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Human prion diseases constitute a group of infectious and invariably fatal neurodegenerative disorders associated with misfolding of the prion protein. Variant Creutzfeldt-Jakob disease (vCJD) is a zoonotic prion disease linked to oral exposure to the infectious agent that causes bovine spongiform encephalopathy (BSE) in cattle. The most recent case of definite vCJD was heterozygous (MV) at polymorphic codon 129 of the prion protein gene PRNP while all of the previous 177 definite or probable vCJD cases who underwent genetic analysis were methionine homozygous (MM). Retrospective prevalence studies conducted on lympho-reticular tissue suggest that the number of asymptomatic vCJD carriers in the United Kingdom might be around 1 in 2000 people. In addition, there have been four known cases of the transmission of vCJD infection via blood transfusion. For these reasons, a sensitive, reliable, and fast diagnostic test is currently needed. We describe a rapid and highly sensitive seeding conversion assay that detects disease-associated prion protein in the brain and cerebrospinal fluid in vCJD after 48-96 h of amplification, with 100% sensitivity and specificity. This method can amplify prions from definite, probable, and possible vCJD cases from patients who are either MM or MV at PRNP-codon 129.

Prion diseases are rare fatal neurological conditions of humans and animals, one of which (variant Creutzfeldt-Jakob disease) is known to be a zoonotic form of the cattle disease bovine spongiform encephalopathy (BSE). What makes one animal prion disease zoonotic and others not is poorly understood, but it appears to involve compatibility between the prion strain and the host prion protein sequence. Concerns have been raised that the United Kingdom sheep flock may have been exposed to BSE early in the cattle BSE epidemic and that serial BSE transmission in sheep might have resulted in adaptation of the agent, which may have come to phenotypically resemble scrapie while maintaining its pathogenicity for humans. We have modeled this scenario in vitro. Extrapolation from our results suggests that if BSE were to infect sheep in the field it may, with time and in some sheep genotypes, become scrapie-like at the molecular level. However, the results also suggest that if BSE in sheep were to come to resemble scrapie it would lose its ability to affect humans.

It is now 18 years since the first identification of a case of vCJD in the UK. Since that time there has been much speculation over how vCJD might impact on human health. To date there have been 177 cases reports in the UK and a further 51 cases worldwide in 11 different countries. Since establishing that BSE and vCJD are of the same strain of agent, we have also shown that there is broad similarity between UK and non-UK vCJD cases on first passage to mice. Transgenic mouse studies have indicated that all codon 129 genotypes are susceptible to vCJD and that genotype may influence whether disease appears in a clinical or asymptomatic form, supported by the appearance of the first case of potential asymptomatic vCJD infection in a PRNP 129MV patient. Following evidence of blood transfusion as a route of transmission, we have ascertained that all blood components and leucoreduced blood, in a sheep model of vCJD have the ability to transmit disease. Importantly, we recently established that a PRNP 129MV patient blood recipient with an asymptomatic infection with limited PrP(Sc) deposition in the spleen could readily transmit disease into mice, demonstrating the potential for peripheral infection in the absence of clinical disease. This, along with the recent appendix survey which identified 16 positive appendices in a study of 32 441 cases, underlines the importance of continued CJD surveillance and maintaining control measures already in place to protect human health.

Following the discovery of a causal link between bovine spongiform encephalopathy (BSE) in cattle and variant Creutzfeldt-Jakob disease (vCJD) in humans, several experimental approaches have been used to try to assess the potential risk of transmission of other animal transmissible spongiform encephalopathies (TSEs) to humans. Experimental challenge of non-human primates, humanised transgenic mice and cell-free conversion systems have all been used as models to explore the susceptibility of humans to animal TSEs. In this review we compare and contrast in vivo and in vitro evidence of the zoonotic risk to humans from sheep, cattle and deer prions, focusing primarily on chronic wasting disease and our own recent studies using protein misfolding cyclic amplification.

The molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined. This is due, in part, to the absence of any well characterized and relevant cultured human cells susceptible to infection with human prions, such as those involved in Creutzfeldt-Jakob disease. In variant Creutzfeldt-Jakob disease, prion replication is thought to occur first in the lymphoreticular system and then spread into the brain. We have, therefore, examined the susceptibility of a human tonsil-derived follicular dendritic cell-like cell line (HK) to prion infection. HK cells were found to display a readily detectable, time-dependent increase in cell-associated abnormal prion protein (PrP(TSE)) when exposed to medium spiked with Creutzfeldt-Jakob disease brain homogenate, resulting in a coarse granular perinuclear PrP(TSE) staining pattern. Despite their high level of cellular prion protein expression, HK cells failed to support infection, as judged by longer term maintenance of PrP(TSE) accumulation. Colocalization studies revealed that exposure of HK cells to brain homogenate resulted in increased numbers of detectable lysosomes and that these structures immunostained intensely for PrP(TSE) after exposure to Creutzfeldt-Jakob disease brain homogenate. Our data suggest that human follicular dendritic-like cells and perhaps other human cell types are able to avoid prion infection by efficient lysosomal degradation of PrP(TSE).

The past 20 years have witnessed a dramatic resurgence of interest in a hitherto obscure neurodegenerative disease, Creutzfeldt-Jakob disease (CJD). This was driven partly by the novelty of the prion hypothesis, which sought to provide an explanation for the pathogenesis of transmissible spongiform encephalopathies, involving a unique epigenetic mechanism, and partly by events in the UK, where an outbreak of a new prion disease in cattle (bovine spongiform encephalopathy or BSE) potentially exposed a large section of the UK population to prion infectivity through a dietary route. The numbers of cases of the resultant novel disease variant CJD (vCJD), have so far been limited and peaked in the UK in the year 2000 and have subsequently declined. However, the effects of BSE and vCJD have been far-reaching. The estimated prevalence of vCJD infection in the UK is substantially higher than the numbers of clinical cases would suggest, posing a difficult dilemma for those involved in blood transfusion, tissue transplantation and cellular therapies. The clinico-pathological phenotype of human prion diseases has come under close scrutiny and molecular classification systems have been developed to account for the different diseases and their phenotypic spectra. Moreover, enhanced human and animal surveillance and better diagnostic tools have identified new human and animal prion diseases. Lastly, as the prion hypothesis has gained widespread acceptance, the concepts involved have been applied to other areas, including extra-chromosomal inheritance in fungi, long-term potentiation in memory formation and the spread of molecular pathology in diverse conditions, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. Studies at the molecular and cellular level have helped to provide a better understanding of human prion diseases, aided pathological diagnosis and helped inform public health decision-making.

