Jonathan D F Wadsworth

Professor John Collinge
email: j.collinge@prion.ucl.ac.uk

Update on human prion disease

The recognition that variant Creutzfeldt-Jakob (vCJD) disease is caused by the same prion strain as bovine spongiform encephalopathy in cattle has dramatically highlighted the need for a precise understanding of the molecular biology of human prion diseases.

Detailed clinical, pathological and molecular data from a large number of human prion disease patients indicate that phenotypic diversity in human prion disease relates in part to the propagation of disease-related PrP isoforms with distinct physicochemical properties. Incubation periods of prion infection in humans can exceed 50 years and therefore it will be some years before the extent of any human vCJD epidemic can be predicted with confidence.

Introduction

Prion diseases are fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), fatal familial insomnia (FFI), kuru and variant CJD (vCJD) in humans [1,[START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF]. Their central feature is the posttranslational conversion of host-encoded, cellular prion protein (PrP C ), to an abnormal isoform, designated PrP Sc [1,[START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF]. This transition appears to involve only conformational change rather than covalent modification and confers PrP Sc with partial resistance to proteolytic degradation and detergent insolubility. Human prion diseases are biologically unique in that the disease process can be triggered through inherited germline mutations in the human prion protein gene (PRNP), infection (by inoculation, or in some cases by dietary exposure) with prion-infected tissue or by rare sporadic events that generate PrP Sc [START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF][START_REF] Wadsworth | Molecular and clinical classification of human prion disease[END_REF][START_REF] Collinge | Molecular neurology of prion disease[END_REF]. Substantial evidence indicates that an abnormal PrP isoform is the principal, if not the sole, component of the transmissible infectious agent, or prion [1,[START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF][START_REF] Legname | Synthetic mammalian prions[END_REF][START_REF] Weissmann | The state of the prion[END_REF]. The mechanism of neurodegeneration that accompanies the accumulation of PrP Sc in the brain remains unknown [START_REF] Mallucci | Rational targeting for prion therapeutics[END_REF] although there is increasing evidence that this may involve an apoptotic mechanism involving inhibition of the proteosome [START_REF] Kristiansen | Disease-related prion protein forms aggresomes in neuronal cells leading to caspase-activation and apoptosis[END_REF]. The existence of multiple strains or isolates of prions, has been difficult to accommodate within a proteinonly model of prion propagation and understanding how a protein-only infectious agent can encode distinct disease phenotypes in humans has been of considerable biological interest. A wealth of experimental evidence now suggests that prion strain diversity is encoded within PrP itself and phenotypic diversity in human prion diseases relates to differing physicochemical properties of abnormal PrP isoforms [START_REF] Telling | Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity[END_REF][START_REF] Collinge | Molecular analysis of prion strain variation and the aetiology of 'new variant[END_REF][START_REF] Parchi | Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Wadsworth | Strain-specific prion-protein conformation determined by metal ions[END_REF][START_REF] Parchi | Classification of sporadic Creutzfeldt-Jakob Disease based on molecular and phenotypic analysis of 300 subjects[END_REF][START_REF] Gambetti | Sporadic and familial CJD: classification and characterisation[END_REF][START_REF] Hill | Molecular classification of sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Zanusso | Identification of distinct Nterminal truncated forms of prion protein in different Creutzfeldt-Jakob disease subtypes[END_REF][START_REF] Lewis | Australian sporadic CJD analysis supports endogenous determinants of molecularclinical profiles[END_REF][START_REF] Safar | Diagnosis of human prion disease[END_REF].

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Aetiology and clinical features of human prion diseases

Human prion diseases can be divided aetiologically into inherited, sporadic and acquired forms [1,[START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF]. Human prion diseases with distinct aetiologies are associated with a range of clinical presentations which are now seen as clinico-pathological syndromes rather than individual disease entities [START_REF] Wadsworth | Molecular and clinical classification of human prion disease[END_REF][START_REF] Collinge | Molecular neurology of prion disease[END_REF][START_REF] Collinge | Inherited prion disease with 144 base pair gene insertion: II: Clinical and pathological features[END_REF].

Sporadic CJD

Approximately 85 % of cases of human prion disease occur sporadically as Creutzfeldt-Jakob disease (sporadic CJD) at a rate of 1-2 cases per million population per year across the world, with an equal incidence in men and women [START_REF] Brown | The epidemiology of Creutzfeldt-Jakob disease: conclusion of a 15-year investigation in France and review of the world literature[END_REF][START_REF] Collins | Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease[END_REF]. The aetiology of sporadic CJD is unknown, although hypotheses include somatic PRNP mutation [START_REF] Brown | The epidemiology of Creutzfeldt-Jakob disease: conclusion of a 15-year investigation in France and review of the world literature[END_REF][START_REF] Collinge | Human prion diseases and bovine spongiform encephalopathy (BSE)[END_REF][START_REF] Wadsworth | Phenotypic heterogeneity in inherited prion disease (P102L) is associated with differential propagation of protease-resistant wild-type and mutant prion protein[END_REF], or the spontaneous conversion of PrP C into PrP Sc as a rare stochastic event [START_REF] Collinge | Human prion diseases and bovine spongiform encephalopathy (BSE)[END_REF]. Polymorphism at residue 129 of human PrP (encoding either methionine (M) or valine (V)) powerfully affects susceptibility to human prion diseases [START_REF] Collins | Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Collinge | Genetic predisposition to iatrogenic Creutzfeldt-Jakob disease[END_REF][START_REF] Palmer | Homozygous prion protein genotype predisposes to sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Windl | Genetic basis of Creutzfeldt-Jakob disease in the United Kingom: a systematic analysis of predisposing mutations and allelic variation in the PRNP gene[END_REF][START_REF] Lee | Increased susceptibility to Kuru of carriers of the PRNP 129 methionine/methionine genotype[END_REF][START_REF] Mead | Balancing selection at the prion protein gene consistent with prehistoric kurulike epidemics[END_REF] About 38% of Europeans are homozygous for the more frequent methionine allele, 51% are heterozygous, and 11% homozygous for valine. Homozygosity at PRNP codon 129 predisposes to the development of sporadic and acquired CJD [START_REF] Collins | Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Collinge | Genetic predisposition to iatrogenic Creutzfeldt-Jakob disease[END_REF][START_REF] Palmer | Homozygous prion protein genotype predisposes to sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Windl | Genetic basis of Creutzfeldt-Jakob disease in the United Kingom: a systematic analysis of predisposing mutations and allelic variation in the PRNP gene[END_REF][START_REF] Lee | Increased susceptibility to Kuru of carriers of the PRNP 129 methionine/methionine genotype[END_REF][START_REF] Mead | Balancing selection at the prion protein gene consistent with prehistoric kurulike epidemics[END_REF]. Most sporadic CJD occurs in individuals homozygous for this polymorphism. This susceptibility factor is also relevant in the acquired forms of CJD, most strikingly in vCJD; all clinical cases studied so far have been homozygous for codon 129 methionine of the PrP gene PRNP (see below).

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Additionally, a PRNP susceptibility haplotype has been identified indicating additional genetic susceptibility to sporadic CJD at or near to the PRNP locus [START_REF] Mead | Sporadic -but not variant -Creutzfeldt-Jakob disease is associated with polymorphisms upstream of PRNP Exon 1[END_REF][START_REF] Vollmert | Significant association of a M129V independent polymorphism in the 5' UTR of the PRNP gene with sporadic Creutzfeldt-Jakob disease in a large German case-control study[END_REF].

