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Biomedical subjects

N Turner

Publications and source records attributed to N Turner.

At least 19 recordsLinked to original sources
PubMed1998

Recombinant alpha-chains of type IV collagen demonstrate that the amino terminal of the Goodpasture autoantigen is crucial for antibody recognition.

Goodpasture's disease, an autoimmune disorder causing severe glomerulonephritis and pulmonary haemorrhage, is characterized by antibodies to the glomerular basement membrane (GBM). The principal target antigen has been identified as the carboxyl terminal non-collagenous (NC1) domain of the alpha3-chain of type IV collagen. Anti-GBM antibodies appear to recognize one major epitope that is common to all patients, and is largely conformational. We have analysed antibody binding to recombinant alpha(IV)NC1 domains using a construct and expression system shown to produce correctly folded antigen that is strongly recognized by autoantibodies. In this system, as with the native antigen, alpha3(IV)NC1 was bound strongly by antibodies from all patients, whereas the closely related alpha1(IV) and alpha5(IV)NC1 domains, similarly expressed, showed no such binding. A series of chimeric NC1 domains, between human alpha3(IV) and alpha1(IV), and between human and rat alpha3(IV), were expressed as recombinant molecules, and were recognized by autoantibodies to varying degrees. Strong binding required the presence of human alpha3(IV) sequence in the amino terminal region of both sets of chimeric molecules. This work strongly suggests that the amino terminal of alpha3(IV)NC1 is critical for antibody recognition, whereas the carboxyl terminal end of alpha3(IV)NC1 has a less important role.

Animals↗
PubMed1997

Characterization of the interaction between the Staphylococcus aureus clumping factor (ClfA) and fibrinogen.

The ability of Staphylococcus aureus to adhere to adsorbed fibrinogen and fibrin is believed to be an important step in the initiation of biomaterial and wound-associated infections. In this study, we show that the binding site in fibrinogen for the recently identified S. aureus fibrinogen-binding protein clumping factor (ClfA) is within the C-terminus of the fibrinogen gamma chain. S. aureus Newman cells expressing ClfA adhered to microtitre wells coated with recombinant fibrinogen purified from BHK cells, but did not adhere to wells coated with a purified recombinant fibrinogen variant where the 4 C-terminal residues of the gamma chain were replaced by 20 unrelated residues. In addition, a synthetic peptide corresponding to the 17 C-terminal amino acids of the fibrinogen gamma chain effectively inhibited adherence of ClfA-expressing cells to fibrinogen. In western ligand blots, a recombinant truncated ClfA protein called Clf33 (residues 221-550) recognized intact recombinant fibrinogen gamma chains, but failed to recognize recombinant fibrinogen gamma chains where the 4 C-terminal amino acids were altered by deletion or substitution. Previous studies have shown that the C-terminal domain of fibrinogen gamma chains contains a binding site for the integrin alphaIIb beta3 (glycoprotein gpIIb/IIIa) receptor on platelets [Kloczewiak, M., Timmons, S., Bednarek, M. A., Sakon, M. & Hawiger, J. (1989) Biochemistry 28, 2915-1919; Farrell, D. H., Thiagarajan, P., Chung, D. W. & Davie, E. W. (1992) Proc. Natl. Acad. Sci. USA 89, 10729-10732; Hettasch, J. M., Bolyard, M. G. & Lord, S. T. (1992) Thromb. Haemostasis 68, 701-706]. We now show that Clf33 inhibits ADP-induced, fibrinogen-dependent platelet aggregation in a concentration-dependent manner and inhibits adhesion of platelets to immobilized fibrinogen under fluid shear stress, indicating that the binding sites for the platelet integrin and the staphylococcal adhesin overlap. The interaction between Clf33 and fibrinogen was further characterized using the BIAcore biosensor. When soluble Clf33 was allowed to bind to immobilized fibrinogen, a Kd of 0.51 +/- 0.19 microM was experimentally determined using equilibrium binding data. It was also shown that the synthetic C-terminal gamma-chain peptide effectively inhibited this interaction.