SUMMARY Creutzfeldt–Jakob disease is a fatal neurodegenerative disease that primarily affects the central nervous system. In this respect, it can be considered alongside the more frequently occurring neurodegenerative diseases, such as Alzheimer's disease. Creutzfeldt–Jakob disease is perhaps the paradigmatic protein misfolding disorder, so comparisons between the mechanisms involved in Creutzfeldt–Jakob disease and other neurodegenerative diseases associated with protein misfolding (such as the tauopathies and synucleinopathies) may also be informative. Like many of these diseases, Creutzfeldt–Jakob disease occurs sporadically or can, more rarely, be associated with mutations. However, Creutzfeldt–Jakob disease can also be acquired and is experimentally transmissible. These properties have had profound public health implications and made the disease of interest to virologists, in addition to those interested in protein misfolding disorders and neurodegeneration. The possible causes for the pronounced phenotypic variation among different forms of Creutzfeldt–Jakob disease are beginning to become understood, and these appear to depend in large measure on the genetics of the host (specifically the sequence of the prion protein gene, PRNP) and the epigenetic aspects of the agent (thought to be a misfolded and aggregated form of the PRNP gene product, termed a prion). This review will examine whether this model in its present form has sufficient complexity and subtlety to account for the clinicopathological variation evident in Creutzfeldt–Jakob disease and will outline the ways in which a more complete and informative molecular definition of human prions are currently being sought. Copyright © 2012 John Wiley & Sons, Ltd.

Sporadic Creutzfeldt-Jakob disease (CJD), the most common human prion disease, is generally regarded as a spontaneous neurodegenerative illness, arising either from a spontaneous PRNP somatic mutation or a stochastic PrP structural change. Alternatively, the possibility of an infection from animals or other source remains to be completely ruled out. Sporadic CJD is clinically characterized by rapidly progressive dementia with ataxia, myoclonus, or other neurologic signs and, neuropathologically, by the presence of aggregates of abnormal prion protein, spongiform change, neuronal loss, and gliosis. Despite these common features the disease shows a wide phenotypic variability which was recognized since its early descriptions. In the late 1990s the identification of key molecular determinants of phenotypic expression and the availability of a large series of neuropathologically verified cases led to the characterization of definite clinicopathologic and molecular disease subtypes and to an internationally recognized disease classification. By showing that these disease subtypes correspond to specific agent strain-host genotype combinations, recent transmission studies have confirmed the biologic basis of this classification. The introduction of brain magnetic resonance imaging techniques such as fluid-attenuated inversion recovery and diffusion-weighted imaging sequences and cerebrospinal fluid biomarker assays for the detection of brain-derived proteins as well as real-time quaking-induced conversion assay, allowing the specific detection of prions in accessible biologic fluids and tissues, has significantly contributed to the improved accuracy of the clinical diagnosis of sporadic CJD in recent years.

Susceptibility to prion infection involves interplay between the prion strain and host genetics, but expression of the host-encoded cellular prion protein is a known prerequisite. Here we consider human embryonic stem cell (hESC) susceptibility by characterizing the genetics and expression of the normal cellular prion protein and by examining their response to acute prion exposure. Seven hESC lines were tested for their prion protein gene codon 129 genotype and this was found to broadly reflect that of the normal population. hESCs expressed prion protein mRNA, but only low levels of prion protein accumulated in self-renewing populations. Following undirected differentiation, up-regulation of prion protein expression occurred in each of the major embryonic lineages. Self-renewing populations of hESCs were challenged with infectious human and animal prions. The exposed cells rapidly and extensively took up this material, but when the infectious source was removed the level and extent of intracellular disease-associated prion protein fell rapidly. In the absence of a sufficiently sensitive test for prions to screen therapeutic cells, and given the continued use of poorly characterized human and animal bioproducts during hESC derivation and cultivation, the finding that hESCs rapidly take up and process abnormal prion protein is provocative and merits further investigation.

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 amplification (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.

Using different prion strains, such as the variant Creutzfeldt-Jakob disease agent and the atypical bovine spongiform encephalopathy agents, and using transgenic mice expressing human or bovine prion protein, we assessed the reliability of protein misfolding cyclic amplification (PMCA) to model interspecies and genetic barriers to prion transmission. We compared our PMCA results with in vivo transmission data characterized by attack rates, i.e., the percentage of inoculated mice that developed the disease. Using 19 seed/substrate combinations, we observed that a significant PMCA amplification was only obtained when the mouse line used as substrate is susceptible to the corresponding strain. Our results suggest that PMCA provides a useful tool to study genetic barriers to transmission and to study the zoonotic potential of emerging prion strains.

Sporadic and variant Creutzfeldt–Jakob disease brain reference materials available from the UK National Institute for Biological Standards and Control have been subjected to further characterisation by Western blot analysis, with particular reference to the co-occurrence of different abnormal disease-associated prion protein (PrPSc) types. The results confirm the presence of genuine type 1 and type 2 protease-resistant PrP (PrPres) in each of the three sporadic Creutzfeldt–Jakob disease reagents, and provide evidence supporting the lower level presence of type 1 PrPres in the variant Creutzfeldt–Jakob disease reagents. We conclude that these reagents provide a valuable resource for future research and development.

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 individuals 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 individuals 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.

Background: Involvement of the eye has been reported in patients with variant Creutzfeldt-Jakob disease (vCJD), but there is disagreement on whether retinal involvement occurs in sporadic Creutzfeldt-Jakob disease (sCJD). Methods: Western blotting, paraffin embedded tissue blotting, and immunohistochemistry were used to test whether the abnormal form of the prion protein (PrPSc) accumulates to detectable levels in the eye in a case of the most common subtype of sCJD (MM1). Results: Low levels of PrPSc were detectable in the retina, localised to the plexiform layers of the central retina. PrPSc was not detectable in other ocular tissues. Conclusions: The abnormal form of the prion protein is present in the retina in the most common sCJD subtype (MM1), albeit at levels lower than those found previously in vCJD and in sCJD of the VV2 subtype.

