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The inadvertent transmission of long incubating, untreatable and fatal neurodegenerative prionopathies, notably iatrogenic Creutzfeldt–Jakob disease, following transplantation of cadaver-derived corneas, pituitary growth, hormones and dura mater, constitutes a historical precedent which has underpinned the application of precautionary principles to modern day advanced cell therapies. To date these have been reflected by geographic or medical history risk-based deferral of tissue donors. Emergent understanding of other prion-like proteinopathies, their potential independence from prions as a transmissible agent and the variable capability of scalably manufacturable stem cells and derivatives to take up and clear or to propagate prions, substantiate further commitment to qualifying neurodegenerative proteinopathy transmission risks. This is especially so for those involving direct or facilitated access to a recipient’s brain or connected visual or nervous system such as for the treatment of stroke, retinal and adult onset neurodegenerative diseases, treatments for which have already commenced. In this review, we assess the prospective global dissemination of advanced cell therapies founded on transplantation or exposure to allogeneic human cells, recap lessons learned from the historical precedents of CJD transmission and review recent advances and current limits in understanding of prion and other neurodegenerative disease prion-like susceptibility and transmission. From these we propose grounds for a reassessment of the risks of emergent advanced cell therapies to transmit neuroproteinopathies and suggestions to ACT developers and regulators for risk mitigation and extension of criteria for deferrals. Electronic supplementary material The online version of this article (10.1007/s00401-018-1941-9) contains supplementary material, which is available to authorized users.

Chronic wasting disease (CWD) is a contagious and fatal neurodegenerative disease and a serious animal health issue for deer and elk in North America. The identification of the first cases of CWD among free-ranging reindeer and moose in Europe brings back into focus the unresolved issue of whether CWD can be zoonotic like bovine spongiform encephalopathy. We used a cell-free seeded protein misfolding assay to determine whether CWD prions from elk, white-tailed deer, and reindeer in North America can convert the human prion protein to the disease-associated form. We found that prions can convert, but the efficiency of conversion is affected by polymorphic variation in the cervid and human prion protein genes. In view of the similarity of reindeer, elk, and white-tailed deer in North America to reindeer, red deer, and roe deer, respectively, in Europe, a more comprehensive and thorough assessment of the zoonotic potential of CWD might be warranted.

Human-to-human transmission of Creutzfeldt–Jakob disease (CJD) has occurred through medical procedures resulting in iatrogenic CJD (iCJD). One of the commonest causes of iCJD was the use of human pituitary-derived growth hormone (hGH) to treat primary or secondary growth hormone deficiency. As part of a comprehensive tissue-based analysis of the largest cohort yet collected (35 cases) of UK hGH-iCJD cases, we describe the clinicopathological phenotype of hGH-iCJD in the UK. In the 33/35 hGH-iCJD cases with sufficient paraffin-embedded tissue for full pathological examination, we report the accumulation of the amyloid beta (Aβ) protein associated with Alzheimer’s disease (AD) in the brains and cerebral blood vessels in 18/33 hGH-iCJD patients and for the first time in 5/12 hGH recipients who died from causes other than CJD. Aβ accumulation was markedly less prevalent in age-matched patients who died from sporadic CJD and variant CJD. These results are consistent with the hypothesis that Aβ, which can accumulate in the pituitary gland, was present in the inoculated hGH preparations and had a seeding effect in the brains of around 50% of all hGH recipients, producing an AD-like neuropathology and cerebral amyloid angiopathy (CAA), regardless of whether CJD neuropathology had occurred. These findings indicate that Aβ seeding can occur independently and in the absence of the abnormal prion protein in the human brain. Our findings provide further evidence for the prion-like seeding properties of Aβ and give insights into the possibility of iatrogenic transmission of AD and CAA. Electronic supplementary material The online version of this article (doi:10.1007/s00401-017-1703-0) contains supplementary material, which is available to authorized users.

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.

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).

