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- Hellmark, Thomas, et al.
(författare)
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Epitope mapping of anti-glomerular basement membrane (GBM) antibodies with synthetic peptides
- 1996
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Ingår i: Clinical and Experimental Immunology. - : Oxford University Press (OUP). - 0009-9104 .- 1365-2249. ; 105:3, s. 504-510
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Tidskriftsartikel (refereegranskat)abstract
- Autoantibodies to the non-collagenous (NC1) domain of the alpha 3(IV)-chain of type IV collagen are found in sera from patients with anti-GBM nephritis. These antibodies have been shown to be pathogenic. In this study the antibody specificity has been investigated in patients with Goodpasture's syndrome and from a patient with atypical anti-GBM antibodies, recognizing the alpha 1(IV)-chain only. Overlapping synthetic peptides, covering the complete NC1 domains of the alpha 1(IV)- and alpha 3(IV)-chains were used in sandwich ELISA and competitive ELISA. None of the Goodpasture sera showed reactivity to the synthetic peptides. However, antibodies from the patient with atypical anti-GBM antibodies recognized a 20 amino acid peptide from the alpha 1(IV)-chain. The reactive peptide was further narrowed down with glycine substitution of the different amino acids. We have localized the epitope to the four last C-terminal amino acids of the alpha 1(IV)-chain, with the sequence 1754-MRRT. The two arginine residues were found to be essential for antibody binding. Threonine is important, while methionine is of less importance. These four amino acids are also determined to be the smallest peptide that could inhibit the binding of the autoantibodies to the native alpha 1(IV)-chain. This study shows that overlapping peptides can be used to map linear epitopes. However, for conformational epitopes such as the Goodpasture epitope, other methods must be used. It would be prognostically important to know the fine specificity of anti-GBM antibodies, since the patient with anti-alpha 1(IV) antibodies had a mild disease, while the Goodpasture patients with anti-alpha 3(IV) antibodies had a rapidly progressive disease.
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- Hellmark, Thomas, et al.
(författare)
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Goodpasture disease. Characterization of a single conformational epitope as the target of pathogenic autoantibodies
- 1999
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 274:36, s. 25862-25868
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Tidskriftsartikel (refereegranskat)abstract
- Goodpasture disease is a prototype autoimmune disease characterized by the formation of autoantibodies against the heterotrimeric basement membrane collagen type IV, which causes a rapidly progressive glomerulonephritis. The pathogenic antibody response is directed to the non-collagenous (NC1) domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1), but not to the homologous region of the alpha1(IV)NC1. To identify the conformation-dependent immunodominant epitope on the alpha3(IV)NC1, a variety of recombinant NC1 domains were constructed by replacing single residues of alpha3(IV) with the corresponding amino acids from the nonreactive alpha1(IV) chain. Replacement mutations were identified that completely destroyed the Goodpasture epitope in the alpha3(IV) chain. Based on the identification of these critical positions, the epitope was finally reconstructed within the frame of the alpha1(IV) chain. The substitution of nine discontinuous positions in the alpha1(IV)NC1 with amino acid residues from the alpha3 chain resulted in a recombinant construct that was recognized by all patients' sera (n = 20) but by none of the sera from healthy controls (n = 10). This provides, for the first time, the molecular characterization of a single immunodominant conformational epitope recognized by pathogenic autoantibodies in a human autoimmune disease, representing the basis for the development of new epitope-specific strategies in the treatment of Goodpasture disease.
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| 5. |
- Hellmark, Thomas, et al.
