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C D Pusey

Publications and source records attributed to C D Pusey.

At least 73 records · Page 4Linked to original sources
PubMed1995

ANCA and predicting relapse in systemic vasculitis.

We studied 60 patients with ANCA-positive systemic vasculitis (SV) to assess the prognostic significance of clinical and serological features at presentation, and the value of sequential monitoring of ANCA, C-reactive protein (CRP) and ESR levels as predictors of disease relapse. Patients were recruited at the time of diagnosis, treated with a standard protocol, and assessed monthly for one year. Clinical remission was achieved in 56/60 (93%), and ANCA became undetectable in 50/60 (83%) after treatment. During the one year follow-up period, disease relapses were seen in 23 (38%) patients. No specific associations were observed between initial disease presentation, initial ANCA level or ANCA antigenic specificity and relapse. However, 13/23 (57%) of relapses were preceded by a rise in ANCA a mean of 7.8 weeks earlier, while at the time of relapse 19/23 (83%) were ANCA-positive. Rises in CRP and ESR occurred in 23/60 (38%) and 14/43 (33%), respectively, but were less closely associated with relapse than ANCA. A sustained rise in ANCA was seen in six patients without relapse while clinical relapse occurred with a negative ANCA in four. Sequential ANCA monitoring at monthly intervals during disease remission is of value, at least during the first year, in the prediction and diagnosis of relapse in SV, and is superior to measurement of CRP or ESR.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1995

Molecular analysis of HLA class II genes in Goodpasture's disease.

A molecular analysis of HLA class II genes was undertaken in order to characterize the previously reported association between HLA-DR2 and glomerulonephritis caused by antibodies to glomerular basement membrane (Goodpasture's disease). Genomic DNA was prepared from 53 patients with Goodpasture's disease and analysed by: (i) Southern blotting using cDNA probes to DRB, DQA and DQB genes, after digestion with TaqI endonuclease; (ii) allele-specific oligonucleotide probing of specifically amplified DNA; and (iii) nucleotide sequencing of relevant alleles. The patients had a greatly increased frequency of DRw15 (a subspecificity of DR2) which was present in 75.5% of patients and 31% of controls (p < 0.0001). The frequency of DR4 was also increased, especially in patients without DRw15. Overall, 90.5% of the patients had either DRw15 or DR4. In contrast, the frequency of DR1 was significantly reduced (patients 5.6%, controls 20.7%, p < 0.01). Differences in the frequencies of DQA and DQB alleles could all be explained by linkage disequilibrium. Nucleotide sequences of relevant alleles were identical to those previously published. Comparison of derived amino acid sequences of expressed DR beta chains showed that the DR beta chains of DRw15 and DR4 shared a six-amino-acid motif from positions 26-31, that included four polymorphic amino acids none of which are shared with DR1. A sequence-specific oligonucleotide detected this amino-acid motif in 45/49 (91.8%) patients tested. Thus, this particular motif, which lies on the floor of the antigen binding groove, has a stronger association with Goodpasture's disease than any individual allele, and may be of pathogenic significance.

Alleles↗
PubMed1995

Development and application of an ELISA for Goodpasture's disease based on sheep alpha 3(IV)NC1 domains.

A high titre of antibodies to collagenase-solubilised human glomerular basement membrane (CS-GBM) is almost pathognomic of Goodpasture's (anti-GBM) disease. In order to develop an assay independent of scarce human material, a molecule of approximately 26 kD corresponding to the C-terminal NC1 domain of the alpha 3 chain of type IV collagen was purified from sheep GBM by gel filtration and reverse phase HPLC. This molecule was antigenic when assessed by inhibition studies, by immunoblotting, and as a ligand on ELISA plates. An ELISA using this sheep alpha 3(IV)NC1 preparation to detect circulating anti-GBM antibodies gave comparable results to the standard RIA using crude CS-GBM. Sera from patients with a variety of nephropathies other than Goodpasture's disease gave negative results. In a prospective study, 170 consecutive sera were analysed by both the ELISA and the RIA. Twenty seven specimens gave positive results in one or both of the assays. Eleven of these were confirmed as true positive results and all were correctly identified by the RIA. Two false negative results in the ELISA occurred in previously treated cases, and both sera were only weakly positive by RIA. The RIA gave 13 false positive results compared with five by ELISA. The ELISA using highly purified sheep antigen is a robust, reliable, and more specific alternative to immunoassays based on crude human antigen preparations.

