Affinity columns containing anti-DNA Id+ human myeloma proteins adsorb human epibodies from intravenous gamma globulin

Objective. To study eluates of intravenous gamma globulin (IVGG) prepared from affinity columns of human cationic IgG myeloma proteins bearing anti-DNA idiotype (Id) markers 16/6, F4, 3I, and 8.12 for possible anti-Id (combining site) blocking activity. Methods. Anti-DNA idiotypic antibody activity was studied in 3 preparations of IVGG containing high, medium, and low levels of IgG anti-F(ab′)₂, and in 4 other commercial IVGG preparations. Affinity-purified IgG anti-DNA (APAD) from systemic lupus erythematosus (SLE) patients was biotinylated, and binding to DNA coated on enzyme-linked immunosorbent assay plates was used to measure anti-DNA antibody activity. IVGG was adsorbed to Sepharose 4B affinity columns linked to a panel of cationic human IgG myeloma proteins positive for anti-DNA Id markers 16/6, F4, 3I, and 8.12. Material adsorbing to such columns was eluted at low pH (2.5) and after neutralization, tested for its ability to inhibit biotinylated APAD reacting with DNA. Results. Only 0.05–0.9% of IVGGs bound firmly to Id affinity columns. These IVGGs were then eluted, using pH 2.5 glycine-saline and eluates neutralized to pH 7.4. Column flowthrough and eluate fractions were compared for their ability to block SLE APAD reacting with DNA. Significant inhibition of SLE APAD combining sites was observed with eluates from anti-DNA Id affinity columns; however, no correlation between IVGG anti-F(ab′)₂ activity and true anti-Id blocking of APAD was apparent. No residual anti-Id activity remained in column flowthrough fractions. No anti-Id blocking activity was recorded for IVGG eluates from human cationic myeloma columns devoid of the 4 anti-DNA Id markers. DNase treatment of IVGG or Id column eluates did not affect anti-Id blocking activity. Thus, all detectable anti-DNA idiotypic antibody capable of blocking SLE anti-DNA combining sites bound to Id+ affinity columns. Column eluates also showed some relative concentration of IgG anti-DNA activity, which was of lower affinity for DNA than antibodies also present in eluates which blocked anti-DNA combining sites. Conclusion. The presence of both anti-DNA and antiidiotypic (anti-combining site) activity in human anti-DNA Id column eluates indicates that epibodies from IVGG are relatively concentrated when this strategy is used. This approach may lead to a new strategy for treatment of SLE nephritis.     

A peptide derived from an autoantibody can stimulate t cells in the (nzb × nzw)f1 mouse model of systemic lupus erythematosus

Objective. To assess the ability of peptides derived from anti-DNA to stimulate syngeneic T lymphocytes and influence lupus in (NZB × NZW)F₁ (NZB/W) mice. Methods. We synthesized (by Geysen pin method) overlapping 12-mer peptides recapitulating the amino acid sequence of the V_H region of a nephritogenic monoclonal IgG2a anti-DNA antibody (A6.1) from an NZB/W mouse. Splenic T cells were cultured with the peptides; multiple experiments assayed 12-mer and 16-mer peptides which contained a triple-base motif (KFKGK). We immunized 20-week-old NZB/W mice with the 12-mer and evaluated them for evidence of nephritis and for quantities of antibodies in plasma and glomeruli. Results. Three clusters of peptides caused proliferation of spleen cells from young, nonimmunized mice. Both the 12-mer FYNQKEKGKATL and the 16-mer GDTFYNQKEKGKATLT peptides stimulated purified T cells. The KXKXK motif occurs in 15% of murine IgV_H (NBRF protein database), compared with 100% (6 of 6) of NZB/W anti-DNA monoclonal antibody. Immunization with the 12-mer peptide increased plasma levels of IgG, anti-DNA, and immune complexes, and levels of anti-DNA in glomeruli; nephritis was accelerated. Conclusion. NZB/W anti-DNA contain peptide sequences in their V_H regions that stimulate self—T cells. At least one motif is frequent in NZB/W anti-DNA. If some activated T cells provide help, this mechanism may contribute to sustained up-regulation of autoantibodies in murine lupus.     

