Anti-cardiolipin antibodies in neurological disorders: cross-reaction with anti-single stranded DNA activity.

Antiphospholipid (PL) antibodies have been detected in sera from patients with chronic neurological diseases associated with disorders of immunity. In an isotype specific radioimmunoassay for anti-cardiolipin (CL) antibodies, we found IgM anti-CL (greater than 2 s.d. above mean of controls) in 17/25 (68%) patients with myasthenia gravis (MG), 8/25 (32%) with the Lambert-Eaton myasthenic syndrome (LEMS), 5/17 (29%) with multiple sclerosis and 3/11 (27%) cases of migraine. IgG anti-CL was only found in low titres in sera from 10 patients with MG and three with LEMS. Significant anti-CL activity could not be detected in sera from nine patients with acute Guillain-Barré Syndrome (GBS), 12 chronic cases of epilepsy, 8/9 with oat cell carcinoma and 9/10 with acute stroke. Further tests on 39 sera with the highest anti-CL activity, from all of the above disease groups, showed a significant correlation between IgM anti-CL and IgM anti-ss DNA activities. In a series of competitive inhibition assays six sera from patients with MG were shown to have a proportion of both specific and cross-reactive IgM anti-CL and IgM anti-ss DNA antibodies. Anti-phospholipid antibodies occur in certain neurological diseases, at lower titres than seen in SLE, yet their cross-reactive binding to ss DNA suggests similar antibacterial origins as have been proposed for lupus auto-antibodies. In the absence of overt infection they might reflect a breakdown of tolerance for non-organ specific membrane antigens in diseases with predominantly organ specific membrane bound putative autoimmunogens.     

The structural requirements for anti-cardiolipin antibody binding in sera from patients with syphilis and SLE

In a search for specific binding patterns of anti-phospholipid (PL) antibody reactivity in sera from patients with systemic lupus erythematosus (SLE), a quantitative assay was used to compare binding curves with those from beef heart cardiolipin (CL) and the following CL analogues: diphosphatidyl propylene glycol (DPPG), which lacks the internal hydroxyl group on the glycerol moiety; acetyl CL (ACL), in which an acetyl group is substituted for the glycerol hydroxyl group; and dimethyl CL (DCL), in which a methyl group is positioned on each phosphate group. In syphilitic sera the plateau level of antibody binding was decreased by 15 and 41% with DPPG and ACL, respectively. Binding to DCL was dramatically suppressed to levels only slightly above baseline. Only the binding curves for CL and DPPG showed saturability, and analysis by Eadie-Scatchard plots showed that the change in binding was primarily due to a more than twofold increase in KD (decreased antibody avidity). Similar patterns were seen with sera from patients with SLE and SLE-like illness, but some uniquely shaped binding curves were observed. Compared to control CL, peak binding levels were 75-88% for DPPG, 16-20% for ACL, and only 1-4% for DCL. These data indicate that the integrity of the CL headgroup, especially at the phosphate moiety, is essential for recognition by anti-CL antibodies from some sources.     

Antibodies directed against ribosomal P proteins cross-react with phospholipids

Anti-ribosomal P protein (anti-P) antibodies are marker antibodies in systemic lupus erythematosus (SLE). Their association with psychiatric or neurological manifestations has been proposed, but remains controversial. Anti-phospholipid antibodies are the hallmark of a syndrome that may comprise a number of neurological manifestations. Thus, anti-P and anti-phospholipid antibodies have both been associated with central nervous system involvement and their co-existence in the same sera was reported. We verified the ability of purified anti-P antibodies to bind different phospholipids and phospholipid-binding proteins in solid-phase assays. Anti-P antibodies from five of eight patients bound cardiolipin (CL) when saturated with fetal calf serum (FCS); in three cases anti-CL antibodies were also detected in the flow-through. No anti-P eluate, nor any corresponding flow-through, bound beta(2)-glycoprotein I alone or prothrombin. Moreover, no anti-P eluate bound CL when the plates were blocked with bovine serum albumin in the absence of FCS. Anti-P antibodies with anti-CL activity bound both ssDNA and dsDNA and also nucleosomes in three patients. Our data indicate a great heterogeneity of anti-P antibodies that appear to be overlapped partially with the other autoantibody populations detected frequently in SLE.     

