Serum antibodies to human leucocyte antigen (HLA)‐E,HLA‐F and HLA‐G in patients with systemic lupus erythematosus (SLE) during disease flares: Clinical relevance of HLA‐F autoantibodies

T lymphocyte hyperactivity and progressive inflammation in systemic lupus erythematosus (SLE) patients results in over‐expression of human leucocyte antigen (HLA)‐Ib on the surface of lymphocytes. These are shed into the circulation upon inflammation, and may augment production of antibodies promoting pathogenicity of the disease. The objective was to evaluate the association of HLA‐Ib (HLA‐E, HLA‐F and HLA‐G) antibodies to the disease activity of SLE. The immunoglobulin (Ig)G/IgM reactivity to HLA‐Ib and β2m in the sera of 69 German, 29 Mexican female SLE patients and 17 German female controls was measured by multiplex Luminex^®‐based flow cytometry. The values were expressed as mean florescence intensity (MFI). Only the German SLE cohort was analysed in relation to the clinical disease activity. In the controls, anti‐HLA‐G IgG predominated over other HLA‐Ib antibodies, whereas SLE patients had a preponderance of anti‐HLA‐F IgG over the other HLA‐Ib antibodies. The disease activity index, Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)‐2000, was reflected only in the levels of anti‐HLA‐F IgG. Anti‐HLA‐F IgG with MFI level of 500–1999 was associated with active SLE, whereas inactive SLE revealed higher MFI (>2000). When anti‐HLA‐F IgG were cross‐reactive with other HLA‐Ib alleles, their reactivity was reflected in the levels of anti‐HLA‐E and ‐G IgG. The prevalence of HLA‐F‐monospecific antibodies in SLE patients was also associated with the clinical disease activity. Anti‐HLA‐F IgG is possibly involved in the clearance of HLA‐F shed from lymphocytes and inflamed tissues to lessen the disease's severity, and thus emerges as a beneficial immune biomarker. Therefore, anti‐HLA‐Ib IgG should be considered as a biomarker in standard SLE diagnostics.     

High Prevalence of Human Anti‐bovine IgG Antibodies as the Major Cause of False Positive Reactions in Two‐Site Immunoassays Based on Monoclonal Antibodies

Abstract

A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n?=?54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities—a catcher and a biotinylated indicator. The monoclonal antibodies were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti‐animal IgG (bovine, mouse, horse, and swine) antibodies and human anti‐bovine serum albumin antibodies were measured using an ELISA design, with direct bridging of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti‐bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n?=?54) were positively correlated to the false positive signals in the PP14 ELISA (r?=?0.923; p?<?0.0001). Antibodies to IgG from other mammalian species (mouse, horse, and swine) were also detected in the donor sera, but levels and frequencies were lower compared to that of HABIA. Furthermore, HABIA were positively correlated to human anti‐bovine serum albumin antibodies in the donor sera (r?=?0.639; p?<?0.0001; n?=?103). HABIA (prevalence 95%) cause false positive reactions due to crossbinding of contaminating bovine IgG and/or crossreaction with mouse IgG in two‐site immunoassays. The apparent presence of human anti‐mouse IgG antibodies (HAMA), described to create false positive results, may be due to a crossreacting fraction of the polyclonal circulating antibodies against bovine IgG.     

Immunoglobulin (Ig)G purified from human sera mirrors intravenous Ig human leucocyte antigen (HLA) reactivity and recognizes one's own HLA types,but may be masked by Fab complementarity‐determining region peptide in the native sera