Peptides from antibodies to DNA elicit cytokine release from peripheral blood mononuclear cells of patients with systemic lupus erythematosus: relation of cytokine pattern to disease duration

Peptides from VH regions of antibodies to DNA drive immune responses in systemic lupus erythematosus (SLE). We studied peptide-induced cytokine release by peripheral blood mononuclear cells (PBMC) of patients, the influence of peptide concentration, disease characteristics and HLA-D haplotypes. Cells secreting cytokines (IFNgamma, IL-2, IL-4 and IL-10) were measured by ELISPOT in PBMC from 31 patients with SLE and 20 matched healthy controls in response to seven peptides (A-G) from the CDR1/FR2 to CDR2/FR3 VH regions of human anti-DNA MAbs. Disease activity was assessed by SELENA-SLEDAI. HLA-DR and -DQ alleles were determined by molecular typing techniques. PBMC from significantly higher proportions of SLE patients than controls responded to VH peptides by generating IFNgamma and IL-10. Type of cytokines released in response to at least one peptide (D) depended on antigen concentration. Cytokine release was not associated with clinical features of SLE except for disease duration. A shift occurred from IFNgamma, IL-4 and IL-10 production in early disease to IL-4 and IL-10 in late disease (suggesting increasing TH2-like responses over time). Three peptides (B, D, G) were more stimulatory in the SLE patients than controls. Although none of the peptides was restricted by any particular MHC class II allele, among responders there was increased prevalence of HLA- DQB1*0201 and/or DRB1*0301, alleles known to predispose to SLE. Thus, responses to some VH peptides are more frequent in SLE and vary with disease duration. Increased responses in individuals with HLA class II genotypes that predispose to SLE suggest that peptide presentation by those molecules permits brisker peripheral blood cell responses to autoantibody peptides, thus increasing risk for disease.     

Selective antibody reactivity with peptides from human endogenous retroviruses and nonviral poly(amino acids) in patients with systemic lupus erythematosus

Objective. To investigate antibody responses to a broad panel of peptides derived from human endogenous retroviruses (HERVs) among unselected patients with systemic lupus erythematosus (SLE). Methods. In sera obtained from 69 patients with SLE and healthy blood donors, immunoassay was used to measure levels of antibody against synthetic peptides derived from HERVs, exogenous retroviruses, and nonviral poly(amino acids). Results. Measurement by immunoassay revealed increased frequencies of antiretroviral antibodies against 2 peptides derived from the env gene of the type C—like class, which includes ERV-9 and HERV-H, and against 2 peptides from the gag region of human T lymphotropic virus type I—related endogenous sequence 1, in patients with SLE. Antibodies to 2 nonviral peptides, polyhistidine and polyproline, were also overrepresented in patient sera. In 1 patient, longitudinal data obtained over a period of 12 years indicated that the concentrations of certain antiretroviral antibodies varied according to disease activity. Conclusion. Reactivity to certain type C HERV—derived antigens was found among patients with SLE. This reactivity could be explained by increased exposure to cross-reactive epitopes from essentially complete type C HERVs.     