Study of procainamide hapten-specific antibodies in rabbits and humans

Procainamide (PA) is the drug most commonly associated with the induction of autoantibodies and drug-related lupus (DRL). While the majority of these patients express autoantibodies, antibodies to the parent drug and metabolites, PA-hydroxylamine (PAHA) or nitroso-PA (NOPA), have not been reported in humans.Hapten-carrier conjugates were prepared using human hemoglobin (HgB) or autologous rabbit erythrocytes with PAHA or NOPA. PA was conjugated to rabbit serum albumin (RSA) or egg albumin (OVA) via diazotization and condensation methods. Rabbits were immunized with hapten conjugates in Freund's adjuvant. These hapten-carrier compounds (5 – 10 μg/ml) were used as test antigens for antibodies in sera from the rabbits and 40 patients on chronic PA treatment, 10 SLE patients, 33 elderly and 20 young normal controls by ELISA. Type I and II collagens were also used as test antigens for human sera.Sera from rabbits immunized with the PA compounds had elevated IgG antibody values to PA, PAHA and NOPA, but no autoantibodies. Absorption of the rabbit sera with the PA compounds reduced the antibody levels; ssDNA and histones failed to inhibit the total binding values. Mean binding to PA - OVA was 0.95 ± 0.41 for PA patients and 1.37 ± 0.26 standard error of means (S.E.M.) in the SLE patients compared to 0.37 ± 0.14 S.E.M. in the normal sera (P⩽0.05); similar binding values to PAHA - HgB and NOPA - HgB were also observed. Sixty-eight percent of the PA patients had antibodies to type II collagen. Elevated binding values to PA compounds were inhibited by absorption of human sera with ssDNA or total histones; absorption with PA or PAHA had no significant effect. These findings suggest that sera from PA patients containing high titers of autoantibodies cross-react in vitro with unrelated antigens.     

The fine specificity of IgG antiguanosine antibodies in systemic lupus erythematosus

The antigen specificity, isotype, and subclass of antinuclear antibodies may be related to their pathogenicity in systemic lupus erythematosus (SLE). Our laboratory found that IgG antibodies that bound the nucleoside, guanosine, occurred frequently in SLE patients. In contrast, sera from healthy subjects contained IgM but not IgG antiguanosine antibodies. The present studies were designed to characterized the fine specificity of IgG antiguanosine antibodies in SLE and compare them with IgM antiguanosine antibodies in normal sera. Serum antinuclear antibodies from six healthy subjects and six SLE patients were isolated by affinity binding to guanosine and measured by an enzyme-linked immunosorbent assay (ELISA). IgM in normal sera, and both IgM and IgG in SLE sera bound guanosine. IgM antiguanosine antibodies in normal sera were polyspecific and bound other nucleosides and 1-methylguanosine but not denatured DNA (ssDNA). In contrast, IgG antiguanosine antibodies from the SLE patients bound guanosine and ssDNA but not other nucleosides or 1-methylguanosine. SLE IgM antiguanosine antibodies had the same fine specificity and bound guanosine and ssDNA but not any of the other nucleosides. These results suggest that SLE IgG and IgM antiguanosine antibodies have fine specificity in contrast to the polyspecific IgM antibodies in normal sera. In addition, subclass analysis indicated that all SLE patients had either IgG1 or IgG3 subclass of antiguanosine antibodies that bind complement. Characterizing the isotype, subclass, and fine antigen specificity of antiguanosine antibodies should assist in evaluating their potential pathogenicity in SLE.     

Monoclonal anti-tuberculosis antibodies react with DNA, and monoclonal anti-DNA autoantibodies react with Mycobacterium tuberculosis.

Classical models of experimental autoimmune diseases, such as adjuvant arthritis entail the use of mycobacteria. Furthermore, BCG immunotherapy may be followed by arthritic symptoms. To test the infection-autoimmunity relationship of mycobacteria, we used monoclonal antibodies raised against M. tuberculosis and against DNA. Murine monoclonal anti-TB antibodies were found to react with ssDNA, dsDNA and other polynucleotides. Monoclonal anti-DNA autoantibodies derived from patients and mice with SLE bound to three glycolipids shared among all mycobacteria and derived from mycobacterial cell wall. Prior incubation of the antibodies with ssDNA and other polynucleotides or with glycolipid antigens inhibited binding. These results indicate that infecting mycobacteria share antigens with human tissue, thus accounting in part for the production of autoantibodies in mycobacterial infections.     