Aggregated and protease-resistant mammalian prion protein (PrPSc) is the primary protein component of infectious prions. Enriched PrPSc preparations are often used to study the mechanisms underlying prion disease. However, most enrichment procedures are relatively nonspecific and tend to yield significant amounts of non-PrPSc components, including various proteins that could confound functional and structural studies. It is thus important to identifythese proteins and assess their potential relevance to prion pathogenesis. Following proteinase K treatment and phosphotungstic acid precipitation of brain homogenate, we have used mass spectrometry to analyze the protein content of PrPSc isolated from prion-infected mice, multiple cases of sporadic Creutzfeldt-Jakob disease (sCJD), and human growth hormone associated casesof iatrogenic CJD (iCJD). Creatine kinase was the primary protein contaminant in all PrPSc samples while many of the other proteins identified were also found in non-CJD controls, suggesting that they are not CJD specific. Interestingly, the Alzheimer’s disease associated peptide amyloid β 1-42 (Aβ1-42) was identified in the majority of the sCJD cases as well as non-CJD age-matched controls while apoliprotein E was found in greater abundance in the sCJD cases. By contrast, while some of the iCJD cases showed evidence of higher molecular weight Aβ oligomers, monomeric Aβ1-42 peptide was not detected by immunoblot and only one case had significant levels of apolipoprotein E. Our data are consistent with the age-associated deposition of Aβ1-42 in older sporadic CJD and non-CJD patients and suggest that both apolipoprotein E and Aβ1-42 abundance can differ depending upon the type of CJD.

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.

An atypical case of prion disease is described in a 54-year-old Dutch man, homozygous for valine at codon 129 of the prion protein gene (PRNP). The clinical phenotype was characterised by progressive dementia, spastic paraplegia and sensorimotor polyneuropathy. The disease duration was 20 months. Genetic analysis of PRNP did not reveal any abnormalities. Neuropathologically, only mild spongiform change and a coarse granular immunohistochemical staining for the abnormal prion protein, PrP(Sc), was observed, with poorly formed plaques in the molecular layer of the cerebellar cortex. However, Western blotting showed low but detectable levels of proteinase K(PK)-resistant PrP(Sc) occurring in an unusual ladder-like profile. These features define a phenotype that corresponds to the recently described protease-sensitive prionopathy (PSPr). Our report on the first Dutch patient with PSPr further expands the spectrum of prionopathies and exemplifies the need to re-evaluate cases of atypical prion disease.

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.

Prion protein type and codon 129 genotype are thought to be major determinants of susceptibility and phenotype in human prion diseases. Using an in-vitro system (protein misfolding cyclic amplification) we have attempted to model human prion protein conversion using the abnormal prion protein associated with each of the major sporadic Creutzfeldt-Jakob disease subtypes, in substrates containing the normal cellular form of the prion protein of each of the three possible human PRNP codon 129 polymorphic genotypes. The prion protein type is converted with fidelity in these amplification reactions, but the efficiency of conversion depends both on the methionine/valine polymorphic status of the sporadic Creutzfeldt-Jakob disease seed and substrate homogenate, and on the abnormal prion protein type.

Demonstration of the abnormal form of the prion protein (PrP) in the brain confirms the diagnosis of human prion disease (PrD). Using immunohistochemistry, we have compared ten monoclonal antibodies in PrD subtypes including sporadic and variant Creutzfeldt‐Jakob disease (CJD), fatal familial insomnia, Alzheimer's disease (AD), and control brains. CJD subgroups were determined using Western blot analysis for the protease‐resistant PrP type in combination with sequencing to determine the genotype at the methionine/valine polymorphism at codon 129 of the prion protein gene. None of the antibodies labeled given subgroups exclusively, but the intensity of immunoreactivity varied among morphologically distinct types of deposit. Fine granular or synaptic PrP deposits stained weakly or not at all with antibodies against the N‐terminus of PrP, and were visible in one case only with 12F10 and SAF54. Coarser and plaque type deposits were immunolabeled with all antibodies. The immunostaining patterns appear characteristic for the disease subgroups. Labeling of certain neurons in all cases irrespective of disease, and staining at the periphery and/or throughout the senile plaques of AD patients were also noted. Antibodies such as 6H4 and 12F10 failed to give this type of labeling and are therefore less likely to recognise non‐pathological PrP material in immunohistochemistry.