Intravenous immunoglobulin (IVIg) reacted with a wide array of human leucocyte antigen (HLA) alleles, in contrast to normal sera, due possibly to the purification of IgG from the pooled plasma. The reactivity of IgG purified from normal sera was compared with that of native sera to determine whether any serum factors mask the HLA reactivity of anti‐HLA IgG and whether IgG purified from sera can recognize the HLA types of the corresponding donors. The purified IgG, unlike native sera, mirrored IVIg reactivity to a wide array of HLA‐I/‐II alleles, indicating that anti‐HLA IgG may be masked in normal sera – either by peptides derived from soluble HLA or by those from antibodies. A < 3 kDa peptide from the complementarity‐determining region (CDR) of the Fab region of IgG (but not the HLA peptides) masked HLA recognition by the purified IgG. Most importantly, some of the anti‐HLA IgG purified from normal sera – and serum IgG from a few donors – indeed recognized the HLA types of the corresponding donors, confirming the presence of auto‐HLA antibodies. Comparison of HLA types with the profile of HLA antibodies showed auto‐HLA IgG to the donors' HLA antigens in this order of frequency: DPA (80%), DQA (71%), DRB345 (67%), DQB (57%), Cw (50%), DBP (43%), DRB1 (21%), A (14%) and B (7%). The auto‐HLA antibodies, when unmasked in vivo, may perform immunoregulatory functions similar to those of therapeutic preparations of IVIg.     

Isolation of human anti-idiotypes broadly cross reactive with anti-dsDNA antibodies from patients with Systemic lupus erythematosus

Antibodies to double stranded (ds)DNA play a central role in clinical diagnosis and disease expression in Systemic lupus erythematosus (SLE). This paper describes the isolation of anti-idiotype reagents (anti/antidsDNA) from four SLE sera and the demonstration of broad and quantitatively similar cross reactivity to both polyclonal and monoclonal anti-dsDNA antibodies isolated from SLE patients. Seven affinity-purified polyclonal and three monoclonal human anti-dsDNA preparations reacted preferentially with anti-idiotype F(ab')(2) coated plates compared to normal immunoglobulin (Ig)G F(ab')(2) coated plates in ELISA. In contrast, autoantibodies of other specificities (anti-Ro/SSA, anti-La/SSB, and anti-U(1)RNP) reacted equally with anti/anti-dsDNA F(ab')(2) and normal IgG F(ab')(2) coated plates. Such anti-idiotypic antibodies could play a significant role in the regulation of anti-dsDNA antibody levels in SLE.     

Relationship between natural and infection‐induced antibodies in systemic autoimmune diseases (SAD): SLE,SSc and RA

Infection or vaccine‐induced T cell‐dependent immune response and the subsequent high‐affinity neutralizing antibody production have been extensively studied, while the connection between natural autoantibodies (nAAbs) and disease‐specific antibodies has not been thoroughly investigated. Our goal was to find the relationship between immunoglobulin (Ig)M and IgG isotype nAAbs and infection or vaccine‐induced and disease‐related autoantibody levels in systemic autoimmune diseases (SAD). A previously described indirect enzyme‐linked immunosorbent assay (ELISA) test was used for detection of IgM/IgG nAAbs against citrate synthase (anti‐CS) and F4 fragment (anti‐F4) of DNA topoisomerase I in 374 SAD samples, with a special focus on systemic lupus erythematosus (SLE) (n = 92), rheumatoid arthritis (n = 73) and systemic sclerosis (n = 157) disease groups. Anti‐measles IgG and anti‐dsDNA IgG/IgM autoantibodies were measured using commercial and in‐house indirect ELISA tests. In all SAD groups the anti‐measles IgG‐seropositive cases showed significantly higher anti‐CS IgG titers (P = 0·011). In anti‐dsDNA IgG‐positive SLE patients, we detected significantly higher levels of anti‐CS and anti‐F4 IgG nAAbs (P = 0·001 and < 0·001, respectively). Additionally, we found increased levels of IgM isotypes of anti‐CS and anti‐F4 nAAbs in anti‐dsDNA IgM‐positive SLE patients (P = 0·002 and 0·016, respectively). The association between IgG isotypes of pathogen‐ or autoimmune disease‐related antibodies and the IgG nAAbs may underscore the immune response‐inducible nature of the diseases investigated. The relationship between protective anti‐dsDNA IgM and the IgM isotype of anti‐F4 and anti‐CS may provide immunoserological evidence for the beneficial roles of nAAbs in SLE patients.     

