Guanine polynucleotides are self‐antigens for human natural autoantibodies and are significantly reduced in the human genome

In the course of investigating anti‐DNA autoantibodies, we examined IgM and IgG antibodies to poly‐G and other oligonucleotides in the sera of healthy persons and those diagnosed with systemic lupus erythematosus (SLE), scleroderma (SSc), or pemphigus vulgaris (PV); we used an antigen microarray and informatic analysis. We now report that all of the 135 humans studied, irrespective of health or autoimmune disease, manifested relatively high amounts of IgG antibodies binding to the 20‐mer G oligonucleotide (G20); no participants entirely lacked this reactivity. IgG antibodies to homo‐nucleotides A20, C20 or T20 were present only in the sera of SLE patients who were positive for antibodies to dsDNA. The prevalence of anti‐G20 antibodies led us to survey human, mouse and Drosophila melanogaster (fruit fly) genomes for runs of T20 and G20 or more: runs of T20 appear > 170 000 times compared with only 93 runs of G20 or more in the human genome; of these runs, 40 were close to brain‐associated genes. Mouse and fruit fly genomes showed significantly lower T20/G20 ratios than did human genomes. Moreover, sera from both healthy and SLE mice contained relatively little or no anti‐G20 antibodies; so natural anti‐G20 antibodies appear to be characteristic of humans. These unexpected observations invite investigation of the immune functions of anti‐G20 antibodies in human health and disease and of runs of G20 in the human genome.     

An antibody profile of systemic lupus erythematosus detected by antigen microarray

Patients with systemic lupus erythematosus (SLE) produce antibodies to many different self‐antigens. Here, we investigated antibodies in SLE sera using an antigen microarray containing many hundreds of antigens, mostly self‐antigens. The aim was to detect sets of antibody reactivities characteristic of SLE patients in each of various clinical states – SLE patients with acute lupus nephritis, SLE patients in renal remission, and SLE patients who had never had renal involvement. The analysis produced two novel findings: (i) an SLE antibody profile persists independently of disease activity and despite long‐term clinical remission, and (ii) this SLE antibody profile includes increases in four specific immunoglobulin G (IgG) reactivities to double‐stranded DNA (dsDNA), single‐stranded DNA (ssDNA), Epstein–Barr virus (EBV) and hyaluronic acid; the profile also includes decreases in specific IgM reactivities to myeloperoxidase (MPO), CD99, collagen III, insulin‐like growth factor binding protein 1 (IGFBP1) and cardiolipin. The reactivities together showed high sensitivity (> 93%) and high specificity for SLE (> 88%). A healthy control subject who had the SLE antibody profile was later found to develop clinical SLE. The present study did not detect antibody reactivities that differentiated among the various subgroups of SLE subjects with statistical significance. Thus, SLE is characterized by an enduring antibody profile irrespective of clinical state. The association of SLE with decreased IgM natural autoantibodies suggests that these autoantibodies might enhance resistance to SLE.     

Self‐dsDNA in the pathogenesis of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic and poly‐aetiological autoimmune disease characterized by the production of antibodies to autologous double‐stranded DNA (dsDNA) which serve as diagnostic and prognostic markers. The defective clearance of apoptotic material, together with neutrophil extracellular traps (NETs), provides abundant chromatin or self‐dsDNA to trigger the production of anti‐dsDNA antibodies, although the mechanisms remain to be elucidated. In SLE patients, the immune complex (IC) of dsDNA and its autoantibodies trigger the robust type I interferon (IFN‐I) production through intracellular DNA sensors, which drives the adaptive immune system to break down self‐tolerance. In this review, we will discuss the potential resources of self‐dsDNA, the mechanisms of self‐dsDNA‐mediated inflammation through various DNA sensors and its functions in SLE pathogenesis.     

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.     