Adenosine Diphosphate↗
PubMed1997

Large scale manufacturing of TXU(anti-CD7)-pokeweed antiviral protein (PAP) immunoconjugate for clinical trials.

We have conjugated the murine monoclonal anti-CD7 antibody TXU to the plant hemitoxin pokeweed antiviral protein (PAP) to construct an effective immunotoxin against CD7 antigen positive hematologic malignancies. The scaled-up production and purification of TXU antibody, PAP toxin, and TXU-PAP immunotoxin permitted the manufacturing of a highly purified clinical-grade TXU-PAP preparation. In clonogenic assays, TXU-PAP elicited selective and potent cytotoxicity against CD7 antigen positive human leukemia cells and killed primary clonogenic leukemic cells from T-lineage acute lymphoblastic leukemia (ALL) patients. To our knowledge, this pre-IND work represents the first effort of producing a clinical-grade PAP immunotoxin for treatment of T-lineage ALL. Since the CD7 antigen is also expressed on AML cells, TXU-PAP could also be useful for the treatment of CD7 positive acute myeloid leukemia (AML) patients.

Antibodies, Monoclonal↗
PubMed1996

A single-chain Fv reactive with the Goodpasture antigen.

Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expressed product. HBR-3 was shown by ELISA, immunoblotting, and immunohistologic techniques, to have the same specificity for alpha 3(IV)NC1 as P1 and autoantibodies from patients with Goodpasture's disease. HBR-3 and P1 were shown to have similar affinity for their mutual ligand. On sections of normal human kidney, the scFv bound only to glomerular basement membrane and distal tubular basement membrane. It did not bind to the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigenic form. We have, therefore, generated a scFv which reproduces the specific binding properties of the parent monoclonal antibody, P1. The potential of HBR-3 as a diagnostic reagent in Alport's syndrome has been demonstrated. The development of this recombinant molecule should permit new approaches to the investigation of Goodpasture's disease.

Amino Acid Sequence↗
PubMed1995

Use of proteinase 3 purified by reverse phase HPLC to detect autoantibodies in systemic vasculitis.

The vasculitis autoantigen proteinase 3 was purified from neutrophil primary granules using reverse phase high performance liquid chromatography. It was shown to be free of important contaminants, was enzymatically active and was antigenic to sera containing antineutrophil cytoplasmic antibodies with a cytoplasmic pattern (C-ANCA) by indirect immunofluorescence. Development of an enzyme-linked immunosorbent assay showed that this preparation could be used to detect autoantibodies in Wegener's granulomatosis and microscopic polyangiitis. Assays based on reverse phase HPLC-purified proteinase 3 will be valuable in diagnosis and monitoring of treatment, and should increase our understanding of the autoimmune response in primary systemic vasculitis.

Amino Acid Sequence↗
PubMed1995

Antibody degradation in wound exudates from blowfly infections on sheep.

Sheep were immunised with ovalbumin and then infected with the sheep blowfly, Lucilia cuprina in order to study immunoglobulin and specific antibody degradation at the wound site. Serum and wound exudates were collected over the infection period and the dry weight and protein content of the exudates were determined. Exudates were analysed by SDS-PAGE and immunoblotting for IgG degradation. Levels of IgG and specific anti-ovalbumin antibodies in the exudates were measured by ELISA. The total weight of exudates increased over the whole period of the infection, while protein content increased in the first 24 h and then remained relatively constant. Immunoglobulin was present 6 h after infection and levels increased with protein content. However, the levels of IgG measured were quite different depending on the secondary antibody used in the ELISA. A monoclonal antibody measured mainly intact IgG while a polyclonal anti-IgG measured intact and degraded IgG. This allowed an estimation that approximately 60% of the IgG in exudates was degraded from 6 h after infection. Assays in vitro showed that L. cuprina larval enzymes degraded sheep antibody. However, measurement of specific anti-ovalbumin levels in exudates suggested that although high levels of antibody were degraded this did not necessarily decrease the level of antigen binding. As a result, IgG degradation may assist and not hinder vaccine development by allowing antibody fragments to penetrate the peritrophic membrane and access gut cell antigens.