Animals↗
PubMed1994

Production and characterization of recombinant Goodpasture antigen in insect cells.

The Goodpasture antigen is the target of anti-basement membrane autoantibodies in Goodpasture's disease, a severe human autoimmune disease characterized by glomerulonephritis and lung hemorrhage. It has been identified as the NC1 domain of the alpha 3 chain of type IV collagen (alpha 3(IV)NC1), a minority component of glomerular basement membrane (GBM). Protocols for obtaining pure human antigen are laborious and low yielding and require cadaver kidneys. Recombinant alpha 3(IV)NC1 produced in Escherichia coli has been insoluble and poorly recognized by patients' autoantibodies. We have used the baculovirus expression system to produce the antigen as a soluble product in Sf9 cells. A transfer vector was constructed from cDNAs encoding the leader peptide, NH2 terminus, and 7 S domain of the human alpha 1 chain of type IV collagen and was joined inframe to the NC1 domain and COOH terminus of the human alpha 3 chain under the control of the polyhedrin promoter. It therefore encodes a hybrid "mini"-collagen chain from which the majority of the central triple helical region has been deleted. The recombinant antigen was seen on SDS-polyacrylamide gel electrophoresis and Western blots of supernatants at its predicted molecular size of 41 kDa and as dimers of 82 kDa. It reacted strongly with human autoantibodies by Western blotting and enzyme-linked immunosorbent assay, inhibited binding of autoantibodies to human GBM, and bound two monoclonal antibodies known to recognize human alpha 3(IV)NC1. A common alternatively spliced variant alpha 3(IV)NC1 mRNA, leading to a truncated NC1 domain of 60 amino acids, was expressed as a fusion protein with the same alpha 1 NH2-terminal sequence. It failed to be exported from the cell and was not recognized by autoantibodies. Other NC1 domains could be expressed in the same way. These recombinant molecules should prove invaluable for the in vitro study of the immunopathogenesis of Goodpasture's disease, and the approach provides a means by which interactions between the different type IV collagen chains found in GBM could be studied in vitro.

Animals↗
PubMed1994

In vivo treatment with a monoclonal antibody to T helper cells in experimental autoimmune glomerulonephritis in the BN rat.

Experimental autoimmune glomerulonephritis (EAG) was induced in brown Norway (BN) rats by a single i.m. injection of homologous glomerular basement membrane (GBM) in Freund's complete adjuvant. This model of anti-GBM disease is characterized by the development, over several weeks, of circulating and deposited anti-GBM antibodies, accompanied by albuminuria. We examined the effects of treatment with MoAb W3/25 (anti-CD4) at different doses, starting at the time of immunization and continued for the duration of the study or for a limited period only. Continued treatment with W3/25, at a dose of 5 or 10 mg/kg intraperitoneally three times per week for 4 weeks, produced a marked reduction in circulating anti-GBM antibodies, absence of detectable deposited antibody and virtual absence of albuminuria. When W3/25 treatment, at 5 or 10 mg/kg, was stopped after 2 weeks, there was still a significant reduction in anti-GBM antibodies and albuminuria at 4 weeks. A similar effect on the disease was achieved when W3/25 was administered only three times during the first week at a dose of 30 mg/kg. Animals injected with W3/25 at a dose of 10 mg/kg through the course of disease showed < 10% W3/25+ cells by FACS analysis of splenic lymphocytes at week 4, while controls and animals treated for shorter periods showed > 30% W3/25+ cells. These results demonstrate that W3/25 can prevent the development of EAG, and that this effect is not dependent on persistent depletion of T cells. Further work is necessary to determine whether anti-T cell therapy is effective in established EAG, and may be worth investigating in human anti-GBM disease.