The mechanism by which a peptide based on complementarity-determining region-1 of a pathogenic anti-DNA auto-Ab ameliorates experimental systemic lupus erythematosus

A peptide based on complementarity-determining region (CDR)-1 of a monoclonal murine anti-DNA Ab that bears the common idiotype, 16/6Id, was synthesized and characterized. The peptide, designated pCDR1, was found to be an immunodominant T-cell epitope in BALB/c mice. The CDR1-based peptide was shown to be capable of inhibiting the in vivo priming of BALB/c mice immunized with the peptide or with the whole anti-DNA 16/6Id(+) mAbs of either mouse or human origin. We show here that administration of pCDR1 (weekly, i.v., 100 microgram/mouse) in aqueous solution for 5 weeks starting at the time of disease induction with the human 16/6Id prevented the development of clinical manifestations of experimental systemic lupus erythematosus (SLE). Further, 10 weekly injections of pCDR1 to BALB/c mice with an established experimental SLE down-regulated clinical manifestations of SLE (e.g., anti-DNA auto-Abs, leukopenia, proteinuria, immune complex deposits in the kidneys) in the treated mice. Prevention of SLE induction was shown to be associated mainly with a decrease in the levels of IL-2, INFgamma, and the proinflammatory cytokine TNFalpha. On the other hand, the secretion of the immunosuppressive cytokine TGFbeta was elevated. Amelioration of the clinical manifestations of an already established experimental SLE correlated with a dramatic decrease in TNFalpha secretion, elevated levels of TGFbeta, and immunomodulation of the Th1 and Th2 type cytokines to levels close to those observed in healthy mice.     

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.     

Pathogenic anti-DNA idiotype (16/6 Id) in systemic lupus erythematosus

Summary Systemic lupus erythematosus (SLE) is regarded as a classical autoimmune disease. Despite this belief, no one has been able to induce the disease in naive animals, neither with DNA nor with anti-DNA antibodies. We report on the induction of SLE in BALB/c mice following immunization with a pathogenic anti-DNA idiotype (16/6 Id) or its anti-Id. We also report on a specific treatment with T suppressor cells specific for the 16/6 Id. The induction of SLE in naive mice with a pathogenic anti-DNA Id suggests an additional mechanism for the diversity of manifestations in this disease.     

The role of the immunoglobulin heavy chain in human anti-dna antibody binding specificity

Objective. To investigate the structural basis for DNA binding of the natural human IgMλ monoclonal antibody KIM4.6. Methods. An IgMλ, non–DNA-reactive variant hybridoma was derived during in vitro subcloning of the anti-DNA antibody KIM4.6. The variable (V)-region heavy (H) and light (L) chain genes expressed by the variant hybridoma were amplified by polymerase chain reaction, cloned, sequenced, and compared with those of the KIM4.6 parent and other DNA-binding and non–DNA-binding antibodies. Results. The VL chain of the variant was identical to that of KIM4.6. In contrast, the VH chain was completely different from the VH chain of the parent but was similar or identical, except in the diversity (D) and joining regions, to the VH chain of the systemic lupus erythematosus (SLE) IgG anti-DNA antibody T14 and SLE IgM nephritogenic anti-DNA antibodies NE-1 and NE-13. Conclusion. The expression of the KIM4.6 VL chain is not sufficient for DNA specificity. The VH chain and its D region play a key role in conferring DNA binding of the KIM4.6 anti-DNA antibody.     

Molecular and genetic characterization of two anti-DNA antibodies derived from patients with systemic lupus erythematosus

Anti-double stranded(ds) DNA antibody is one of markers of systemic lupus erythematosus (SLE). Two human monoclonal anti-DNA antibody – producing cell lines were established from two SLE patients. One cell line secreted IgG isotype antibody (KSUG) and the other secreted IgM isotype antibody (KSUN). The light chains of the two immunoglobulins were lambda chains. The nucleotide sequences for the immunoglobulin variable region genes of the two antibodies were determined and compared to germline sequences. The heavy and lambda light chains of KSUG were V_H3 family and V λ IIIb, respectively. The heavy and lambda light chains of KSUN were V_H4 family and V λ IX, respectively. Antibody KSUG, IgG isotype, showed somatic mutations, whereas KSUN, IgM isotype, used the germline gene without mutation. These findings reconfirm the current paradigms that IgM anti-DNA antibodies are produced by utilizing germline genes whereas IgG anti-DNA antibodies are produced by somatic mutations. Received: 13 June 1997 / Accepted 22 December 1997     