Variant Creutzfeldt-Jakob disease (vCJD) differs from other human prion diseases in that the pathogenic prion protein PrP(Sc) can be detected to a greater extent at extraneuronal sites throughout the body, principally within lymphoid tissues. However, a recent study using a high-sensitivity Western blotting technique revealed low levels of PrP(Sc) in skeletal muscle from a quarter of Swiss patients with sporadic CJD (sCJD). This posed the question of whether PrP(Sc) in muscle could also be detected in vCJD, sCJD, and iatrogenic (iCJD) patients from other populations. Therefore, we have used the same high-sensitivity Western blotting technique, in combination with paraffin-embedded tissue blotting, to screen for PrP(Sc) in muscle tissue specimens taken at autopsy from 49 CJD patients in the United Kingdom. These techniques identified muscle PrP(Sc) in 8 of 17 vCJD, 7 of 26 sCJD, and 2 of 5 iCJD patients. Paraffin-embedded tissue blotting analysis showed PrP(Sc) in skeletal muscle in localized anatomical structures that had the morphological and immunohistochemical characteristics of nerve fibers. The detection of PrP(Sc) in muscle tissue from all forms of CJD indicates the possible presence of infectivity in these tissues, suggesting important implications for assessing the potential risk of iatrogenic spread via contaminated surgical instruments.

Human prion diseases are rare neurodegenerative disorders that can occur as sporadic, familial or acquired disorders. Within each of these categories there is a wide range of phenotypic variation that is not encountered in other neurodegenerative disorders. The identification of the prion protein and its key role in the pathogenesis of this diverse group of diseases has allowed a fuller understanding of factors that influence disease phenotype. In particular, the naturally occurring polymorphism at codon 129 in the prion protein gene has a major influence on the disease phenotype in sporadic, familial and acquired prion diseases, although the underlying mechanisms remain unclear. Recent technical advances have improved our ability to study the isoforms of the abnormal prion protein in the brain and in other tissues. This has lead to the concept of molecular strain typing, in which different isoforms of the prion protein are proposed to correspond to individual strains of the transmissible agent, each with specific biological properties. In sporadic Creutzfeldt-Jakob disease there are at least six major combinations of codon 129 genotype and prion protein isotype, which appear to relate to distinctive clinical subgroups of this disease. However, these relationships are proving to be more complex than first considered, particularly in cases with more than a single prion protein isotype in the brain. Further work is required to clarify these relationships and to explain the mechanism of neuropathological targeting of specific brain regions, which accounts for the diversity of clinical features within human prion diseases.

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.

BACKGROUND: A highly sensitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) and flow cytometry techniques have previously been developed and employed to characterize soluble cellular prion protein (PrPc) expression in whole blood and separated components from healthy adult blood donors. No previous studies with these techniques have evaluated the concentration and expression of PrP in the blood of patients with variant Creutzfeldt-Jakob disease (vCJD). STUDY DESIGN AND METHODS: For blood from vCJD patients, sporadic CJD (sCJD) patients, non-CJD neurological controls, and healthy adults, PrPc was measured by DELFIA and cell-associated PrP was measured by flow cytometry. RESULTS: DELFIA analysis identified a significant reduction in the concentration of PrPc in the whole blood of vCJD (p = 0.012) and non-CJD neurological patients (p = 0.0004) compared with healthy adults. A significant elevation was found in plasma PrPc in sCJD patients compared with healthy adult (p = 0.022) and neurological controls (p = 0.050). Flow cytometry found no significant differences between groups in expression of PrP on platelets and lymphocytes, nor in sensitivity of cellular PrP to proteinase K. Neurological controls show significantly less PrP on red cells than healthy adults. CONCLUSION: There are differences in free and cell-associated PrP found in blood of CJD patients and control groups, some of which might be useful with other tests in disease profiling as an aid to diagnoses.

