Antibodies against A peptide sequence located in the linker region of the HMG-1/2 box domains in sera from patients with juvenile rheumatoid arthritis

Objective. To extend our work on the mapping of B cell epitopes on nucleosomal high mobility group (HMG) proteins in the sera of patients with juvenile rheumatoid arthritis (JRA). Methods. Seventy-seven pauciarticular-onset JRA serum samples from antinuclear antibody (ANA)- positive patients and 42 polyarticular-onset JRA patient sera found to react with HMG-2 by immunoblotting were used in this study. To identify B cell epitopes on HMG-2, recombinant HMG-2 protein fragments were used in enzyme-linked immunosorbent assay (ELISA) and in competition ELISA experiments with a set of overlapping synthetic peptides. Fine epitope mapping was achieved by oligopeptide synthesis, followed by immunoblotting. Results. Pauciarticular, but not polyarticular, JRA patient sera were found to recognize a lysine-rich major epitope (KKGKKKDP), which is located in the linker region of the HMG box domains of the HMG-2 nonhistone chromosomal protein. No significant immunoreactions were observed in sera from ANA-negative JRA patients and in sera from children with nonrheumatic diseases, indicating that this epitope seems to be specific for pauciarticular-onset JRA. Conclusion. In addition to our previous finding that JRA sera will react with a defined epitope on HMG-17, pauciarticular JRA patient sera were also found to recognize a defined epitope on the HMG-2 protein, thus suggesting the importance of this epitope in the etiology of JRA.     

Autoantibodies to the chromosomal protein HMG-17 in juvenile rheumatoid arthritis

Objective. To determine the antibody profiles in sera from patients with juvenile rheumatoid arthritis (JRA). Methods. Immunoblotting using nuclear extracts and recombinant high-mobility group (HMG) nonhistone chromosomal proteins. Results. Antibodies directed against HMG-17 were found in 47% of antinuclear antibody (ANA)–positive patients with pauciarticular-onset JRA and in 16% of ANA-positive patients with polyarticular-onset JRA. HMG-17 values of 6% and 8%, respectively, were detected in ANA-negative patients with JRA and in those with nonrheumatic diseases. Conclusion. There is evidence for a high prevalence of anti—HMG-17 antibodies in sera of patients with pauciarticular-onset JRA.     

Autoantibodies to the chromosomal protein HMG-17 in juvenile rheumatoid arthritis.

OBJECTIVE. To determine the antibody profiles in sera from patients with juvenile rheumatoid arthritis (JRA). METHODS. Immunoblotting using nuclear extracts and recombinant high-mobility group (HMG) nonhistone chromosomal proteins. RESULTS. Antibodies directed against HMG-17 were found in 47% of antinuclear antibody (ANA)-positive patients with pauciarticular-onset JRA and in 16% of ANA-positive patients with polyarticular-onset JRA. HMG-17 values of 6% and 8%, respectively, were detected in ANA-negative patients with JRA and in those with nonrheumatic diseases. CONCLUSION. There is evidence for a high prevalence of anti-HMG-17 antibodies in sera of patients with pauciarticular-onset JRA.     

Autoantibodies to nonhistone chromosomal proteins HMG-1 and HMG-2 in sera of patients with juvenile rheumatoid arthritis

IgG antibodies against the high mobility group (HMG) nonhistone chromosomal proteins HMG-1 and/or HMG-2 were detected in the sera of 49 (39%) of 126 antinuclear antibody (ANA)-positive patients with juvenile rheumatoid arthritis (JRA), by immunoblotting. Clinical diagnosis classified these patients in 2 major groups, 105 with pauciarticular-onset JRA and 21 with polyarticular-onset JRA. Anti-HMG-1 and/or anti-HMG-2 antibodies were found in 8 (25%) of 32 pauciarticular-onset JRA patients with uveitis and in 34 (47%) of 73 patients without uveitis, whereas anti-HMG-1 and/or anti-HMG-2 antibodies were found in 4 (24%) of 17 children with polyarticular-onset JRA without uveitis. Among 53 sera from ANA-negative JRA patients, 3 (6%) were positive for anti-HMG-1 and/or anti-HMG-2 antibodies, whereas no reactivity to HMG-1 or HMG-2 proteins was observed in 48 sera from age-matched children with nonrheumatic diseases.     