A peptide mimic blocks the cross‐reaction of anti‐DNA antibodies with glomerular antigens

Anti‐DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross‐reacting with renal antigens. Previously, we demonstrated that the binding affinity of anti‐DNA antibodies to self‐antigens is isotype‐dependent. Furthermore, significant variability in renal pathogenicity was seen among a panel of anti‐DNA isotypes [derived from a single murine immunoglobulin (Ig)G3 monoclonal antibody, PL9‐11] that share identical variable regions. In this study, we sought to select peptide mimics that effectively inhibit the binding of all murine and human anti‐DNA IgG isotypes to glomerular antigens. The PL9‐11 panel of IgG anti‐DNA antibodies (IgG1, IgG2a, IgG2b and IgG3) was used for screening a 12‐mer phage display library. Binding affinity was determined by surface plasmon resonance. Enzyme‐linked immunosorbent assay (ELISA), flow cytometry and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP, or ‘ALW’) which binds to all PL9‐11 IgG isotypes. Preincubation with the ALW peptide reduced the binding of the PL9‐11 anti‐DNA antibodies to DNA, laminin, mesangial cells and isolated glomeruli significantly. Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to anti‐DNA antibodies by alanine scanning. Finally, ALW inhibited the binding of murine and human lupus sera to dsDNA and glomeruli significantly. In conclusion, by inhibiting the binding of polyclonal anti‐DNA antibodies to autoantigens in vivo, the ALW peptide (or its derivatives) may potentially be a useful approach to block anti‐DNA antibody binding to renal tissue.     

Clinical application of anti double-stranded and single-stranded DNA antibodies measurements by enzyme-linked immunosorbent assay (ELISA) using E. coli plasmid DNA]

Anti double-stranded (ds) and single-stranded (ss) DNA IgG antibodies were detected in patients with various connective tissue diseases by RECOMBIGEN ELISA Anti ds-DNA Kit and ELISA Anti ss-DNA Kit (Nippon DPC Co) using E. coli plasmid DNA. High levels of anti ds-DNA and anti ss-DNA antibodies were mostly found and distributed in patients with systemic lupus erythematosus(SLE) and allied disorders. The sensitivity of Anti ds-DNA Kit was higher than that of current ELISA kit using calf thymus DNA antigen. In SLE patients with anti DNA antibodies sixty per cent of sera were reacted with both ds- and ss-DNA antigens, and forty per cent of sera were reacted only with ss-DNA antigen; there was no sample reacted positively with ds-DNA antigen alone. RECOMBIGEN ELISA Anti ds-DNA Kit and ELISA Anti ss-DNA Kit are highly sensitive and useful methods to detect anti ds- and/or ss-DNA IgG antibodies in patients with SLE.     

Crossreactivity of Human Anti-dsDNA Antibodies to Phosphorylcholine: Clues to Their Origin

The presence of anti-double stranded DNA (dsDNA) antibodies is a serological diagnostic feature of systemic lupus erythematosus (SLE), an autoimmune rheumatic disorder. Studies by several investigators have suggested that a response to a microbial antigen can lead to the induction of SLE-like autoimmunity, in both humans and mice, since anti-dsDNA antibodies have been shown to crossreact with foreign antigens. In particular, anti-DNA antibodies have been shown to crossreact with phosphorylcholine (PC), a dominant epitope on pneumococcal cell wall polysaccharide. We have investigated the binding characteristics of human polyclonal anti-DNA antibodies from the sera of SLE patients. In this study we show that the DNA binding of polyclonal serum derived antibodies can be partially inhibited by phosphorylcholine (PC). The binding of affinity-purified anti-DNA antibodies from the sera of patients with SLE was also found to be inhibited by PC. We further demonstrated that the serum IgG1 (T dependent) anti-DNA response was more likely to crossreact with PC than the IgG2 (T independent) response to DNA. The studies suggest there may be a T dependent and T independent response to DNA with the T dependent response displaying more crossreactivity with microbial antigen.     