Summary. Variant Creutzfeldt–Jakob disease (CJD) is a novel acquired human prion disease apparently resulting from exposure to the bovine spongiform encephalopathy (BSE) agent. Variant CJD differs from other human prion diseases in that the disease-associated form of the prion protein and infectivity are readily detectable in lymphoid tissues throughout the body. Lymphoid tissues and lymphocytes are implicated in the peripheral pathogenesis of prion diseases (where infectivity may be detected during the preclinical phase of the illness), giving rise to concerns that blood and blood products may also contain infectious particles, representing a possible source of iatrogenic spread of variant CJD. This concern has been reinforced following the experimental transmission of BSE in a sheep model by transfusion of blood and buffy coat from animals in the preclinical phase of the illness, and the recent identification of a UK case of variant CJD in a patient who had received packed red blood cells that had been donated by an individual who subsequently died from variant CJD. Studies in animal models suggest that most prion infectivity in blood may be cell-associated, with lower levels in the plasma, and there is evidence to suggest that any infectivity present may be reduced during the process of plasma fractionation. However, the possibility that plasma or blood products could transmit the disease cannot be excluded. Further studies are required to develop more sensitive means to detect disease-associated prion protein in blood; such techniques could be employed for screening purposes to reduce exposure to contaminated products and to assist with risk management in potentially exposed individuals.

Variant CJD is a novel human prion disease that represents the first known occasion in which animal prion diseases have been transmitted to humans. There are many uncertainties concerning vCJD, including the mechanism of transmission between species, the extent of human exposure to the BSE agent, the infectious dose for humans, and the future burden of human disease. It is hoped that continuing scientific research may lead to answers to some of these questions and that further understanding of the mechanism of prion replication may lead to the development of effective treatment. Indeed a recent publication has suggested that the drugs quinacrine or chloropromazine may be candidates for the treatment of human prion diseases [42].

The neuropathological and biochemical features of the 89 histologically confirmed cases of variant Creutzfeldt-Jakob disease (vCJD) diagnosed up to the end of October 2001 in the UK are reviewed. Histology of the central nervous system, lymphoid tissues and other organs was accompanied by immunocytochemistry and Western blot analysis of the disease-associated form of the prion protein (PrP(RES)). All patients with vCJD were methionine homozygotes at codon 129 of the PrP gene. The pathology of vCJD showed relatively uniform morphological and immunocytochemical characteristics, which were distinct from other forms of CJD. PrP(RES) accumulation was widespread in lymphoid tissues in vCJD, but was not identified in other non-neural tissues. PrP(RES) in vCJD brain tissue showed a uniform glycotype pattern distinct from sporadic CJD. Given the increasingly widespread occurrence of bovine spongiform encephalopathy in Europe and Asia, there is a major need for widespread CJD surveillance. This should be accompanied by a multidisciplinary laboratory approach to the investigation and diagnosis of all forms of CJD, with the need to investigate autopsy tissues from suspected cases by the histological and biochemical techniques described herein.

Introduction Variably protease sensitive prionopathy (VPSPr) is a recently described, sporadic human prion disease that is pathologically and biochemically distinct from the currently recognised sporadic Creutzfeldt-Jakob disease (sCJD) subtypes. The defining biochemical features of the abnormal form of the prion protein (PrPSc) in VPSPr are increased sensitivity to proteolysis and the presence of an N- and C-terminally cleaved ~8 kDa protease resistant PrPSc (PrPres) fragment. The biochemical and neuropathological profile of VPSPr has been proposed to resemble either Gerstmann–Sträussler–Scheinker syndrome (GSS) or familial CJD with the PRNP-V180I mutation. However, in some cases of VPSPr two protease resistant bands have been observed in Western blots that co-migrate with those of type 2 PrPres, suggesting that a proportion of the PrPSc present in VPSPr has properties similar to those of sCJD. Results Here, we have used conformation dependent immunoassay to confirm the presence of PrPSc in VPSPr that is more protease sensitive compared with sCJD. However, CDI also shows that a proportion of PrPSc in VPSPr resists PK digestion of its C-terminus, distinguishing it from GSS associated with ~8 kDa PrPres, and showing similarity to sCJD. Intensive investigation of a single VPSPr case with frozen tissue from multiple brain regions shows a broad, region-specific spectrum of protease sensitivity and differential stability of PrPSc in the absence of PK treatment. Finally, using protein misfolding cyclic amplification and real-time quaking induced conversion, we show that VPSPr PrPSc has the potential to seed conversion in vitro and that seeding activity is dispersed through a broad range of aggregate sizes. We further propose that seeding activity is associated with the ~19 and ~23 kDa PrPres rather than the ~8 kDa fragment. Conclusions Therefore, PrPSc in VPSPr is heterogeneous in terms of protease sensitivity and stability to denaturation with the chaotrope GdnHCl and includes a proportion with similar properties to that found in sCJD. Electronic supplementary material The online version of this article (doi:10.1186/s40478-014-0152-4) contains supplementary material, which is available to authorized users.

Objective: To describe a unique case of Gerstmann-Straussler-Scheinker (GSS) disease caused by a novel prion protein ( PRNP ) gene mutation and associated with strongly positive voltage-gated potassium channel (VGKC)-complex antibodies (Abs). Methods: Clinical data were gathered from retrospective review of the case notes. Postmortem neuropathologic examination was performed, and DNA was extracted from frozen brain tissue for full sequence analysis of the PRNP gene. Results: The patient was diagnosed in life with VGKC-complex Ab–associated encephalitis based on strongly positive VGKC-complex Ab titers but no detectable LGI1 or CASPR2 Abs. He died despite 1 year of aggressive immunosuppressive treatment. The neuropathologic diagnosis was GSS disease, and a novel mutation, P84S, in the PRNP gene was found. Conclusion: VGKC-complex Abs are described in an increasingly broad range of clinical syndromes, including progressive encephalopathies, and may be amenable to treatment with immunosuppression. However, the failure to respond to aggressive immunotherapy warns against VGKC-complex Abs being pathogenic, and their presence does not preclude the possibility of prion disease.

Chronic wasting disease in elk might be a threat to human health., The risks posed to human health by individual animal prion diseases cannot be determined a priori and are difficult to address empirically. The fundamental event in prion disease pathogenesis is thought to be the seeded conversion of normal prion protein to its pathologic isoform. We used a rapid molecular conversion assay (protein misfolding cyclic amplification) to test whether brain homogenates from specimens of classical bovine spongiform encephalopathy (BSE), atypical BSE (H-type BSE and L-type BSE), classical scrapie, atypical scrapie, and chronic wasting disease can convert normal human prion protein to the abnormal disease-associated form. None of the tested prion isolates from diseased animals were as efficient as classical BSE in converting human prion protein. However, in the case of chronic wasting disease, there was no absolute barrier to conversion of the human prion protein.