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.

The function of the normal conformational isoform of prion protein, PrP(C), remains unclear although lines of research have suggested a role in the cellular response to oxidative stress. Here we investigate the expression of PrP(C) in hypoxic brain tissues to examine whether PrP(C) is in part regulated by neuronal stress. Cases of adult cerebral ischemia and perinatal hypoxic-ischemic injury in humans were compared with control tissues. PrP(C) immunoreactivity accumulates within neuronal processes in the penumbra of hypoxic damage in adult brain, and within neuronal soma in cases of perinatal hypoxic-ischemic injury, and in situ hybridization analysis suggests an up-regulation of PrP mRNA during hypoxia. Rodents also showed an accumulation of PrP(C) in neuronal soma within the penumbra of ischemic lesions. Furthermore, the infarct size in PrP-null mice was significantly greater than in the wild type, supporting the proposed role for PrP(C) in the neuroprotective adaptive cellular response to hypoxic injury.

Objective To study the distribution of disease-associated prion protein (PrP) in oral and dental tissues in variant CJD. Design Prospective single centre autopsy based study. Setting Within the National CJD Surveillance Unit, UK, 2000-2002. Materials and methods Patients with suspected variant CJD undergoing autopsy where permission to remove tissues for research purposes had been obtained from the relatives. Fixed and frozen autopsy tissues from the brain, trigeminal ganglion, alveolar nerve, dental pulp, gingiva, salivary gland, tongue and tonsils were studied by Western blot, PET blot and immunocytochemistry to detect disease-associated PrP. Results Disease-associated PrP was only detected in the brain, trigeminal ganglia and tonsils. Conclusions The failure to detect disease-associated PrP in most dental and oral tissues will help inform ongoing risk assessments for dental surgery in relation to the possible iatrogenic transmission of variant CJD via dental instruments.

Conformational conversion of proteins in disease is likely to be accompanied by molecular surface exposure of previously sequestered amino-acid side chains. We found that induction of beta-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine. Antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize the pathological isoform of the prion protein but not the normal cellular isoform, as assessed by immunoprecipitation, plate capture immunoassay and flow cytometry. Antibody binding to the pathological epitope is saturable and specific, and can be created in vitro by partial denaturation of normal brain prion protein. Conformation-selective exposure of Tyr-Tyr-Arg provides a probe for the distribution and structure of pathologically misfolded prion protein, and may lead to new diagnostics and therapeutics for prion diseases.

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.

Prion diseases are characterized by the presence of the abnormal prion protein PrP Sc , which is believed to be generated by the conversion of the α-helical structure that predominates in the normal PrP isoform into a β-sheet structure resistant to proteinase K (PK). In human prion diseases, two major types of PrP Sc , type 1 and 2, can be distinguished based on the difference in electrophoretic migration of the PK-resistant core fragment. In this study, protein sequencing was used to identify the PK cleavage sites of PrP Sc in 36 cases of prion diseases. We demonstrated two primary cleavage sites at residue 82 and residue 97 for type 1 and type 2 PrP Sc , respectively, and numerous secondary cleavages distributed along the region spanning residues 74–102. Accordingly, we identify three regions in PrP Sc : one N-terminal (residues 23–73) that is invariably PK-sensitive, one C-terminal (residues 103–231) that is invariably PK-resistant, and a third variable region (residues 74–102) where the site of the PK cleavage, likely reflecting the extent of the β-sheet structure, varies mostly as a function of the PrP genotype at codon 129.

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 …

Between 1970 and 2003, seven cases of human dura mater-associated Creutzfeldt-Jakob disease (CJD) were identified in the UK. Furthermore, we identified a case of CJD in a porcine dura graft recipient. The mean incubation period of the human dura mater cases was 93 (range 45-177) months. The clinico-pathological features of the cases are described and compared with cases previously reported in the world literature.