Classical sporadic CJD presents as a rapidly progressive multifocal dementia usually with myoclonus. The onset is usually in the 45-75 year age group with median age at death of 68 years [START_REF] Collins | Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease[END_REF]. The clinical progression is typically over weeks progressing to akinetic mutism with a median disease duration of 5 months [START_REF] Collins | Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease[END_REF]. Prodromal features, present in around a third of cases, include fatigue, insomnia, depression, weight loss, headaches, general malaise and ill-defined pain sensations. In addition to mental deterioration and myoclonus, frequent additional neurological features include extrapyramidal signs, cerebellar ataxia, pyramidal signs and cortical blindness. Raised cerebrospinal fluid 14-3-3 protein, neuronal specific enolase (NSE), and S-100, although not specific for CJD, may be helpful diagnostically in the appropriate clinical context [START_REF] Collins | Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Jimi | High levels of nervous system-specific proteins in cerebrospinal fluid in patients with early stage Creutzfeldt-Jakob disease[END_REF][START_REF] Zerr | Cerebrospinal fluid concentration of neuron-specific enolase in diagnosis of Creutzfeldt-Jakob disease[END_REF][START_REF] Hsich | The 14-3-3 brain protein in cerebrospinal fluid as a marker for transmissible spongiform encephalopathies[END_REF][START_REF] Collinge | New Diagnostic Tests for Prion Diseases[END_REF][START_REF] Otto | S-100 protein concentration in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease[END_REF][START_REF] Knight | Creutzfeldt-Jakob disease: a rare cause of dementia in elderly persons[END_REF]. The electroencephalogram (EEG) may show characteristic pseudoperiodic sharp wave activity that is helpful in diagnosis but present only in around 60% of cases [START_REF] Collins | Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease[END_REF]. In contrast cortical signal changes on diffusion weighted MRI are extremely helpful in the diagnosis of CJD [START_REF] Kropp | Diffusion-weighted MRI in patients with Creutzfeldt-Jakob disease[END_REF][START_REF] Matoba | Creutzfeldt-Jakob disease: serial changes on diffusion-weighted MRI[END_REF][START_REF] Shiga | Diffusion-weighted MRI abnormalities as an early diagnostic marker for Creutzfeldt-Jakob disease[END_REF][START_REF] Young | Diffusion-weighted and fluid-attenuated inversion recovery imaging in Creutzfeldt-Jakob disease: high sensitivity and specificity for diagnosis[END_REF][START_REF] Tschampa | Radiological assessment of Creutzfeldt-Jakob disease[END_REF][START_REF] Macfarlane | A review of the neuroimaging findings in human prion disease[END_REF]. Neuropathological confirmation of CJD is by demonstration of spongiform change, neuronal loss and astrocytosis. PrP amyloid plaques are usually not present in CJD [START_REF] Budka | Neuropathology of prion diseases[END_REF] although PrP immunohistochemistry, is nearly always positive [START_REF] Budka | Neuropathology of prion diseases[END_REF][START_REF] Budka | Neuropathological diagnostic criteria for Creutzfeldt-Jakob disease (CJD) and other human spongiform encephalopathies (Prion diseases)[END_REF][START_REF] Bell | Prion protein immunocytochemistry -UK five centre consensus report[END_REF].

Atypical forms of sporadic CJD are well recognised. 10% of cases of CJD have a much more prolonged clinical course with a disease duration of over 2 years [START_REF] Brown | Creutzfeldt-Jakob disease of long duration: clinicopathological characteristics, transmissibility, and differential diagnosis[END_REF]. Around 10%

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of CJD cases present with cerebellar ataxia rather than cognitive impairment, so-called ataxic CJD [START_REF] Gomori | The ataxic form of Creutzfeldt-Jakob disease[END_REF]. Heidenhain's variant of CJD refers to cases in which cortical blindness predominates with severe involvement of the occipital lobes. The panencephalopathic type of CJD refers to cases with extensive degeneration of the cerebral white matter in addition to spongiform vacuolation of the gray matter and has been predominately reported from Japan [START_REF] Gomori | The ataxic form of Creutzfeldt-Jakob disease[END_REF][START_REF] Mizutani | Neuropathology of Creutzfeldt-Jakob disease in Japan. With special reference to the panencephalopathic type[END_REF][START_REF] Matsusue | White matter lesions in panencephalopathic type of Creutzfeldt-Jakob disease: MR imaging and pathologic correlations[END_REF]. Amyotrophic variants of CJD have been described with prominent early muscle wasting. However, most cases of dementia with amyotrophy are not experimentally transmissible and their relationship with CJD is unclear [START_REF] Salazar | Syndromes of amyotrophic lateral sclerosis and dementia: relation to transmissible Creutzfeldt-Jakob disease[END_REF][START_REF] Morita | Presenile dementia combined with amyotrophy: a review of 34 Japanese cases[END_REF].

Inherited prion disease

Approximately 15% of human prion diseases are associated with autosomal dominant pathogenic mutations in PRNP [START_REF] Gambetti | Sporadic and familial CJD: classification and characterisation[END_REF][START_REF] Collinge | Human prion diseases and bovine spongiform encephalopathy (BSE)[END_REF][START_REF] Collinge | Prion Diseases[END_REF][START_REF] Kovacs | Mutations of the prion protein gene phenotypic spectrum[END_REF][START_REF] Mead | Prion disease genetics[END_REF]. How pathogenic mutations in PRNP cause prion disease has yet to be resolved, however, in most cases the mutation is thought to lead to an increased tendency of PrP C to form PrP Sc , although there is evidence to suggest that this may not be solely attributable to decreased thermodynamic stability of mutated PrP C [START_REF] Riek | Prion protein NMR structure and familial human spongiform encephalopathies[END_REF][START_REF] Swietnicki | Familial mutations and the thermodynamic stability of the recombinant human prion protein[END_REF]. Experimentally manipulated mutations of the prion gene can lead to spontaneous neurodegeneration without the formation of detectable protease resistant PrP [START_REF] Muramoto | Heritable disorder resembling neuronal storage disease in mice expressing prion protein with deletion of an α-helix[END_REF][START_REF] Hegde | A transmembrane from of the prion protein in neurodegenerative disease[END_REF]. These findings raise the question of whether all inherited forms of human prion disease invoke disease through the same mechanism, and in this regard it is currently unknown whether all are transmissible by inoculation [START_REF] Collinge | Prion Diseases[END_REF].

Over 30 autosomal dominant pathogenic PRNP mutations have been described [START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF][START_REF] Wadsworth | Molecular and clinical classification of human prion disease[END_REF][START_REF] Kovacs | Mutations of the prion protein gene phenotypic spectrum[END_REF][START_REF] Mead | Prion disease genetics[END_REF]. In the appropriate clinical setting identification of a pathogenic PRNP

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7 mutation provides diagnosis of inherited prion disease and sub-classification according to mutation; PRNP analysis is also used for presymptomatic genetic testing in affected families [START_REF] Collinge | Molecular neurology of prion disease[END_REF][START_REF] Collinge | Prion Diseases[END_REF][START_REF] Mead | Prion disease genetics[END_REF][START_REF] Collinge | Presymptomatic detection or exclusion of prion protein gene defects in families with inherited prion diseases[END_REF]. Traditionally, inherited prion diseases have been classified by the presenting clinical syndrome, falling into three main sub-divisions of either GSS, CJD or FFI. GSS classically presents as a chronic cerebellar ataxia with pyramidal features with dementia occurring much later in a clinical course that is typically longer than that seen in classical CJD [START_REF] Collinge | Prion Diseases[END_REF]. Fatal familial insomnia (FFI) is characterised by progressive untreatable insomnia, dysautonmoia and dementia, selective thalamic degeneration and is most commonly associated with a missense mutation at codon 178 of PRNP [START_REF] Medori | Fatal familial insomnia, a prion disease with a mutation at codon 178 of the prion protein gene[END_REF],

although sporadic FFI with no causative mutation in PRNP has been reported [START_REF] Mastrianni | Fatal sporadic insomnia (FSI): Fatal familial insomnia (FFI) phenotype without a mutation of the prion protein (PrP) gene[END_REF][START_REF] Montagna | Familial and sporadic fatal insomnia[END_REF].