Animals↗
PubMed1995

Evaluation of proposed methods to update human testing of self-contained breathing apparatus.

The current Man Test protocols used by the National Institute for Occupational Safety and Health for the certification testing of self-contained breathing apparatus (SCBA) do not provide continuous real-time information on the performance of these devices during actual use. In addition, current protocols do not test human subjects at the same absolute work rates but at rates that vary according to the subjects' body weights. This study was conducted to evaluate revised "Use Test" protocols proposed by the U.S. Bureau of Mines, which are normalized to subjects' body weights. No differences in duration were observed among the three body weight categories for the multiple work rate. Use Test 2. It was concluded that the proposed Use Test protocols could form the basis for eventual recommendations to revise the current Man Tests for SCBA performance evaluation.

Adult↗
PubMed1995

The use of topical 5% lignocaine ointment for the relief of pain associated with post-operative nasal packing.

Packing of the nasal cavity remains a common routine precautionary measure following septal surgery. The nasal pack and its removal 24 h later are often cited by patients as the most painful aspects of septal surgery. We present the results of a randomized, prospective controlled trial of the use of topical 5% lignocaine ointment as a method of pain relief following post-operative nasal packing. Post-operative pain as measured using a visual analogue scale at 3 h post-operatively was halved in patients receiving a lignocaine impregnated nasal pack compared with those having a standard vaseline gauze pack (P < 0.05). Pain scores at 6 h post-operatively and at pack removal were also reduced, but these failed to reach significance. No patients suffered reactionary haemorrhage. The use of topical lignocaine ointment is safe and may have a place in the relief of pain due to post-operative nasal packing.

Administration, Topical↗
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↗
PubMed1995

Reaccreditation in general practice. How New Zealand approaches the solution.

Both the College of Family Physicians at Canada and the Royal New Zealand College of General Practitioners require family physicians to complete at least minimum amounts of continuing medical education each year to maintain active membership. The recently introduced reaccreditation program in New Zealand is unique in its approach to encouraging family doctors to assess and improve the services they provide their patients and could be of interest to Canadian family doctors planning their own continuing medical education.

Certification↗
PubMed1994

Solvent/detergent-treated plasma suppresses shear-induced platelet aggregation and prevents episodes of thrombotic thrombocytopenic purpura.

Two children with congenital chronic relapsing thrombotic thrombocytopenic purpura (TTP) have episodes every 3 weeks. These relapses can be prevented by the infusion of normal fresh-frozen plasma (FFP) without concurrent plasmapheresis. We conducted a study to determine whether the exposure of normal plasma to agents that inactivate human immunodeficiency virus and other viruses destroys the component necessary for the effective treatment of this type of TTP that requires only plasma infusion to prevent or reverse relapses. Clinical responsiveness and von Willebrand factor (vWF)-mediated fluid shear stress-induced platelet aggregation were evaluated before and after the infusion of 10 mL/kg FFP or solvent [tri(n-butyl)phosphate]/detergent (Triton X-100)-treated plasma (S/D plasma). Platelet aggregation at shear stresses of 90 to 180 dyne/cm2 (similar to those in the partially occluded microcirculation) imposed for 30 seconds was excessive using the citrated platelet-rich plasma of both patients, and was associated with the presence of unusually large vWF forms in patient platelet-poor plasma. Infusion with either FFP or S/D plasma at 3-week intervals caused the platelet count to increase to (or above) normal within 1 week (on 12 of 12 occasions); the disappearance or diminution of unusually large vWF forms within 1 hour (on 6 of 10 occasions studied); and the reversal within 1 to 4 hours of excessive shear-induced platelet aggregation (on 8 of 9 occasions studied). We conclude that a component in normal plasma resistant to S/D treatment is responsible for preventing thrombocytopenia and TTP episodes, and for controlling excessive shear-induced aggregation in these patients. Our results suggest that excessive in vivo platelet aggregation in chronic relapsing TTP and excessive in vitro vWF-mediated shear-induced aggregation may be similar phenomena.

