Enhanced Creutzfeldt-Jakob disease surveillance in the older population: Assessment of a protocol for screening brain tissue donations for prion disease.

Human prion diseases, including Creutzfeldt-Jakob disease (CJD), occur in sporadic, genetic, and acquired forms. Variant Creutzfeldt-Jakob disease (vCJD) first reported in 1996 in the United Kingdom (UK), resulted from contamination of food with bovine spongiform encephalopathy. There is a concern that UK national surveillance mechanisms might miss some CJD cases (including vCJD), particularly in the older population where other neurodegenerative disorders are more prevalent. We developed a highly sensitive protocol for analysing autopsy brain tissue for the misfolded prion protein (PrPSc ) associated with prion disease, which could be used to screen for prion disease in the elderly. Brain tissue samples from 331 donors to the Edinburgh Brain and Tissue Bank (EBTB), from 2005 to 2022, were analysed, using immunohistochemical analysis on fixed tissue, and five biochemical tests on frozen specimens from six brain regions, based on different principles for detecting PrPSc . An algorithm was established for classifying the biochemical results. To test the effectiveness of the protocol, several neuropathologically confirmed prion disease controls, including vCJD, were included and blinded in the study cohort. On unblinding, all the positive control cases had been correctly identified. No other cases tested positive; our analysis uncovered no overlooked prion disease cases. Our algorithm for classifying cases was effective for handling anomalous biochemical results. An overall analysis suggested that a reduced biochemical protocol employing only three of the five tests on only two brain tissue regions gave sufficient sensitivity and specificity. We conclude that this protocol may be useful as a UK-wide screening programme for human prion disease in selected brains from autopsies in the elderly. Further improvements to the protocol were suggested by enhancements of the in vitro conversion assays made during the course of this study.

Variably protease-sensitive prionopathy mimicking frontotemporal dementia.

Sporadic prion diseases are fatal neurodegenerative disorders characterized clinically by rapidly progressive dementia and myoclonus. Variably protease-sensitive prionopathy (VPSPr) is a recently identified sporadic human prion disorder that may present with a lengthy atypical clinical history. Here, we describe a case of VPSPr in a patient with a long history of suspected frontotemporal dementia (FTD). A 61-year-old man presented with speech difficulties, including naming objects and constructing multipart sentences, while there was no difficulty in comprehension. Movement abnormalities included slightly jerky pursuit, minor dysmetria of saccades and brisk reflexes. There was no family history of dementia. Later he developed swallowing difficulties and the possibility of FTD with motor neuron disease was suspected. He died at the age of 71 and his brain was donated to the London Neurodegenerative Diseases Brain Bank. The brain (1004 g) showed mild to moderate atrophy, predominantly in the frontal lobe. Histology revealed moderate spongiform microvacuolation mostly affecting the frontal and parietal cortices, but also present focally in the basal ganglia and the cerebellum. Only mild Alzheimer pathology was found by extensive immunohistochemistry, in keeping with BrainNet Europe stage II. Trans-activation response DNA-binding protein 43 kDa and α-synuclein immunostains were negative. Immunostaining for prion protein (PrP) showed granular/synaptic positivity in a patchy distribution, mainly within the deeper cortex, and also revealed microplaques in the cerebellum and basal ganglia. Western blotting confirmed a low molecular weight protease-resistant PrP band with a faint ladder-like pattern in the absence of types 1 and 2 isoforms. These features are diagnostic of VPSPr. VPSPr can mimic various neurodegenerative conditions; diagnosis requires both PrP immunohistochemistry and Western blotting. The presence of patchy spongiform change in the absence of other neurodegenerative pathology should raise suspicion of VPSPr, even in elderly patients with a lengthy clinical history.

Amyloid-ß accumulation in the CNS in human growth hormone recipients in the UK.

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 (Aß) 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. Aß 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 Aß, 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 Aß 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 Aß and give insights into the possibility of iatrogenic transmission of AD and CAA.

UK Iatrogenic Creutzfeldt-Jakob disease: investigating human prion transmission across genotypic barriers using human tissue-based and molecular approaches.

Creutzfeldt-Jakob disease (CJD) is the prototypic human prion disease that occurs most commonly in sporadic and genetic forms, but it is also transmissible and can be acquired through medical procedures, resulting in iatrogenic CJD (iCJD). The largest numbers of iCJD cases that have occurred worldwide have resulted from contaminated cadaveric pituitary-derived human growth hormone (hGH) and its use to treat primary and secondary growth hormone deficiency. We report a comprehensive, tissue-based and molecular genetic analysis of the largest series of UK hGH-iCJD cases reported to date, including in vitro kinetic molecular modelling of genotypic factors influencing prion transmission. The results show the interplay of prion strain and host genotype in governing the molecular, pathological and temporal characteristics of the UK hGH-iCJD epidemic and provide insights into the adaptive mechanisms involved when prions cross genotypic barriers. We conclude that all of the available evidence is consistent with the hypothesis that the UK hGH-iCJD epidemic resulted from transmission of the V2 human prion strain, which is associated with the second most common form of sporadic CJD.