Human Polyreactive IgM Monoclonal Antibodies with Blocking Activity Against Self-Reactive IgG

Natural IgM antibodies have been found to be involved in the control of IgG reactivity in normal serum. The authors investigated the blocking activity of four human IgM monoclonal antibodies (BY-2, BY-7, BY-10 and IRM-7) derived from B-cells from blood samples of three renal dialysis patients, which had shown multispecific properties similar to those observed for natural polyreactive autoantibodies. To achieve this, competitive inhibition assays were performed with these MoAbs on the binding of IgG purified from a healthy control, three patients with SLE, and two patients with autoimmune thyroiditis, to histone, dsDNA, RNP and thyroglobulin. MoAbs inhibited binding of self-reactive IgG to histone and dsDNA, but not to thyroglobulin or RNP, of natural and active or inactive phase disease-associated autoreactive IgG. The inhibitory effect of the MoAbs was mediated by V-region dependent interactions with autoreactive IgG, as shown by the ability of these MoAbs to block the binding of F(ab')₂ fragments of autoreactive IgG to antigens (histone and dsDNA). The blocking of autoantibody activity was dose-dependent with maximal inhibition occurring at a specific molar ratio between the patient's IgG and a given MoAb. In contrast, MoAbs did not inhibit binding of IgG alloantibodies present in the sera of four polytransfused renal dialysis patients to target antigens on the surface of different cells. These results support the concept of a functional idiotypic network regulating autoimmune responses, and suggest that the IgM MoAbs under study may be natural polyreactive antibodies belonging to the physiological network of autoantibodies with highly connected V-regions, capable of binding and functionally neutralizing V-regions of natural and pathologic autoantibodies.     

Intramolecular epitope spreading among anti-caspase-8 autoantibodies in patients with silicosis,systemic sclerosis and systemic lupus erythematosus,as well as in healthy individuals

Dysregulation of apoptosis through the Fas-Fas ligand pathway is relevant in autoimmune disease onset. We recently reported elevated serum levels of sFas in patients with silicosis, systemic sclerosis (SSC) and systemic lupus erythematosus (SLE), and proposed a block of apoptosis in the pathogenesis. The disturbance of apoptosis in lymphocytes including autoreactive clones could induce autoantibody production. Since autoantibodies directed against unknown antigens are present in the sera of these patients, the sera samples were examined for the presence of autoantibodies directed to caspase-8. Using Western blotting, autoantibodies against caspase-8 were detected in healthy individuals and in over 60% of patients. Using epitope mapping employing 12 amino acid polypeptides with SPOTs system, a minimum of 4 epitopes and a maximum of 13 were found, which implied that epitope spreading was in progress. It is noteworthy that two important catalytic cystein residues were included within the epitopes; firstly the active site cystein Cys287, and secondly Cys360 located in the unique pentapeptide motif QACQG. Using recombinant human caspase-8 linked protein chip array, autoantibodies were identified and molecular weight determined. The antibodies were mainly IgG; 80% were subclass IgG1(lambda); 20% were IgG4(kappa). Despite the ratio of human light chain kappa:lambda = 2:1, the predominance of IgG1(lambda) is noticeable. Anti-caspase-8 autoantibodies are detectable in healthy individuals and in patients suffering silicosis, SSc or SLE. A few epitopes were detected in healthy individuals compared to those suffering autoimmune diseases, indicating the intramolecular epitope spreading. Relationship of autoantibodies and the clinical background of the patients requires clarification.     

Characterization of autoantibodies to endothelial cells in systemic sclerosis (SSc): association with pulmonary fibrosis

To determine the prevalence and the characterization of antibodies to endothelial cells in patients with SSc, serum samples from 80 patients with SSc, 20 patients with systemic lupus erythematosus (SLE), and 20 healthy control subjects were examined by ELISA using cultured human umbilical vein endothelial cells (HUVEC), indirect immunofluorescence analysis (IIF), and immunoblotting using cytoplasmic extract of HUVEC. IgG and/or IgM isotype anti-endothelial cell antibodies (AECA) were demonstrated by ELISA in 43 of 80 patients with SSc (54%), in 15 of 20 patients with SLE (75%), and in none of 20 healthy control subjects. Immunofluorescence analysis on HUVEC substrate showed homogeneous cytoplasmic staining. Immunoblotting demonstrated that these patients had antibodies directed to one or several antigens of approximately 60, 90, 110 and 140 kD, and the most common responses were to the 90-kD antigen. By the immunofluorescence method using HUVEC, affinity-purified anti-90-kD antibodies showed identical cytoplasmic staining to that produced by sera positive for AECA. Furthermore, AECA were closely correlated with pulmonary fibrosis in patients with SSc. These findings suggest that patients with SSc have abnormal antibodies to endothelial cell antigens, and support the hypothesis that endothelial dysfunction is involved in the development of this disease.     

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.     