(författare)
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Identification of a clinically relevant immunodominant region of collagen IV in Goodpasture disease
- 1999
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Ingår i: Kidney International. - : Elsevier BV. - 1523-1755 .- 0085-2538. ; 55:3, s. 936-944
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The characteristic feature of Goodpasture disease is the occurrence of an autoantibody response to the noncollagenous domain of the alpha3 chain of type IV collagen [alpha3(IV)NC1] in the alveolar and glomerular basement membrane. These antibodies are associated with the development of a rapidly progressive glomerulonephritis, with or without lung hemorrhage, whereas autoantibodies specific for the other alpha chains of the heterotrimeric type IV collagen probably do not cause disease. In this study, we have investigated whether differences in fine specificity of autoimmune recognition of the alpha3(IV)NC1 correlate with clinical outcome. METHODS: For mapping of antibody binding to type IV collagen, chimeric collagen constructs were generated in which parts of the alpha3(IV)NC1 domain were replaced by the corresponding sequences of homologous nonreactive alpha1(IV). The different recombinant collagen chimeras allowed the analysis of antibody specificities in 77 sera from well-documented patients. RESULTS: One construct that harbors the aminoterminal third of the alpha3(IV)NC1 was recognized by all sera, indicating that it represents the dominant target of the B-cell response in Goodpasture disease. Seventy percent of the samples recognized other parts of the molecule as well. However, only reactivity to the N-terminus of the alpha3(IV)NC1 correlated with prognosis, that is, kidney survival after six months of follow-up. CONCLUSION: The results indicate the crucial importance of antibody recognition of this particular domain for the pathogenesis of Goodpasture disease, thereby opening new avenues for the development of better diagnostic and therapeutic procedures.
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- Hertervig, Erik, et al.
(författare)
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Anti-neutrophil cytoplasmic antibodies in chronic inflammatory bowel disease. Prevalence and diagnostic role
- 1995
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Ingår i: Scandinavian Journal of Gastroenterology. - : Informa UK Limited. - 1502-7708 .- 0036-5521. ; 30:7, s. 693-698
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Anti-neutrophil cytoplasmic antibodies (ANCA), originally found to be associated with vasculitis, have been reported to be present in chronic inflammatory bowel disease. Most often the ANCA staining pattern is of the perinuclear type (p-ANCA), although nuclear and cytoplasmic stainings are seen. Single studies have shown some of the antibodies to react with lactoferrin or cathepsin G; however, most studies have not been able to determine a main antigenic specificity. We studied the prevalence of ANCA in sera from 155 patients with ulcerative colitis, 128 patients with Crohn's disease, and 51 patients with coeliac disease. The presence of ANCA was correlated to disease activity, extent, and age of onset of the diseases. Furthermore, we tried to characterize the antigen specificity by enzyme-linked immunosorbent assay (ELISA), using elastase, lactoferrin, myeloperoxidase, proteinase 3, and cathepsin G as antigens. METHODS: The sera were screened for ANCA by indirect immunofluorescence. Anti-nuclear antibodies (ANA) were analysed on HEp2 cells, and ELISA for specific ANCA was performed using the antigens mentioned. RESULTS: Most of the sera with positive immunofluorescence had the p-ANCA type of pattern. Seventy-eight of 155 (50.3%) of the patients with ulcerative colitis were ANCA-positive, compared with 31 of 128 (24.2%) of patients with Crohn's disease (p < 0.001). However, in the subgroup with Crohn's colitis, 16 of 44 (36.4%) were ANCA-positive. Only 4 of 51 patients (7.7%) with coeliac disease showed positive immunofluorescence (p < 0.001 compared with ulcerative colitis). Less than 10% of the samples were positive in the specific ELISA assays; thus other than the most well known granule proteins can be the target for ANCA in ulcerative colitis. CONCLUSION: ANCA occur significantly more often in ulcerative colitis than in Crohn's disease. However, the prevalence of ANCA is rather high in Crohn's colitis. ANCA are thus of limited value in differentiating Crohn's colitis from ulcerative colitis. ANCA found in inflammatory bowel disease are different from those associated with vasculitis. The antigen(s) responsible remain to be determined.
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| 7. |
- Segelmark, Mårten, et al.
(författare)
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Binding and inhibition of myeloperoxidase (MPO): a major function of ceruloplasmin?