A case of sporadic Creutzfeldt-Jakob disease (sCJD) is described in a young Dutch protein prion gene (PRNP) codon 129 valine homozygote. Certain clinical and molecular features of this case overlap those of variant CJD. The case highlights possible difficulties in the differential diagnosis of vCJD and the more rare sCJD subtypes based on molecular features alone.

The neuropathological and biochemical features of 33 cases of variant Creutzfeldt-Jakob disease (vCJD) diagnosed up to the end of 1998 are analysed in relation to the 646 cases of suspected CJD referred to the CJD Surveillance Unit laboratory from 1990 to 1998. Morphological studies of the central nervous system, lymphoid tissues and other organs were accompanied by immunocytochemistry; Western blot analysis of PrPRES was performed on frozen brain tissue. The findings were analysed in relation to clinical and genetic data. The pathology of vCJD showed morphological and immunocytochemical characteristics distinct from other cases of CJD. PrP accumulation was widespread in lymphoid tissues in vCJD, but was not identified in other non-neural tissues. PrPRES accumulation in vCJD brain tissue showed a uniform glycotype pattern distinct from sporadic CJD. All analysed cases of vCJD were methionine homozygotes at codon 129 of the PrP gene. No evidence currently exists to suggest that cases of CJD diagnosed in individuals who are MV or VV at codon 129 of the PrP gene represent 'human bovine spongiform encaphalopathy (BSE)'. Continued surveillance is required to further investigate this possibility, with the need to investigate autopsy tissues from suspected cases by histological and biochemical techniques.

Surveillance for infection by endoscopy for variant CJD and other human prion diseases The agents responsible for transmissible spongiform encephalopathies or prion diseases target the central nervous system, but their unique nature and pathophysiology has meant that prion diseases have made an impact in disciplines as diverse as dentistry and transfusion medicine, in large measure because of their resistance to decontamination by conventional means.1 In 1996, a new variant form of Creutzfeldt–Jakob disease (CJD) was described in the UK that preferentially affects young adults, resulting in a disease that often presents with psychiatric symptoms progressing to ataxia, dementia and terminating in akinetic mutism.2 The cause of this disease was proposed to be oral exposure to the bovine encephalopathy (BSE) agent, and subsequent studies have only served to strengthen this link, with no credible alternative explanation having been proposed.3–6 All human prion diseases are progressive and uniformly fatal neurodegenerative conditions, and, in the case of the acquired forms (iatrogenic CJD and kuru), associated with potentially very long incubation periods.7 Host genetics substantially influence these diseases: familial prion diseases are closely associated with mutations in the prion protein gene ( PRNP ), and the methionine /valine polymorphism at PRNP codon 129 appears to influence susceptibility, incubation period and in some respects disease phenotype.7 Our understanding of the pathogenesis of prion diseases has been greatly aided by the development of methods to detect and localise abnormal forms of the prion protein (PrPSc), which is the major, if not the sole, component of the infectious agent.7 Variations of standard immunohistochemical methods and Western blotting that use protease digestion and other discriminant steps to distinguish between the ubiquitous normal form of the prion protein (PrPC) and PrPSc have been used. With such tools, the distribution of this protease-resistant …

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent of the human prion diseases, which are fatal and transmissible neurodegenerative diseases caused by the infectious prion protein (PrPSc). The origin of sCJD is unknown, although the initiating event is thought to be the stochastic misfolding of endogenous prion protein (PrPC) into infectious PrPSc. By contrast, human growth hormone-associated cases of iatrogenic CJD (iCJD) in the United Kingdom (UK) are associated with exposure to an exogenous source of PrPSc. In both forms of CJD, heterozygosity at residue 129 for methionine (M) or valine (V) in the prion protein gene may affect disease phenotype, onset and progression. However, the relative contribution of each PrPC allotype to PrPSc in heterozygous cases of CJD is unknown. Using mass spectrometry, we determined that the relative abundance of PrPSc with M or V at residue 129 in brain specimens from MV cases of sCJD was highly variable. This result is consistent with PrPC containing an M or V at residue 129 having a similar propensity to misfold into PrPSc thus causing sCJD. By contrast, PrPSc with V at residue 129 predominated in the majority of the UK human growth hormone associated iCJD cases, consistent with exposure to infectious PrPSc containing V at residue 129. In both types of CJD, the PrPSc allotype ratio had no correlation with CJD type, age at clinical onset, or disease duration. Therefore, factors other than PrPSc allotype abundance must influence the clinical progression and phenotype of heterozygous cases of CJD., Author Summary In Creutzfeldt-Jakob disease (CJD), heterozygosity at residue 129 for methionine or valine in normal prion protein may affect disease phenotype, onset and progression. However, the relative contribution of each prion protein allotype to the infectious, disease associated form of prion protein (PrPSc) is unknown. Here we report the novel observation that in heterozygous cases of sporadic CJD the PrPSc allotype ratio is highly variable. This case-by-case variability is consistent with the origin of sporadic CJD being the spontaneous, but random, misfolding of either host prion protein allotype into infectious PrPSc. By contrast, in heterozygous cases of iatrogenic CJD in the United Kingdom resulting from exposure to contaminated human growth hormone, the PrPSc allotype ratio is much more homogeneous and consistent with exposure to infectious PrPSc containing valine at residue 129. Surprisingly, the PrPSc allotype ratio did not correlate with disease onset or duration in either disease type. Thus, factors other than PrPSc allotype ratio likely influence the clinical progression of heterozygous cases of CJD. Moreover, our results suggest that the ratio of methionine to valine in PrPSc may be a means of determining the origin of prion infection.

Early epidemiological studies on sporadic Creutzfeldt–Jakob disease did not identify blood transfusion as a risk factor for the disease. However, the emergence of variant Creutzfeldt–Jakob disease (vCJD) in 1996 and the identification of PrPSc in lymphoid tissues in this novel disorder led to concerns that transmission of infectivity by blood transfusion might be a possibility. These concerns were fully realised in 2004, when the first case of vCJD associated with transmission by blood transfusion was identified in a recipient who was a methionine homozygote at codon 129 in the prion protein gene, as in all other vCJD patients. Other similar cases have subsequently emerged, along with cases of asymptomatic vCJD infection in a blood transfusion recipient and a plasma product recipient, both of whom were heterozygous at codon 129 of the prion protein gene. This chapter reviews the experimental evidence for the transmission of prion infectivity by blood transfusion in a range of experimental models, discusses the evidence for the transmission of vCJD by blood transfusion and plasma products and considers the future possibilities for the development and potential uses of blood-based screening tests for human prion diseases.