Chemokines in synovial inflammation in rheumatoid arthritis: basic and clinical aspects

Abstract

The mechanism by which anti-DNA antibodies cause glomerulonephritis in systemic lupus erythematosus (SLE) has not been fully elucidated. However, not a few recent studies suggest the pivotal role of nucleosomal histones in the binding of immune complexes to the glomerular basement membrane. Some antibodies cross-react with both double-stranded (ds) DNA and nucleosome, but antibodies exclusively specific to the nucleosome have also been described. In lupus model mice, anti-nucleosome antibodies are reported to emerge even before the production of anti-dsDNA antibodies. BK virus T antigen, bacterial DNA, a DNA binding protein nucleobindin and autoreactive helper T cells specific to nucleosome or anti-DNA antibody-derived peptides have been shown to induce or enhance anti-DNA antibody production in normal or lupus-prone animals. These new experimental models are expected to be helpful in the exploration of the true antigen that elicits autoimmune reactions in SLE.     

Antibacterial and antipeptide antibodies in Japanese and Finnish patients with rheumatoid arthritis

It has been suggested that Proteus infection may be involved in the pathogenesis of rheumatoid arthritis (RA). Bacterial and peptide immune responses in patients with RA and other control subjects were investigated in two geographically different populations. Serum samples from Finnish patients with early (n=72) and advanced (n=27) RA and 30 Finnish healthy controls, as well as from Japanese RA patients from two different locations: Tokyo (n=30) and Otsu (n=30), 18 patients with systemic lupus erythematosus (SLE) and 23 Japanese healthy controls were all screened for the total, and class-specific (IgG, IgA and IgM) antibodies against Proteus mirabilis, Escherichia coli and Serratia marcescens by indirect immunofluorescence assay. These samples were also tested for the determination of levels of isotypic antibodies against the shared epitope involving 16-mer synthetic peptides containing the EQRRAA or ESSRAL sequences and compared to scrambled control peptide by using an enzyme-labeled immunosorbent assay method. Significantly elevated levels of IgG and IgM antibodies to P. mirabilis and antibodies against both EQRRAA and ESSRAL peptides were detected in sera of Finnish patients with early and advanced RA, and in Japanese patients from Otsu or Tokyo compared to their corresponding control groups. In contrast, no difference either in the total or in any of the isotypic antibodies were observed between these groups when serum samples were screened against each of E. coli and S. marcescens or against the control peptide. Furthermore, there was a significant correlation between the antibody levels against Proteus bacteria only and both EQRRAA and ESRRAL peptides. Our findings support the possibility for specific involvement of P. mirabilis in the etiopathogenesis of RA even in early cases.Abbreviations ELISA Enzyme linked immunosorbent assay - IIF Immunofluorescence - RA Rheumatoid arthritis - SLE Systemic lupus erythematosus     

Is alpha-actinin a target for pathogenic anti-DNA antibodies in lupus nephritis?

OBJECTIVE: Following recent reports that pathogenic murine anti-DNA antibodies bind to alpha-actinin, it was obviously of interest to assess the ability of human pathogenic anti-double-stranded DNA (anti-dsDNA) antibodies to bind this antigen. Both human monoclonal anti-DNA antibodies and antibodies affinity purified from the sera of patients with systemic lupus erythematosus (SLE) were investigated. METHODS: An enzyme-linked immunosorbent assay was established to measure immunoglobulin binding to alpha-actinin. Antibodies binding dsDNA were purified from the sera of SLE patients who either had active renal disease or had never had renal disease. Serum samples were selected at times when the patients' sera exhibited high IgG binding to dsDNA. The binding of supernatants from 3 high-affinity human anti-dsDNA IgG hybridomas (RH14, B3, and DIL-6) and 7 human IgM anti-DNA hybridomas was also investigated. RESULTS: A greater proportion of anti-dsDNA IgG-binding antibodies purified from patients with renal disease bound to alpha-actinin than did those purified from the sera of patients without renal disease. The specificity of binding to the 100-kd alpha-actinin molecule was confirmed by Western blotting. The pathogenic human antibodies RH14 and B3 bound strongly to alpha-actinin, while nonpathogenic DIL-6 bound very weakly. RT84, the IgM antibody that binds dsDNA with the highest affinity, exhibited the greatest binding to alpha-actinin. CONCLUSION: The results of our study support the findings of previous studies using murine anti-DNA monoclonal antibodies, which suggest that pathogenic anti-dsDNA antibodies cross-react with alpha-actinin.     

