Anti-GBM antibodies in Goodpasture syndrome; anatomy of an epitope.
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Anti-Glomerular Basement Membrane Disease↗
Biomedical subjects
M Segelmark
Publications and source records attributed to M Segelmark.
Autoantibodies against components of renal basement membrane.
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Antibody Specificity↗
Systemic necrotizing vasculitides in severe alpha1-antitrypsin deficiency.
We describe the clinical presentation and outcome in a series of eight patients with systemic necrotizing vasculitis and severe alpha1-antitrypsin (AAT) deficiency followed up at three Swedish hospitals during 1968-92. We also review six other cases reported in the literature during the same period. Diagnosis of severe AAT deficiency was based on the presence of the PiZZ phenotype, or low plasma total trypsin inhibitory capacity, or a low plasma AAT concentration (10-40% of the normal mean value) and presence of the PiSZ or PiFZ phenotype. The diagnosis of systemic vasculitis was biopsy-verified in all eight patients. Pretreatment laboratory findings, treatment protocol, and outcome were reviewed in each of the 14 patients. Of the eight patients in the Swedish series, six had systemic vasculitis of the microscopic polyangiitis form, one had Wegener's granulomatosis, and another had Henoch-Schönlein purpura. In the series as a whole (n = 14), median age at diagnosis was 48 years (range 44-84), the median number of affected organs was eight, and all 14 patients had skin involvement, and either renal or joint involvement (in most cases both); 71% (10/14) had emphysema; 57% (8/14) had hepatic abnormalities (two having cirrhosis, two fibrosis, and one multiple aneurysms in hepatic arteries); one patient who presented with acute ulcerative colitis developed manifest vasculitic syndrome three years later; and 64% (9/14) died, the major cause of death being renal failure. This syndrome, characterized by multiple organ involvement and fatal outcome, has been underdiagnosed. Physicians should be alert to the presence of the PiZ AAT deficiency gene in patients with systemic vasculitis, especially when the course is progressive or when the patient also has emphysema or cirrhosis. Awareness of those features may aid prompt recognition and enable early treatment.
Adult↗
The PiZ gene of alpha 1-antitrypsin as a determinant of outcome in PR3-ANCA-positive vasculitis.
We have previously demonstrated that a strong correlation exists between systemic vasculitis with proteinase 3-anti-neutrophil cytoplasm antibodies (PR3-ANCA) and heterozygosity for alpha 1-antitrypsin (alpha 1-AT) deficiency, PiZ. In the present study we characterized the PiZ-positive subgroup by laboratory findings, clinical features and outcome. The series studied consisted of 18 PiZ-positive and 81 PiZ-negative PR3-ANCA patients, comparable in sex ratio, age, C-reactive protein concentrations and renal function at diagnosis, and treatment. PiZ-positive patients had a more disseminated disease as reflected by the number of affected organs (P < 0.01). We found no group differences in relapse tendency. Overall mortality was 39% (7 of 18) in the PiZ-positive and 16% (13 of 81) in the non-PiZ group (P = 0.048). When survival analysis was restricted to 66 patients included in the study at disease onset, the group difference was significant (P = 0.016). The results suggest that the subnormal response of plasma alpha 1-AT seen in PiZ-heterozygotes enhances the risk of dissemination of the vasculitic process and the risk of a fatal outcome. We consider alpha 1-AT phenotyping to be justified in cases of PR3-ANCA-positive vasculitis. Treatments decreasing plasma alpha 1-AT (such as plasmapheresis without plasma replacement) may be deleterious.
Adult↗
Screening for anti-neutrophil cytoplasmic antibodies (ANCA): is indirect immunofluorescence the method of choice?
Detection of ANCA has become an important tool for the diagnosis and monitoring of disease activity in Wegener's granulomatosis (WG). Unfortunately, a group of sera positive by the standard method for ANCA detection, indirect immunofluorescence (IIF), are negative when more specific tests with purified proteins are used. In order to examine this discrepancy we examined groups of sera selected for being (i) C-ANCA-positive by IIF; (ii) positive in proteinase 3 (PR3)-ANCA ELISA; and (iii) from 24 patients with WG. The following assays were used: IIF, PR3-ANCA ELISA and capture PR3-ANCA ELISA using MoAbs against PR3. Furthermore, since granule enzymes are released during coagulation, we also measured ANCA in complex with PR3. To test if granule enzyme release had any influence on ANCA detection, both serum and EDTA-plasma were collected from a patient with active WG. No difference, however, was found. In the IIF-positive group (n = 60) 68% of the sera were positive in PR3-ANCA ELISA, 86% in capture PR3-ANCA ELISA and 80% were positive for the PR3/IgG-ANCA complex. In the PR3-ANCA ELISA group (n = 105) 88% of the sera were positive by IIF, 98% in capture PR3-ANCA ELISA and 53% in the PR3/IgG-ANCA assay. To evaluate the tests clinically sera from 24 patients with WG were examined. In the remission group (n = 10) two patients were positive by IIF, four in the PR3-ANCA ELISA, and five in the capture PR3-ANCA ELISA. Fourteen had active disease, and in this group 11/14 were positive by IIF, 10/14 in PR3-ANCA ELISA and 12/14 by capture-ELISA. The correlation between IIF and capture PR3-ANCA ELISA titre (r = 0.72, P = 0.0095) was better than between PR3-ANCA ELISA and IIF (r = 0.56, P = 0.043). It is concluded that the capture PR3-ANCA ELISA is more sensitive than PR3-ANCA ELISA, and that the capture ELISA can be used for screening of PR3-ANCA.