Real-time quaking-induced conversion (RT-QuIC) is an assay in which disease-associated prion protein (PrP) initiates a rapid conformational transition in recombinant PrP (recPrP), resulting in the formation of amyloid that can be monitored in real time using the dye thioflavin T. It therefore has potential advantages over analogous cell-free PrP conversion assays such as protein misfolding cyclic amplification (PMCA). The QuIC assay and the related amyloid seeding assay have been developed largely using rodent-passaged sheep scrapie strains. Given the potential RT-QuIC has for Creutzfeldt–Jakob disease (CJD) research and human prion test development, this study characterized the behaviour of a range of CJD brain specimens with hamster and human recPrP in the RT-QuIC assay. The results showed that RT-QuIC is a rapid, sensitive and specific test for the form of abnormal PrP found in the most commonly occurring forms of sporadic CJD. The assay appeared to be largely independent of species-related sequence differences between human and hamster recPrP and of the methionine/valine polymorphism at codon 129 of the human PrP gene. However, with the same conditions and substrate, the assay was less efficient in detecting the abnormal PrP that characterizes variant CJD brain. Comparison of these QuIC results with those previously obtained using PMCA suggested that these two seemingly similar assays differ in important respects.

Disease-associated prion protein (PrP(Sc)) can be distinguished from the cellular isoform (PrP(C)) by conformation-dependent immunoassay (CDI). This technique exploits the presence of an epitope, accessible in PrP(C), but only unmasked by denaturation in PrP(Sc). In this study, we investigated PrP(Sc) in different brain regions in variant and sporadic Creutzfeldt-Jakob disease (CJD) by using CDI, and directly compared the results with those obtained using the more commonly employed protease digestion and Western blotting. In general, there was good agreement between the results, although there were certain discrepancies in relative abundance when the regional distribution in variant CJD cases was considered. The results largely confirmed the previously described targeting of different brain regions by variant and sporadic CJD. Additionally, the combination of protease digestion and CDI detection demonstrated, for the first time, the presence of PrP(Sc) in variant CJD brains that is susceptible to proteolysis under standard conditions.

The phenotypic and strain-related properties of human prion diseases are, according to the prion hypothesis, proposed to reside in the physicochemical properties of the conformationally altered, disease-associated isoform of the prion protein (PrP Sc ), which accumulates in the brains of patients suffering from Creutzfeldt-Jakob disease and related conditions, such as Gerstmann-Straussler-Scheinker disease. Molecular strain typing of human prion diseases has focused extensively on differences in the fragment size and glycosylation site occupancy of the protease-resistant prion protein (PrP res ) in conjunction with the presence of mutations and polymorphisms in the prion protein gene ( PRNP ). Here we report the results of employing an alternative strategy that specifically addresses the conformational stability of PrP Sc and that has been used previously to characterize animal prion strains transmitted to rodents. The results show that there are at least two distinct conformation stability states in human prion diseases, neither of which appears to correlate fully with the PrP res type, as judged by fragment size or glycosylation, the PRNP codon 129 status, or the presence or absence of mutations in PRNP . These results suggest that conformational stability represents a further dimension to a complete description of potentially phenotype-related properties of PrP Sc in human prion diseases.

Prion strains are defined by their biological properties after transmission to wild-type mice, specifically by their incubation periods and patterns of vacuolar pathology (‘lesion profiles’). Preliminary results from transmissions of variant Creutzfeldt–Jakob disease (vCJD) to wild-type mice provided the first compelling evidence for the close similarity of the vCJD agent to the agent causing bovine spongiform encephalopathy (BSE). Complete results from this investigation, including the transmission characteristics of vCJD from brain and peripheral tissues of 10 cases (after primary transmission and subsequent mouse-to-mouse passage), have now been analysed. All 10 vCJD sources resulted in consistent incubation periods and lesion profiles, suggesting that all 10 patients were infected with the same strain of agent. Incubation periods suggested that infectious titres may be subject to regional variation within the brain. Comparison of incubation periods and lesion profiles from transmission of brain and peripheral tissues showed no evidence of tissue-specific modification in the biological properties of the agent. Analysis of the protease-resistant prion protein (PrPres) by Western blotting from primary and subsequent passages in mice showed a glycosylation pattern closely resembling that of vCJD in humans, the so-called BSE ‘glycoform signature’. Minor variations in PrPres fragment size were evident between mouse strains carrying different alleles of the gene encoding PrP both in primary transmissions and on further passages of vCJD brain. Overall, the results closely resembled those of previously reported transmissions of BSE in the same mouse strains, consistent with BSE being the origin of all of these vCJD cases.

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)).

BACKGROUND: 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 individuals. Protein misfolding cyclic amplification (PMCA), a method for the amplification of minute amounts of disease-associated abnormal prion protein (PrPSc) 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. STUDY DESIGN AND METHODS: With the use of seed sources from individuals 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 amplification efficiency and freeze-thaw on a substrate's ability to support amplification and the degree of amplification achieved by serial PMCA (sPMCA) were investigated. RESULTS: Seed/substrate PRNP codon 129 compatibility was found to have a major influence on PrPSc amplification efficiency. Individual 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 PrPSc 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 PrPSc production detected in the unseeded PLT substrate. CONCLUSIONS: 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.

Background: The identification of transmission of variant Creutzfeldt-Jakob disease (vCJD) by blood transfusion has prompted investigation to establish whether there has been any alteration in the vCJD agent following this route of secondary transmission. Any increase in virulence or host adaptation would require a reassessment of the risk analyses relating to the possibility of a significant secondary outbreak of vCJD. Since there are likely to be carriers of the vCJD agent in the general population, there is a potential for further infection by routes such as blood transfusion or contaminated surgical instruments.Methodology: We inoculated both wild-type and transgenic mice with material from the first case of transfusion associated vCJD infection.Principal Findings: The strain transmission properties of blood transfusion associated vCJD infection show remarkable similarities to the strain of vCJD associated with transmission from bovine spongiform encephalopathy (BSE).Conclusions: Although it has been hypothesized that adaptation of the BSE agent through secondary passage in humans may result in a greater risk of onward transmission due to an increased virulence of the agent for humans, our data presented here in two murine models suggest no significant alterations to transmission efficiency of the agent following human-to-human transmission of vCJD.