Complement-fixing properties of antinuclear antibodies distinguish drug-induced lupus from systemic lupus erythematosus

The immunofluorescence antinuclear antibody (ANA) test has been widely used to monitor autoimmune disease, but its value for diagnostic purposes is compromised by low specificity and high prevalence in disease-free individuals. The capacity of autoantibodies to fix serum complement proteins when bound to antigen is an important effector function because this property is associated with acute and chronic inflammatory processes. The current study evaluates the complement-fixing properties of antinuclear antibodies (CANA) in three well-defined and clinically-related patient groups: systemic lupus erythematosus (SLE), drug-induced lupus (DIL) and drug-induced autoimmunity (DIA). Of 20 patients diagnosed with SLE, 90% displayed complement-fixing ANA while this feature was present in only two of 18 patients with DIL and no patients with DIA without associated disease even though the mean ANA titres were similar among these patient groups. CANA was significantly correlated with anti-Sm activity. Because SLE but not DIL or DIA can be a life-threatening disease associated with complement consumption in vivo, these results demonstrate that measurement of CANA is a diagnostically useful tool and may have immunopathologic implications.     

Antineutrophil cytoplasmic antibodies in systemic lupus erythematosus

Objective. To examine the prevalence, subspecificities, and clinical associations of antineutrophil cytoplasmic antibodies (ANCA) in patients with systemic lupus erythematosus (SLE). Methods. One hundred fifty-seven sera from 120 patients with SLE were examined for classic (c) and perinuclear (p) pattern ANCA by indirect immunofluorescence. Antibody subspecificities were determined by enzyme-linked immunosorbent assay (ELISA). Serologic results were correlated with clinical manifestations as categorized by the BILAG (British Isles Lupus Assessment Group) index. Results. ANCA were found in 40 of the 157 sera (25%). Only a pANCA, not a cANCA, pattern of fluorescence was seen. By ELISA testing, 16 sera reacted to lactoferrin, 8 to elastase, and 4 to lysozyme. There was no reactivity to proteinase 3 (PR3) or myeloperoxidase (MPO). No correlation of pANCA, or any of the ANCA subspecificities, with organ system involvement, as categorized by the BILAG index, was found. Notably, there was no correlation of ANCA results with lupus vasculitis. Conclusion. The absence of cANCA, anti-PR3, and anti-MPO shows that with appropriate assay conditions, ANCA testing assists in the differentiation between SLE and the ANCA-associated vasculitides. The lack of a correlation between pANCA or any ANCA subspecificity and clinical manifestations suggests that ANCA do not identify particular clinical subsets among SLE patients, including those with lupus vasculitis.     

Common occurrence of an antiidiotypic antibody that recognizes T14+ anti-DNA antibodies in patients with systemic lupus erythematosus

Objective. To investigate whether antibodies to a T14 anti-DNA antibody can be found in patients with systemic lupus erythematosus (SLE). Methods. Seventy-six serum samples (37 from patients with SLE) were randomly selected from among sera submitted for routine antinuclear antibody testing. Short, overlapping peptides based on the partial V_H (variable region of the heavy chain) sequence of the T14 antibody were synthesized on multipins and screened for reactivity with SLE sera. In addition, selected peptides from T14 and related proteins were synthesized in bulk and screened for reactivity with both SLE and control sera. A monoclonal antibody was generated to determine the prevalence of the T14 idiotype (T14+ Id) in the different study populations. Results. Antibodies were detected by a peptide based on the third complementarity-determining region (CDR3) of the T14 protein in 15 (41%) of 37 patients with SLE or 15 (54%) of 28 who had anti-DNA antibodies, in 3 (9%) of 34 patients without anti-DNA antibodies (9 of whom had SLE), and in 6 (10%) of 57 healthy controls. In SLE sera, the antiidiotypic (anti-Id) responses (IgM and IgG) correlated well with the anti-DNA responses (IgG), and both responses correlated well with the T14+ Id activity in SLE sera. Control peptides based on the 18/2 (16/6+ Id) and S107 proteins detected low antibody activities in SLE sera, attributable to cross-reactivity with the T14 peptide. A peptide based on an unrelated human antibody was not reactive with these sera. Conclusion. Anti-Id antibodies directed to T14 V_HCDR3 were found commonly in the sera of patients with SLE, and they appeared to be induced by the anti-DNA antibodies present in the sera. Based on these findings, these secondary antibodies may be pathogenic in SLE.     