Cluster analysis of human autoantibody reactivities in health and in type 1 diabetes mellitus: a bio-informatic approach to immune complexity

Informatic methodologies are being applied successfully to analyze the complexity of the genome. But beyond the genome, the immune system reflects the state of the body in health and disease. Traditionally, immunologists have reduced the immune system, where possible, to one-to-one relationships between particular antigens and particular antibodies or T-cell clones. Autoimmune diseases, caused by an immune attack against a body component, are usually investigated by following the response to single self-antigens. In this study, we apply informatics to analyze patterns of autoantibodies rather than single species of autoantibodies. This study was designed not to replace traditional approaches to immune diagnosis, but to test whether meaningful patterns of autoantibodies might exist. Using an unbiased solid-phase ELISA antibody test, we detected serum IgG and IgM antibodies in the sera of 20 healthy persons and 20 persons with type 1 diabetes mellitus binding to an array of 87 different antigens, mostly self-antigens. The healthy subjects manifested autoantibodies to a variety of self-antigens, many known to be associated with autoimmune diseases. We investigated the patterns of these autoantibodies using a coupled two-way clustering algorithm developed for analyzing data from gene arrays. We now report that the reactivity patterns of autoantibodies to particular subsets of self-antigens exhibited non-trivial structure, which significantly discriminated between healthy persons and persons with type 1 diabetes. The results show that despite the wide prevalence of autoantibodies, the patterns of reactivity to defined subsets of self-antigens can provide information about the state of the body.     

Structural properties of the glycoplasmanylinositol anchor phospholipid of the complement membrane attack complex inhibitor CD59.

Peripheral blood B cells from patients with systemic autoimmune disease and healthy volunteers were immortalized using EBV and the frequencies of B cell precursors that produced immunoglobulin class-specific antibodies against anti-nRNP, a specific marker for mixed connective tissue disease, were assessed using limiting dilution analysis. The frequencies of EBV-induced B cell precursors that produced IgG anti-nRNP were correlated closely with the serum titres of the corresponding autoantibodies, which indicates that B cell precursors that produced potentially pathogenic autoantibodies could be immortalized from the peripheral blood of the patients by EBV. In contrast, the frequency of EBV-induced B cell precursors that produced IgM anti-nRNP in patients with systemic autoimmune disease was comparable to that in healthy volunteers and greater than those that produced IgG and IgA anti-nRNP. Moreover, many of the clones that produced IgM antibodies against nRNP reacted with other autoantigens, such as double-stranded DNA, single-stranded DNA and rabbit IgG. These polyreactive IgM antibodies are believed to belong to the 'natural antibodies', to be coded by the germline immunoglobulin V genes, and to react with evolutionarily conserved structural cellular components, including nRNP. Our finding that nRNP is one of the target antigens for this polyreactive autoantibody may lead to the elucidation of the origin of the pathogenic IgG and IgA anti-nRNP antibodies found in sera from patients with systemic autoimmune diseases.     

Pathogenic anti-DNA antibodies in SLE: idiotypic families and genetic origins

We have adopted an idiotypic approach to study the double stranded DNA (dsDNA) binding antibodies of systemic lupus erythematosus (SLE). Three anti-idiotypic reagents, 8.12, 3I, and F4, identify cross reactive idiotypes that are each expressed on anti-dsDNA antibodies in the sera of many patients with SLE. These idiotypic antibodies are implicated in the pathogenesis of SLE as they are present in immune complex deposits in the kidneys of patients with SLE glomerulonephritis. The autoantibody associated idiotypes are also expressed on antibodies that do not bind DNA. We are investigating the origin of the pathogenic anti-dsDNA antibodies of SLE by comparing the autoantibodies, the antibodies to foreign antigens, and the myeloma proteins that express each SLE associated idiotype. In conjunction with serological analysis of these idiotypic systems, molecular genetic studies indicate that both the 8.12 and the 3I autoantibody associated idiotypes may be germline encoded, while the F4 idiotype is generated by somatic mutation. The data further suggest that the antigenic specificity of the pathogenic anti-DNA antibodies of SLE is acquired through somatic mutation of germline immunoglobulin genes. By studying the regulation of genes capable of encoding pathogenic autoantibodies, in both SLE patients and non-autoimmune individuals, we may be able to elucidate the pathogenesis of autoimmune disease and begin to design more effective therapeutic interventions.     