Variably protease-sensitive prionopathy is a newly described human prion disease of unknown aetiology lying out with the hitherto recognized phenotypic spectrum of Creutzfeldt-Jakob disease. Two cases that conform to the variably protease-sensitive prionopathy phenotype have been identified prospectively in the U.K. since the first description of the condition in 2008 in the U.S.A. To determine the incidence and phenotype of variably protease-sensitive prionopathy within a single well-defined cohort, we have conducted a retrospective review of patients referred to the National Creutzfeldt-Jakob Disease Research & Surveillance Unit during the period 1991-2008. The approach taken was to screen frozen brain tissue by western blotting for the form of protease-resistant prion protein that characterizes variably protease-sensitive prionopathy, followed by neuropathological and clinical review of candidate cases. Cases diagnosed as sporadic Creutzfeldt-Jakob disease with atypical neuropathology were also reviewed. Four hundred and sixty-five cases were screened biochemically, yielding four candidate cases of variably protease-sensitive prionopathy. One was discounted on pathological and clinical grounds, and one was a known case of variably protease-sensitive prionopathy previously reported, leaving two new cases, which were confirmed biochemically and neuropathologically as variably protease-sensitive prionopathy. A third new case that lacked frozen tissue was recognized retrospectively on neuropathological grounds alone. This means that five cases of variably protease-sensitive prionopathy have been identified (prospectively and retrospectively) during the surveillance period 1991-2011 in the U.K. Assuming ascertainment levels equivalent to that of other human prion diseases, these data indicate that variably protease-sensitive prionopathy is a rare phenotype within human prion diseases, which are themselves rare. Biochemical investigation indicates that the abnormal protease-resistant prion protein fragment that characterizes variably protease-sensitive prionopathy is detectable at low levels in some cases of sporadic Creutzfeldt-Jakob disease and conversely, that the form of abnormal prion protein that characterizes sporadic Creutzfeldt-Jakob disease can be found in certain brain regions of cases of variably protease-sensitive prionopathy, indicating molecular overlaps between these two disorders.

Objective:Current cerebrospinal fluid (CSF) tests for sporadic Creutzfeldt–Jakob disease (sCJD) are based on the detection of surrogate markers of neuronal damage such as CSF 14-3-3, which are not specific for sCJD. A number of prion protein conversion assays have been developed, including real time quaking-induced conversion (RT-QuIC). The objective of this study is to investigate whether CSF RT-QuIC analysis could be used as a diagnostic test in sCJD.Methods:An exploratory study was undertaken that analyzed 108 CSF samples from patients with neuropathologically confirmed sCJD or from control patients. Of the 108 CSF samples, 56 were from sCJD patients (30 female, 26 male; aged 31–84 years; mean age, 62.3 ± 13.5 years), and 52 were from control patients (26 female, 26 male; aged 43–84 years; mean age, 67.8 ± 10.4 years). A confirmatory group of 118 patients was subsequently examined that consisted of 67 cases of neuropathologically confirmed sCJD (33 female, 34 male; aged 39–82 years; mean age, 67.5 ± 9.0 years) and 51 control cases (26 female, 25 male; aged 36–87 years; mean age, 63.5 ± 11.6 years).Results:The exploratory study showed that RT-QuIC analysis had a sensitivity of 91% and a specificity of 98% for the diagnosis of sCJD. These results were confirmed in the confirmatory study, which showed that CSF RT-QuIC analysis had a sensitivity and specificity of 87% and 100%, respectively.Interpretation:This study shows that CSF RT-QuIC analysis has the potential to be a more specific diagnostic test for sCJD than current CSF tests. ANN NEUROL 2012;72:278–285.

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.

