ORCID Profile
0000-0003-2034-8613
Current Organisation
The University of Edinburgh
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Publisher: Wiley
Date: 23-01-2009
DOI: 10.1111/J.1537-2995.2008.01954.X
Abstract: Four recent cases of transfusion-related transmission of variant Creutzfeldt-Jakob disease (vCJD) highlight the need to develop a highly sensitive and specific screening test to detect infectivity in the blood of asymptomatic infected in iduals. Protein misfolding cyclic lification (PMCA), a method for the lification of minute amounts of disease-associated abnormal prion protein (PrP(Sc)) to readily detectable levels, could be incorporated into such a test provided that a suitable substrate source for routine use in human PMCA reactions can be found. With the use of seed sources from in iduals with variant and sporadic CJD, the use of human platelets (PLTs) as a PMCA substrate source was evaluated. The effects of seed/substrate prion protein gene (PRNP) codon 129 genotype compatibility on lification efficiency and freeze-thaw on a substrate's ability to support lification and the degree of lification achieved by serial PMCA (sPMCA) were investigated. Seed/substrate PRNP codon 129 compatibility was found to have a major influence on PrP(Sc) lification efficiency. In idual substrates, of the same PRNP codon 129 genotype, could be pooled and stored frozen for use in subsequent PMCA reactions. A consistent 10-fold increase in PrP(Sc) detection sensitivity was achieved after each round of sPMCA, resulting in a 10,000-fold increase in detection sensitivity after four rounds, with no evidence of de novo PrP(Sc) production detected in the unseeded PLT substrate. Providing issues of seed/substrate PRNP codon 129 compatibility are taken into consideration human PLTs are a suitable, readily available, renewable substrate source for use in human PMCA applications.
Publisher: Elsevier BV
Date: 09-2005
DOI: 10.1016/J.BBRC.2005.07.045
Abstract: A method for the extraction and purification of PrP(C), in its native monomeric form, from outdated human platelet concentrates is described. Both calcium ionophore platelet activation and lysis in Triton X-100 were evaluated as methods for the extraction of soluble platelet PrP(C) in its monomeric form. Following platelet activation, the majority of released PrP(C) was detected as a disulphide linked high molecular weight complex, which under reducing conditions could be separated into what appear to be stable non-disulphide linked PrP dimers or PrP covalently linked to another as yet unidentified protein. This phenomenon appears to be unique to activation since only monomeric PrP(C) was detected following lysis of resting platelets. Subsequently, PrP(C) was purified from the Triton X-100 lysate by sequential cation ion exchange and Cu2+ affinity chromatography. From 10 L of outdated platelet concentrate, we were able to recover 1.29 mg PrP(C) at a purity of 92%.
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 12-2009
Publisher: Informa Healthcare
Date: 02-2008
Abstract: Variant Creutzfeldt-Jakob disease (vCJD) is a transmissible neurodegenerative prion disease that continues to present a unique problem for medical diagnostics. Uncertainties remain over the prevalence of vCJD in the UK population and its incubation period in in iduals of different genotypes. Although the infectious agent that causes vCJD is widely distributed in the peripheral tissues of patients and those carrying the disease, it does not provoke any host immune response that would be amenable to detection. The recent realisation that it can be transmitted by blood transfusion, and that in iduals are infectious long before the appearance of symptoms, have increased the need for a blood-screening assay. This paper reviews progress that has been made in the development of potential tests and the protocols that have been devised for their evaluation.
Publisher: Springer Science and Business Media LLC
Date: 10-06-2011
DOI: 10.1007/S00401-010-0708-8
Abstract: A key event in the pathogenesis of prion diseases is the conversion of the normal cellular isoform of the prion protein into the disease-associated isoform, but the mechanisms operating in this critical event are not yet fully understood. A number of novel approaches have recently been developed to study factors influencing this process. One of these, the protein misfolding cyclical lification (PMCA) technique, has been used to explore defined factors influencing the conversion of cellular prion protein in a cell-free model system. Although initially developed in animal models, this technique has been increasingly applied to human prion diseases. Recent studies have focused on the role of different isoforms of the disease-associated human prion protein and the effects of the naturally occurring polymorphism at codon 129 in the human prion protein gene on the conversion process, improving our understanding of the interaction between host and agent factors that influence the wide range of phenotypes in human prion diseases. This technique also allows a greatly enhanced sensitivity of detection of disease-associated prion protein in human tissues and fluids, which is potentially applicable to disease screening, particularly for variant Creutzfeldt-Jakob disease. The PMCA technique can also be used to model human susceptibility to a range of prions of non-human origin, which is likely to prove of considerable future interest as more novel and potentially pathogenic prion diseases are identified in animal species that form part of the human food chain.