Fine specificity of autoantibodies to calreticulin: epitope mapping and characterization

Extracellular calreticulin (CRT) as well as anti‐CRT antibodies have been reported in patients with various autoimmune disorders and CRT has been implicated in ‘epitope spreading’ to other autoantigens such as the Ro/SS‐A complex. In addition, antibodies against parasite forms of the endoplasmic reticulum chaperone, CRT, have been found in patients suffering from onchocerciasis and schistosomiasis. In this study, we screened sera for anti‐CRT antibodies from patients with active and inactive systemic lupus ertythematosus (SLE) and primary or secondary Sjögren’s syndrome. Approximately 40% of all SLE patients were positive for anti‐CRT antibodies. The antigenic regions of CRT were determined using full length CRT and fragments of CRT prepared in yeast and Escherichia coli, respectively. Synthetic 15mer peptides corresponding to the major autoantigenic region of CRT (amino acids 1–289), each one overlapping by 12 amino acids, were used to map the B cell epitopes on the CRT protein recognized by autoimmune sera. Major antigenic epitopes were found to be associated with the N‐terminal half of the protein in 69% of the SLE sera from active disease patients, while the C‐domain was not antigenic. Major epitopes were found to be reactive with antibodies in sera from SLE patients with both active and inactive disease, spanning different regions of the N and P‐domains. Sera from both healthy and disease controls and primary Sjögren’s syndrome patients were non‐reactive to these sequences. Limited proteolysis of CRT with two major leucocyte serine proteases, elastase and cathepsin G, demonstrated that an N‐terminal region of CRT is resistant to digestion. Interestingly, some of the epitopes with the highest reactivity belong to the fragments of the protein which bind to C1q and inhibit complement activation. Whether C1q association with CRT is a pathological or protective interaction between these two proteins is currently under investigation.     

Autoantibodies to crude human leucocyte interferon (IFN), native human IFN, recombinant human IFN-alpha 2b and human IFN-gamma in healthy blood donors.

Recently, naturally occurring antibodies to IFN-alpha were discovered in a few systemic lupus erythematosus (SLE) and cancer patients; however, in most patients monitored for anti-IFN antibodies before treatment, no antibodies were found. In an attempt to explain the 'IFN-blocking effect' that we observed in all serum samples we investigated 200 sera from healthy blood donors. We isolated the globulin fraction, and used rabbit anti-human IgG and IgM columns, protein A columns and T-gel affinity chromatography to isolate human IgG and IgM. All sample fractions were tested in a biological IFN neutralization assay by means of a sensitive MTT-assay. We found that normal human serum contained autoantibodies to crude human leucocyte IFN, native human fibroblast IFN, recombinant human leucocyte IFN-alpha 2b and recombinant human IFN-gamma, and that these naturally occurring antibodies were biologically active immunoglobulins of IgG and IgM type. These anti-IFN antibodies were also present in purified human normal immunoglobulin pools. We conclude that all humans have naturally occurring anti-interferon antibodies in their serum, and it is a tempting theory that human cytokines and lymphokines are, at least partly, regulated by immunoglobulins.     