Remarkably, some families show extensive phenotypic variability which can encompass both CJD-and GSS-like cases as well as other cases which do not conform to either CJD or GSS phenotypes [START_REF] Collinge | Inherited prion disease with 144 base pair gene insertion: II: Clinical and pathological features[END_REF][START_REF] Kovacs | Mutations of the prion protein gene phenotypic spectrum[END_REF][START_REF] Mead | Prion disease genetics[END_REF][START_REF] Collinge | Diagnosis of Gerstmann-Straussler syndrome in familial dementia with prion protein gene analysis[END_REF][START_REF] Collinge | Prion dementia without characteristic pathology[END_REF][START_REF] Mallucci | Inherited prion disease with an alanine to valine mutation at codon 117 in the prion protein gene[END_REF][START_REF] Mead | Inherited prion disease with six octapeptide repeat insertional mutation--molecular analysis of phenotypic heterogeneity[END_REF]. Progressive dementia, cerebellar ataxia, pyramidal signs, chorea, myoclonus, extrapyramidal features, pseudobulbar signs, seizures and amyotrophic features can be seen in variable combinations. Such atypical prion diseases may lack the classical histological features of a spongiform encephalopathy entirely although PrP immunohistochemistry is usually positive [START_REF] Collinge | Prion dementia without characteristic pathology[END_REF]. The existence of phenotypic overlap between individuals with different mutations and even in family members with the same PRNP mutation indicates that accurate classification of inherited human prion diseases should be based upon mutation alone [START_REF] Collinge | Inherited prion disease with 144 base pair gene insertion: II: Clinical and pathological features[END_REF][START_REF] Collinge | Terminology of Prion Disease[END_REF]. Because of the extensive phenotypic variability seen in inherited prion disease and its ability to mimic other neurodegenerative conditions, notably Alzheimer's disease, PRNP analysis should be considered in all patients with undiagnosed dementing and ataxic disorders [START_REF] Collinge | Inherited prion disease with 144 base pair gene insertion: II: Clinical and pathological features[END_REF][START_REF] Collinge | Diagnosis of Gerstmann-Straussler syndrome in familial dementia with prion protein gene analysis[END_REF][START_REF] Collinge | Prion dementia without characteristic pathology[END_REF][START_REF] Collinge | Human Prion Diseases: Aetiology and Clinical Features[END_REF].

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Acquired prion disease

Although the human prion diseases are transmissible diseases, acquired forms have, until recently, been confined to rare and unusual situations.

Iatrogenic CJD

The two most frequent causes of iatrogenic CJD occurring through medical procedures have arisen as a result of implantation of dura mater grafts and treatment with growth hormone derived from the pituitary glands of human cadavers [START_REF] Brown | Friendly fire" in medicine: hormones, homografts, and Creutzfeldt-Jakob disease[END_REF][START_REF] Brown | Iatrogenic Creutzfeldt-Jakob disease at the millennium[END_REF]. Less frequent incidences of human prion disease have resulted from iatrogenic transmission of CJD during corneal transplantation, contaminated electroencephalographic (EEG) electrode implantation and surgical operations using contaminated instruments or apparatus [START_REF] Brown | Friendly fire" in medicine: hormones, homografts, and Creutzfeldt-Jakob disease[END_REF][START_REF] Brown | Iatrogenic Creutzfeldt-Jakob disease at the millennium[END_REF]. The clinical presentation in iatrogenic forms of human prion disease appear to be related to their aetiology and in particular the route of exposure to human prions [START_REF] Collinge | Prion Diseases[END_REF][START_REF] Brown | Iatrogenic Creutzfeldt-Jakob disease at the millennium[END_REF][START_REF] Belay | Transmissible spongiform encephalopathies in humans[END_REF]. Peripheral routes of infection are typically associated with longer incubation periods and usually present with a kuru-like syndrome in which ataxia rather than dementia is the prominent early clinical feature. In contrast, patients with dura mater graft-related exposure to human prions, in which infectivity is placed in closed proximity to the brain, typically have a clinical presentation similar to that of sporadic CJD [START_REF] Heath | Dura materassociated Creutzfeldt-Jakob disease: experience from surveillance in the UK[END_REF],

although exceptions with unusual clinical features have been reported [START_REF] Lane | Creutzfeldt-Jakob disease in a pregnant woman with an implanted dura mater graft[END_REF].

Kuru A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 9
The most well-known example of acquired prion disease in humans is kuru, transmitted by cannibalism among the Fore linguistic group of the Eastern Highlands in Papua New Guinea [START_REF] Mead | Balancing selection at the prion protein gene consistent with prehistoric kurulike epidemics[END_REF][START_REF] Collinge | Prion Diseases[END_REF][START_REF] Alpers | Epidemiology and clinical aspects of kuru[END_REF][START_REF] Collinge | Kuru in the 21st century--an acquired human prion disease with very long incubation periods[END_REF]. The central clinical feature of kuru is progessive cerebellar ataxia and in sharp contrast to sporadic CJD, dementia is late and may be absent. A prodrome and three clinical stages consisting of an ambulatory stage, a sedentary stage and a tertiary stage have been described [START_REF] Collinge | Prion Diseases[END_REF][START_REF] Alpers | Epidemiology and clinical aspects of kuru[END_REF][START_REF] Collinge | Kuru in the 21st century--an acquired human prion disease with very long incubation periods[END_REF]. Remarkably, kuru demonstrates that incubation periods of infection with human prions can exceed 50 years [START_REF] Collinge | Kuru in the 21st century--an acquired human prion disease with very long incubation periods[END_REF]. PRNP codon 129 genotype has shown a pronounced effect on kuru incubation periods and susceptibility, and most elderly survivors of the kuru epidemic are heterozygotes [START_REF] Lee | Increased susceptibility to Kuru of carriers of the PRNP 129 methionine/methionine genotype[END_REF][START_REF] Mead | Balancing selection at the prion protein gene consistent with prehistoric kurulike epidemics[END_REF][START_REF] Collinge | Kuru in the 21st century--an acquired human prion disease with very long incubation periods[END_REF]. The clear survival advantage for codon 129 heterozygotes provides a powerful basis for selection pressure in the Fore [START_REF] Mead | Balancing selection at the prion protein gene consistent with prehistoric kurulike epidemics[END_REF][START_REF] Collinge | Kuru in the 21st century--an acquired human prion disease with very long incubation periods[END_REF]. However, an analysis of worldwide haplotype diversity and allele frequency of coding and non-coding polymorphisms of PRNP suggests that balancing selection at this locus (in which there is more variation than expected because of heterozygote advantage) is much older and more geographically widespread [START_REF] Mead | Balancing selection at the prion protein gene consistent with prehistoric kurulike epidemics[END_REF]. Evidence for balancing selection has been shown in only a few human genes. With biochemical and physical evidence of cannibalism on five continents, one explanation is that cannibalism resulted in prion disease epidemics in human prehistory, thus imposing balancing selection on PRNP [START_REF] Mead | Balancing selection at the prion protein gene consistent with prehistoric kurulike epidemics[END_REF].