Is α‐actinin a target for pathogenic anti‐DNA antibodies in lupus nephritis?

Objective

Following recent reports that pathogenic murine anti‐DNA antibodies bind to α‐actinin, it was obviously of interest to assess the ability of human pathogenic anti–double‐stranded DNA (anti‐dsDNA) antibodies to bind this antigen. Both human monoclonal anti‐DNA antibodies and antibodies affinity purified from the sera of patients with systemic lupus erythematosus (SLE) were investigated.

Methods

An enzyme‐linked immunosorbent assay was established to measure immunoglobulin binding to α‐actinin. Antibodies binding dsDNA were purified from the sera of SLE patients who either had active renal disease or had never had renal disease. Serum samples were selected at times when the patients' sera exhibited high IgG binding to dsDNA. The binding of supernatants from 3 high‐affinity human anti‐dsDNA IgG hybridomas (RH14, B3, and DIL‐6) and 7 human IgM anti‐DNA hybridomas was also investigated.

Results

A greater proportion of anti‐dsDNA IgG–binding antibodies purified from patients with renal disease bound to α‐actinin than did those purified from the sera of patients without renal disease. The specificity of binding to the 100‐kd α‐actinin molecule was confirmed by Western blotting. The pathogenic human antibodies RH14 and B3 bound strongly to α‐actinin, while nonpathogenic DIL‐6 bound very weakly. RT84, the IgM antibody that binds dsDNA with the highest affinity, exhibited the greatest binding to α‐actinin.

Conclusion

The results of our study support the findings of previous studies using murine anti‐DNA monoclonal antibodies, which suggest that pathogenic anti‐dsDNA antibodies cross‐react with α‐actinin.

     

Some autoantibodies to Ro/SS-A and La/SS-B are antiidiotypes to anti–double-stranded DNA

Objective. To determine the relationship of anti–Ro/SS-A and anti–La/SS-B antibodies to anti–double-stranded DNA (anti-dsDNA) in sera from patients with systemic lupus erythematosus. Methods. Sera with anti–Ro/SS-A alone (n = 5) or those with anti–Ro/SS-A and anti–La/SS-B (n = 7) were absorbed with purified Ro/SS-A and La/SS-B, respectively. The absorbed sera were then tested for reactivity with MOLT-4 extract by Western blot and dsDNA by enzyme-linked immunosorbent assay (ELISA). With selected sera, anti-dsDNA was isolated on DNA cellulose columns and anti–Ro/SS-A and anti–La/SS-B were isolated on antigen-affinity columns. Reactivity between anti-dsDNA and autologous anti–Ro/SS-A or anti–La/SS-B, as well as inhibition by cognate antigens, was studied. Results. After absorption, all sera showed reactivity with small nuclear RNP A and D bands in Western blots, and some showed reactivity with dsDNA by ELISA. Anti-dsDNA populations (n = 4) were purified on dsDNA cellulose columns. Anti–Ro/SS-A (n = 1) and anti–La/SS-B (n = 3) were affinity purified from the same sera as the anti-dsDNA. In all cases, anti-dsDNA bound autologous anti–Ro/SS-A and anti–La/SS-B much more strongly than it bound normal pooled IgG. Moreover, dsDNA, but not RNA, blocked these interactions. In addition, Ro/SS-A blocked anti–Ro/SS-A and La/SS-B blocked anti–La/SS-B in these same interactions. Conclusion. In sera with anti–Ro/SS-A and anti–La/SS-B, there are subpopulations of these antibodies that bind and mask anti-dsDNA. We hypothesize that these anti–Ro/SS-A and anti–La/SS-B antibodies are antiidiotypes to idiotypes on anti-dsDNA and that they both mask and down-regulate these anti-dsDNA antibodies.     