Antiribosomal P antibodies in pediatric patients with systemic lupus erythematosus and psychosis

Objective. To study antibodies directed against ribosomal P proteins, a sensitive and specific marker of depression and psychosis in systemic lupus erythematosus (SLE), in pediatric patients with SLE. Methods. One hundred six serum samples were obtained from 79 patients with SLE. Sixty age- and sex-matched control sera were obtained, and 12 samples were obtained from children with primary psychosis. Antibodies to recombinant ribosomal P (rRP) protein were detected using a standard enzyme-linked immunosorbent assay. Results. All 12 children with non–SLE-associated psychosis had normal levels of anti-rRP antibodies. Elevated levels of anti-rRP were found in 11 of 64 pediatric SLE patients without a history of psychosis (17%). In the group of 13 SLE patients with psychosis, 5 (38%) had increased anti-rRP antibody levels during the time of acute psychosis, and which significantly decreased during remission. In addition, most of the SLE patients without a history of psychosis had a good correlation between anti-rRP antibody levels and disease activity. The patients with psychosis had significantly less renal involvement than the patients without a history of psychosis. This apparent protection from renal disease was not related to the presence or absence of either antiribosomal P or anti-DNA antibodies. Conclusion. Elevated serum levels of antibodies to ribosomal P protein can distinguish SLE-associated psychosis from primary psychosis of childhood. In SLE, elevated antiribosomal P antibody levels were not specific for psychosis. Serial assays were useful for monitoring the disease activity.     

Association of alpha-actinin-binding anti-double-stranded DNA antibodies with lupus nephritis

OBJECTIVE: Anti-double-stranded DNA (anti-dsDNA) antibodies may contribute to the pathogenesis of glomerulonephritis (GN) by cross-reacting with alpha-actinin in murine models and in some patients with systemic lupus erythematosus (SLE). We therefore sought to determine possible disease associations with serologic and clinical features and to characterize this new autoantibody specificity. METHODS: One hundred patients with SLE were recruited into this multicenter study, as well as 100 rheumatic disease controls and 2,100 healthy blood donors. Clinical disease was evaluated by the SLE Disease Activity Index (SLEDAI; excluding the anti-DNA component). Anti-dsDNA antibodies were detected by conventional enzyme-linked immunosorbent assay (ELISA) and by a commercial enzyme immunoassay (EIA). Anti-alpha-actinin antibodies were detected by ELISA, and their specificity was confirmed by Western blotting and by indirect immunofluorescence using rat kidney sections and mesangial cells as substrates. Highly positive sera were selected for absorption experiments and were affinity-purified for cross-reactivity studies and measurement of antibody avidity. RESULTS: Sera from 62 of the SLE patients had anti-dsDNA antibodies; 21 of these sera also had anti-alpha-actinin antibodies, as compared with 1 of the 38 sera without anti-dsDNA antibodies. Of the 22 patients with anti-alpha-actinin antibodies, 10 had GN, as compared with 14 of the 78 without anti-alpha-actinin antibodies (P < 0.01). In patients with GN, anti-alpha-actinin, but not anti-dsDNA, antibodies correlated with the SLEDAI score (minus the anti-DNA component) and with treatment. The fraction of serum anti-dsDNA antibodies that cross-reacted with alpha-actinin exhibited high avidity for dsDNA, as determined using a commercial EIA for high-avidity anti-dsDNA antibodies and an in-house conventional ELISA. CONCLUSION: The alpha-actinin-binding antibodies are significantly associated with GN in SLE. Whether such autoantibodies may anticipate the development of this complication of SLE remains to be verified.     