Variant CJD

The appearance of a novel human prion disease, variant CJD (vCJD), in the United Kingdom from 1995 onwards [START_REF] Will | A new variant of Creutzfeldt-Jakob disease in the UK[END_REF], and the experimental confirmation that this is caused by the same prion strain as that causing BSE in cattle [START_REF] Collinge | Molecular analysis of prion strain variation and the aetiology of 'new variant[END_REF][START_REF] Hill | The same prion strain causes vCJD and BSE[END_REF][START_REF] Bruce | Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent[END_REF][START_REF] Asante | BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein[END_REF], has led to widespread concern that exposure to the epidemic of BSE poses a distinct and conceivably a severe threat to public health in the United Kingdom and other countries [START_REF] Collinge | Molecular neurology of prion disease[END_REF][START_REF] Collinge | Variant Creutzfeldt-Jakob disease[END_REF]. The extremely prolonged and variable incubation periods seen with prion diseases when crossing a species barrier means that it will be some years before the parameters of any human epidemic can be predicted with confidence [START_REF] Collinge | Kuru in the 21st century--an acquired human prion disease with very long incubation periods[END_REF][START_REF] Collinge | Variant Creutzfeldt-Jakob disease[END_REF][START_REF] Hilton | Prevalence of lymphoreticular prion protein accumulation in UK tissue samples[END_REF][START_REF] Frosh | Analysis of 2000 consecutive UK tonsillectomy specimens for disease-related prion protein[END_REF][START_REF] Hilton | Pathogenesis and prevalence of variant Creutzfeldt-Jakob disease[END_REF]. In the meantime, we are faced with the possibility that significant numbers in the population may be incubating this disease and that they might pass it on to others via blood transfusion, blood products, tissue and organ transplantation and other iatrogenic routes [START_REF] Collinge | Variant Creutzfeldt-Jakob disease[END_REF][START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay[END_REF][START_REF] Llewelyn | Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion[END_REF][START_REF] Peden | Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient[END_REF][START_REF] Wroe | Clinical presentation and pre-mortem diagnosis of blood transfusion associated variant Creutzfeldt-Jakob Disease[END_REF] .

To-date, the clinical presentation of human BSE infection has only been recognised as vCJD in PRNP codon 129 methionine homozygotes. The neuropathological features of vCJD are somewhat different from those seen in classical (sporadic or iatrogenic) CJD, and are characterised by the presence of abundant florid PrP plaques [START_REF] Will | A new variant of Creutzfeldt-Jakob disease in the UK[END_REF][START_REF] Ironside | Neuropathology and molecular biology of variant Creutzfeldt-Jakob disease[END_REF] and the propagation of type 4 PrP Sc in brain [START_REF] Collinge | Molecular analysis of prion strain variation and the aetiology of 'new variant[END_REF][START_REF] Hill | Molecular classification of sporadic Creutzfeldt-Jakob disease[END_REF] (Figure 1). The early clinical presentation of vCJD resembles kuru more than classical CJD and consists of behavioral and psychiatric disturbances, peripheral sensory disturbance and cerebellar ataxia. protein is not helpful, and may often be negative [START_REF] Green | Use of 14-3-3 and other brain-specific proteins in CSF in the diagnosis of variant Creutzfeldt-Jakob disease[END_REF]. The most useful non-invasive investigation in a dvanced cases of vCJD has been MR neuroimaging, particularly the FLAIR sequence [START_REF] Collie | Diagnosing variant Creutzfeldt-Jakob disease with the pulvinar sign: MR imaging findings in 86 neuropathologically confirmed cases[END_REF]. Early case reports noted bilateral increased signal in the posterior thalamus (pulvinar) on T2 weighted images [START_REF] Chazot | New variant of Creutzfeldt-Jakob disease in a 26-year-old French man[END_REF]. A retrospective review of MR scans from 36 histologically confirmed cases of vCJD using mainly other forms of human prion disease subjects as controls suggested that the "pulvinar sign" occurred frequently in advanced cases of vCJD [START_REF] Zeidler | The pulvinar sign on magnetic resonance imaging in variant Creutzfeldt-Jakob disease[END_REF]. However, histologically confirmed cases of vCJD with minimal or absent pulvinar changes on MR neuroimaging at a mean 10.5 months during an illness of mean 15 months duration were identified in t his series. The absence of characteristic MR findings does therefore not exclude a diagnosis of vCJD and more recently figures of 81% sensitivity and 94% specificity have been reported in a series of patients including 27 cases of vCJD diagnosed by tonsil biopsy [START_REF] Siddique | Tonsil biopsy in the investigation of suspected variant Creutzfeldt-Jakob disease -a cohort study of 50 pts[END_REF]. As these studies suggest, the pulvinar sign is not specific for vCJD and these MRI appearances are described in sporadic CJD [START_REF] Rossetti | Mimicry of variant Creutzfeldt-Jakob disease by sporadic Creutzfeldt-Jakob disease: importance of the pulvinar sign[END_REF], paraneoplastic limbic encephalitis [START_REF] Mihara | The "pulvinar sign" in a case of paraneoplastic limbic encephalitis associated with non-Hodgkin's lymphoma[END_REF] and in a number of rare conditions [START_REF] Yoshimura | A case of benign intracranial hypertension with bilateral reversible thalamic lesions on magnetic resonance imaging[END_REF][START_REF] Hahn | Unusual MRI findings after status epilepticus due to cat-scratch disease[END_REF][START_REF] Harding | Progressive neuronal degeneration of childhood with liver disease (Alpers' disease) presenting in young adults[END_REF][100]. In contrast, a firm tissue based diagnosis of vCJD can be

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made during life by tonsil biopsy, with demonstration of a characteristic sub-type of PrP Sc (see below).

Molecular basis of phenotypic variability in human prion disease

The marked clinical heterogeneity observed in human prion diseases has yet to be explained. However, it has been clear for many years that distinct isolates, or strains, of prions can be propagated in the same host and these are biologically recognised by To-date we have identified four major types of human PrP Sc associated with sporadic and acquired human prion diseases that can be differentiated on immunoblots after limited proteinase K digestion of brain homogenates [START_REF] Collinge | Molecular analysis of prion strain variation and the aetiology of 'new variant[END_REF][START_REF] Wadsworth | Strain-specific prion-protein conformation determined by metal ions[END_REF][START_REF] Hill | Molecular classification of sporadic Creutzfeldt-Jakob disease[END_REF] (Figure 1). PrP Sc types 1-3 are seen in classical (sporadic or iatrogenic) CJD brain, while type 4 PrP Sc is uniquely seen in vCJD brain [START_REF] Collinge | Molecular analysis of prion strain variation and the aetiology of 'new variant[END_REF][START_REF] Wadsworth | Strain-specific prion-protein conformation determined by metal ions[END_REF][START_REF] Hill | Molecular classification of sporadic Creutzfeldt-Jakob disease[END_REF]]. An earlier classification of PrP Sc types seen in classical CJD described only two banding patterns [START_REF] Parchi | Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease[END_REF] with PrP Sc types 1 and 2 that we describe corresponding with the type 1 pattern of Gambetti and colleagues, and our type 3 Clearly, new experimental methods may be required in order to fully document the spectrum of abnormal PrP isoforms seen in inherited prion disease. In this context it is important to note that not all inherited prion disease may invoke disease through the same mechanism. For example the PRNP A117V mutation gives rise to transmembrane forms of PrP (PrP ctm ) which it is proposed may invoke a neurological disease without generation of PrP Sc [START_REF] Hegde | A transmembrane from of the prion protein in neurodegenerative disease[END_REF]. It is also not presently known whether all inherited prion diseases are transmissible by inoculation.