Lymphocyte Antigens in Neuropsychiatric Systemic Lupus Erythematosus

Objective. To examine the relationships among specific lymphocyte antigenic reactivities of lupus sera and central nervous system complications of systemic lupus erythematosus (SLE), lymphocytotoxic antibody (LCA) positivity, and specific cognitive impairment. Methods. Sera from 115 patients with SLE were examined for the presence of IgM- and IgG-class auto-antibodies binding to surface target antigens on lymphocytes, by immunoblotting and microdroplet lymphocytotoxicity studies. Seventy-three of these patients also underwent detailed neuropsychological testing within the same time period. Results. Significant associations were found between reactivities to several lymphocyte antigenic moieties and neuropsychiatric SLE (NPSLE) or cognitive impairment. Specifically, immunoblot reactivities to 31–32-kd, 50–52-kd, 54–56-kd, and 97–98-kd targets were associated with clinical NPSLE; there was a significant association between reactivity to the 50–52-kd moiety in particular and cognitive impairment. There were also associations between LCA and immunoblot reactivity. Furthermore, the previously reported association between LCA positivity and specific visuospatial cognitive impairment was confirmed with data obtained from 2 different batteries of neuropsychological tests. Conclusion. In some cases, specific antigenic targets of LCA-containing sera may be implicated in the pathogenesis of NPSLE.     

Drug-induced lupus erythematosus secondary to nafcillin: the first reported case

With an estimated incidence of 15–30,000 cases per year in the United States, drug-induced lupus erythematosus (DIL) is an uncommon iatrogenic condition. The number of implicated medications increases each year. We report the first case of DIL secondary to nafcillin administration in a patient with a prosthetic aortic valve and methicillin-sensitive Staphylococcus aureus who developed a facial rash and lower back pain.The opinions expressed herein are those of the authors and do not reflect official opinion of the US Department of the Navy or US Department of Defense.     

Antibodies to Carbonic Anhydrase in Systemic Lupus Erythematosus and Other Rheumatic Diseases

Objective. Autoantibodies to CA were demonstrated in the sera of patients with systemic lupus erythematosus (SLE) and some other rheumatic diseases. This study was undertaken to define the isoform and species specificity of these reactions, as well as to develop a method for detecting immune complexes. Methods. Antibodies to CA were sought by enzyme-linked immunosorbent assay (ELISA) and by Western immunoblotting. Results. An increased prevalence of CA autoantibodies was detected, by both methods, in patients with SLE, scleroderma, and polymyositis, compared with controls. In SLE patients, CA autoantibodies occurred preferentially in those with anti—U1 RNP or anti-U1 RNP and Ro/SS-A. Some sera reacted with only the CA 1 or CA II isoform, while ˜50% of sera that were CA positive reacted with both isoforms. The autoantibodies reacted preferentially with the human enzymes, rather than the bovine CA, both on Western blot and by ELISA. Selected IgG F(ab')₂ fragments from anti-CA—containing sera specifically inhibited the enzyme activity of CA, and the CA inhibitor acetazolamide partially inhibited the binding of anti-CA to CA. Thus, at least a part of autoanti-CA is directed toward the active site of CA. Finally, CA molecules were detected as immune complexes in sera from selected anti-CA-positive patients. Conclusion. Autoantibodies to CA represent a previously unrecognized autoantibody to an abundant intracellular protein of the human erythrocyte.     

Drug-induced lupus following treatment with infliximab in rheumatoid arthritis

After introduction of infliximab for the treatment of rheumatoid arthritis (RA), there have been many reports of patients developing asymptomatic higher rate of antinuclear antibodies and anti-dsDNA antibodies than in non-infliximab-treated patients. However, only five clinical drug-induced lupus (DIL) cases have been documented following treatment with infliximab, in RA and in Crohn's diseases. We report a case of a 69-year-old female with a 5 year history of RA, whowas successfully treated with low-dose methotrexate (MTX) and infliximab (initially 3 mg/kg and from the fourth infusion 5 mg/kg) for 23 weeks. Before the sixth infusion, she was diagnosed with DIL by both clinical features (fever > 38 degrees C, recurrence of active synovitis, myalgia, erythematous rash and general malaise) and laboratory findings (antinuclear antibodies 1:160, anti-double-stranded DNA positive by ELISA assay, decreased serum complement C3 andC4, hypergammaglobulinaemia, increased erythrocyte sedimentation rate). After discontinuation of treatment and therapy with oral prednisone, lupus resolved within 8 weeks.     

