Expression and characterization of recombinant rat α3(IV)NC1 and its use in induction of experimental autoimmune glomerulonephritis

Clinical and Experimental Immunology

Haxby

Juggapah

et al. 2008

SummaryExperimental autoimmune glomerulonephritis (EAG) can be induced in Wistar Kyoto (WKY) rats by immunization with the non-collagenous domain (NC1) of the alpha 3 chain of type IV collagen, a3(IV)NC1. In patients with Goodpasture's disease, the major B cell epitope is located at the N-terminus of a3(IV)NC1. In order to investigate whether B and T cell responses in EAG are directed towards immunodominant peptides within the same region of rat a3(IV)NC1, we immunized WKY rats with recombinant rat a3 (

Section: Assessment Of Disease After Immunization With Synthetic Peptmentioning

confidence: 99%

Identification of a nephritogenic immunodominant B and T cell epitope in experimental autoimmune glomerulonephritis

Clinical and Experimental Immunology

Haxby

Juggapah

et al. 2008

“…8,11,13 Briefly, urine samples from experimental animals were subjected to immunoelectrophoresis at 60 v in an electrophoresis tank containing Barbitone buffer (BDH Laboratory Supplies, Poole, UK), pH 9.5, for 6 hours, using a 1% agarose gel (BDH Laboratory Supplies) containing rabbit anti-sera to rat albumin raised in our laboratory. Results were calculated using rat serum albumin standards (which were run at the same time) and expressed in mg per 24 hours.…”

Section: Albuminuriamentioning

confidence: 99%

“…8,11,13 Briefly, recombinant rat ␣3(IV)NC1 was coated onto ELISA plates (Life Technologies, Paisley, UK) at a concentration of 5 g/ml by overnight incubation at 4°C. An optimum dilution of test or control sera, as determined in preliminary experiments, was then applied for 1 hour at 37°C.…”

Section: Circulating Anti-␣3(iv)nc1 Antibody Concentrationsmentioning

confidence: 99%

“…8,11,13 Tissue was embedded in OCT II embedding medium (Miles Inc., Elkhart, IN) on cork disks, snap-frozen in isopentane (BDH Laboratory Supplies), pre-cooled in liquid nitrogen, and stored at Ϫ70°C. Cryostat sections were cut at 5-m in thickness and were incubated with fluorescein isothiocyanate-labeled rabbit anti-rat IgG (Serotec Ltd.).…”

Section: Deposition Of Igg On the Gbmmentioning

confidence: 99%

“…T cells have been shown to be present in the glomeruli of animals with EAG, 11,13 to proliferate in response to ␣3(IV)NC1, 12,17 and to transfer disease to naive recipients. 9,18 Glomerular T cells from rats with EAG show restricted T-cell receptor CDR3 spectratypes, demonstrating that they are an oligoclonal antigen-driven population.…”

mentioning

confidence: 99%

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Mucosal Tolerance Induced by an Immunodominant Peptide from Rat α3(IV)NC1 in Established Experimental Autoimmune Glomerulonephritis

The American Journal of Pathology

Abbott

Karegli

et al. 2009

Self Cite

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar Kyoto (WKY) rats by immunization with the noncollagenous domain of the ␣ 3 chain of type IV collagen, ␣3(IV)NC1. Recent studies have identified an immunodominant peptide, pCol (24-38), from the N-terminus of rat ␣3(IV)NC1; this peptide contains the major B-and T-cell epitopes in EAG and can induce crescentic nephritis. In this study, we investigated the mechanisms of mucosal tolerance in EAG by examining the effects of the nasal administration of this peptide after the onset of disease. A dose-dependent effect was observed: a dose of 300 g had no effect, a dose of 1000 g resulted in a moderate reduction in EAG severity, and a dose of 3000 g produced a marked reduction in EAG severity accompanied by diminished antigen-specific, T-cell proliferative responses. These results demonstrate that mucosal tolerance in EAG can be induced by nasal administration of an immunodominant peptide from the N-terminus of ␣3(IV)NC1 and should be of value in designing new therapeutic strategies for patients with Goodpasture's disease and other autoimmune disorders.

The evolution of crescentic nephritis and alveolar haemorrhage following induction of autoimmunity to glomerular basement membrane in an experimental model of Goodpasture's disease

The Journal of Pathology

Moss

Duda

et al. 2003

Self Cite

Goodpasture's, or anti-glomerular basement membrane (GBM), disease presents with rapidly progressive glomerulonephritis and lung haemorrhage, and is caused by autoimmunity to the NC1 domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1). This study examines the development of crescentic nephritis and alveolar haemorrhage in a model of Goodpasture's disease, experimental autoimmune glomerulonephritis (EAG), induced in WKY rats by immunization with rat GBM in adjuvant. An increase in circulating anti-GBM antibodies and albuminuria was observed by week 2, which increased further by weeks 3 and 4, while a decrease in creatinine clearance was observed by week 2, which decreased further by weeks 3 and 4. The kidneys of animals with EAG showed linear deposits of IgG on the GBM and a transient glomerular infiltration by CD4+ T cells at week 2. By week 3 there were large deposits of fibrin in Bowman's space, and glomerular infiltration by CD8+ T cells and macrophages, accompanied by focal necrotizing glomerulonephritis with crescent formation. Ultrastructural studies showed glomerular endothelial cell swelling and epithelial cell foot process effacement at week 2. As the lesion progressed, capillary loops became occluded and the mesangium became expanded by mononuclear cells. By week 3 there was detachment of the endothelium from the GBM, and accumulation of fibrin beneath the disrupted endothelial cells and in Bowman's space. Occasional breaks were observed in the continuity of the basement membrane, and cytoplasmic projections from infiltrating mononuclear cells could be seen crossing the capillary wall between the lumen and the crescent. The lungs of animals with EAG showed patchy binding of IgG to the alveolar basement membrane (ABM) at week 2, and infiltration of the interstitium by CD8+ T cells and macrophages by weeks 3 and 4, accompanied by both interstitial and alveolar haemorrhage. Ultrastructural studies showed focal mononuclear cell infiltrates in alveolar walls at week 2. Occasional breaks were observed in the basement membrane and adjacent endothelium by weeks 3 and 4, together with accumulation of surfactant and erythrocytes within the alveolar spaces. This study defines for the first time the relationship between the immunological and pathological events during the evolution of EAG, and provides the basis for further work on the pathogenesis of Goodpasture's disease.