A C C E P T E D M A N U S C
Efforts to produce an unified international classification and nomenclature of human PrP Sc types has been complicated by the fact that the N-terminal conformation of some PrP Sc subtypes seen in sporadic CJD can be altered in vitro via changes in metal-ion occupancy [START_REF] Wadsworth | Strain-specific prion-protein conformation determined by metal ions[END_REF][START_REF] Hill | Molecular classification of sporadic Creutzfeldt-Jakob disease[END_REF] [START_REF] Safar | Diagnosis of human prion disease[END_REF].

The hypothesis that alternative conformations or assembly states of PrP provide the molecular substrate for clinicopathological heterogeneity seen in human prion diseases (and that this relates to the existence of distinct human prion strains) has been strongly

supported by transmission experiments to conventional and transgenic mice. Transgenic mice expressing only human PrP with either valine (V) or methionine (M) at residue 129 have shown that this polymorphism constrains both the propagation of distinct human

A C C E P T E D M A N U S C R I P T

PrP Sc conformers and the occurrence of associated patterns of neuropathology [START_REF] Collinge | Molecular analysis of prion strain variation and the aetiology of 'new variant[END_REF][START_REF] Hill | The same prion strain causes vCJD and BSE[END_REF][START_REF] Asante | BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein[END_REF]131,132]. Biophysical measurements suggest that this powerful effect of residue 129 on prion strain selection is likely to be mediated via its effect on the conformation of PrP Sc or its precursors or on the kinetics of their formation, as it has no measurable effect on the folding, dynamics or stability of PrP

Peripheral pathogenesis in human prion disease

Concomitant with studies on PrP Sc from brain has been investigation of PrP Sc in peripheral tissues in human prion diseases. These studies have established that the pathogenesis of vCJD differs significantly from that of other forms of CJD. PrP Sc is readily detectable in lymphoreticular tissues in vCJD and not in classical CJD or inherited prion disease [START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay[END_REF]108,[137][138][139][140][141][142]. The fact that tonsillar prion infection has not been detected in iatrogenic CJD associated with use of human cadaveric derived pituitary hormone [138] or kuru (our unpublished data) argues that the distinctive pathogenesis of vCJD relates to the effect of prion strain rather than to a peripheral route of infection.

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Depending upon the density of lymphoid follicles, PrP Sc concentrations in vCJD peripheral tissues can vary enormously, with levels relative to brain as high as 10% in tonsil [START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay[END_REF]138] or as low as 0.002 % in rectum [START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay[END_REF]. A distinctive PrP Sc type, designated type 4t, is seen in both ante-mortem and post-mortem tonsil from patients with vCJD [START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay[END_REF]138] (Figure 1) including secondary vCJD infection resulting from blood transfusion [START_REF] Wroe | Clinical presentation and pre-mortem diagnosis of blood transfusion associated variant Creutzfeldt-Jakob Disease[END_REF]. Type 4t PrP Sc in tonsil differs in the proportions of the PrP glycoforms from type 4

PrP Sc seen in vCJD brain [START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay[END_REF]138] implying the superimposition of tissue and strain specific effects on PrP glycosylation [START_REF] Collinge | Variant Creutzfeldt-Jakob disease[END_REF]138]. As infection of lymphoreticular tissues is thought to precede neuroinvasion, and indeed has been detected in archived surgical samples removed prior to development of vCJD [START_REF] Hilton | Prevalence of lymphoreticular prion protein accumulation in UK tissue samples[END_REF]143], tonsil biopsy is likely to allow firm diagnosis at the early clinical stage or indeed pre-clinically [START_REF] Collinge | Molecular neurology of prion disease[END_REF]. Early diagnosis obviates the need for further investigation, which may include brain biopsy, necessary to exclude alternative potentially treatable conditions and will be increasingly important with the advent of putative treatments and clinical trials where the aim must be to intervene early before extensive CNS damage has occurred [START_REF] Collinge | Molecular neurology of prion disease[END_REF]. To-date tonsil biopsy has shown has shown 100 % sensitivity and specificity for diagnosis of clinical vCJD [START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay[END_REF][START_REF] Siddique | Tonsil biopsy in the investigation of suspected variant Creutzfeldt-Jakob disease -a cohort study of 50 pts[END_REF]137,138] (and our unpublished data)

The distinctive peripheral pathogenesis of vCJD has provided the basis for prevalence screening of the general population for vCJD infection. Two anonymous screens of lymphoreticular tissues, removed during routine surgery, have been performed [START_REF] Hilton | Prevalence of lymphoreticular prion protein accumulation in UK tissue samples[END_REF][START_REF] Frosh | Analysis of 2000 consecutive UK tonsillectomy specimens for disease-related prion protein[END_REF].

Tonsil appears a more sensitive reporter of vCJD prion infection than appendix [144] and it is of concern that three positives appendix specimens were reported from a

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19 retrospective screen of around 12 000 largely appendix samples [START_REF] Hilton | Prevalence of lymphoreticular prion protein accumulation in UK tissue samples[END_REF]. A pilot scale prospective study of 2000 tonsils found no positives, despite use of high sensitivity methods [START_REF] Frosh | Analysis of 2000 consecutive UK tonsillectomy specimens for disease-related prion protein[END_REF]. However, this negative result cannot provide reassurance that significant community infection is unlikely because of the relatively small sample size, demographic and age-related factors and unknown test sensitivity during the prolonged incubation period [START_REF] Frosh | Analysis of 2000 consecutive UK tonsillectomy specimens for disease-related prion protein[END_REF]. Accordingly, a national large scale study is now being organized by the UK Department of Health which aims to screen 100,000 tonsils for disease-associated PrP to estimate prevalence of vCJD prion infection in the UK population.

Secondary transmission of vCJD prions

The unknown but potentially high prevalence of clinically silent infection with BSE prions allied with the distinctive pathogenesis of vCJD has led to significant concerns that there may be substantial risks of iatrogenic transmission of vCJD prions via blood products and contaminated surgical and medical instruments [START_REF] Collinge | Variant Creutzfeldt-Jakob disease[END_REF][START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay[END_REF]142,145].

Indeed three cases of transfusion-associated vCJD prion infection have already been reported from amongst a very small cohort of known exposed individuals [START_REF] Llewelyn | Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion[END_REF][START_REF] Peden | Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient[END_REF][START_REF] Wroe | Clinical presentation and pre-mortem diagnosis of blood transfusion associated variant Creutzfeldt-Jakob Disease[END_REF]. Prions resist many conventional sterilisation procedures and surgical stainless steel-bound prions transmit disease with remarkable efficiency when implanted into mice [146,147].

Precautionary risk reduction measures have already been taken with respect to blood and blood products [START_REF] Llewelyn | Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion[END_REF]148] however major concerns remain relating to possible iatrogenic transmission of vCJD prions via contaminated surgical instruments. Transmission of vCJD to conventional mice involves a transmission barrier that severely limits the ability

A C C E P T E D M A N U S C R I P T

to detect low titre infectivity [START_REF] Hill | The same prion strain causes vCJD and BSE[END_REF][START_REF] Bruce | Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent[END_REF][START_REF] Asante | BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein[END_REF]149]. Pending development of a transmission barrierfree model for bioassay of vCJD prions, high sensitivity immunodetection of PrP Sc has been used to provide an upper limit on PrP Sc levels in peripheral tissues, including blood, to inform risk assessment models [START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay[END_REF]141,142]. High concentrations of PrP Sc appear to be largely confined to the central nervous system and lymphoreticular tissues in vCJD.