DETECTION OF ANTI-DNA ANTIBODIES: A COMPARISON BETWEEN TWO FARR ASSAYS, CRITHIDIA LUCILIAE AND A HUMAN CHROMOSOMAL SUBSTRATE ASSAY

Four commercially available assays were compared with a ¹⁴CDNA Farr assay for their ability to detect anti-DNA antibodiesin 119 sera from 109 patients with systemic lupus erythematosus(SLE) and 25 control sera. The ¹⁴C DNA Farr assay was the mostsensitive and specific assay (SLE, 57% positive, controls, 0%).A commercial ¹²⁵I DNA Farr assay was significantly less sensitive(SLE, 39% positive). The fluorescence human chromosomal preparationassay was as sensitive as the ¹⁴C DNA Farr assay (SLE, 58% positive)but less specific (controls, 8% positive). The immunofluorescenceCrithidia luciliae assay was specific, but less sensitive (SLE,37% positive) than the MC DNA Farr assay. Adaptation of Crithidiato immunoperoxidase did not alter its sensitivity or performance.These results confirm that the ¹⁴C DNA Farr assay, locally refinedand performed by experienced hands, is the most sensitive andspecific assay for anti-DNA antibodies. The ¹²⁵I DNA Farr wasno more sensitive than the Crithidia assay but considerablymore laborious. The human chromosomal preparation may be suitableas a rapid screening test for anti-DNA antibodies. KEY WORDS: Anti-DNA antibodies, Systemic lupus erythematosus, Crithidia luciliae, Fair assay     

Sex hormone involvement in the induction of experimental systemic lupus erythematosus by a pathogenic anti-DNA idiotype in naive mice

The effect of sex hormones on the induction of experimental systemic lupus erythematosus (SLE) with a human anti-DNA (16/6 Id +) antibody, was studied. We found that injection of the pathogenic idiotype to BALB/c females and orchiectomized males treated with estrogen caused a rapid outburst of the disease 3 months after immunization, while nonestrogen treated mice developed the disease 5 months after immunization. The flare of SLE disease was characterized by raised levels of autoantibodies in the sera to dsDNA, histones, cardiolipin, Sm, RNP, SSA (Ro), SSB (La) and an emergence of high titers of mouse antibody carrying the 16/6 Id. These enhanced antibody levels were associated with an increase in erythrocyte sedimentation rate, proteinuria and leukopenia. Immunofluorescent studies confirmed the existence of immune complexes in the afflicted kidneys. Testosterone treated BALB/c females and orchiectomized males developed a classical response to the human anti-DNA antibody (16/6 Id +), but failed to develop fulminant SLE-like disease. Our data demonstrate the importance of sex hormones on the induction of experimental SLE-like disease in mice with no genetic tendency to autoimmunity.     

Studies of anti-F(ab')2 antibodies and possible immunologic control mechanisms in systemic lupus erythematosus

Studies of anti-DNA and anti-F(ab')2 antibodies were performed by enzyme-linked immunosorbent assay in 51 patients with systemic lupus erythematosus (SLE). Patients with severe, uncontrolled disease showed high levels of anti-DNA and low levels of anti-F(ab')2 antibodies. Patients with quiescent SLE usually showed high levels of anti-F(ab')2 and low levels of anti-DNA antibodies. Isolated anti-F(ab')2 antibodies from autologous SLE remission serum or from the sera of unaffected siblings of SLE patients showed maximum inhibition in test systems using affinity-purified SLE anti-DNA antibodies reacting with single-stranded DNA.     