Pathogenic Anti-DNA Antibodies in SLE: Idiotypic Families and Genetic Origins

We have adopted an idiotypic approach to study the double stranded DNA (dsDNA) binding antibodies of systemic lupus erythematosus (SLE). Three anti-idiotypic reagents, 8.12, 31, and F4, identify cross reactive idiotypes that are each expressed on anti-dsDNA antibodies in the sera of many patients with SLE. These idiotypic antibodies are implicated in the pathogenesis of SLE as they are present in immune complex deposits in the kidneys of patients with SLE glomerulonephritis. The autoantibody associated idiotypes are also expressed on antibodies that do not bind DNA. We are investigating the origin of the pathogenic anti-dsDNA antibodies of SLE by comparing the autoantibodies, the antibodies to foreign antigens, and the myeloma proteins that express each SLE associated idiotype. In conjunction with serological analysis of these idiotypic systems, molecular genetic studies indicate that both the 8.12 and the 31 autoantibody associated idiotypes may be germline encoded, while the F4 idiotype is generated by somatic mutation. The data further suggest that the antigenic specificity of the pathogenic anti-DNA antibodies of SLE is acquired through somatic mutation of germline immunoglobulin genes. By studying the regulation of genes capable of encoding pathogenic autoantibodies, in both SLE patients and non-autoimmune individuals, we may be able to elucidate the pathogenesis of autoimmune disease and begin to design more effective therapeutic interventions.     

Over-expression of TATA binding protein (TBP) and p53 and autoantibodies to these antigens are features of systemic sclerosis, systemic lupus erythematosus and overlap syndromes

The aim of this study was to determine the expression levels of p53 and TATA binding protein (TBP) and the presence of autoantibodies to these antigens in Asian Indian patients with systemic sclerosis (SSc), overlap syndromes (OS) and systemic lupus erythematosus (SLE). Fifty patients with SSc, 20 with OS, including mixed connective tissue diseases (MCTD), 20 with SLE, 10 disease controls (DC) and 25 controls (C) were studied. The over-expression of p53 and TBP antigen was determined quantitatively by sandwich enzyme-linked immunosorbent assay (ELISA), varies between four- and sevenfold higher in patients with SSc, OS and SLE, in comparison to DC and C. The expressed protein antigens were not present as free antigens but as immune-complexes. Autoantibodies to p53 were detected by ELISA in 78% subjects with SSc, 100% with OS and 80% with SLE. Autoantibodies to TBP were observed in 28% patients with SSc, 25% with OS and 15% with SLE. In comparison to healthy controls, the titre of antibodies to p53 was significantly higher in patients with SSc (P = 0.00001) than the patients with OS (P = 0.00279) and SLE (P = 0.00289), whereas the titre of antibodies to TBP was higher in patients with OS (P = 0.00185) than the SLE (P = 0.00673) and the SSc (P = 0.00986) patients. Autoantibodies to p53 and TBP were detected in all these patients and the levels of these two autoantibodies showed weak negative correlation with each other. We propose that the over-expression of these antigens might be due to hyperactive regulatory regions in the p53 and TBP gene.     

Elevated immunoglobulin G antibodies to the proline-rich amino-terminal region of Epstein-Barr virus nuclear antigen-2 in sera from patients with systemic connective tissue diseases and from a subgroup of Sjögren's syndrome patients with pulmonary involvements

Associations of Epstein-Barr virus (EBV) and autoimmune diseases have been hypothesized. We have analysed IgG antibodies to EBV nuclear antigen (EBNA)-2 in sera from Japanese patients with autoimmune systemic connective tissue diseases (CTD), exemplified by systemic lupus erythematosus (SLE), primary Sjogren's syndrome (SS), rheumatoid arthritis (RA), systemic sclerosis (SSc) and secondary SS (classical CTDs complicated with SS). An enzyme-linked immunosorbent assay (ELISA) which uses glutathione-S-transferase polypeptides fused to EBV nuclear antigen (EBNA)-2 and EBNA-1 was developed. Ratios of IgG antibody reactivity to whole IgG concentrations of sera were calculated to normalize EBNA-2 and EBNA-1 antibody levels to the hypergammaglobulinaemia that occurs in CTD. The ELISA optical density OD(450) readings of IgG antibodies to both the amino-terminal aa 1-116 of EBNA-2 and carboxyl-terminal aa 451-641 of EBNA-1 were elevated significantly in patients with SLE, primary SS, RA, SSc and secondary SS when compared to EBNA-1. The OD readings were divided by serum IgG concentrations to normalize for the hypergammaglobulinaemia. The specific levels of IgG antibodies to the amino-terminal region of EBNA-2 were elevated in patients with SLE, primary SS or RA, as well as those with secondary SS complicated with SLE or RA. The EBNA-2 amino-terminal region contains a polyproline tract and a proline-rich sequence and has considerable amino acid sequence homology with many cellular proline-rich proteins. High ratios of EBNA-2 aa 1-116 to EBNA-1 aa 451-641 IgG antibody levels which probably suggest reactivation of EBV latent infection were associated significantly with pulmonary involvement in SS patients. These results are consistent with the hypothesis that the sequence similarity between the amino-terminal region of EBNA-2 and proline-rich cellular proteins is associated with pathogenesis in a subpopulation of CTD patients, possibly by the molecular mimicry-epitope shift mechanism.     