Heterogeneity of anti-idiotypic antibodies to anti-DNA antibodies in humans.

Serum antibodies in some patients with systemic lupus erythematosus (SLE) were found to have specificity to idiotypes (Id) of 0-81 (human monoclonal anti-single-stranded DNA (ssDNA) antibody) but not to Id of NE-1 (human monoclonal anti-double-stranded DNA (dsDNA) antibody) or pooled human IgM. The interaction of the antibodies and 0-81 was blocked by the co-existence of free ssDNA. Some of SLE sera also showed preferential binding to Id determinants of NE-1, which included the antigen-binding sites of the dsDNA antibody. Some other SLE sera reacted with both Id of 0-81 and NE-13. Thus, there was heterogeneous population among human anti-Id autoantibodies to anti-DNA antibodies. The anti-Id activity was commonly detected in inactive SLE sera, and less frequently in normal controls, suggesting some regulatory role for anti-Id antibodies in the production of autoantibodies.     

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.     

Lymphocyte autoantibodies and alloantibodies in HIV-positive haemophilia patients.

In order to elucidate the fine specificity of anti-cardiolipin antibodies (ACA) in patients with SLE compared to patients with syphilis (SY) various inhibition experiments were performed. Seven SLE sera and eight SY sera positive for ACA were diluted and preincubated with either cardiolipin VDRL-antigen, mitochondial particles, dsDNA, ssDNA or dilution buffer. The sera were subsequently assayed for residual ACA activity of IgG or IgM class using a sensitive ELISA technique. Significant inhibition of IgM ACA activity in SLE sera was found with cardiolipin, VDRL-antigen and mitochondrial particles. Cardiolipin inhibited binding to a significantly higher extent than the other antigens. In SY sera significant inhibition of the IgM ACA activity was found with all antigens used. The strongest inhibition was seen using VDRL-antigen. Inhibition of IgG ACA activity could only be clearly estimated in SY sera where VDRL-antigen was found to be a much stronger inhibitor than the rest, purified cardiolipin being the weakest. Only two out of seven SLE sera were IgG ACA positive which made a clear conclusion impossible but a strong inhibitory capability of pure cardiolipin and a weaker inhibition with VDRL-antigen was found. This study disclosed a difference between SLE and SY sera showing strong reactivity of ACA in SLE sera with purified cardiolipin, contrasting to ACA in SY sera which predominantly reacted with cardiolipin in the liposome environment, as found in the VDRL-antigen and in mitochondrial particles.     

Linear epitopes of two different autoantigens-La/SSB and myelin basic protein--with a high degree of molecular similarity,cause different humoral immune responses

The region 147-154aa of La/SSB presents 83% sequence similarity with the 139-146aa region of the human myelin basic protein (MBP). The aim of this study was to investigate the prevalence and significance of antibodies against both epitopes in sera from patients with systemic autoimmune diseases, and to compare the humoral responses produced after rabbit immunization. Peptides 147-154aa of La/SSB and 139-146aa of the MBP were attached on tetrameric sequential oligopeptide carriers and used for immunizations of New Zealand White rabbits. Antibodies to immunizing peptides, as well as to the peptides corresponding to other previously defined La/SSB epitopes (289-308aa, 349-364aa), to the intact human MBP (hMBP) and to the recombinant human La/SSB (rechLa) proteins, were identified using specific ELISA assays. Sera from 45 patients with Sjogren's syndrome (pSS), 49 with Systemic Lupus Erythematosus (SLE), and 18 with Rheumatoid Arthritis (RA) were tested against the two peptides and the hMBP. Twenty-two per cent of sera from pSS patients, 27% of SLE patients, and none from RA sera reacted with the La epitope; 27% from pSS sera, 22% of SLE sera, and 17% of RA sera gave a positive reaction against the MBP peptide. Finally, 19% of pSS, 30% of SLE, and 38% of RA sera reacted with the hMBP. Thirty-five days after immunization of rabbits with the La epitope, antibodies were produced against all three La/SSB peptides, the MBP peptide, and the hMBP and rechLa proteins. Rabbits immunized with the MBP peptide produced antibodies against the immunizing peptide and the mimicking peptide of La shortly after immunization, whilst antibodies against the other La epitopes and the two intact proteins were produced later. Inhibition experiments in rabbit sera with high reactivity against hMBP, using the MBP peptide as inhibitor, revealed that 80% of serum reactivity was abolished. In conclusion, a significant proportion of human autoimmune sera reacted with both La and MBP derived peptides, as well as with hMBP. La 147-154aa peptide, when used for animal immunizations, induces a fast epitope spreading involving both La and MBP. In contrast, the mimicking MBP epitope induces a delayed response against the other La epitopes. Thus, despite the fact that these peptides present molecular similarity, they induce different immune responses.     