Antibodies, Antineutrophil Cytoplasmic↗
Some patients with anti-myeloperoxidase autoantibodies have a C-ANCA pattern.
Rapidly progressive glomerulonephritis with or without other signs of systemic vasculitis is often accompanied by antibodies to myeloperoxidase. Such antibodies normally produce a perinuclear pattern on ethanol-fixed neutrophils (perinuclear anti-neutrophil cytoplasm antibodies (P-ANCA)) at indirect immunofluorescence. We report here sera from three patients that are anti-myeloperoxidase-positive in ELISA that instead produce a cytoplasmic pattern (classical anti-neutrophil cytoplasmic antibodies (C-ANCA)), a pattern normally seen in conjunction with antibodies to proteinase 3. These sera did not react with proteinase 3. For two of the sera the specificity of the anti-myeloperoxidase reaction was confirmed with inhibition-ELISA experiments and with immunoblotting. A mouse anti-myeloperoxidase MoAb that produces a cytoplasmic pattern is also described. Competition ELISA experiments show that this antibody and anti-myeloperoxidase sera with cytoplasmic pattern recognize epitopes that are separate from epitopes recognized by another perinuclear pattern producing anti-myeloperoxidase MoAb. 'Cytoplasmic pattern' epitopes as well as 'perinuclear pattern' epitopes can be found on all three major myeloperoxidase isoforms, after separation by ion exchange chromatography. Affinity chromatography, using the cytoplasmic pattern producing anti-myeloperoxidase monoclonal antibody, shows that the epitope recognized by this MoAb is present on all myeloperoxidase molecules. This epitope is not confined to any special subpopulation. These findings indicate that all myeloperoxidase do not relocate after ethanol fixation, and that C-ANCA and P-ANCA epitopes exist simultaneously on the same myeloperoxidase molecule. We propose that the two immunofluorescence patterns arise due to different availabilities of the epitopes in the microenvironment where myeloperoxidase is present.
Aged↗
Strong link between the alpha 1-antitrypsin PiZ allele and Wegener's granulomatosis.
OBJECTIVES: To ascertain whether a relationship exists between the PiZ alpha 1-antitrypsin (alpha 1AT) variant and antineutrophil cytoplasm antibodies (ANCA)-positive vasculitis in a large group of Swedish patients, and whether analysis for the presence of the PiZ variant might be useful for diagnostic or prognostic purposes. DESIGN: Retrospective cross-sectional study. SETTING: The Department of Internal Medicine, Malmö General Hospital, and the Department of Nephrology, University of Lund, Sweden. SUBJECTS AND MAIN OUTCOME MEASURES: Serum samples from 105 proteinase 3-ANCA-positive patients were analysed using an ELISA with a monoclonal antibody specific for the PiZ-gene product. Complete clinical data were retrieved for 84% (88/105) of the patients, for diagnosis and survival analysis. RESULTS: We identified 17 heterozygotes and one homozygote (P < 0.0001). All 88 patients with available clinical data were considered to have some form of microscopic vasculitis including 66 (75%) diagnosed as having Wegener's granulomatosis (WG), of whom 15 (23%) were PiZ heterozygotes (odds ratio 6.0, 95% confidence interval 3-10). There were no significant differences between PiZ carriers and noncarriers in sex distribution, mean age at onset of disease, interval between onset and inclusion in the study, or in median duration of follow-up (P > 0.2 for all comparisons). During follow-up, 38% (6/16) of the PiZ heterozygotes died, compared with 17% (11/66) of noncarriers of the variant (P= 0.02), which suggests that PiZ heterozygosity may be a marker of poor prognosis. PiZ heterozygotes with systemic vasculitis would not appear to be identifiable by their pretreatment plasma alpha 1AT concentrations, as all such patients in the present study had concentrations within or above the normal range. CONCLUSION: We conclude that heterozygotes for the PiZ variant of the alpha 1AT gene are at greater risk of than the general population of developing WG. Knowledge of such a genetic factor may not only aid our understanding of the mechanism involved in this illness but may also serve as significant prognostic factor in following the course of the disease.
Adolescent↗
ANCA and IgG subclasses.
We studied IgG subclass distribution of ANCA in 247 patients with ELISA. We used monoclonal subclass specific antibodies and purified MPO and proteinase 3 as antigen. We found that IgG1 and IgG4 yield high values in both groups. We also found that high IgG2 titers were rare among the anti-proteinase 3 positive, while it was common among anti-MPO sera. In the assays for IgG3 the situation was the opposite.
Antibodies, Antineutrophil Cytoplasmic↗
IgG subclasses of antineutrophil cytoplasm autoantibodies (ANCA).
