest that the EIA is a sensitive and specific method for detection and measurement of anti-dsDNA antibodies. Several clinical applications of the EIA are discussed.     

Correlations between antinucleosome antibodies and anti-double-stranded DNA antibodies, C3, C4, and clinical activity in lupus patients

OBJECTIVE: To examine the correlations between antinucleosome antibodies and anti-double-stranded (ds) DNA antibodies, complement (C) 3 and 4 levels, and clinical activities in SLE patients. METHODS: Antinucleosome antibodies and anti-dsDNA antibodies were detected by enzyme-linked immunosorbent assays (ELISA). The levels of C3 and C4 were measured by nephelometry. Clinical activities were determined by SLE Disease Activity Index (SLEDAI). RESULTS: Of 65 SLE patients, the prevalence of antinucleosome antibodies were higher than anti-ds DNA antibodies (52.3 vs 36.9%, respectively, p < 0.05). Similar results were obtained in 45 active SLE patients, 64.4% for antinucleosome antibodies and 46.7% for anti-ds DNA antibodies. Of 34 patients lacking anti-ds DNA antibodies, 16 (47.1%) were shown antinucleosome antibodies. Activity of antinucleosome antibodies was significantly correlated with the SLEDAI scores and inversedly correlated with the C3 levels but not with the C4 levels. CONCLUSION: Antinucleosome antibodies could be one of the earliest and most sensitive markers in diagnosis of SLE, particularly in anti-dsDNA antibodies-negative patients. More importantly, antinucleosome antibodies is correlated with clinical activities and C3 levels.     

Reassessment of measurement of anti double-stranded DNA antibodies by Farr's assay using double-stranded DNA from E. coli and application of DNA binding Activities in high salt solution]

Farr's assay using double-stranded (ds) DNA from E. coli is a most sensitive and specific method for the detection of anti ds-DNA antibodies in patients with systemic lupus erythematosus (SLE). Because of the lack of sufficient DNA antigens, however, final antibody titers were hardly determined when the sera contained antibodies titered more than 100U/ml (or more than 60 per cent by DNA binding activities). In such sera DNA binding activities were measured by using ds-DNA tracer adjusted final concentration of NaCl to 125 mM. Higher binding activities measured by high-salt tracer are obtained significantly in SLE patients groups with nephrotic syndrome, proteinuria, cast, renal failure, diffuse proliferative nephritis, low serum complement levels, anemia and/or low IgG/IgA levels compared with the patients who lacked these clinical findings. In contrast the patients with digital rash or cramp showed significantly lower high-salt binding activities. The patients with pleuropericarditis tended to have lower bindings. The non-lupus patients including MCTD also had lower levels. These clinical characteristics could not be evaluated by standard Farr's assay. High-salt bindings suggest the presence of high avidity antibodies and also partly may mean the high levels of low avidity antibodies. The application of high-salt binding activities, thus, is a useful tool for the evaluation of clinical characteristics of SLE patients who had high levels of anti ds-DNA antibodies by standard Farr's assay.     

Is the target of anti-cardiolipin antibodies the same in Gougerot-Sj?gren syndrome and lupus erythematosus disseminatus?

Forty-seven patients were diagnosed as having systemic lupus erythematosus (SLE) and 34 patients primary Sj?gren's syndrome (SS); 30 controls were also studied. Anti cardiolipin (CL), anti double-stranded DNA (ds DNA) and anti single-stranded DNA (ss DNA) antibodies were determined by the enzyme-linked immunosorbent assay. Elevated anti-CL antibody levels were detected in 47.8 p. 100 (n = 46) of patients with SLE and in 85.3 p. 100 (n = 34) of patients with SS, but only once in controls. Elevated ss DNA were detected in 91.5 p. 100 (n = 47) of patients with SLE and in 18.3 p. 100 of patients with SS but never in controls. Elevated anti-ss DNA were detected in 93.3 p. 100 and 97.1 p. 100 respectively of patients with SLE and SS and in 3.3 p. 100 of controls. There was no correlation between anti-CL and thrombosis, circulating lupus anti coagulant or VDRL. The most striking association, however, was between anti-CL and anti ss-DNA antibodies in SLE. There was no correlation between anti-CL and anti ds-DNA antibodies in SLE patients. Anti CL antibodies were correlated both to ss-DNA and anti ds-DNA in SS patients. Absorption of positive anti-CL antibodies sera were done on DNA (ss-DNA and ds-DNA) affinity column chromatography: anti-CL antibodies were absorbed only by ss DNA in SLE and by both ss DNA and ds DNA in SS.     