Immunoglobulin A anti‐phospholipid antibodies in Swedish cases of systemic lupus erythematosus: associations with disease phenotypes,vascular events and damage accrual

Immunoglobulin (Ig) G‐ and IgM‐class anti‐cardiolipin antibodies (aCL) and lupus anti‐coagulant (LA) are included in the 1997 update of the American College of Rheumatology (ACR‐97) systemic lupus erythematosus (SLE) criteria. Despite limited evidence, IgA‐aCL and IgA anti‐β₂‐glycoprotein‐I (anti‐β₂GPI) were included in the 2012 Systemic Lupus International Collaborating Clinics criteria. The present study aimed to evaluate IgG‐/IgA‐/IgM‐aCL and anti‐β₂GPI occurrence in relation to disease phenotype, smoking habits, pharmacotherapy, anti‐phospholipid syndrome (APS) and organ damage among 526 Swedish SLE patients meeting ACR‐97. Patients with rheumatoid arthritis (n = 100), primary Sjögren’s syndrome (n = 50) and blood donors (n = 507) served as controls. Anti‐phospholipid antibodies (aPL) were analysed by fluoroenzyme‐immunoassays detecting aCL/anti‐β₂GPI. Seventy‐six (14%) SLE cases fulfilled the Sydney APS‐criteria, and ≥ 1 aCL/anti‐β₂GPI isotype (IgG/IgA/IgM) occurred in 138 SLE patients (26%). Forty‐five (9%) of the SLE cases had IgA‐aCL, 20 of whom (4%) lacked IgG‐/IgM‐aCL. Seventy‐four (14%) tested positive for IgA anti‐β₂GPI, 34 (6%) being seronegative regarding IgG/IgM anti‐β₂GPI. Six (1%) had APS manifestations but were seropositive regarding IgA‐aCL and/or IgA anti‐β₂GPI in the absence of IgG/IgM‐aPL and LA. Positive LA and IgG‐aPL tests were associated with most APS‐related events and organ damage. Exclusive IgA anti‐β₂GPI occurrence associated inversely with Caucasian ethnicity [odds ratio (OR) = 0·21, 95% confidence interval (CI) = 0·06–0·72) and photosensitivity (OR = 0·19, 95% CI = 0·05–0·72). Nephritis, smoking, LA‐positivity and statin/corticosteroid‐medication associated strongly with organ damage, whereas hydroxychloroquine‐medication was protective. In conclusion, IgA‐aPL is not rare in SLE (16%) and IgA‐aPL analysis may have additional value among SLE cases with suspected APS testing negative for other isotypes of aPL and LA.     

Human antibodies against formaldehyde-human serum albumin conjugates or human serum albumin in individuals exposed to formaldehyde

Sera from patients undergoing hemodialysis with formaldehyde (F)-sterilized dialyzers were studied to determine if antibodies against F conjugated to human serum albumin (HSA) could be detected. F-human serum albumin (F-HSA) conjugates were prepared using ratios of F to HSA that did not precipitate the HSA. The F-HSA conjugates migrate differently electrophoretically than HSA with an increased negative charge of F-HSA as compared with HSA. The F-HSA was used in an ELISA. The results demonstrated that in certain sera, IgG, IgM, IgA and IgE antibodies against F-HSA could be measured. In the highest titered sera, it was shown that the IgG antibody was not directed against F alone or F-lysine but against an antigenic grouping of F-HSA. No correlation of either IgG or IgE antibodies with immune complex or allergic reactions was found in this series of dialysis patients. Some sera from dialysis patients had antibody activity against HSA. Sera from 2 physicians with rhinitis after F exposure had no antibody activity against F-HSA or HSA. Two nurses with a history of F-induced asthma had no IgG antibodies but did have IgE antibodies against F-HSA and HSA. This spectrum of immunologic responses is analogous to responses in dogs immunized with F or F dog albumin. We have not been able to identify anti HSA antibodies in patients reactive to other hapten-HSA compounds and it is suggested that anti HSA antibodies in F-exposed humans may relate to the F exposure.     