Anticyclic citrullinated peptide antibodies in patients with mixed connective tissue disease

Abstract

The clinical significance of anticyclic citrullinated peptide (CCP) antibodies in patients with mixed connective tissue disease (MCTD) was assessed. Altogether, 86 sera from MCTD patients, 96 from rheumatoid arthritis (RA) patients, 42 from systemic lupus erythematosus (SLE) patients, 23 from systemic sclerosis (SSc) patients, 21 from poymyositis/dermatomyositis (PM/DM) patients, and 17 from those with Sjögren’s syndrome (SjS) were tested for anti-CCP antibodies using an enzyme-lined immunosorbent assay. Among the 96 RA patients, anti-CCP antibodies were detected in 85%, with the frequency being significantly higher than in MCTD, SLE, SSc, PM/DM, and SjS patients (9%, 14%, 13%, 14%, and 18%, respectively; P < 0.001). Among eight MCTD patients who fulfilled the diagnostic criteria for RA, only 50% had anti-CCP antibodies, and the prevalence was significantly lower than for all RA patients (p < 0.01). All eight patients who fulfilled the criteria for RA had overlap of SLE and SSc, except one patient, whereas the four anti-CCP-positive patients who did not fulfill the criteria for RA had SjS without overlapping features of SLE and SSc; moreover, most of their antibody titers were low. These results suggested that anti-CCP antibodies are associated with RA in MCTD patients, but careful diagnosis of RA is required if patients with low titers of anti-CCP antibodies lack overlapping SLE and SSc.     

What is the primary antigen in systemic lupus erythematosus?

The mechanism by which anti-DNA antibodies cause glomerulonephritis in systemic lupus erythematosus (SLE) has not been fully elucidated. However, not a few recent studies suggest the pivotal role of nucleosomal histones in the binding of immune complexes to the glomerular basement membrane. Some antibodies cross-react with both double-stranded (ds) DNA and nucleosome, but antibodies exclusively specific to the nucleosome have also been described. In lupus model mice, anti-nucleosome antibodies are reported to emerge even before the production of anti-dsDNA antibodies. BK virus T antigen, bacterial DNA, a DNA binding protein nucleobindin and autoreactive helper T cells specific to nucleosome or anti-DNA antibody-derived peptides have been shown to induce or enhance anti-DNA antibody production in normal or lupus-prone animals. These new experimental models are expected to be helpful in the exploration of the true antigen that elicits autoimmune reactions in SLE.     

ANTI-DNA ANTIBODIES IN THE PRIMARY ANTIPHOSPHOLIPID SYNDROME (PAPS)

Primary antiphospholipid syndrome (PAPS) is considered a distinctentity from SLE and patients with PAPS are generally regardedas being dsDNA antibody negative. Levels of IgG and IgM ss andds DNA antibodies were measured by ELISA in 30 patients whofulfilled the criteria for the diagnosis of PAPS. We comparedthese patients with 20 normal controls and seven patients withidiopathic SLE. We also examined all the sera for anti-nuclearantibodies by Hep-2 cells and for dsDNA antibodies by Crithidia. We found that 16 patients with PAPS had antibodies to ss and/ordsDNA. Only three of the 16 positive patients had both IgG andIgM anti-DNA antibodies. Twelve patients had anti-nuclear antibodies,but only two were weakly positive for dsDNA antibodies by Crithidiaimmunofluorescence. Eleven out of 30 patients with PAPS hadIgM anti-dsDNA antibodies compared to two out of the seven SLEpatients. The PAPS patients with anti-DNA antibodies were clinicallyindistinguishable from the PAPS patients without antibodiesagainst DNA. Our results show that 53% of patients with PAPS had antibodiesto DNA which supports the view that PAPS and SLE are probablyoverlapping disorders. KEY WORDS: ELISA, Primary antiphospholipid syndrome, Anti-DNA antibodies