DNA antibodies and complement in SLE patients

Sixty-seven patients with systemic lupus erythematosus (SLE) were followed up for 3-19 months (mean 12) in a prospective study. The activity of SLE was estimated on clinical grounds and correlated with DNA antibody and complement levels. The disease reactivations consisted mostly of articular and cutaneous symptoms. There were 17 relapses and 22 complicating infections during the follow-up period. The levels of antibodies to native, double-stranded (ds) DNA (P less than 0.001) and antibodies to denatured, single-stranded (ss) DNA of IgG class (P less than 0.001) and C3 (P less than 0.001) correlated best with disease activity, which was estimated on the clinical symptoms and signs. These assays were not reliable, however, in predicting minor exacerbations. The levels of IgM class ss-DNA antibodies were significantly higher in SLE patients without nephritis than in SLE nephritis patients. In most cases, the combination of IgG class ss-DNA antibody and complement (C3 and CH50) determinations differentiated SLE relapse from infection.     

A study of anti-poly (ADP-ribose) antibodies and an anti-DNA antibody idiotype and other immunological abnormalities in lupus family members.

The genetic background of systemic lupus erythematosus (SLE) has been reexamined in a study of the serum of 31 lupus patients and 80 asymptomatic first degree relatives by measuring a common, cross reacting anti-DNA antibody idiotype designated 134, antibodies to poly(ADP-ribose), serum C3, circulating immune complexes, and antinuclear antibodies (ANA). Over 30% of the relatives had raised 134 and anti-poly(ADP-ribose) levels, and 9% had ANA titres greater than 1/20. In contrast, only one relative had a low serum C3 level. These results confirm that immunogenetic abnormalities associated with the production of autoantibodies and particular idiotypes must exist amongst lupus relatives as well as the patients. The production of autoantibodies, however, is not necessarily matched to the clinical expression of SLE.     

Anti-DNA antibodies of IgA class in patients with systemic lupus erythematosus

Summary Sera obtained from 53 patients with systemic lupus erythematosus (SLE) were investigated for the presence of immunoglobulin class-specific antibodies against native (ds)DNA and denatured (ss)DNA. The methods employed were the Crithidia luciliae test and an enzymelinked immunosorbent assay (ELISA), respectively. Anti-dsDNA antibodies of IgG class were seen in 42%, IgM-anti-dsDNA antibodies in 43%, and IgA-anti-dsDNA antibodies in 30% of the patients. There was an association between the presence of both IgG- and IgA anti-dsDNA antibodies and the activity of the disease. Patients with active nephritis also had anti-dsDNA antibodies of IgG and IgA class significantly more often than patients with inactive nephritis or without renal disease. IgG-anti-ssDNA antibodies were seen in 89%, IgM-anti-ssDNA antibodies in 51%, and IgA-anti-ssDNA antibodies in 66% of the patients. Patients with nephritis had low levels of antibodies to ssDNA of IgM class. We suggest that immunoglobulin class-specific anti-DNA antibodies should de determined in the diagnosis and monitoring of SLE.     

DNA sequence and conformation specificity of lupus autoantibodies. Preferential binding to the left-handed Z-DNA form of synthetic polynucleotides

The binding specificity of 16 sera from systemic lupus erythematosus (SLE) patients was studied by enzyme-linked immunosorbent assay (ELISA), using 4 native DNAs of different guanine-cytosine (G-C) content and a group of synthetic polynucleotides. All the SLE sera showed increased binding to poly(dA-dC).poly(dG-dT), compared with calf thymus DNA in the right-handed B conformation. No significant differences were noted in binding of selected SLE sera to the native DNAs that differed in G-C content or superhelicity of DNA. With poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC), the majority of SLE sera showed a preferential binding to the salt-induced Z form, compared with the B form. In addition, an average twelve-fold increase was found in binding to Z-form brominated poly(dG-dC).poly(dG-dC) compared with B-form poly(dG-dC).poly(dG-dC), when the polymers were coated on the plates in 0.15M NaCl. The preferential binding of SLE sera to poly(dA-dC).poly(dG-dT) and to Z-DNA may be important in the formation of circulating immune complexes and subsequent vascular damage, or may provide a clue to the mechanism of production of anti-DNA antibodies in this disease.     