Sera that had been positive in routine ELISA for ANCA were studied retrospectively for the IgG subclass distribution of these autoantibodies. An ELISA previously developed for measurement of IgG subclasses of anti-GBM antibodies was modified for this purpose. Of a total of 247 sera, 114 were found to be positive in at least one of the assays for IgG subclasses of anti proteinase 3, 72 of these patients were men and 42 were women, giving a ratio of 1.8. Also 134 sera were positive in at least one of the IgG subclass assays for antimyeloperoxidase (MPO), with a male/female ratio of 0.97. The ANCA seem to consist mainly of IgG1 and IgG4 autoantibodies. Among the anti-MPO group, IgG2 is relatively common and IgG3 is scarce. Contrasting with this, IgG3 is relatively common in the antiproteinase 3 group. In this group high IgG2 titres are rare. Twelve sera were found to be positive for both autoantigens. Clinical data were studied for 44 patients. Prognosis for old patients was found to be poor. Patients with inactive disease were often positive in only one subclass assay, while patients with active disease were positive in two or more subclass assays (P < 0.01).
Adolescent↗
Antigen restriction and IgG subclasses among anti-GBM autoantibodies.
Thirty-seven sera positive for anti-GBM antibodies (antibodies against the 'Goodpasture antigen' = NC1-domain of collagen IV) in routine analysis were studied for antigen restriction and IgG subclasses. Four different polypeptides, the four alpha-chains (alpha 1, alpha 2, alpha 3 and alpha 4) of human collagen IV NC1, were used as coating in ELISA assays. Autoantibodies were detected with monoclonal antibodies towards human IgG subclasses. All patients had antibodies to the alpha 3 chain, and most patients reacted much more to the alpha 3 peptide than to any of the other three peptides. This shows that the NC1 domain of the alpha 3 chain of collagen IV is the major target for anti-GBM antibodies. A minor group of patients, 6 of 37, showed no antigen restriction and had only moderate titres. It remains to be studied, however, whether they have antibodies to another epitope and a different clinical picture. All four subclasses of IgG antibodies were present, but IgG1 antibodies dominated. A group, consisting of females mainly, had relatively high titres of IgG4 antibodies to the NC1 domain of the alpha 3 chain of collagen IV.
Anti-Glomerular Basement Membrane Disease↗
How and why should we detect ANCA?
Antineutrophil cytoplasmic antibodies (ANCA) have become an established tool for the diagnosis of systemic vasculitis. The major role for ANCA testing is in diagnosing renal insufficiency of unknown origin, where a positive test indicates whether the patient will benefit from immunosuppressive treatment or not. A negative test result almost completely rules out the presence of systemic vasculitis. In this clinical setting the major antigens for ANCA are proteinase 3 and myeloperoxidase, and antibodies to these antigens can best be tested by ELISA. In other clinical settings like inflammatory bowel disease, arthritis and so on, several other ANCA specificities have been described and the IIF test is preferred. However, the clinical value of these somewhat more esoteric specificities is doubtful. New developments in assay techniques and better knowledge of specific epitopes will lead to tools for the improved diagnosis as well as follow up of patients during treatment, as has already been seen with the capture assay for PR3-ANCA.
Antibodies, Antineutrophil Cytoplasmic↗
Pseudomonas-induced lung damage in cystic fibrosis correlates to bactericidal-permeability increasing protein (BPI)-autoantibodies.
OBJECTIVE: Lung damage is the most common cause of death in cystic fibrosis (CF). It is induced by bacterial colonization and inflammatory activity perpetuates its course. Autoantibodies directed against BPI (bactericidal permeability increasing protein), called BPI-ANCA, have recently been associated with cystic fibrosis. Here we confirm this association and evaluate the relation between ANCA and total IgG level as they relate to bacterial colonization, pulmonary function, and musculoskeletal symptoms. METHODS: BPI-ANCA, MPO-ANCA, and PR3-ANCA were measured with ELISA in 46 adult patients with CF. Total IgG was determined by immunoturbidimetry. Results were correlated to bacterial colonization, lung function and musculoskeletal symptoms. RESULTS: BPI-ANCA was found in 33 patients. In the whole group, both BPI-ANCA and total IgG were inversely correlated to lung function, but in patients chronically colonized with Pseudomonas aeruginosa (P. aeruginosa), BPI-ANCA alone was correlated to lung damage (p = 0.01). Median lung function, measured as forced expiratory volume in 1 second, in P. aeruginosa colonized patients with high levels of BPI-ANCA was 43% of the predicted value. In BPI-ANCA negative, the corresponding figure was 83%. In patients not colonized with P. aeruginosa, this relation was less evident. No correlation between ANCA and musculoskeletal symptoms was seen. CONCLUSION: P. aeruginosa induced lung damage in CF patients is associated with the presence of BPI-ANCA. P. aeruginosa colonized patients without BPI-ANCA have almost normal lung function. We suggest that BPI-ANCA discriminate P. aeruginosa colonized CF patients with severe lung damage from those whose disease is less destructive. Vasculitis like symptoms in CF are not ANCA associated.
Adult↗