Anticyclic citrullinated peptide antibodies in patients with mixed connective tissue disease

Abstract

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

Novel autoantibodies against muscle-cell membrane proteins in patients with myositis

Objective. To search for autoantibodies against muscle cell-specific surface membrane antigens in patients with inflammatory myopathies. Methods. A cell enzyme-linked immunosorbent assay (cell ELISA) using a human rhabdomyosarcoma cell line (TE-671) was developed and performed in serial dilutions with either nonfixed or fixed cells. A total of 141 different patient sera were tested: 90 from patients with various rheumatic diseases, 12 from patients with cardiomyopathies, 25 from patients with other muscular diseases, and 14 from patients who had undergone major surgery or who had other noninflammatory diseases. As controls, 20 sera were obtained from healthy donors. Results were correlated using immunofluorescence staining and flow cytometry. Results. Using the nonfixed cell ELISA, the proportions of positive sera from the patient groups with rheumatic diseases were 71% with polymyositis (PM), 15% with dermatomyositis (DM), 18% with systemic sclerosis (SSc), 15% with systemic lupus erythematosus (SLE), and 7% with rheumatoid arthritis. Sera from healthy donors, as well as sera from patients with nonrheumatic diseases, did not show significant reactivities. When other cell lines, including a chondrosarcoma, a bladder carcinoma, a pancreas carcinoma, and human foreskin fibroblasts, were used as substrates, positive sera did not react in the cell ELISA. Results obtained with the cell ELISA system using nonfixed cells were confirmed by flow cytometry and immunofluorescence staining. A strong protein band of 50 kd was detected on plasma membrane preparations from TE-671 muscle cells in 33% of PM sera (n = 12). Conclusion. In most sera from patients with PM, DM, and some other rheumatic diseases (i.e., SSc and SLE), autoantibodies directed against muscle-cell surface antigens can be detected. Since these molecules are localized in the muscle-cell surface membrane, autoantibodies directed against these antigens could play a major role in the pathogenesis of PM.     

Etiology and mechanisms of drug-induced lupus.

Systemic rheumatic symptoms occur with widely different frequencies as a side effect of long-term therapy with some 39 medications currently in use. Because symptoms are nonspecific, subjective, and protean, diagnosis of drug-induced lupus (DIL) requires awareness of this risk of chronic medication. However, laboratory features and the characteristic of full recovery after discontinuing treatment are helpful in differentiating drug-induced from spontaneous lupus or other syndromes. Drug-induced lupus is probably mediated by reactive drug metabolites, not the ingested medications, and susceptibility to neutrophil-mediated oxidative transformation is a property of ten lupus-inducing drugs reported so far. Mechanisms for DIL modeled after drug hypersensitivity reactions are unsupported experimentally and inconsistent with the features of DIL. However, several new lines of investigation using mouse models have opened up promising leads into the origin of autoreactive T cells and disease development in DIL.     

Association of anti–ribosomal P protein antibodies with neuropsychiatric systemic lupus erythematosus

Objective. Although it has been reported that anti–ribosomal P protein antibodies (anti-P) are highly specific for lupus psychosis, there have been discrepancies among the studies regarding the clinical correlation of these antibodies with systemic lupus erythematosus (SLE). The present study was therefore carried out to reappraise the association of anti-P and neuropsychiatric SLE. Methods. Highly purified synthetic ribosomal P peptides of the carboxyl-terminal 22-amino acid sequence were conjugated to human serum albumin (HSA) with the use of glutaraldehyde. Anti-P in sera from 75 patients with SLE (26 without central nervous system disease [non-CNS], 28 with lupus psychosis, and 21 with nonpsychotic CNS involvement [nonpsychotic CNS lupus]) were analyzed with an enzyme-linked immunosorbent assay (ELISA), using HSA–ribosomal P peptide conjugates as antigens. Anti-P levels were quantitated by subtracting the nonspecific binding activities to HSA. Results. The ELISA was found to be specific for anti-P, as determined by comparison with the results of Western blotting using extracts of HEp-2 cells. Serum anti-P levels were significantly elevated in patients with lupus psychosis, including organic brain syndrome and nonorganic psychosis, compared with those with non-CNS SLE or those with nonpsychotic CNS lupus. There were no significant differences in serum anti-P levels between patients with organic brain syndrome and those with nonorganic psychosis. Anti–P antibodies were not detected in the cerebrospinal fluid of patients with either lupus psychosis or nonpsychotic CNS lupus. Conclusion. Our results, obtained from the highly specific ELISA using HSA–ribosomal P peptide conjugates, confirm the correlation of serum anti-P with lupus psychosis. The data also suggest that differences in the purity of the ribosomal P peptides used might be a major reason for the conflicting results in the literature regarding the association of anti-P with lupus psychosis.     