While a range of surgically important tissues have levels of PrP Sc 10 4 -10 5 fold lower than found in brain [START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay[END_REF], the demonstration of prion infectivity in vCJD rectum despite barely detectable levels of PrP Sc is consistent with PrP Sc /prion infectivity ratios in vCJD being closely similar to experimental rodent prion strains [145]. These findings strongly endorse the value of PrP Sc analysis in vCJD for defining risk reduction strategies to limit secondary transmission of vCJD prions and reinforce concerns that iatrogenic transmission of vCJD prions might occur through contaminated endoscopes, biopsy forceps or surgical instruments [142,145,147,150,151].

Predictions of the eventual size of a vCJD epidemic have varied widely, although some recent estimates, based on current cases of vCJD, suggest that the total epidemic may be relatively small [152]. However, key uncertainties, notably with respect to major genetic effects on the incubation period [START_REF] Mead | Prion disease genetics[END_REF]105], suggest the need for caution. Importantly, such models cannot estimate the number of infected individuals, which remains unknown. The mean incubation period for kuru has been estimated to be around 12 years [START_REF] Collinge | Kuru in the 21st century--an acquired human prion disease with very long incubation periods[END_REF] with a similar estimate in iatrogenic CJD associated with the use of human-cadaver-derived pituitary growth hormone [START_REF] Brown | Iatrogenic Creutzfeldt-Jakob disease at the millennium[END_REF]. However, the maximum incubation periods in kuru can exceed 50 years [START_REF] Collinge | Kuru in the 21st century--an acquired human prion disease with very long incubation periods[END_REF]. The transmission barrier of BSE between cattle and human beings

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21 is unknown and cannot be directly measured. However, the cattle-to-mouse barrier for BSE has been well characterized experimentally by comparative endpoint titration. BSE prions transmit readily to laboratory mice, including after oral dosing [153]. The murine LD 50 (lethal dose causing 50% mortality) in C57Bl/6 mice is about 500-fold higher than that in cattle [154]; this barrier also results in a three-fold to four-fold increase in mean incubation period. Mean incubation periods of human BSE infection of 30 years or more should therefore be regarded as possible, if not probable, with the longest incubation periods approaching (and perhaps exceeding) the typical human lifespan [START_REF] Collinge | Kuru in the 21st century--an acquired human prion disease with very long incubation periods[END_REF]. The mean incubation periods of secondary vCJD (involving human-to-human transmission) would however be expected to have considerably shorter mean incubation periods than in primary vCJD resulting from exposure to BSE prions. The incubation periods seen in the first clinical cases of secondary vCJD are 6 and 6.5 years [START_REF] Llewelyn | Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion[END_REF][START_REF] Wroe | Clinical presentation and pre-mortem diagnosis of blood transfusion associated variant Creutzfeldt-Jakob Disease[END_REF] suggesting the shortest incubation periods of primary vCJD will be much longer, particularly as the oral route of transmission is also typically associated with longer incubation periods than parenteral routes. A preliminary estimate of the shortest incubation periods of primary vCJD, based on the youngest cases seen, is around 12 years which would be consistent with such extrapolation from the shortest incubation periods in secondary vCJD [START_REF] Collinge | Kuru in the 21st century--an acquired human prion disease with very long incubation periods[END_REF]. pathogenesis related to route of exposure (intravenous rather than the oral route of exposure to BSE prions presumed in primary vCJD). It is also possible that the absence of a species barrier in secondary (human to human) infection may affect phenotype.

However the detection of type 4t PrP Sc in the tonsil of the most recent codon 129MM

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 23 transfusion case [START_REF] Wroe | Clinical presentation and pre-mortem diagnosis of blood transfusion associated variant Creutzfeldt-Jakob Disease[END_REF] argues against these interpretations, and suggests that absence of tonsillar PrP Sc in the MV blood recipient may be due to the effect of PRNP genotype and perhaps selection of a distinctive prion strain. These findings highlight the importance of the need for continued surveillance and investigation of all forms of human prion disease within the UK and other populations with extensive dietary exposure to BSE prions.

  distinctive clinical and pathological features[START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF]101,102]. It is therefore likely that a proportion of clinicopathological heterogeneity seen in sporadic CJD and other human prion diseases relates to the propagation of distinct human prion strains. Within the framework of the protein-only hypothesis of prion propagation, distinct clinical and neuropathological phenotypes are thought to be determined by the propagation of distinct PrP Sc isoforms with divergent physicochemical properties[1,[START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF][START_REF] Telling | Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity[END_REF][START_REF] Collinge | Molecular analysis of prion strain variation and the aetiology of 'new variant[END_REF][START_REF] Parchi | Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease[END_REF]103,104].Furthermore the propagation of distinct abnormal PrP isoforms may be determined by the host genome[START_REF] Asante | BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein[END_REF]105,106].

Figure 1 Figure 2 Figure 1 A

 121 Figure 1 Characterization of disease related prion protein in human prion disease.

Figure 2 A

 2 Figure 2

  features (cerebellar signs, chorea, dystonia, myoclonus, upper motor neuron signs and visual symptoms), develop late in the course of the illness[START_REF] Spencer | First hundred cases of variant Creutzfeldt-Jakob disease: retrospective case note review of early psychiatric and neurological features[END_REF]. The duration of disease is longer in vCJD with mean patient survival times of about 14 months[START_REF] Knight | Creutzfeldt-Jakob disease: a rare cause of dementia in elderly persons[END_REF], compared with about 5 months for classical CJD[START_REF] Collins | Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease[END_REF]. Moreover, whereas classical CJD is predominantly a late onset disease with a median age at death of 68 years[START_REF] Collins | Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease[END_REF], the median age of onset of vCJD is 26 years[START_REF] Knight | Creutzfeldt-Jakob disease: a rare cause of dementia in elderly persons[END_REF][START_REF] Spencer | First hundred cases of variant Creutzfeldt-Jakob disease: retrospective case note review of early psychiatric and neurological features[END_REF]. The EEG is not helpful in the diagnosis of vCJD, whilst generalised slowing is usually present, the characteristic periodic changes associated with classical CJD are not. The CSF 14-3-3

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	the commonest neurological
	M A N U S C R I P T
	A C C E P T E D
	Neurological symptoms have preceded psychiatric symptoms in a minority of cases. No
	common early neurological features have been reported, but paraesthesiae and/or pain in
	the limbs is seen in around half of the cases. However a significant proportion of patients
	exhibited neurological symptoms within 4 months of clinical onset, and these included
	poor memory, pain, sensory symptoms, unsteadiness of gait and dysarthia.
	Disorientation, hallucinations, paranoid ideation, confabulation, impaired self-care, and
	11

Common early psychiatric features include depression, withdrawal, anxiety, insomnia, and apathy.