Blood Transfusion↗
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

Gene expression of plasminogen activator inhibitor 1 in transplant kidneys complicated by renal vein thrombosis: a combined study by in-situ hybridization and immunohistochemistry.

In an attempt to understand the pathogenesis of renal vein thrombosis occurring early after renal transplantation, gene expression of plasminogen activator inhibitor-1 (PAI-1) was investigated by an in-situ hybridization technique. The cases examined were six transplant kidneys complicated by renal vein thrombosis, four 'normal' kidneys and five time-matched transplant kidneys not complicated by renal vein thrombosis but showing acute tubular necrosis, infection, or normal histology. The cell types expressing PAI-1 mRNA were also studied by combined in-situ hybridization and immunohistochemical double staining techniques. Our results showed that PAI-1 mRNA was expressed in transplant kidneys complicated by renal vein thrombosis but there was no detectable expression in 'normal' kidneys, nor in time-matched transplant kidneys not complicated by thrombosis. Double staining showed that PAI-1 mRNA was predominantly expressed by capillary endothelial cells, particularly around large- or medium-sized renal arteries and small nerves. Smooth-muscle cells in the wall of major or medium-sized renal arteries also showed positive expression of PAI-1 in three of six thrombosed transplants. However, endothelium in the major renal vein showed relatively little signal. The pattern was different from that in rejection. The possible relevance of these findings is discussed.

Adult↗
PubMed1993

The treatment of epiphora with balloon dacryocystoplasty.

We examine a new treatment for epiphora: balloon dilatation of the nasolacrimal duct, or dacryocystoplasty. The procedure, which was performed as an out-patient procedure under local anaesthetic, was carried out on 20 patients with epiphora, 17 due to anatomical and 3 due to functional obstruction. There was an overall success rate of 60%, with complete relief of symptoms in 4 patients and a symptomatic improvement in another 8 (follow-up 2-9 months, mean 4.2 months). All patients tolerated the procedure well with minimal per-operative and post-operative discomfort. A higher success rate was seen in those patients with either a low anatomical or a functional obstruction. The procedure can be easily repeated and, if unsuccessful, still allows a dacryocystorhinostomy to be performed at a later date.

Adolescent↗
PubMed1992

Proton transport and Na+/H+ exchange in vesicles isolated from sockeye salmon (Oncorhynchus nerka) kidneys during migration from salt to fresh water.

Renal epithelial function, proton flux and sodium stimulated proton flux, was observed in vesicles isolated from the brush border of the proximal tubule of Sockeye Salmon (Oncorhynchus nerka) during migration. Brush border membrane vesicles (BBMV) were isolated from the body kidney of Sockeye Salmon using aggregation/differential centrifugation techniques. Vesicle purity was tested using a series of epithelial and basal lateral markers including alkaline phosphatase, maltase, gamma-glutamyl transferase (GGTP), Mg(2+)-activated ATP-ase, Na(+)+K(+)-activated ATPase, and 5'-nucleotidase and the lysosomal marker acid phosphatase. An enrichment/depletion factor for each marker was determined by comparison of purified BBMV with kidney homogenate. Vesicles exhibit an enrichment factor for alkaline phosphatase, GGTP, maltase, Mg(2+)-activated ATP-ase, Na(+)+K(+)-activated ATPase, and 5'-nucleotidase. A depletion factor was observed for acid phosphatase. Vesicle integrity was tested by measuring the time course of proton flux in the presence of a pH gradient. Amiloride sensitive sodium stimulated proton flux was observed in these vesicles. The presence of sodium caused a saturable increase in the rate of proton flux, indicating the activity of a sodium/proton antiport protein in BBMV.

Acclimatization↗