Publisher: Wiley
Date: 09-08-2007
DOI: 10.1002/PATH.2204
Abstract: Variant Creutzfeldt-Jakob disease (vCJD) poses a serious risk of secondary transmission and the need to detect infectivity in asymptomatic in iduals is therefore of major importance. Following infection, it is assumed that minute amounts of disease-associated prion protein (PrP(Sc)) replicate by conversion of the host cellular prion protein (PrP(C)). Therefore, methods of rapidly reproducing this conversion process in vitro would be valuable tools in the development of such tests. We show that one such technique, protein misfolding cyclic lification (PMCA), can lify vCJD PrP(Sc) from human brain tissue, and that the degree of lification is dependent upon the substrate PRNP codon 129 polymorphism. Both human platelets and transgenic mouse brain are shown to be suitable alternative substrate sources, and lified PrP(Sc) can be detected using a conformation-dependent immunoassay (CDI), allowing the detection of putative proteinase K sensitive forms of PrP(Sc).
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-12-2008
Publisher: Wiley
Date: 02-09-2011
Publisher: Wiley
Date: 02-09-2011
DOI: 10.1111/J.1365-2990.2011.01162.X
Abstract: To determine premortem and post mortem factors affecting quality and yield of RNA isolated from the unique archived brain material in the UK National Creutzfeldt-Jakob Disease Surveillance Unit Brain and Tissue Bank and to compare this to control brain tissue with no neurological disease. In parallel and in replicate, RNA was prepared from the frontal parasagittal or subfrontal cortex of s les dissected from half brains (frozen intact) or from brain s les snap frozen or placed in RNALater. A total of 350 RNA s les from 78 human autopsy cases, 21 variant Creutzfeldt-Jakob disease, 26 other neurological diseases and 31 non-neurological diseases were studied. There was no difference in the quality or yield of RNA isolated from variant Creutzfeldt-Jakob disease, other neurological disease and non-neurological disease brains. RNA preparations from archived frozen half brains or snap frozen autopsy s les were generally of poor quality (RNA integrity number 5). Age at death, gender, post mortem interval and freezer storage time had no effect on RNA quality. Reasonable-quality RNA can be isolated from s les dissected from archived frozen human half brains but repeated s ling results in RNA degradation. Better-quality RNA is obtained from s les placed in RNALater than from snap frozen s les. The quality and yield of RNA are not affected by age at death, gender, post mortem interval of >6 h or freezer storage time.
Publisher: Wiley
Date: 05-03-2009
Publisher: Mary Ann Liebert Inc
Date: 02-2009
Abstract: The human prion diseases, such as variant Creutzfeldt-Jakob disease (vCJD), are characterized by the conversion of the normal cellular prion protein (PrP(C)) into an abnormal disease associated form (PrP(Sc)). Monoclonal antibodies (MAbs) that recognize these different PrP isoforms are valuable reagents both in the diagnosis of these diseases and in prion disease research in general but we know of no attempts to raise MAbs against native human PrP(C). We immunized prion protein gene ablated (PrP(-/-)) mice with native human PrP(C) purified from platelets (pHuPrP) generating a predominantly IgG isotype anti-pHuPrP polyclonal antibody response in all mice. Following fusion of splenocytes from the immunized mice with SP2/0 myeloma cells, we were able to identify single cell clone and cryopreserve 14 stable hybridoma cell lines producing MAbs that reacted with pHuPrP. The properties of these MAbs (such as isotype, binding to native/denatured pHuPrP, and HuPrP epitopes recognized) are described. Furthermore, several of these MAbs showed a selectivity in their ability to immunoprecipitate disease associated PrP(Sc) and its corresponding protease resistant core (PrP(res)).
Publisher: Elsevier BV
Date: 09-2014
Location: United Kingdom of Great Britain and Northern Ireland
Location: France
Location: United States of America
No related grants have been discovered for Mark Head.