Aims: The panencephalopathic type of Creutzfeldt-Jakob disease (PECJD) has extensive abnormalities in cerebral white matter as well as the cortex. PECJD has rarely been described in Caucasians and debate continues on its classification and pathogenesis. We describe our experience of PECJD over a 14-year period of surveillance for CJD in the Netherlands and the UK. Methods: Between 1993 and 2006, nine cases of PECJD were identified. Clinical, histological and biochemical characteristics of all patients were analysed and compared; all cases were classified clinically as sporadic CJD. Results: The median age at onset was 57.8 years and median disease duration was 22 months. The average brain weight was 887 g. Most patients showed a two-stage clinical course with initial rapid deterioration to a state of akinetic mutism, which then persisted over a longer time scale. Neuropathological findings were characterized by severe global atrophy with status spongiosus. Cerebral white matter involvement tended to be associated with either disease duration or severity of cerebral cortical lesions. Five patients could be classified into the MM1 subtype of sporadic CJD, one patient into the MM2 subgroup and another into the MV2 subgroup. Two patients were heterozygous at codon 129 in the prion protein gene and contained both type 1 and type 2 PrPres isoforms in the brain. Conclusions: We believe that white matter pathology in PECJD represents an end-stage pattern that reflects secondary degeneration due to widespread cortical neuronal loss that occurs in the early part of the disease, rather than representing a primary lesion.

Objective: Variant Creutzfeldt-Jakob disease (vCJD) was first reported in the United Kingdom in 1996. Since then, the majority of cases have been observed in the United Kingdom where there was a major epidemic of bovine spongiform encephalopathy. France was the second country affected. To address the hypothesis of the involvement of a common strain of agent, we have compared clinical, neuropathological, and biochemical data on vCJD patients from both countries. Methods: In France and the United Kingdom, epidemiological and clinical data were obtained from analysis of medical records and direct interview of the family of the patients using the same standardized questionnaire in both countries. When brain material was available, we performed with similar methods a comparative Study of brain lesions and PrPres glycoform ratios in both vCJD populations. Results: Clinical data, genetic background, neuropathological finding, and biochemical findings in the 185 patients observed in France (n = 23) and the United Kingdom (n = 162) were similar except for age at death. Currently, blood transfusion is a risk factor identified only in the United Kingdom. Interpretation: The close similarity between the cases of vCJD in France and the United Kingdom supports the hypothesis that a common strain of infectious agent is involved in both countries. The 5-year delay in the peak that we observed in France compared with the United Kingdom fits well with the increase in the importation of beef products to France from the United Kingdom between 1985 and 1995.

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.

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.

Background: One definite, one probable and several possible transmissions of sporadic Creutzfeldt–Jakob disease (sCJD) have followed corneal transplantation. We report an incident in the UK in 1997 in which both corneas and scleras from a donor, subsequently confirmed to have had sCJD, were transplanted. The final clinical outcome for two surviving recipients is still not yet known. Case report: In 1997, a 56-year-old woman died from biopsy-proven carcinoma of the bronchus. Both eyes were donated for transplantation. Shortly before she died, she had developed neurological symptoms thought to be due to brain metastases. However, the final result of a neurological post-mortem examination revealed evidence of sCJD. By this time the corneas had been transplanted, one 3 months previously into a 40-year-old man for keratoconus and the other 4 months previously into an 85-year-old woman for Fuchs’ dystrophy. In addition, both scleras had been transplanted into a 36-year-old man undergoing oculoplastic reconstructive surgery. The surgeons and patients were informed and removal of tissue was advised but undertaken in only two of the patients. Immunohistochemistry failed to demonstrate the presence of the abnormal form of the prion protein in explanted tissue. Conclusions: Eight years after the event, two patients remain free of symptoms suggestive of iatrogenic CJD (http://www.cjd.ed.ac.uk/criteria.htm). The third having died aged 92 years, some 7 years after surgery, showing signs of dementia not considered indicative of iatrogenic CJD. Nevertheless this adverse incident attracted substantial publicity. Coupled with continuing concerns in the UK about person-to-person transmission of variant CJD, this has lead to a number of important consequences in donor eye retrieval, ocular tissue banking and transplantation.

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.