- 1997
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Ingår i: Clinical and Experimental Immunology. - : Oxford University Press (OUP). - 0009-9104 .- 1365-2249. ; 108:1, s. 167-174
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Tidskriftsartikel (refereegranskat)abstract
- Interactions between plasma proteins and MPO were studied. The protein fraction of normal plasma and serum was shown to exhibit an inhibitory effect on the peroxidase activity of MPO. Most of the inhibitory effect could be retained on an MPO-coupled affinity chromatography column. In particular, a protein with apparent mol. wt of 130 kD showed affinity for MPO. The protein was identified as ceruloplasmin by N-terminal amino acid sequencing and immunochemistry. During separation procedures the peroxidase inhibitory effect was limited to ceruloplasmin-containing fractions of plasma. Purified ceruloplasmin inhibited the peroxidase activity of MPO in a concentration-dependent manner, and exhibited selective binding to MPO-coated microtitre plates. This binding could be inhibited by MPO dissolved in buffer. Correspondingly the binding of MPO to ceruloplasmin-coated plates could be blocked by ceruloplasmin in solution, showing a physical interaction to occur between the two proteins under physiological conditions. We also found affinity to exist between MPO and C3 (and its C3d-containing fragments). However, C3 and C3 fragments did not inhibit the peroxidase reaction in vitro. We propose that ceruloplasmin takes part in the clearance and inactivation of MPO, in vivo. We also speculate that impaired inactivation of MPO may have a pathophysiological role in inflammatory diseases characterized by autoantibodies to MPO, such as rapidly progressive glomerulonephritis with P-ANCA (perinuclear anti-neutrophil cytoplasmic antibodies).
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| 8. |
- Segelmark, Mårten, et al.
(författare)
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The PiZ gene of α1-antitrypsin as a determinant of outcome in PR3-ANCA-positive vasculitis
- 1995
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Ingår i: Kidney International. - : Elsevier BV. - 0085-2538. ; 48:3, s. 844-850
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Tidskriftsartikel (refereegranskat)abstract
- We have previously demonstrated that a strong correlation exists between systemic vasculitis with proteinase 3-anti-neutrophil cytoplasm antibodies (PR3-ANCA) and heterozygosity for α1-antitrypsin (α1-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 α1-AT seen in PiZ-heterozygotes enhances the risk of dissemination of the vasculitic process and the risk of a fatal outcome. We consider α1-AT phenotyping to be justified in cases of PR3-ANCA-positive vasculitis. Treatments decreasing plasma α1-AT (such as plasmapheresis without plasma replacement) may be deleterious.
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- Westman, Kerstin W.A., et al.
(författare)
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Clinical evaluation of a capture ELISA for detection of proteinase-3 antineutrophil cytoplasmic antibody : Technical note
- 1998
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Ingår i: Kidney International. - : Elsevier BV. - 0085-2538. ; 53:5, s. 1230-1236
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Tidskriftsartikel (refereegranskat)abstract
- Detection of antineutrophil cytoplasmic antibodies (ANCA) has become a useful tool in the diagnosis of Wegener's granulomatosis and microscopic polyangiitis. However, the results obtained with indirect immunofluorescence (IIF) and by ELISA for ANCA demonstration do not always correlate. A possible explanation for this finding could be that proteins are denatured during the process of antigen purification or during coating onto the solid phase. To avoid this possibility, a monoclonal antibody to PR3 that is precoated on the plate can be used. In the present study we have used the monoclonal antibody (MoAb) 4A3 for the capture of PR3 in an ELISA, and a clinical evaluation of the diagnostic properties of the new capture ELISA has been made. The sensitivity of the capture PR3-ANCA ELISA was 85% in a material of c-ANCA positive sere. A specificity of 90% was obtained in analyses from patients having various forms of glomerulonephritis. There was a significantly higher diagnostic sensitivity of the capture PR3-ANCA ELISA (85%) compared to c- ANCA by IIF (58%) in patients with Wegener's granulomatosis with renal involvement. Capture PR3-ANCA and direct ELISA for MPO-ANCA together gave a diagnostic sensitivity of 98%, versus 75% using IIF. In conclusion, the capture PR3-ANCA ELISA seems to be a valuable tool in the diagnosis of Wegener's granulomatosis with renal involvement. Preliminary data suggest that the technique may have an advantage over direct ELISA for PR3-ANCA, as well as in the follow-up of c-/PR3-ANCA associated vasculitides. However, further prospective studies are needed to clarify this premise.
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