Cross-recognition between histones and La/SSB may account for anti-DNA reactivity in SLE patients

Antibodies to La/SSB are detected in sera of patients with primary Sjogren's syndrome (pSS) and systemic lupus erythematosus (SLE). The vast majority of anti-La/SSB positive sera contain antibodies directed towards a linear B-cell epitope of La/SSB spanning the sequence 349-364aa (pep349-364). The aim of this study was to evaluate the fluctuation of antibody levels to major B-cell epitopes of La/SSB over time and investigate for their possible crossreactions. Sequential sera from 15 SLE and 15 pSS patients, followed from 3 to 10 years were obtained. All patients with SLE were positive for anti-Ro/SSA, anti-La/SSB and anti-dsDNA antibodies and patients with pSS were positive for anti-Ro/SSA and anti-La/SSB antibodies. Sera from 30 patients with SLE without anti-La/SSB antibodies and 30 healthy individuals served as disease and negative control respectivelly. All sera tested for the presence of anti-pep349-364 antibodies, using a specific ELISA. Specific anti-pep349-364 IgG was purified from sera of SLE patients and evaluated for cross reactivity against dsDNA and histones. In all SLE sera the levels of anti-pep349-364 antibodies varied in time and fluctuated in parallel with anti dsDNA antibodies. Anti-pep349-364 IgG purified from 7 SLE patients. Five out of 7 were found to react with calf thymus DNA in ELISA. All purified (7/7) anti-pep349-364 IgG preparations reacted with histone H1 and failed to produce a positive immunofluorescence pattern in Crithidia luciliae anti-dsDNA assay which lacks histones. Competative inhibition experiments demonstrated that histone H1 could inhibit completely the binding of anti-pep349-364 IgG to pep349-364 while pep349-364 inhibited by 70% the binding of anti-pep349-364 IgG to histone H1. These findings indicate that a subgroup of SLE patients possess cross-reacting anti-histone H1 antibodies and anti-pep349-364 antibodies, which can be faulty considered as anti-dsDNA reactivity in regular ELISA techniques.     

Immunogenicity of singlet oxygen modified human DNA: implications for anti-DNA antibodies in systemic lupus erythematosus

Reactive oxygen species (ROS)-modified DNA has been shown to be a better and more discriminating immunogen than native DNA (nDNA) for the production of anti-DNA autoantibodies in SLE (systemic lupus erythematosus). Among ROS, the role of hydroxyl radical (.OH) in the induction of damage and modification of nDNA has been extensively studied while such documentation implicating singlet oxygen ((1)O(2)) in inducing immunogenicity in nDNA leading to the production of anti-double-stranded (ds) DNA autoantibodies in SLE is not yet available. This prospective study was undertaken to evaluate the immunogenicity of healthy human dsDNA modified with (1)O(2) generated by methylene blue plus radiant light. Female rabbits were immunized with (1)O(2)-modified human dsDNA to raise anti-dsDNA antibodies. (1)O(2)-modified anti-dsDNA rabbit immune sera and the (1)O(2)-modified anti-dsDNA rabbit purified immunoglobulin G (IgG) were tested against a variety of dsDNA antigenic substrates through direct enzyme-linked immunosorbent assay (ELISA). The immunogenicity of (1)O(2)-modified human dsDNA was further evaluated by studying its immunoreactivity with SLE patients' sera and SLE patients' purified anti-dsDNA IgG. As compared to healthy human sera, (1)O(2)-modified anti-dsDNA rabbit immune sera as well as the (1)O(2)-modified anti-dsDNA rabbit purified IgG demonstrated a strong affinity towards (1)O(2)-modified human dsDNA(.)(1)O(2)-modified human dsDNA proved to be potentially more immunogenic against SLE patients' whole sera and SLE patients' purified IgG as compared to healthy human sera. Our findings suggest that (1)O(2) may also be inducing immunogenicity in native dsDNA resulting in the production of anti-dsDNA autoantibodies as seen in SLE patients.     