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.     

High prevalence of anti-cardiolipin and other autoantibodies in a healthy elderly population.

Serum samples from 64 apparently healthy individuals (32 men and 32 women, mean age 81.0 years) were examined for the prevalence of several autoantibodies, including rheumatoid factor (RF), antinuclear antibodies (ANA), antibodies to extracted cellular antigens Ro (SSA), La (SSB), Sm, U1nRNP and Scl-70. IgG and IgM isotype-specific ELISA methods were applied for the detection of antibodies to ssDNA (anti-ssDNA), to dsDNA (anti-dsDNA) and to cardiolipin (anti-CL). The sera of this elderly population were found to contain a plethora of autoantibodies; RF was detected in 14.1%, ANA in 31.3% and anti-Ro (SSA) in 1.6% of the individuals. Precipitating antibodies to La (SSB), Sm, U1nRNP and Scl-70 were absent, while 15.6% of the sera displayed precipitating antibodies to a common undefined human spleen antigen. ELISA methods revealed anti-ssDNA in 17.2% of the individuals, anti-dsDNA in 14.1% and anti-CL in an extremely high incidence (51.6%). Notably, the above autoantibodies were exclusively of IgG isotype. Tests of 261 sera from healthy non-elderly individuals disclosed only anti-CL (IgG and IgM isotypes) in 2.3% of them. The levels of IgA and IgG immunoglobulins were increased in 23.4% and 29.7% of the elderly subjects, respectively. IgM was elevated in 3.1%, but it was also found decreased in 9.4%. This study documents the high incidence of autoantibodies in the healthy elderly, including for the first time, anti-CL antibodies. Furthermore, the relative impairment in IgM autoantibody production observed, possibly indicates the involution of the senescent immune system.     

Anti-DNA Antibodies Induced by BK Virus Inoculations

The ability of BK virus to induce anti-DNA antibodies in rabbits, and the ability of these antibodies to bind natural eukaryotic DNA and synthetic polynucleotides have been analysed. The specificity of the binding was assayed by inhibition of anti-dsDNA and -ssDNA ELISA tests with dsDNA, ssDNA, and synthetic single-stranded as well as double-stranded polynucleotides. The anti-dsDNA activity of two rabbit antisera was effectively inhibited by dsDNA and ssDNA and poly(dAdT)-poly(dAdT). The other nucleotide antigens produced relatively less inhibition. The anti-ssDNA binding was most efficiently inhibited by the homologous antigen, whereas inhibition by dsDNA only reached approximately 70% of the maximum as defined by ssDNA as inhibitor. This indicates the existence of a selective anti-ssDNA antibody population and a population recognizing both ssDNA and dsDNA within the sera. Cross-reaction of the induced anti-DNA antibodies with phospholipid antigens, such as cardiolipin, phosphatidylic acid, and bacterial cell surface, could not be demonstrated. We conclude that antibodies resulting from inoculation with BK virus specifically bind to dsDNA and ssDNA and possess a high affinity for the synthetic duplex poly(dAdT). In this way, they have some similarities with anti-DNA antibodies encountered in SLE (systemic lupus erythematosus) in both man and mouse.     