Studies of human polyclonal and monoclonal antibodies binding to lupus autoantigens and cross-reactive antigens

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune rheumatic disease serologically characterized by production of a variety of autoantibodies. Antibodies to double-stranded (ds) DNA are considered to be a diagnostic marker in SLE and their presence often correlates with active disease. The murine R4A anti-dsDNA antibody was found to cross-react with a peptide, D/EWD/EYS/G (R4A peptide), identified by analysing decapeptides selected from a peptide library. The R4A peptide inhibited binding of antibody to dsDNA and antibody deposition in kidneys in vivo. In other previous work, mice immunized with the peptide in a decapeptide form bound to a polylysine backbone, multiple antigenic peptide, were found to develop both anti-DNA and anticardiolipin antibodies. METHODS: To determine if human anti-DNA antibodies bind R4A peptide, we investigated the binding of monoclonal and polyclonal anti-dsDNA and anticardiolipin antibodies to the R4A peptide from patients with SLE. RESULTS: DNA binding by four immunoglobulin (Ig) G and two IgM human monoclonal anti-DNA antibodies was inhibited by the R4A peptide. While monomeric peptide was unable to inhibit affinity-purified polyclonal anti-DNA antibodies, serum anti-DNA reactivity was inhibited by an octameric form of the peptide in 10 SLE patients. CONCLUSIONS: Human anti-DNA reactivity includes the same fine specificity as that present in murine anti-DNA reactivity. Peptide binding might be a useful surrogate marker for SLE.     

Autoantibodies to lipoprotein lipase and dyslipidemia in systemic lupus erythematosus

OBJECTIVE: To demonstrate the binding of bovine lipoprotein lipase (LPL) by IgG from sera obtained from patients with systemic lupus erythematosus (SLE) and other rheumatic diseases, and the relationship of anti-LPL to triglyceride levels in SLE. METHOD: Binding of LPL by IgG from sera obtained from patients with SLE and other rheumatic diseases was measured by an enzyme-linked immunosorbent assay technique. Lipid profiles for fasting blood samples obtained from SLE patients and control subjects were determined. RESULTS: Sera obtained from 105 patients with SLE were assessed for reactivity with LPL, and 49 (47%) of the results were positive. Sera obtained from patients with rheumatoid arthritis (RA) (n = 80), Sj?gren's syndrome (n = 30), polymyositis and dermatomyositis (n = 30), and progressive systemic sclerosis (n = 31) were also studied, and 10 (13%), 3 (10%), 12 (40%), and 13 (42%), respectively, were positive for reactivity with LPL. It was determined that all affinity-purified anti-double-stranded DNA (dsDNA) antibodies and 4 of 5 monoclonal anti-dsDNA antibodies bound to LPL. The binding of IgG depleted of anti-dsDNA to LPL indicates a second anti-LPL activity in SLE. Measurements of fasting lipid levels in SLE patients with anti-LPL revealed a strong positive correlation of antibody levels and total serum triglycerides, apolipoprotein B, and apolipoprotein E concentrations. CONCLUSION: Antibodies to LPL occurred in 47% of SLE patients and in a similar percentage of patients with polymyositis or systemic sclerosis. The prevalence of these antibodies was less in patients with RA or Sj?gren's syndrome. It is hypothesized that the elevated triglyceride levels in SLE patients are in part attributable to anti-LPL, and this lipid abnormality could contribute to the premature atherosclerosis known to be present in patients with SLE.     