Similar frequency of autoantibodies against 70-kD class heat-shock proteins in healthy subjects and systemic lupus erythematosus patients

Stress or heat-shock proteins may be involved in the initiation and perpetuation of autoimmune diseases. In order to investigate a possible role of autoantibodies against the 70-kD family of heat-shock proteins in systemic lupus erythematosus (SLE), sera of SLE patients and healthy subjects were tested for the presence of IgG and IgM antibodies to 70-kD class proteins. These proteins were purified by affinity chromatography on ATP-agarose and used in Western blotting studies. The data obtained revealed that antibodies to the 72-kD and the 73-kD heat-shock proteins occurred with similar frequencies both in healthy subjects and SLE patients. Thus, approximately 20% of the sera in each group contained IgG antibodies, and IgM antibodies were detected in about 30% of the sera tested. Moreover, in SLE patients no association between the occurrence and litre of these antibodies and disease activity was found. These data suggest that antibodies to the 70-kD class heat-shock proteins are naturally occurring and argue therefore against an involvement of these antibodies in the pathogenesis of SLE.     

Antibody reactivity against single stranded DNA of various species in normal children and in children with diffuse connective tissue diseases

The aim of this work was to study possible differences in the humoral response against autologous and heterologous (bacterial and mammalian) ssDNA in children with diffuse connective tissue diseases (DCTD) compared with age matched controls. We found that IgM anti ssDNA were significantly increased in systemic lupus eritematosus (SLE) and in juvenile arthritis (JA), but not in juvenile dermatomyositis (JDM). IgG anti ssDNA were significantly elevated only in children with SLE. We next evaluated the binding specificity to human and bacterial ssDNA by inhibition assays. We found that SLE and JA sera recognised epitopes shared in common to endogenous and bacterial ssDNA. In contrast, in normal subjects IgG binding to bacterial DNA was not inhibited by human DNA, while IgG anti human ssDNA were cross reactive with the bacterial antigen. These data suggest that natural antibodies (IgM) producing cells are activated in some but not all DCTD, and that normal children have different reactivity against autologous and heterologous ssDNA with respect to SLE and JA patients.     

Serum concentration of immunoglobulin G‐type antibodies against the whole Epstein–Barr nuclear antigen 1 and its aa35–58 or aa398–404 fragments in the sera of patients with systemic lupus erythematosus and multiple sclerosis

Several studies suggest that infection by Epstein–Barr virus (EBV) might be one of the environmental factors which facilitates the development of autoimmune disorders in genetically susceptible individuals. Recent data indicate that high anti‐Epstein–Barr nuclear antigen 1 (EBNA)‐1 immunoglobulin (Ig)G titre is a strong risk factor for multiple sclerosis (MS) in patients both with and without the main genetic predisposing trait, human leucocyte antigen (HLA)‐DRB1*15:01. Because no similar studies have been published in systemic lupus erythematosus (SLE) patients, we determined the HLA‐DRB1*15:01 carrier state and the serum titres against the whole EBNA‐1 and its small fragments aa35–58 and aa398–404 in 301 SLE patients, 135 MS patients and in 345 healthy controls. The carrier state of the HLA‐DRB1*15:01 allele was deduced from genotyping of a tagSNP (rs3135388) by applying a Taqman‐based assay. The serum concentrations of antibodies to EBNA‐1 and its aa35–58 or aa398–404 fragments were determined using a commercial assay (ETI‐EBNA‐G) and home‐made enzyme‐linked immunosorbent assays, respectively. The serum concentration of anti‐EBNA‐1 antibodies was significantly (P < 0·001) higher both in MS and SLE patients than in controls. Similar significant differences were found both in HLA‐DRB1*15:01 carriers and non‐carriers. Furthermore, titres of antibodies against the aa35–58 EBNA‐1 fragment were elevated both in MS and SLE patients. By contrast, the levels of aa398–404 EBNA‐1 antibodies were elevated significantly only in the SLE patients. These findings indicate that high anti‐EBNA‐1 IgG titres are HLA‐DRB1*15:01‐independent risk factors not only for MS, but also for SLE, while high antibody titres against the aa398–404 fragment are characteristic for SLE.     