Molecular typing is of considerable importance for the surveillance and epidemiology of human transmissible spongiform encephalopathies (TSEs). It relies on the detection of distinct protease-resistant prion protein (PrP Sc ) core fragments that differ in molecular mass and/or glycoform ratio. In this collaborative study, we tested the inter-laboratory agreement in TSE molecular typing. Sixteen characterized brain specimens from sporadic TSEs and variant Creutzfeldt-Jakob disease (vCJD) cases were distributed blindly to seven laboratories for molecular characterization by a defined protocol and classification. Agreement between laboratories in the classification of samples was excellent. In particular, there were no differences in the distinction between PrP Sc type 1, type 2A, and type 2B with one exception, which eventually was identified as a case with types 1 and 2 co-occurrence. This shows that the general technique and particular classification system used here are robust and represent a reliable basis for diagnostic and epidemiologic purposes. The subtle further distinction of subtypes among type 1 and type 2 groups requires high-sensitivity gel electrophoresis protocols that are unsuitable for routine diagnostic needs and must be reserved for research investigations. Further research is necessary on the identification and significance of co-occurrence of PrP Sc types 1 and 2 within one brain.

Publisher Summary This chapter describes prion diseases as a group of fatal neurodegenerative diseases that affect humans and agricultural, domestic, and free-roaming mammals. They may have acquired or genetic causes and can also occur in a sporadic fashion. The chapter also emphasizes that prion diseases are devastating illnesses that have caught the public attention in recent years due to the emergence of, and uncertainties associated with, new forms of prion disease, such as bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) and their medical, public health, and economic significance. Neuropathology is essential for a definitive diagnosis of prion disease and is invaluable in defining the remarkable range of phenotypic variation occurring in this group of disorders, which far exceeds those in other human neurodegenerative diseases. The chapter concludes that continuing surveillance for human prion diseases is required to further characterize these parameters, and in particular to monitor the incidence and spread of vCJD across the world.

Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently subclassified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K (PK) digested abnormal prion protein (PrP(res)) identified on Western blotting (type 1 or type 2). These biochemically distinct PrP(res) types have been considered to represent potential distinct prion strains. However, since cases of CJD show co-occurrence of type 1 and type 2 PrP(res) in the brain, the basis of this classification system and its relationship to agent strain are under discussion. Different brain areas from 41 sCJD and 12 iatrogenic CJD (iCJD) cases were investigated, using Western blotting for PrP(res) and two other biochemical assays reflecting the behaviour of the disease-associated form of the prion protein (PrP(Sc)) under variable PK digestion conditions. In 30% of cases, both type 1 and type 2 PrP(res) were identified. Despite this, the other two biochemical assays found that PrP(Sc) from an individual patient demonstrated uniform biochemical properties. Moreover, in sCJD, four distinct biochemical PrP(Sc) subgroups were identified that correlated with the current sCJD clinico-pathological classification. In iCJD, four similar biochemical clusters were observed, but these did not correlate to any particular PRNP 129 polymorphism or western blot PrP(res) pattern. The identification of four different PrP(Sc) biochemical subgroups in sCJD and iCJD, irrespective of the PRNP polymorphism at codon 129 and the PrP(res) isoform provides an alternative biochemical definition of PrP(Sc) diversity and new insight in the perception of Human TSE agents variability.

Cerebral and cardiac amyloid deposits have been reported after scrapie infection in transgenic mice expressing variant prion protein (PrPC) lacking the glycophosphatidylinositol anchor. The amyloid fibril protein in the systemic amyloid deposits was not characterized, and there is no clinical or pathological association between prion diseases and systemic amyloidosis in humans. Nevertheless, in view of the potential clinical significance of these murine observations, we tested both human amyloidotic tissues and isolated amyloid fibrils for the presence of PrPSc, the prion protein conformation associated with transmissible spongiform encephalopathy (TSE). We also sequenced the complete prion protein gene, PRNP, in amyloidosis patients. No specific immunohistochemical staining for PrPSc was obtained in the amyloidotic cardiac and other visceral tissues of patients with different types of systemic amyloidosis. No protease-resistant prion protein, PrPres, was detectable by Western blotting of amyloid fibrils isolated from cardiac and other systemic amyloid deposits. Only the complete normal wild-type PRNP gene sequence was identified, including the usual distribution of codon 129 polymorphisms. These reassuringly negative results do not support the idea that there is any relationship of prions or TSE with human systemic amyloidosis, including cardiac amyloid deposition. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Variant Creutzfeldt-Jakob disease (vCJD) poses a serious risk of secondary transmission and the need to detect infectivity in asymptomatic individuals 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 amplification (PMCA), can amplify vCJD PrP(Sc) from human brain tissue, and that the degree of amplification 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 amplified PrP(Sc) can be detected using a conformation-dependent immunoassay (CDI), allowing the detection of putative proteinase K sensitive forms of PrP(Sc).

By using high-sensitivity Western blotting and immunohistochemistry, pituitary glands from patients with sporadic and variant Creutzfeldt–Jakob disease (sCJD and vCJD, respectively) were analysed for the presence of the protease-resistant form of the prion protein (PrPres). PrPres was detected in a greater proportion of vCJD pituitaries than sCJD pituitaries and was localized predominantly in the neurohypophysis. PrPres was also detected in a recurrent pituitary adenoma from an sCJD patient. Immunohistochemical analysis showed sparse positive labelling, predominantly in folliculostellate cells, in vCJD and sCJD adenohypophyses. The PrPres glycosylation pattern in the vCJD neurohypophyses showed a predominance of the unglycosylated band, which differed markedly from patterns found in all other vCJD tissues. The presence of PrPres in the pituitary of CJD patients at autopsy suggests that human growth hormone-related iatrogenic CJD may have indeed resulted from infectivity in collected pituitaries rather than necessarily from contamination of pituitary pools by adjacent brain tissue.

Sporadic Creutzfeldt–Jakob disease (sCJD) is the commonest form of human prion disease, with an annual mortality rate of ∼1–1.5 million cases per annum in most countries where systematic surveillance has been established (Ladogana et al ., 2005). The combined data from these studies have allowed analysis of hundreds of cases of this rare disease, as shown by Collins and colleagues, in this issue of Brain (Collins et al ., 2006). The phenotypic spectrum of sCJD is broad, including several clinical subgroups that have been recognized for many years, amongst which are the Brownell–Oppenheimer and Heidenhain variants. The basis of this clinicopathological variation in sCJD has been studied for over 10 years, with the general consensus that the two major phenotypic determinants are the presence of either methionine or valine at the polymorphic codon 129 of the patient's prion protein gene ( PRNP ), and the physico-chemical properties of the abnormal prion protein (PrPSc) that accumulates in the brain during the course of the disease (Ironside et al ., 2005). The former is readily and unambiguously determined by restriction length polymorphism analysis or gene sequencing, but the latter is generally approached indirectly by determining the size of the protease resistant core fragment of the PrPSc by western blotting of detergent-extracted and proteinase K-digested homogenates of CJD brain tissue. Two rival classification systems for the PrPSc isoforms accumulating in the brain in sCJD and other human prion disorders exist (Parchi et al ., 1999, Hill et al ., 2003). Although much has been made of their differences, a neutral observer would be struck by the considerable extent of overlap between the two systems. The Hill et al . (2003) system recognizes three PrPSc size classes, whereas two major classes (with subtypes) are recognized by Parchi et al . (1999) …