Albuminuria↗
PubMed1994

Tissue-specific distribution of the Goodpasture antigen demonstrated by 2-D electrophoresis and western blotting.

The target of autoantibodies in Goodpasture's disease, the Goodpasture antigen has recently been characterized as the NC1 domain of the alpha 3 chain of type IV collagen. In order to study the Goodpasture antigen in different organs, NC1 domains were isolated from basement membranes (BM) of human glomeruli (GBM), tubules (TBM), alveoli (ABM), placenta (PBM) and aorta (VBM). NC1 preparations were separated by 2-D electrophoresis, and silver stained or immunoblotted to determine the subunit structure and antigenicity of different basement membranes. All basement membranes contained monomeric components of MW 26 kDa and 24 kDa, and associated dimers, corresponding to the 2-D location of alpha 1(IV) and alpha 2(IV) chains respectively. However, GBM, ABM, and to a lesser extent TBM possessed an extra set of monomeric components of MW 28 kDa and associated dimers corresponding to the proposed location of alpha 3 (IV) and alpha 4 (IV) chains. 2-D-separated polypeptides were Western blotted with autoantibodies from patients with Goodpasture's disease, a monoclonal antibody to the Goodpasture antigen (P1) and a monoclonal antibody to the bovine alpha 3 (IV) chain. The predominant binding of all these reagents was to cationic 28 kDa monomers of GBM, ABM and TBM, corresponding to the alpha 3 (IV) chain, although autoantibodies and Pl also bound to neutral 28 kDa monomers, corresponding to the alpha 4 (IV) chain. Autoantibodies bound weakly to more neutral components of PBM and VBM, but neither monoclonal antibody bound to these basement membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Glomerular Basement Membrane Disease↗
PubMed1993

The value of indirect immunofluorescence and solid phase techniques for ANCA detection. A report on the first phase of an international cooperative study on the standardization of ANCA assays. EEC/BCR Group for ANCA Assay Standardization.

This study describes the results of phase I of an international effort to develop and standardize assays for the detection of anti-neutrophil cytoplasmic antibodies (ANCA). 12 sera, four of which were selected for their potential to cause problems in the detection of various ANCA specificities, were analyzed in the standard indirect immunofluorescence (IIF) test and in ELISAs for ANCA routinely performed in the seven participating laboratories. The IIF methodology differed with respect to the dilution of the serum being screened and the concentration of the conjugate used. Results from sera with high ANCA titers were similar, although the quantitative values could not be compared. In sera containing rheumatoid factor and anti-nuclear antibodies (ANA), ANCA-unrelated staining patterns were observed. Six antigen preparations were used in ELISA for the detection of cANCA. In ELISA with purified proteinase-3 all three cANCA sera were positive, but not anti-myeloperoxidase (MPO) or anti-lactoferrin (LF) positive sera. The other assays were less sensitive or gave inconsistent results. Various preparations of purified MPO and LF used in ELISA were readily recognized by anti-MPO and anti-LF positive sera. From this study it can be concluded that the IIF test, although performed with different methods, shows comparable results using strongly positive sera. In general solid phase assays for cANCA detection are not well standardized and need improvement although the purified proteinase-3 ELISA is possibly an exception. MPO and LF can be used in ELISA procedures for the detection of pANCA-related antibodies.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1993

The role of T-helper lymphocytes in priming for experimental autoimmune glomerulonephritis in the BN rat.