New epitopes and function of anti‐M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome

M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjögren's syndrome (SS) carried inhibitory autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti‐M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human‐M3R including the N‐terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme‐linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti‐M3R antibody‐positive SS, ‐negative SS and controls for 12 h. After loading with Fluo‐3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca²⁺ concentrations [(Ca²⁺)i] were measured. Antibodies to the N‐terminal, first, second and third loops were detected in 42·9% (18 of 42), 47·6% (20 of 42), 54·8% (23 of 42) and 45·2% (19 of 42) of SS, while in 4·8% (two of 42), 7·1% (three of 42), 2·4% (one of 42) and 2·4% (one of 42) of controls, respectively. Antibodies to the second loop positive SS‐IgG inhibited the increase of (Ca²⁺)i induced by cevimeline hydrochloride. Antibodies to the N‐terminal positive SS‐IgG and antibodies to the first loop positive SS‐IgG enhanced it, while antibodies to the third loop positive SS‐IgG showed no effect on (Ca²⁺)i as well as anti‐M3R antibody‐negative SS‐IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti‐M3R antibodies on salivary secretion might differ based on these epitopes.     

Epitope mapping with synthetic peptides of 52-kD SSA/Ro protein reveals heterogeneous antibody profiles in human autoimmune sera.

The reactivity of autoantibodies present in the sera of 489 patients with Sjögren's syndrome (SS), systemic lupus erythematosus (SLE) and other autoimmune diseases was investigated by ELISA using recombinant 52-kD SSA/Ro protein (rRo52) and 39 overlapping synthetic peptides representing the entire sequence of Ro52. We report that IgG antibodies reacting with rRo52 were present in the sera of a large number of patients with SS (67% of patients with primary SS and 46% of patients with SS associated with SLE), whereas they were less frequent (10-25%) in SLE, rheumatoid arthritis (RA), juvenile chronic arthritis (JCA) and mixed connective tissue disease (MCTD), and absent in scleroderma. Among the 39 peptides tested, five were recognized by sera from 30-65% of patients with SS, namely peptides representing residues 2-11, 107-122, 107-126, 277-292 and 365-382. Patients with JCA had raised levels of IgG antibodies reacting with peptides 2-11 and 365-382, and 51% of patients with MCTD had raised levels of IgG antibodies reacting with peptide 365-382. None of the five peptides was recognized by more than 20% of sera from patients with SLE and RA. Interestingly, and of importance in the field of diagnostic tests based on peptides, the reactivity of antibodies to the Ro52 synthetic peptides varied greatly according to the origin of sera. Inhibition experiments using either patients' sera or antibodies induced in rabbits against Ro52 peptides showed that the four domains 2-11, 107-122, 277-292 and 365-382 are accessible on the surface of the Ro52 protein. These regions may thus be involved in the induction of specific antibodies in autoimmune patients.     

Evaluation of the cross-reaction between anti-DNA and anti-cardiolipin antibodies in SLE and experimental animals.

The cross-reaction between anti-DNA and anti-cardiolipin IgG antibodies and its relation to the standard test for syphilis was studied with sera and monoclonal antibodies derived from human patients and mice with systemic lupus erythematosus (SLE). Syphilitic sera of humans and rabbits infected with the spirochete Treponema pallidum were also tested in this study. In addition, rabbits were immunized with ssDNA and cardiolipin and the cross-reactions of the induced antibodies were studied in two different assay systems. The results of these experiments suggest: that the anti-DNA and anti-cardiolipin IgG autoantibodies in SLE sera constitute separate antibody populations and, therefore, cardiolipin cannot play a role in the induction of immune response to DNA in SLE; that in immunized experimental animals there is a significant level of cross-reaction between anti-DNA and anti-cardiolipin-the detection of this cross-reaction depends on highly amplified solid phase assay systems which measure low affinity antibodies and that there is no correlation between the activity of syphilitic sera in the serologic test for syphilis and their binding to pure cardiolipin-this implies that cardiolipin may not be the dominant ingredient in this test as previously proposed.