Molecular typing of the abnormal form of the prion protein (PrP(Sc)) has come to be regarded as a powerful tool in the investigation of the prion diseases. All evidence thus far presented indicates a single PrP(Sc) molecular type in variant Creutzfeldt-Jakob disease (termed type 2B), presumably resulting from infection with a single strain of the agent (bovine spongiform encephalopathy). Here we show for the first time that the PrP(Sc) that accumulates in the brain in variant Creutzfeldt-Jakob disease also contains a minority type 1 component. This minority type 1 PrP(Sc) was found in all 21 cases of variant Creutzfeldt-Jakob disease tested, irrespective of brain region examined, and was also present in the variant Creutzfeldt-Jakob disease tonsil. The quantitative balance between PrP(Sc) types was maintained when variant Creutzfeldt-Jakob disease was transmitted to wild-type mice and was also found in bovine spongiform encephalopathy cattle brain, indicating that the agent rather than the host specifies their relative representation. These results indicate that PrP(Sc) molecular typing is based on quantitative rather than qualitative phenomena and point to a complex relationship between prion protein biochemistry, disease phenotype and agent strain.

Human prion diseases can occur as an idiopathic disorder (sporadic Creutzfeldt–Jakob disease) or can be acquired, as is the case for variant Creutzfeldt–Jakob disease. These disorders are characterized by the accumulation of a protease-resistant form of the host-encoded prion protein termed PrPSc in the brains of affected individuals. PrPSc has been proposed to be the principal, if not sole, component of the infectious agent, with its accumulation in the central nervous system the primary event leading to neurodegeneration. A major question remains as to whether self-propagating structural differences in PrPSc might account for the clinicopathological diversity evident in Creutzfeldt–Jakob disease and whether different prion protein types underlie the existence of different strains of causative agent. Here, we describe the results of a large-scale biochemical study of PrPSc from autopsy-proved cases of variant Creutzfeldt–Jakob disease (n = 59) and compare these with cases of sporadic Creutzfeldt–Jakob disease (n = 170) in the United Kingdom over the period 1991 to 2002. The results show PrPSc in variant Creutzfeldt–Jakob disease to be remarkably stereotyped. In contrast, considerable heterogeneity in PrPSc exists both between and within cases of sporadic Creutzfeldt–Jakob disease. Ann Neurol 2004;55:851–859

Background: Immunocytochemical accumulation of prion protein (PrP) in lymphoid tissues is a feature of variant Creutzfeldt–Jakob disease (vCJD) that has been used both to aid in the diagnosis of patients and as a basis of large scale screening studies to assess the prevalence of preclinical disease in the UK. However, the specificity of this approach is unknown. Aim: To assess the specificity of lymphoreticular accumulation of PrP for vCJD by examining a range of human diseases. Methods: Paraffin wax embedded lymphoreticular tissues from patients with several reactive conditions (58 cases), tumours (27 cases), vCJD (54 cases), and other human prion diseases (56 cases) were assessed. PrP accumulation was assessed by immunocytochemistry using two different monoclonal anti-PrP antibodies and a sensitive detection system. Results: All cases of vCJD showed widespread lymphoreticular accumulation of PrP; however, this was not seen in the other conditions examined. Conclusion: Lymphoreticular accumulation of PrP, as assessed by immunocytochemistry, appears to be a highly specific feature of vCJD.

The neuropathological features of human prion diseases are spongiform change, neuronal loss, astrocytic proliferation and the accumulation of PrP(Sc), the abnormal isoform of prion protein (PrP). The pattern of brain involvement is remarkably variable and is substantially influenced by the host PrP genotype and PrP(Sc) isotype. Variant Creutzfeldt-Jakob disease (vCJD) is a novel human prion disease which results from exposure to the bovine spongiform encephalopathy (BSE) agent. The neuropathology of vCJD shows consistent characteristics, with abundant florid and cluster plaques in the cerebrum and cerebellum, and widespread accumulation of PrP(res) on immunocytochemistry. These features are distinct from all other types of human prion disease. Spongiform change is most marked in the basal ganglia, while the thalamus exhibits severe neuronal loss and gliosis in the posterior nuclei. These areas of thalamic pathology correlate with the areas of high signal seen in the thalamus on magnetic resonance imaging (MRI) examination of the brain. Western blot analysis of PrP(Sc) in the brain in vCJD tissue shows a uniform isotype, with a glycoform ratio characterized by predominance of the diglycosylated band, distinct from sporadic CJD. PrP(Sc) accumulation in vCJD is readily detectable outside the brain, in contrast with other forms of human prion disease, particularly in the lymphoid system and in parts of the peripheral nervous system. This has raised concern about the possible iatrogenic transmission of vCJD by contaminated surgical instruments, or blood. All cases of vCJD are methionine homozygotes at codon 129 of the prion protein gene (PRNP). Continued surveillance is required to investigate cases of vCJD in the UK and other countries where BSE has been reported, particularly as cases of 'human BSE' in individuals who are MV or VV at codon 129 of the PrP gene have not yet been identified. Histological, genetic and biochemical techniques are essential tools for the adequate diagnosis and investigation of human prion diseases.

Summary. Variant Creutzfeldt–Jakob disease (vCJD) is a novel acquired human prion disease resulting from human exposure to the agent causing bovine spongiform encephalopathy (BSE). vCJD differs from all other human prion diseases in that the disease-associated form of the prion protein and infectivity are present in lymphoid tissues throughout the body. Lymphoid tissues and lymphocytes are implicated in the peripheral pathogenesis of prion diseases (where infectivity may be detected during the preclinical phase of the illness), giving rise to concerns that blood and blood products may also contain infectivity, thus representing a possible source of iatrogenic spread of vCJD. These concerns have been reinforced by the recent transmission of BSE in an experimental sheep model by blood transfusion from an infected animal in the preclinical phase of the illness. Studies in other animal models suggest that most infectivity in blood may be cell-associated, with lower levels in the plasma, and there is evidence to indicate that any infectivity present may be reduced during the process of plasma fractionation. At present, the attempts to detect disease-associated prion protein and infectivity in buffy coat from vCJD patients have been negative, but these studies have been limited in size and in the sensitivity of the detection systems employed. Further studies are required to develop more sensitive means of detection of disease-associated prion protein in blood; such techniques could also be employed for screening purposes, both individually and to help ascertain more precisely the likely numbers of future cases of vCJD.

