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- Kalaaji, M, et al.
(författare)
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Critical comparative analyses of anti-alpha-actinin and glomerulus-bound antibodies in human and murine lupus nephritis
- 2006
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Ingår i: Arthritis and Rheumatism. - : Wiley. - 1529-0131 .- 0004-3591. ; 54:3, s. 914-926
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Tidskriftsartikel (refereegranskat)abstract
- Objective. Although anti-double-stranded DNA (anti-dsDNA) antibodies are important in lupus nephritis, the question regarding which glomerular structures (a-actinin, nucleosomes, or others) are recognized by nephritogenic anti-dsDNA antibodies is still controversial. In this study, we determined which glomerular structures are recognized by monoclonal and in vivo-bound nephritogenic antibodies. Methods. Western blotting was used to analyze the ability of nephritogenic anti-dsDNA antibodies to recognize glomerular and nucleosomal structures. Sera from patients with lupus nephritis, sera from random antinuclear antibody-positive patients, and paired antibodies from sera and kidney eluates from nephritic (NZB X NZW)F-1 mice were analyzed for activity against proteins identified by monoclonal nephritogenic antibodies, and against a-actinin, dsDNA, nucleosomes, histone H1, heparan sulfate, DNase I, and type IV collagen. Immunoelectron microscopy was used to determine the glomerular localization of a-actinin and in vivo-bound autoantibodies in nephritic (NZB X NZW)F-1 mouse kidneys. Results. Anti-alpha-actinin antibodies were observed in human and murine lupus nephritis sera and in sera from patients without systemic lupus erythematosus and were not detected in kidney eluates from nephritic mice. Antibodies to dsDNA and histone H1 were detected in all eluates. Western blot analyses revealed that nephritogenic anti-dsDNA antibodies recognized a 32-kd band, identified as histone H1. Competitive enzyme-linked immunosorbent assay demonstrated that nephritogenic monoclonal antibodies, and dominant antibodies eluted from nephritic kidneys, cross-reacted with dsDNA and H1. This cross-reactive anti-H1 specificity was largely absent in sera from those mice. Immunoelectron microscopic analysis of nephritic (NZB X NZW)F-1 mouse kidneys revealed that antibodies eluted from kidneys, but not anti-alpha-actinin antibodies, bound to distinct nephritis-associated electron-dense structures linked to glomerular basement membranes. Conclusion. Cross-reactive anti-dsDNA/anti-histone H1 antibodies, but not anti-alpha-actinin antibodies, are central among those deposited in nephritic glomeruli.
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| 2. |
- Kalaaji, M., et al.
(författare)
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Glomerular apoptotic nucleosomes are central target structures for nephritogenic antibodies in human SLE nephritis
- 2007
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Ingår i: Kidney International. - : Elsevier BV. - 1523-1755 .- 0085-2538. ; 71:7, s. 664-672
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies to double-stranded (dsDNA) are associated with systemic lupus erythematosus (SLE) and directly involved in human lupus nephritis. Information about their glomerular target antigens is inconsistent, and whether availability of target antigens, antibody specificity or avidity are nephritogenic parameters, is not determined. In this study, we analyzed renal tissue from anti-dsDNA antibody-positive lupus patients with nephritis by morphological and immunological assays, including immune electron microscopy (IEM) and colocalization IEM, an EM-based confocal microscopy assay. IEM demonstrated that antibody deposits were confined to electron dense structures (EDS) in glomerular membranes. These autoantibodies colocalized with nucleosome-binding anti-dsDNA/-histone/-transcription factor antibodies. To confirm the colocalization IEM-data, we developed a colocalization terminal deoxynucleotidyltransferase (TdT) biotin-dUTP nicked end-labeled (TUNEL) IEM assay where extracellular DNA was traced by TdT-mediated introduction of biotinylated nucleotides and autoantibodies by IEM. Results consistently demonstrated that DNA colocalized with autoantibodies in glomerular membrane-associated EDS. The colocalization IEM and colocalization TUNEL IEM assays thus demonstrate that intra-glomerular membrane-associated nucleosomes are targeted by anti-dsDNA autoantibodies in human lupus nephritis. The data provide a new approach to understand basic molecular and immunological processes accounting for antibody-mediated nephritis in human SLE.
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