Evidence for direct anti-heparin-sulphate reactivity in sera of SLE patients

Recently it has been suggested that anti-dsDNA antibodies (Abs) promote tissue damage in systemic lupus erythematosus (SLE) by cross-reactivity with highly negatively charged tissue components such as heparan sulphate (HS), the major glycosaminoglycan of the glomerular basement membrane (GBM). Other authors, however, support the theory of DNA-anti-dsDNA immune complex deposition in situ. To further elucidate the possible role of HS antibodies, we developed a new ELISA system with heparan sulphate bound to solid phase. SLE patients (n=40) showed a higher reactivity against HS (mean=28.4, SD=34.3) as compared to normal donors (n=28, mean=15.2, SD=6.3) and patients with rheumatoid arthritis (n=35, mean=14.3, SD=6.4). The addition of native dsDNA or HS to SLE sera was followed by a dose-dependent reduction in anti-HS reactivity. In contrast, in an anti-dsDNA ELISA, no reduction was observed when HS was added to SLE sera. An increase in reactivity was observed when SLE sera with and without a prior incubation with dsDNA were digested with DNAse I or II. After the purification of serum samples by protein A sepharose under dissociative conditions, seven out of eight SLE patients showed an increase in anti-HS reactivity. No correlation of the anti-HS Abs was found with organ involvement or other serological parameters. We concluded, that there is evidence for a direct anti-HS Ab reactivity in SLE sera. A part of these antibodies seems to show low avidity anti-dsDNA cross-reactivity.     

Autoantibodies and target antigens in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides

In this study of antineutrophil cytoplasmic antibody (ANCA)-associated diseases, we determined the prevalence of other autoantibodies and the antigen specificities of ANCA. ANA were common, occurring in 7 of 36 (19%) patients with Wegener's granulomatosis, in 16 of 34 (47%) patients with microscopic polyarteritis, in 6 of 11 (55%) patients with segmental necrotising glomerulonephritis and in 8 of 18 (44%) of those with ANCA-associated systemic vasculitis without renal involvement. ANA were associated more often with pANCA and microscopic polyarteritis than with cANCA (P<0.05). Patterns were speckled (n=23), homogeneous (n=10) or nucleolar (n=4). Anticardiolipin antibodies were also common, occurring in 10 of 25 (40%) patients with Wegener's granulomatosis, in 8 of 14 (57%) patients with microscopic polyarteritis and in 6 of 18 (33%) of those with a systemic vasculitis. However, anticardiolipin antibodies did not correlate with the presence of ANCA in any of the disease groups. Anti-GBM antibodies were demonstrated in only 2 of 25 (8%) patients with Wegener's granulomatosis, in I patient with microscopic polyarteritis (1/14, 7%) and in 1 with segmental nectrotising glomerulonephritis (1/11, 9%). All four patients with anti-GBM antibodies had either cANCA or pANCA. In the second part of the study, the target antigens of ANCA were determined in Wegener's granulomatosis, microscopic polyarteritis, systemic vasculids, inflammatory bowel disease, rheumatoid arthritis and systemic lupus erythematosus (SLE). Of the 19 sera with cANCA, 13 (68%) were directed against proteinase 3; other antigens were myeloperoxidase (1/19,5%), elastase and lactoferrin together (1/19, 5%), lysozyme (1/19, 5%) or unknown (3/19,16%). Of the 12 (58%) sera from patients with Wegener's granulomatosis who had cANCA, 7 bound to proteinase 3. Antimyeloperoxidase antibodies were present in 14 of 45 (31%) sera with pANCA; other antigens were proteinase 3 (5/45,11 %), elastase (3/45, 78%), lactoferrin (1/45, 2%), cathepsin G (5/45, 11 %) or unknown (17/45, 38%). Antimyeloperoxidase antibodies were common in microscopic polyarteritis (6/14, 43%) and systemic vasculitis (5/16, 31%). However, the majority of target antigens in systemic vasculitis and rheumatoid arthritis with pANCA were not determined. Atypical ANCA were present in four patients, one with inflammatory bowel disease (1/8, 13%) and three with SLE (3/15, 20%). The specificities were cathepsin G, cathepsin G plus lactoferrin, or unknown in two sera. A recent report has suggested that bactericidal /permeability-increasing protein may be the target in patients with inflammatory bowel disease.