The prion diseases, or transmissible spongiform encephalopathies, are a group of fatal neurodegenerative disorders which occur in animals and man [4]. The first of these to be identified was the disease in sheep known as scrapie, which is endemic in many parts of the world including Europe and the USA. Scrapie has been extensively studied in experimental models to investigate the nature of the unique transmissible agents responsible for this group of disorders. Early studies revealed that the scrapie agent had a unique spectrum of physical and chemical properties, Table 1 Human prion diseases Category Disease First reported Idiopathic Sporadic CJD 1920-1921 Sporadic fatal insomnia 1999 Inherited Familial CJD 1924 Gerstmann-Straussler-Scheinker syndrome 1928-1936 Fatal familial insomnia 1986 Acquired Kuru 1957 Iatrogenic CJD 1974 Variant CJD 1996 with a marked resistance to conventional forms of microbial inactivation. In 1982, Prusiner published the prion hypothesis [62], which states that the transmissible agent responsible for this group of disorders is composed of a modified host protein, prion protein, which in humans is encoded by a gene on chromosome 20. Accumulation of the abnormal isoform of the prion protein (PrPres) in the brain is a characteristic feature of all prion diseases, and the detection of this abnormal protein is now of major importance in their diagnosis [40, 58, 59].

These results open the door to more physiological models of a prophylactic or therapeutic use of blocking peptides. A diverse range of compounds has been investigated for experimental TSE prophylaxis or therapy13xCongo red prolongs the incubation period in scrapie-infected hamsters. Ingrosso, L. et al. J. Virol. 1995; 69: 506–508PubMedSee all References, 14xProphylactic potential of pentosan polysulphate in transmissible spongiform encephalopathies. Farquhar, C. et al. Lancet. 1999; 353: 117Abstract | Full Text | Full Text PDF | PubMed | Scopus (90)See all References, 15xProlongation of scrapie incubation period by an injection of dextran sulphate 500 within the first month before or after injection. Farquhar, C. et al. J. Gen. Virol. 1986; 67: 463–473Crossref | PubMedSee all References, 16xAmphotericin B delays both scrapie agent replication and PrPres accumulation in Syrian hamster scrapie. McKenzie, D. et al. J. Virol. 1994; 68: 7534–7536PubMedSee all References, 17xMS-8209, a water soluble amphotericin B derivative, affects both scrapie agent replication and PrPres accumulation in Syrian hamster scrapie. Adjou, K.T. et al. J. Gen. Virol. 1999; 80: 1079–1085PubMedSee all References, 18xEffectiveness of anthracycline against experimental prion disease in Syrian hamsters. Tagliavini, F. et al. Science. 1997; 276: 1119–1122Crossref | PubMed | Scopus (149)See all References, 19xScrapie-induced neuronal loss is reduced by treatment with basic fibroblast growth factor. Fraser, J.R. et al. Neuroreport. 1997; 8: 2405–2409Crossref | PubMedSee all References (Table 1Table 1). Although most appear to influence the incubation period in experimental models, some (including pentosan polysulphate and MS-8209) have been shown to inhibit TSE infection when administered prophylactically under certain experimental conditions. A single study in which basic fibroblast growth factor was administered towards the end of the incubation period following intraocular injection of scrapie found enhanced survival of retinal photoreceptors, suggesting a potential late-stage therapeutic effect. These compounds are under further evaluation, but the conceptually elegant approach of Chesebro and colleagues10xSpecific inhibition of in vitro formation of protease-resistant prion protein by synthetic peptides. Chabry, J. et al. J. Biol. Chem. 1998; 273: 13203–13207Crossref | PubMed | Scopus (133)See all References, 11xSpecies-independent inhibition of prion protein (PrP) formation by a peptide containing conserved PrP sequence. Chabry, J. et al. J. Virol. 1999; 73: 6245–6250PubMedSee all References also merits further study in vitro, and perhaps eventually in vivo. Given the probable widespread exposure of the UK human population to the BSE agent (which, in some cases, has resulted in variant CJD after an unknown but presumably lengthy incubation period), any compound which offers prophylactic potential should be rigorously explored. However, the logistics and ethics of implementing such a regime, even if of proven efficacy, will be formidable.Table 1Compounds which have a demonstrable prophylactic or potential therapeutic effect in experimental TSEaaCompoundExampleRef.Anionic dyes (amyloid stain)Congo red13xCongo red prolongs the incubation period in scrapie-infected hamsters. Ingrosso, L. et al. J. Virol. 1995; 69: 506–508PubMedSee all References13Sulphated glycansPentosan polysulphate14xProphylactic potential of pentosan polysulphate in transmissible spongiform encephalopathies. Farquhar, C. et al. Lancet. 1999; 353: 117Abstract | Full Text | Full Text PDF | PubMed | Scopus (90)See all References14Dextran sulphate 50015xProlongation of scrapie incubation period by an injection of dextran sulphate 500 within the first month before or after injection. Farquhar, C. et al. J. Gen. Virol. 1986; 67: 463–473Crossref | PubMedSee all References15Polyene antibioticsAmphotericin B16xAmphotericin B delays both scrapie agent replication and PrPres accumulation in Syrian hamster scrapie. McKenzie, D. et al. J. Virol. 1994; 68: 7534–7536PubMedSee all References16MS-820917xMS-8209, a water soluble amphotericin B derivative, affects both scrapie agent replication and PrPres accumulation in Syrian hamster scrapie. Adjou, K.T. et al. J. Gen. Virol. 1999; 80: 1079–1085PubMedSee all References17Anthracycline4-iodo-4-deoxy-doxorubicin18xEffectiveness of anthracycline against experimental prion disease in Syrian hamsters. Tagliavini, F. et al. Science. 1997; 276: 1119–1122Crossref | PubMed | Scopus (149)See all References18Growth factorsBasic fibroblast growth factor19xScrapie-induced neuronal loss is reduced by treatment with basic fibroblast growth factor. Fraser, J.R. et al. Neuroreport. 1997; 8: 2405–2409Crossref | PubMedSee all References19aAbbreviation: TSE, transmissible spongiform encephalopathy.