  R I P T to their type 2 pattern[START_REF] Parchi | Classification of sporadic Creutzfeldt-Jakob Disease based on molecular and phenotypic analysis of 300 subjects[END_REF]107]. While type 4 PrP Sc is readily distinguished from the PrP Sc types seen in classical CJD by a predominance of the diglycosylated PrP glycoform, type 4 PrP Sc also has a distinct proteolytic fragment size[START_REF] Hill | Molecular classification of sporadic Creutzfeldt-Jakob disease[END_REF] although this is not recognized by the alternative classification which designates type 4PrP Sc as type 2b[107].In addition to the identification of distinct human PrP Sc types associated with sporadic and acquired prion disease, molecular strain typing has provided insights into the phenotypic heterogeneity seen inherited human prion diseases[START_REF] Kovacs | Mutations of the prion protein gene phenotypic spectrum[END_REF]108]. In agreement with existing evidence that human prion strain diversity is generated through variance in PrP The propagation of pathological isoforms of wild type PrP may also make a significant contribution to phenotypic variability in inherited prion disease[23,115- 117]. These collective data indicate that PrP Sc isoforms may be generated in inherited prion disease with unique physicochemical properties, reflected by sensitivity to proteinase K digestion or PrP Sc / prion infectivity ratios that are very different from the PrP Sc types propagated in sporadic and acquired forms of human prion disease. For

	ACCEPTED MANUSCRIPT
	example in P102L inherited prion disease, it is now apparent that three isoforms of protease resistant PrP with divergent physicochemical properties can be propagated. Two distinct abnormal conformers derived from PrP 102L generate protease-resistant fragment size corresponding A C C E P T E D fragments of either ca. 21-30 kDa or 8 kDa [23,108-110,118], while abnormal conformers of wild type PrP appear to generate proteolytic fragments of only ca. 21-30 kDa [23]. Glycoform ratios of ca. 21-30 kDa proteolytic fragments generated from PrP M A N U S C R I P T 102L and wild type PrP are not only distinct from each other, but are also distinct from
	13

Sc conformation and glycosylation, cases of inherited prion disease caused by point mutaions have glycoform ratios of PrP Sc fragments distinct from seen in both classical CJD [108-112] and vCJD [108]. Individuals with the same PRNP mutation can also propagate PrP Sc with distinct fragment sizes [108,109,113]. Notably however the detection of PrP Sc in the molecular mass range of ca. 21-30 kDa is by no means a consistent feature. Instead, some cases, in particular those in which amyloid plaques are a prominent feature, show smaller protease resistant fragments of ca. 7-15 kDa [53,108-110,113,114].

those generated from wild type PrP in sporadic or acquired CJD

[START_REF] Wadsworth | Phenotypic heterogeneity in inherited prion disease (P102L) is associated with differential propagation of protease-resistant wild-type and mutant prion protein[END_REF]

. Differences in neuropathological targeting of these distinct disease-related PrP species, together with differences in their abundance and potential neurotoxicity, provides a molecular mechanism for generation of multiple phenotypes in P102L inherited prion disease.

  However in the PrP codon 129MV genotype, none of these phenotypes resemble the neuropathological phenotype of vCJD as the propagation of type 4 PrP Sc is dissociated from the occurrence of abundant florid PrP plaques [132]. While caution must be exercised in extrapolating from animal models, even where faithful recapitulation of molecular and pathological phenotypes is possible [79,131] (Figure 2), our data, together with closely similar findings of Manson and colleagues [136], argue that primary human BSE prion infection, and secondary infection with vCJD prions by iatrogenic routes, may not be restricted to a single disease phenotype. Stratification of all human prion disease cases by PrP Sc type will enable rapid recognition of any change in relative frequencies of particular PrP Sc sub-types in relation to either BSE exposure patterns or iatrogenic sources of vCJD prions [2,79,131,132].

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	[132].
	C [133]. These data are consistent with a conformational selection model of prion transmission barriers [2,80,131,134] (a model also recently supported by work with yeast prions [135]) and strongly support the "protein only" hypothesis of infectivity by suggesting that prion strain variation is encoded by a combination of PrP conformation and glycosylation [2]. M A N U S C R I P T
	A C C E P T E D
	homozygous for human PrP M129 propagate either type 2 or 4 PrP Sc with respective
	neuropathologies consistent with human sporadic CJD or vCJD [79,131], whereas
	transgenic mice homozygous for human PrP V129 either propagate type 5 PrP Sc and a
	distinct pattern of neuropathology, or develop clinical prion disease in the absence of
	detectable PrP Sc [77,131]. Depending on the source of the inoculum, multiple disease
	phenotypes are also seen in human PrP codon 129MV heterozygous transgenic mice
	17

These findings also provide a molecular basis for PRNP codon 129 as a major locus influencing both prion disease susceptibility and phenotype in humans. Notably, our transgenic modeling studies indicate that there is no common preferred PrP Sc conformation for V129 and M129 human PrP that can be generated as a result of exposure to the vCJD/BSE prion strain

[131]

. Depending on the origin of the inoculum and the PrP codon 129 genotype of the host, primary or secondary transmission of BSEderived prions can result in four distinct prion disease phenotypes. Transgenic mice

  of a neurological disorder; the brain showed no pathological features of vCJD[START_REF] Peden | Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient[END_REF]. It is unclear if this individual would have gone on to develop clinical prion disease had they lived longer and if so would have had the phenotype of vCJD. Subclinical or carrier states of prion infection are recognised in animal models[134,155], including BSE or vCJD prion-inoculated transgenic mice expressing human prion proteins[START_REF] Asante | BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein[END_REF]131,132,136].Modelling susceptibility in such "humanised" transgenic mice suggests such genotypes will be susceptible but may develop disease phenotypes distinct from vCJD because of selection and propagation of different prion strain types[131,132]. The absence of detectable brain PrP Sc in the 129 MV blood transfusion patient precluded molecular strain typing of prions. Although splenic PrP Sc from this case resembled that seen in vCJD, this required use of phosphotungstic acid precipitation of PrP Sc which interferes with molecular strain typing[START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay[END_REF]. In addition, type 5 PrP Sc , seen in vCJD-inoculated transgenic mice expressing human PrP 129V, but not yet documented in humans, has a similar glycoform profile[START_REF] Hill | The same prion strain causes vCJD and BSE[END_REF]131,132]. It is possible that this patient was propagating a prion strain distinct from that causing vCJD. Importantly, Peden et al noted that they were unable to detect PrP Sc in lymphoid follicles in tonsil, appendix or large intestine from this patient, although abnormal PrP was detected in spleen and in a cervical lymph node. As tonsillar involvement has to date been invariably seen in clinically affected cases of vCJD, Peden et al speculated that this case may represent a distinctive

	M A N U S C R I P T
	A C C E P T E D
	It remains unclear if individuals with the other PRNP codon 129 genotypes, VV and MV,
	will also succumb to clinical disease with longer mean incubation periods after primary
	infection with BSE prions or secondary transmission of vCJD prions. The PRNP 129MV
	blood transfusion recipient had evidence of prion infection of lymphoreticular tissues at
	autopsy but died from a ruptured abdominal aortic aneurysm with no reported evidence

Acknowledgments

We are grateful to Ray Young for preparation of the figures.

A C C E P T E D

M A N U S C R I P T


PhD Andrew D Blann
email: a.blann@bham.ac.uk

Platelets: the universal killer?

Keywords: Platelets, thrombosis, P selectin

published or not. The documents may come

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Have platelets caused the greater proportion of human mortality and morbidity? What is their role in the cardiovascular, connective tissue and neoplastic disease that will strike the majority of the Western world and an increasing proportion of those in the developing world?