Serum or breast milk immunoglobulins mask the self‐reactivity of human natural IgG antibodies

B cells producing IgG antibodies specific to a variety of self‐ or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein‐destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab’)₂ fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self‐reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.     

Relationship between antibodies to dsDNA and to soluble cellular antigens and histologically defined glomerulonephritis in patients with SLE

To better define the relationships between circulating autoantibodies and renal involvement in systemic lupus erythematosus (SLE), antibodies to both dsDNA and soluble cellular antigens were detected in sera from a large series of SLE patients. Significantly higher dsDNA binding activities and lower complement levels at onset were found in patients with renal disease; however, this was uniquely due to subjects with diffuse or focal proliferative glomerulonephritis. Patients with membranous nephropathy (MGN) showed very low dsDNA binding activities (6/9 of them being negative for dsDNA antibodies) and normal mean C3 and C4 levels. A comparison between patients with proliferative nephritis and patients without renal involvement with high dsDNA binding activities revealed significantly lower complement levels in the former group. No significant difference was observed in the prevalence of antibodies to soluble cellular antigens between patients with or without renal disease; however, nRNP antibody was two-fold more frequent in patients with MGN than in all other subgroups. This study highlights the close relationship between concurrently high anti-dsDNA and low complement levels and proliferative glomerulonephritis in SLE, and suggests that subjects with MGN may represent a subgroup of SLE patients showing peculiar serological features. Different mechanisms possibly involved in the pathogenesis of MGN in SLE are discussed.     

Decreased levels of autoantibody against histone deacetylase 3 in patients with systemic sclerosis

Systemic sclerosis (SSc) is characterized by immunological abnormalities, especially the production of autoantibodies against various cellular components. Treatment with histone deacetylase (HDAC) inhibitors prevents collagen accumulation in a mouse SSc model. Additionally, autoantibody against HDAC-3 is produced in colon cancer patients, while HDAC-1 and HDAC-2 do not elicit autoantibody response. To determine the presence and levels of antibodies (Abs) against HDAC-3 in SSc. Anti-HDAC-3 Ab was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using human recombinant HDAC-3. The HDAC-3 activity was evaluated by ELISA using the fluorimetric HDAC lysyl substrate that comprises an acetylated lysine side chain. Contrary to our hypothesis that autoimmune background in SSc induced the production of autoantibody against HDACs, IgG and IgM anti-HDAC-3 Ab levels in SSc patients were significantly lower than in normal controls (p < 0.0005 and 0.001, respectively). Furthermore, decreased levels of IgG anti-HDAC-3 Ab were specific to SSc, since IgG anti-HDAC-3 Ab levels in patients with dermatomyositis (DM) and those with systemic lupus erythematosus (SLE) were similar and slightly increased relative to normal controls, respectively. Immunoblotting analysis showed that anti-HDAC-3 Ab was detected in normal controls and patients with DM or SLE, while it was absent in SSc patients. The HDAC-3 activity was significantly inhibited by IgG isolated from sera of normal controls, whereas such inhibitory effect was not observed by IgG isolated from sera of SSc patients. These results indicate the lack of anti-HDAC-3 autoantibody in SSc patients, which is produced in healthy individuals as well as DM and SLE patients, suggesting that this autoantibody might function as protective Ab.     