Altered metabolism of poly(adp-ribose) in the peripheral blood lymphocytes of patients with systemic lupus erythematosus

A method was developed to measure poly(ADP-ribose) metabolism in peripheral blood lymphocytes. The technique involved the isolation of lymphocytes on Ficoll gradients, followed by lysis with 5M NaCl. The synthesis and degradation of poly(ADP-ribose) in this crude lysate, measured by the incorporation of ³H-labeled NAD into acid-precipitable counts, was compared in 18 patients with systemic lupus erythematosus (SLE), 10 patients with rheumatoid arthritis, and in 10 control patients without rheumatoid arthritis. Patients with SLE showed a 70% decrease in poly(ADP-ribose) synthesis (P < 0.001); this decreased synthesis persisted even with the addition of histones or DNase. We present possible explanations of the role of poly(ADP-ribose) in SLE.     

Enhanced binding of lupus sera to the polyamine-induced left-handed Z-DNA form of polynucleotides

The natural polyamines putrescine, spermidine, and spermine are small polyvalent cations present in all living cells. Spermidine and spermine are excellent promoters of left-handed Z-DNA, an immunogenic form of DNA that binds readily with anti-DNA antibodies in the sera of patients with systemic lupus erythematosus (SLE). We studied the binding of a panel of 16 SLE sera to poly(dA-dC).poly(dG-dT) and poly(dG-m5dC).poly(dG-m5dC) in the presence and absence of spermidine and spermine using an enzyme-linked immunosorbent assay. The majority of SLE sera showed a 50-150% mean increase in optical density values when incubated with the polynucleotides and either 0.25 mM spermidine or 0.025 mM spermine than when incubated with the polynucleotides alone. Under these conditions, the polynucleotides assumed the Z-DNA form. Since polyamines are ubiquitous cellular components and since potential Z-DNA-forming alternating purine-pyrimidine sequences are widely dispersed in native DNA, the increased binding of SLE sera to polyamine-induced Z-DNA suggests a pathogenic role for these compounds in SLE.     

A COMPARISON OF AUTOANTIBODIES AND COMMON DNA ANTIBODY IDIOTYPES IN SLE PATIENTS AND THEIR SPOUSES

In order to determine whether environmental influence per semight influence autoantibody production, sera from the spousesof 20 SLE patients wereexamined. No antibodies to cardiolipin,poly (ADP-ribose), or ENA were detected and none had detectablerheumatoid factor. One weakly positive ANA reaction was noted,one had anti-DNA antibodies (by RIA and ELISA) and in two serathe common DNA antibody ldiotype 16/6 was found. The idiotypewas not, however, present on either anti-DNA or anti-K30 antibodies.Although long-term analyses arc required, it is evident thatsharing the same environment with patients who commonly expressa wide range of autoantibodies and common idiotypes-rarely leadsto their expression in non-autoimmune subjects KEY WORDS: Genetic influence, Familial influence, Autoimmunity, Anti-DNA antibodies, Anti-K30 antibodies.     

Antibodies to UV light denatured DNA in systemic lupus erythematosus: detection by filter radioimmunoassay and clinical correlations.

Antibodies to ultraviolet light denatured DNA (UV DNA) have been measured in patients with systemic lupus erythematosus (SLE) and normal subjects, using a millipore filter radioimmunoassay. High levels of UV DNA binding were only found in patients with SLE. The presence of UV DNA antibodies correlated well with the presence of native DNA antibodies, although immunodiffusion studies and inhibition techniques showed these antibodies to be immunologically distinct in many cases. Forty-one per cent of the SLE patients had had photosensitivity at some stage of their disease, but there was a poor correlation between this symptom and the presence of UV DNA antibodies. Although UV DNA is known to be a potent immunogen, none of the results from this study suggests that antibodies to UV DNA are more than another example of the broad spectrum of antinuclear antibodies seen in SLE.     

Complement fixation by anti-dsDNA antibodies in SLE: measurement by radioimmunoassay and relationship with disease activity.

We have developed a sensitive, solid phase radioimmunoassay (RIA) to quantify the amount of complement (C') fixation by anti-double-stranded DNA (anti-dsDNA) antibodies and have studied sera from 48 patients with systemic lupus erythematosus (SLE). 46% of the patients were positive in this assay. There was no correlation with serum C' levels, and only weak correlation with anti-dsDNA activity as measured by a solid phase RIA. An association was shown between positive values for C' fixation by anti-dsDNA antibodies and the presence of active lupus (p less than 0.01); there was a similar association with the presence of active renal disease in those patients with raised DNA binding (p less than 0.01). Our results suggest that quantitative measurement of C' fixation by anti-dsDNA antibodies may provide information about the pathogenicity of such antibodies in patients with SLE and be a better guide to potential end organ damage than the conventional measurement of DNA binding.     