In our model of experimental autoimmune glomerulonephritis (EAG), BN rats given a single IM injection of homologous glomerular basement membrane (GBM) in FCA develop anti-GBM autoantibodies with focal glomerulonephritis. To investigate the role of lymphocytes in the induction of EAG, we examined the effects of antigen rechallenge and of adoptive cell transfer from donors with nephritis to naive recipients. Groups of animals were rechallenged with GBM in FCA following either low (non-nephritogenic) doses of GBM, or following resolution of EAG. This resulted in an enhanced anti-GBM antibody response in both groups, suggesting the presence of GBM specific T or B memory cells. To investigate this possibility, spleen cells from animals with EAG were transferred into lightly irradiated recipients. There was no significant rise in anti-GBM antibody levels after transfer. However, subsequent challenge with GBM in FCA resulted in an enhanced anti-GBM antibody response by 2 weeks when compared with recipients of normal spleen cells. Cells capable of priming for EAG developed by week 4 after immunization of donors and persisted in the recipients for at least 24 weeks. To investigate which cell type was responsible for this effect, we depleted or positively selected donor spleen cells prior to transfer, using Dynabeads coated with monoclonal antibodies to Th or B lymphocytes. Depletion of Th cells, but not B cells, reduced the enhanced anti-GBM antibody response of recipients challenged with GBM in FCA. Positively selected Th cells, but not B cells, resulted in an enhanced anti-GBM antibody response similar to that in positive controls. These results demonstrate the presence of immunological memory for the autoantigen and show that priming for EAG is mediated by Th lymphocytes.

Animals↗
PubMed1993

Myeloperoxidase binds to vascular endothelial cells, is recognized by ANCA and can enhance complement dependent cytotoxicity.

Myeloperoxidase (MPO) and proteinase-3 (Pr-3) can bind to vascular endothelial cells (EC) and are available for recognition by autoantibodies present in P-ANCA or C-ANCA containing sera, respectively. The bound MPO also retains its enzymic functions and effectively interacts with hydrogen peroxide to mediate detachment of endothelial cells from their substratum. EC bound MPO-anti-MPO complexes can contribute to the complement-dependent EC injury demonstrated by some P-ANCA sera.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1993

Significance of autoantibodies to purified proteinase 3 in systemic vasculitis.

Proteinase 3 was purified from human neutrophils and used in ELISA to examine sera from 150 patients with systemic vasculitis. It was found to be the major target of autoantibodies in Wegener's granulomatosis, and anti-proteinase 3 antibodies were also found in some patients with microscopic polyarteritis. Anti-proteinase 3 antibodies were associated with a chronic relapsing course.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1993

Relationship between disease activity and ANCA level by ELISA in the long-term management of vasculitis.

Solid-phase immunoassays were used to examine the relationship between ANCA concentration and disease activity during follow-up of 63 patients with systemic vasculitis. ANCA levels fell with induction therapy. Relapses during long-term follow-up were generally associated with high or rising ANCA concentrations, although the temporal relationship between clinical relapse and changes in ANCA was variable.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1993

Renal transplantation following immunoadsorption in highly sensitized recipients.

Five highly sensitized patients, with panel reactivity greater than 80% for 1.75-5 years, were treated by extracorporeal staphylococcal protein-A immunoadsorption, prednisolone, and cyclophosphamide. The five patients underwent treatment of 18-40 (mean 31) liters of plasma, respectively in 4-7 (mean 5.6) sessions. This reduced the titer of cytotoxic antibodies to sensitizing antigens to < 1/8 in all cases and abolished reactivity to crossreacting antigens. Two patients required retreatment following resynthesis of cytotoxic antibodies. All five patients have been transplanted, and four of these now have stable serum creatinines of 168 mumol/L at 34 months, 208 mumol/L at 29 months, 96 mumol/L at 5 months, and 125 mumol/L at 3 months posttransplantation. One patient had primary graft dysfunction due to acute tubular necrosis; the kidney was removed after eight weeks and showed cortical necrosis without evidence of acute rejection.

Adult↗