Autoantibodies to a collagen-specific molecular chaperone, heat-shock protein 47, in systemic sclerosis

Heat-shock proteins are highly conserved and immunogenic proteins, which may be involved in the initiation and perpetuation of autoimmune diseases. Heat-shock protein 47 (HSP47) is expressed by collagen-secreting cells such as fibroblasts and serves as a collagen-specific molecular chaperone that plays a crucial role in collagen metabolism. Abnormal collagen accumulation and autoimmunity are characteristics of systemic sclerosis (SSc). We determined the presence and prevalence of autoantibodies to HSP47 in patients with SSc and also in tight-skin (TSK/+) mice, an animal model for SSc. Anti-HSP47 autoantibodies were present in SSc patients with a frequency of 26%, while patients with systemic lupus erythematosus, those with dermatomyositis, those with keloid and healthy subjects did not have anti-HSP47 antibodies. IgG1 and IgG2 were the major Ig isotypes of the autoantibodies. Patients positive for anti-HSP47 had a significantly shorter duration of disease than those who were negative. Anti-HSP47 autoantibodies were also positive in 79% of TSK/+ mice. Thus, autoantibodies to HSP47 were present in the sera from SSc patients as well as those from TSK mice, and may be associated with the pathogenesis of SSc.     

Detection of antibodies to the Epstein-Barr virus nuclear antigens in the sera from patients with systemic lupus erythematosus

The sera from 65 patients with systemic lupus erythematosus (SLE) were examined by the immunoblotting method to detect antibodies to Epstein-Barr virus (EBV)-associated antigens, especially EBV nuclear antigens (EBNA), and compared with the sera from 66 healthy subjects roughly age- and sex-matched to the patients. Most sera from patients with SLE defined three major EBV-associated antigens with molecular weights (MW) of 70,000 (70K), 90K and 140K in Raji cells, which must correspond to the EBNA-1, 2, and 3, respectively. Approximately 70% of the sera from SLE patients demonstrated the antibodies to the 90K and 140K antigens, whereas the positive rates of these two antibodies were less than 10% in the sera from healthy subjects. The differences of these positive rates of the antibodies between SLE patients and healthy subjects were statistically highly significant. Antibody to EBNA-1 was conspicuously detected in the sera from both SLE patients and healthy subjects, although the difference between the two groups was still significant. The possible role of EBV infection was discussed on the basis of the pathogenesis of SLE.     

Prevalence of systemic autoimmune rheumatic diseases and clinical significance of ANA profile: data from a tertiary hospital in Shanghai,China

It is necessary and useful to explore prevalence of various systemic autoimmune rheumatic diseases (SARDs) in patients with suspicion of having SARDs and to characterize antinuclear antibodies (ANA) profile for identifying different populations (SARDs and non‐SARDs). A total of 5024 consecutive patients with available medical records were investigated, whose sera had been tested for ANA profile, including ANA, anti‐dsDNA and anti‐extractable nuclear antigen (ENA) antibodies, between 31 January 2012 and 26 March 2014. Only 594 (11.8%) patients were diagnosed with SARDs of those suspected with SARDs. The prevalence of systemic lupus erythematosus (SLE) was highest (3.2%), followed by rheumatoid arthritis (RA) (2.5%), primary Sjögren's syndrome (pSS) (1.7%), ankylosing spondylitis (AS) (1.5%), etc. Of females, SLE also showed the highest prevalence (6%), while of males, AS showed the highest prevalence (1.9%). The prevalence of most SARDs was closely associated with age, except mixed connective tissue disease (MCTD), and the variation characteristics among different age groups were different among various SARDs. The prevalence of ANA was significantly increased in most SARD patients [especially in SLE, systemic sclerosis (SSc) and MCTD]. For anti‐ENA antibodies, in contrast to some autoantibodies associated with multiple SARDs (e.g. anti‐SSA, SSB, nRNP), others were relatively specific for certain diseases, such as anti‐dsDNA, Sm, histone, nucleosome and Rib‐P for SLE, anti‐SCL‐70 for SSc and anti‐Jo‐1 for polymyositis/dermatomyositis (PM/DM). Of note, ANA profile appeared to be of little significance for AS, ANCA‐associated vasculitis (AAV), polymyalgia rheumatic (PMR), adult‐onset Still's disease (ASD) and Behcet's disease (BD). The younger were more likely to have the presence of anti‐dsDNA, Sm, histone or Rib‐P for SLE, and anti‐SSA for RA or MCTD. No significant differences for frequencies of ANA and anti‐ENA autoantibodies were found between sexes in most SARDs, with the exception of RA and AS. The present study suggests that, of patients with SARDs‐like clinical manifestations, the proportion of those with true SARDS is small, for most of whom tests for autoantibodies are necessary and useful to help make a prompt and precise diagnosis.