Left-handed "Z" DNA antibodies in rheumatoid arthritis and systemic lupus erythematosus

A solid phase radioimmunoassay was used to evaluate antibodies to 13 nucleic acid antigens for their ability to distinguish between systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and control sera. The antigens included 5 synthetic duplex DNA, several single-stranded nucleic acids and 2 left-handed "Z" DNA. Many of the 45 SLE patients' sera showed distinctive patterns of binding to the various antigens, but this could not be correlated with disease type or activity. In general, the duplex DNA antigens gave the best sensitivity and specificity for the detection of SLE but the antigen poly(dA) may be superior. Antibodies to "Z" DNA were found in some SLE patients but surprisingly, the binding was significantly higher in the RA group than in the SLE group. This is the first nucleic acid antigen to show more specificity for RA than for SLE.     

Multiple serologic reactions and their relationship to clinical activity in systemic lupus erythematosus

Analysis of the binding of serum from 56 systemic lupus erythematosus patients to native DNA (nDNA), denatured DNA (dDNA), poly I, poly(dT), RNA, and cardiolipin revealed multiple antigen binding in many of the sera. Raised levels of antibodies (IgG and/or IgM) to denatured DNA were found in the highest percentage (68%) of patients. A small subset of patients with multiple raised IgM antibodies, renal disease, and vasculitic skin rash was identified. No correlation between multiple serologic activity and clinical disease was found.     

Specificity of antibodies to bacterial DNA in the sera of healthy human subjects and patients with systemic lupus erythematosus.

OBJECTIVE: To elucidate the epitope structure to DNA by identifying antigenic determinants on bacterial DNA bound by anti-DNA antibodies from normal human subjects (NHS) and patients with systemic lupus erythematosus (SLE). METHODS: Sera from NHS and patients with SLE were tested by ELISA for the presence of antibodies to single stranded DNA from calf thymus, Micrococcus lysodeikticus, Staphylococcus epidermidis, Clostridium perfringens, and Klebsiella pneumoniae. To assess binding to conserved and nonconserved determinants, sera were absorbed on DNA-cellulose affinity columns bearing each of the bacterial DNA and then tested for binding to the other DNA antigens. RESULTS: Absorption of SLE sera with any of the bacterial DNA caused a loss of binding to all other bacterial DNA as well as calf thymus DNA. In contrast, absorption of NHS sera with bacterial DNA caused a loss of binding to the DNA on the affinity column with much less effect on binding to the other DNA antigens. CONCLUSION: These results indicate a marked difference in the specificity of antibodies to bacterial DNA in NHS and patients with SLE. The binding of SLE anti-DNA to predominantly conserved determinants suggests that a shift in patterns of anti-DNA specificity may be associated with the autoimmune state.     

Profiles of antibodies to histones, DNA and IgG in patients with systemic rheumatic diseases determined by ELISA

The occurrence of antibodies against the total histone complex and the histone fraction H1, antibodies against denatured (ss) DNA and the synthetic double stranded polynucleotide poly dAT, as well as rheumatoid factors (RF) was determined in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and Sj?gren's syndrome (SS) using enzyme linked immunosorbent assays (ELISA). Antihistone antibodies could be demonstrated at a frequency of about 17% in the patients with systemic rheumatic disease with no differences between the groups, even if there was a tendency for anti-H1 antibodies to occur more often in the SLE and SS patients than in the RA patients. Some of the antihistone antibody activity seen in the RA patients seems to be due to crossreactive RF. All patient groups showed significant IgG anti-ssDNA antibody activity compared to the controls, but the highest antibody levels were seen in the SLE patients. IgG antipoly dAT antibodies occurred significantly more often and at higher levels in the SLE patients than in the other patient groups. Although the individual tests did not readily distinguish the 3 diseases from each other, the antibody profiles were different. Patients with SS had the broadest reactivity, and the SLE patients had antibodies predominantly restricted to polynucleotides.