Humoral response against myelin antigens in two strains of rats with different susceptibility to experimental allergic encephalomyelitis (EAE).

Lewis (Lw) rats are susceptible and Wistar (Wr) rats are usually resistant to the induction of experimental allergic encephalomyelitis (EAE). In this study we analyze the humoral response to myelin antigens, providing evidence for different B cell response to myelin basic protein (MBP) and other myelin proteins between these two strains of rats with different susceptibility to EAE. In fact, IgG anti-MBP titers in Wr rats were markedly higher than in Lw ones. Moreover, an inverse relationship between the amount of antigen injected to induced EAE and the level of anti-MBP antibodies was observed in Wr rats, while IgG anti-MBP varied in a positive dose-depending manner in sera from Lw rats. Also, sera from Wr rats analyzed by immunoblotting showed a strong reactivity with MBP and other myelin proteins, but sera from Lw rats reacted only with MBP. Evaluation of IgA and IgM against MBP in Wr rats showed again higher titers of these isotypes when compared with the titers observed in Lw rats. The distribution of IgG subclasses in sera from both strains indicated that Wr produced low titers of specific IgG1, while Lw rats did not produce specific IgG1. However, Wr rats showed high levels of IgG2a, IgG2b and IgG2c subclasses while lesser titers of these isotypes were observed in Lw animals. These findings indicate that both strains have the capacity to develop antibodies against portions of the MBP molecule, but antibody production is greater in the resistant Wistar rats suggesting a B cell activation in these animals, that could be related to their lower susceptibility.     

Interpretation of the Raji cell assay in sera containing anti-nuclear antibodies and immune complexes.

The Raji cell assay is regarded as a test for the detection and quantitation of immune complexes. It is frequently positive in sera from patients with SLE. We have demonstrated a relationship between Raji cell binding and antibodies to DNA and soluble cellular antigens. In five sera containing high titres of antibodies of known single specificity, most of the Raji cell binding occurred in the 7S IgG fraction where the majority of anti-nuclear antibody was also found. When each of these sera was incubated with its specific antigen, Raji cell binding increased. Subsequent fractionation showed that this binding was in the high molecular weight fraction (greater than 200,000 daltons) and that Raji cell binding and antibody activity were abolished in the 7S fraction. These data confirm that Raji cell bind immune complexes but also indicate that 7S anti-nuclear antibodies may interact directly with Raji cells by an unknown mechanism. Therefore, in sera of patients with anti-nuclear antibodies, binding to Raji cells does not necessarily imply the presence of immune complexes alone.     

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.     

Inhibitor(s) of natural anti-cardiolipin autoantibodies.

IgG fractions were purified on Sepharose anti-human IgG column from eight sera of healthy donors, having no anti-cardiolipin (aCL) activity as measured by anti-cardiolipin ELISA assay (aCL-ELISA). All the IgG fractions, after elution with 4.9 M MgCl2, reacted with CL. The antigen-binding characteristics of the IgG fractions purified from normal human serum (NHS) were similar to those of IgG fractions purified from sera of four patients with the anti-phospholipid syndrome (APLS). Competition assay confirmed the specificity of the binding of the purified IgG fractions to CL. The same results have been achieved with IgG fractions purified on Sepharose Protein-A column. The binding to CL was completely inhibited by either whole NHS and sera from various animal species, or by beta 2-glycoprotein I (beta 2-GPI). Our results support the notion of the existence of both natural anti-CL antibodies and serum inhibitor(s) in sera of healthy individuals. It is conceivable that in part the pathogenesis of APLS entails defects in the natural inhibitors of aCL antibodies.     