Historically, p athophysiological aspects of platelet biology have focussed on their relatively passive and reactive role s in minimising blood loss after injury (desirable thrombosis), in contributing to the final stages of cardiovascular disease and its risk factors (largely in the inappropriate formation of life-threatening undesirable thrombosis ), and on attempts in the out-patient and General Practice clinic, the cardiac catheter suite, and on the ward to reduce the risk of these thromboses with anti-coagulant and anti-platelet agents. Indeed, some data (e.g. adhesion of platelets to the endothelium prior to the development of manifest atherosclerotic lesions) have prompted the placement of platelets much closer to the centre of cardiovascular disease, hypothesising that platelets actually trigger atherosclerosis ( 1-3). But perhaps the most compelling evidence of the importance of this cell are the beneficial results of trials of anti-platelet drugs, of which the princ iples are aspirin and clopidogrel (4-6).

Our colleagues in oncology have long had an interest in coagulation and the platelet, and thus, in thrombosis. For example, patients with cancer are at risk of venous thromboembolism, have raised plasma fibrinogen, von Willebrand factor and fibrin degradation products, and thrombocytosis can be used as an independent prognostic indicator [START_REF] Lip | Cancer and the pro-thrombotic state[END_REF]. Indeed, several commentators have mused that hypercoagulability preceeds cancer [START_REF] Nierodzik | Hypercoagulability preceding cancer[END_REF]. However, a more cancer-specific role for the platelet in this type of disease has been proposed by Pinedo et al [START_REF] Pinedo | Involvement of platelets in tumour angiogenesis?[END_REF], who suggest that differences in the release of pro-angiogenic (e.g. platelet derived growth factor, angiopoietin) and anti-antigenic (e.g. platelet factor 4, thrombospondin) factors by this cell modifies the angiogenic balance of a tumour and its ability to metastasise. Data supporting this hypothesis has been produced by several groups, who have showed not only

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that platelets carry the pro-angiogenic vascular endothelial growth factor but also release it upon activation by thrombin 10,11). Similarly, Caine et al have reported that the inflammatory cytokine IL-6 will cause the dose-dependent release of VEGF from normal platelets in vitro [START_REF] Caine | Platelet activation, coagulation and angiogenesis in breast and prostate carcinoma[END_REF], whilst Rosselli et al [START_REF] Roselli | Vascular endothelial growth factor (VEGF-A) plasma levels in non-small cell lung cancer: relationship with coagulation ands platelet activation markers[END_REF] have demonstrated a strong correlation between VEGF and platelet counts as well as with a plasma marker of platelet activation (soluble P selectin) in both squamous and adenoma variants of non-small cell lung cancer, suggesting that the platelet could be an important source of this growth factor . It is therefore possible (at least in theory) that platelets may be important in angiogenesis and in wound healing.

Thrombocytosis and abnormalities in platelet function ha ve long been a recognised feature of connective tissue diseases such as rheumatoid arthritis and systemic lupus erythematosus and, of course, aspirin is for many the first line of treatment [START_REF] Hutchinson | Thrombocytosis in rheumatoid arthritis[END_REF][START_REF] Budman | Hematologic aspects of systemic lupus erythematosus. Current concepts[END_REF][START_REF] Ertenli | Pathologic thrombopoiesis of rheumatoid arthritis[END_REF]. Increased numbers of platelet microparticles (fragments of cytoplasm) with pro-thrombotic potential (17) may c ontribute to the increased risk of disease in this group. However, the latter stages of the disease certainly involve thrombosis, such that the terminal events are cardiovascular (e.g. pulmonary embolism, myocardial infarction and stroke). Accordingly, rheumatoid d isease may indeed be seen as an additional risk factor for cardiovascular disease in the same light as is diabetes and hypertension. Finally, inflammation (in the absence of a connective tissue component) has been strongly promoted as a player in the complex pathophysiology of atherosclerosis, and this extends to the platelet [START_REF] Caine | Platelet activation, coagulation and angiogenesis in breast and prostate carcinoma[END_REF][START_REF] Libby | Inflammation and thrombosis: The clot thickens[END_REF][START_REF] Ross | Atherosclerosis-an inflammatory disease[END_REF].

Thus the platelet and coagulopathy are target for each of the major disease processes that afflict our species (Table 1) , but what tools do we have to study the p athophysiology of this cell? A major problem with the study of the isolated platelet (when obtained from venous

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blood for studies such as aggregometry or adhesion) is that their fragile nature leads to the strong likelihood of an ex-vivo (and therefore artifactual) effect. Complex analyses such as are offered by flow cytometry may also provide useful, although possibly also artefactual, data, although this approach, by its nature, does not le nd itself to high-throughput research.

However, flow cytometry, directed towards surface molecules such as CD63 and gpIIb/IIIa (which binds fibrinogen) , can be performed on whole blood, thus avoiding the preparation artefact of a purified population of platelets. A third opportunity to study this cell is by its secreted products. When activated ( by, for example, thrombin or adenosine diphosphate), platelets release the contents of their alpha granules, such as beta thromboglobulin and platelet factor four, which can be measured in plasma by enzyme linked immunosorbent assay (ELISA). A parallel event in this degranulation is the appearance, at the cell surface, of the adhesion molecule CD62P, also known as P selectin, which can be detected by flow cytometry. However, using ELISA, a form of P selectin can also be found in the plasma (i.e.

soluble P-selectin), and this product is becoming recognised as a valuable tool for assessing platelet activation in vivo and in vitro [START_REF] Blann | The adhesion molecule P-selectin and cardiovascular disease[END_REF]. This molecule may also, under certain circumstances, be found on the surface of the endothelial cell, leading to confusion in some minds regarding the origin of the plasma form. However, is now clear that the platelet is the major source of soluble P selectin (20, and references therein).

These tools therefore, provide the opportunity to study the pathophysiology of thrombosis.

However, some crucial questions can only be answered in animal model (presuming, of course, that the biology of the particular species reflects that of our species, which is frequently not the case ( 21)), and in this respect the P selectin knock-out mouse has proved fascinating [START_REF] Robinson | Multiple, targeted deficiencies in selectins reveal a predominant role for P-selectin in leukocyte recruitment[END_REF]. Nevertheless, some tools cannot easily be carried across species, and monoclonal antibodies (mAbs) are good example of this problem as those directed towards

  to react to the same molecules of common laboratory animals, frustrating the clinical scientists. But one source of frustration has recently been relieved by the production of a series of mAb's specific for rat P selectin. In the current issue of this Journal, Hirose et al report the develo pment and application of such reagents from hybridoma cell lines using rat platelets stimulated with thrombin as immunogens[START_REF] Hirose | Elevation of rat plasma P-selectin in acute rat lung injury[END_REF]. They use it to demonstrate that soluble P selectin could serve as a marker of lung inflammation and thus, by extrapolation, a new tool for studying thrombosis in this and other settings. An additional point of interest in the large variation in soluble P selectin between different strains of rat -a phenomenon not (yet) reported in our own species. These anti-rat P -selectin antibodies therefore may well open up a new field of potential research in this most common pathophysiology of human diseases. Cardiovascular disease § Platelets are present in atheroma § Rupture of the atheroma, alongside circulating thrombus , are responsible for acute coronary syndromes such as myocardial infarction and angina § Platelets contribute to deep vein thrombosis and pulmonary embolism § Anti-platelet drugs effective in postponing major clinical events such as myocardial infarction and stroke Cancer § Platelets carry vascular endothelial growth factor (although it is unclear whether or not this is clinically relevant) § Patients with existing cancer are at risk of venous thrombosis § Patients with an unexplained venous thrombosis are often found later to have an asymptomatic cancer Connective tissue disease § Raised platelet count often present in rheumatoid arthritis § Increased numbers of pro-thrombotic platelet microparticles in systemic lupus erythematosus § Connective tissue disease inevitable progresses to the cardiovascular system and thus major event such as venous and arterial thrombosis