The Trypanosoma lewisi immunofluorescence test: A new simple technique for simultaneous determination of total antinuclear antibodies and the detection of antibodies to double-stranded DNA

An indirect immunofluorescence technique was developed for the detection of antibodies to dsDNA and the simultaneous assessment of antinuclear antibodies ‘in toto’ (ANA). This assay was based upon the use as substrate of smears of peripheral blood derived from rats infected with Trypanosoma lewisi. T. lewisi possesses a giant kinetoplast posteriorly to the nucleus. Enzyme digestion and absorption experiments provided strong evidence that T. lewisi kinetoplast contains dsDNA uncontaminated by other nuclear antigens. The T. lewisi immunofluorescent test was evaluated on a total of 130 sera (30 from patients with SLE) and compared with radioimmunoassays for antibodies to dsDNA ([¹²⁵I]dsDNA-RIA) and antibodies to ssDNA ([¹²⁵I]ssDNA-RIA). Excellent correlation was found between kinetoplast immunofluorescence and [¹²⁵I]dsDNA-RIA, whereas no non-SLE sera showing significant ssDNA binding activity gave kinetoplast staining. With a single exception, only SLE sera reacted with T. lewisi kinetoplast. Sera containing auto-antibodies other than ANA did not induce fluorescene of any part of the parasite, including the flagellum and its base. These results indicated that the T. lewisi immunofluorescence test is specific and reliablem and combines the advantages of Crithidia luciliae with those of Trypanosoma gambiense. It may be used routinely for evaluating of total ANA and simultaneous detection of antibodies against dsDNA.     

Characterization of the Endothelial Surface Proteins Recognized by Anti-Endothelial Antibodies in Primary and Secondary Autoimmune Vasculitis

The antigenic structures recognized by anti-endothelial cell antibodies (AECA) in sera from 10 Wegener's granulomatosis (WG) and 12 systemic lupus erythematosus (SLE) patients with signs of vasculitis were characterized by immunoprecipitation of selectively radiolabeled surface membrane proteins from human umbilical vein endothelial cells. Electrophoretic analysis of the immunoprecipitated proteins revealed reactivities against endothelial antigens ranging in size from 200 to 25 kDa. AECA antigens were not cell specific, since the same sera also reacted, at least in part, with radiolabeled human fibroblast surface proteins. The majority of WG patients displayed a constant precipitation pattern of five proteins (180, 155, 125, 68, and 25 kDa). On the contrary, AECA from SLE sera reacted with a more heterogeneous series of endothelial proteins. A group of four proteins, however, was also found in the majority of SLE sera: 200, 180, 155, and 25 kDa. In addition, some endothelial antigens were immunoprecipitated only by WG (125 kDa) or by SLE sera (200 kDa), suggesting a different endothelial reactivity in different vasculitic processes. The reaction did not involve intracellular proteins as demonstrated by the lack of reactivity of SLE sera negative for AECA but positive for anti-cytoplasmic or anti-nuclear antibodies. These data confirming that AECA recognize surface endothelial determinants further support a potential pathogenetic role for these antibodies in autoimmune vasculitis.     

Catalytic antibodies in clinical and experimental pathology: human and mouse models

Most of the data accumulated through studies on natural catalytic autoantibodies indicate that production scales up markedly in pathological abnormalities. We have previously described an increased level of DNA-hydrolyzing autoantibodies in the sera of patients with various autoimmune disorders [systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma], HIV infection and lymphoproliferative diseases accompanied by autoimmune manifestations. In the present study, we show that an increased level of catalytic activity of autoantibodies can be observed in the sera of autoimmune mice, thus providing a fundamental insight into the medical relevance of abzymes. Polyclonal autoantibodies purified from sera of NZB/W, MRL-lpr/lpr and SJL/J mice show proteolytic and DNA-hydrolyzing activities, as opposed to those harvested from non-autoimmune BALB/c mice. The expressiveness of the catalytic activity was strongly dependent on the age of the animal. The highest levels of catalytic activity were found in the sera of mice aged between 8 and 12 months; the lowest level was typical of younger animals whose age ranged from 6 to 8 weeks. Specific inhibition assays of the catalytic activities were performed to throw light on the nature of the abzyme activity. Within a cohort of aging animals, a strong correlation between marked autoimmune abnormalities and levels of catalytic activities has been established. Nonimmunized SJL/J mice revealed specific immune responses to myelin basic protein (MBP), skeletal muscle myosin (skMyo) and cardiac myosin (Myo), and highly purified antibodies from their serum show specific proteolytic attack against the target antigens. This finding prompted us to undertake a more detailed study of specific antibody-mediated proteolysis in diseased humans. A targeted catalytic response was originally demonstrated against MBP and Myo in multiple sclerosis and myocarditis patients, respectively.