Rapidly progressive dementia with thalamic degeneration and peculiar cortical prion protein immunoreactivity, but absence of proteinase K resistant PrP: a new disease entity?

BACKGROUND: Human prion diseases are a group of rare fatal neurodegenerative conditions with well-developed clinical and neuropathological diagnostic criteria. Recent observations have expanded the spectrum of prion diseases beyond the classically recognized forms. RESULTS: In the present study we report six patients with a novel, apparently sporadic disease characterised by thalamic degeneration and rapidly progressive dementia (duration of illness 2-12 months; age at death: 55-81 years). Light and electron microscopic immunostaining for the prion protein (PrP) revealed a peculiar intraneuritic distribution in neocortical regions. Proteinase K resistant PrP (PrPres) was undetectable by Western blotting in frontal cortex from the three cases with frozen tissue, even after enrichment for PrPres by centrifugation or by phosphotungstic acid precipitation. Conformation-dependent immunoassay analysis using a range of PK digestion conditions (and no PK digestion) produced only very limited evidence of meaningful D-N (denatured/native) values, indicative of the presence of disease-associated PrP (PrPSc) in these cases, when the results were compared with appropriate negative control groups. CONCLUSIONS: Our observation expands the spectrum of conditions associated with rapidly progressive dementia and may have implications for the understanding of the pathogenesis of prion diseases.

Variably protease-sensitive prionopathy in the UK: a retrospective review 1991-2008.

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.

A novel seven-octapeptide repeat insertion in the prion protein gene (PRNP) in a Dutch pedigree with Gerstmann-Sträussler-Scheinker disease phenotype: comparison with similar cases from the literature.

Human prion diseases can be sporadic, inherited or acquired by infection and show considerable phenotypic heterogeneity. We describe the clinical, histopathological and pathological prion protein (PrP(Sc)) characteristics of a Dutch family with a novel 7-octapeptide repeat insertion (7-OPRI) in PRNP, the gene encoding the prion protein (PrP). Clinical features were available in four, neuropathological features in three and biochemical characteristics in two members of this family. The clinical phenotype was characterized by slowly progressive cognitive decline, personality change, lethargy, depression with anxiety and panic attacks, apraxia and a hypokinetic-rigid syndrome. Neuropathological findings consisted of numerous multi- and unicentric amyloid plaques throughout the cerebrum and cerebellum with varying degrees of spongiform degeneration. Genetic and molecular studies were performed in two male family members. One of them was homozygous for valine and the other heterozygous for methionine and valine at codon 129 of PRNP. Sequence analysis identified a novel 168 bp insertion [R2-R2-R2-R2-R3g-R2-R2] in the octapeptide repeat region of PRNP. Both patients carried the mutation on the allele with valine at codon 129. Western blot analysis showed type 1 PrP(Sc) in both patients and detected a smaller ~8 kDa PrP(Sc) fragment in the cerebellum in one patient. The features of this Dutch kindred define an unusual neuropathological phenotype and a novel PRNP haplotype among the previously documented 7-OPRI mutations, further expanding the spectrum of genotype-phenotype correlations in inherited prion diseases.

Further characterisation of the prion protein molecular types detectable in the NIBSC Creutzfeldt-Jakob disease brain reference materials.

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 (PrP(Sc)) types. The results confirm the presence of genuine type 1 and type 2 protease-resistant PrP (PrP(res)) in each of the three sporadic Creutzfeldt-Jakob disease reagents, and provide evidence supporting the lower level presence of type 1 PrP(res) in the variant Creutzfeldt-Jakob disease reagents. We conclude that these reagents provide a valuable resource for future research and development.

Human platelets as a substrate source for the in vitro amplification of the abnormal prion protein (PrP) associated with variant Creutzfeldt-Jakob disease.

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 (PrP(Sc)) to readily detectable levels, could be incorporated into such a test provided that a suitable substrate source for routine use in human PMCA reactions can be found. 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 PrP(Sc) 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 PrP(Sc) detection sensitivity was achieved after each round of sPMCA, resulting in a 10,000-fold increase in detection sensitivity after four rounds, with no evidence of de novo PrP(Sc) production detected in the unseeded PLT substrate. 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.

Detection of type 1 prion protein in variant Creutzfeldt-Jakob disease.

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.

Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7.

Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.

Heterogeneous surface expression of EspA translocon filaments by Escherichia coli O157:H7 is controlled at the posttranscriptional level.

Type III secretion systems of enteric bacteria enable translocation of effector proteins into host cells. Secreted proteins of verotoxigenic Escherichia coli O157 strains include components of a translocation apparatus, EspA, -B, and -D, as well as "effectors" such as the translocated intimin receptor (Tir) and the mitochondrion-associated protein (Map). This research has investigated the regulation of LEE4 translocon proteins, in particular EspA. EspA filaments could not be detected on the bacterial cell surface when E. coli O157:H7 was cultured in M9 minimal medium but were expressed from only a proportion of the bacterial population when cultured in minimal essential medium modified with 25 mM HEPES. The highest proportions of EspA-filamented bacteria were detected in late exponential phase, after which filaments were lost rapidly from the bacterial cell surface. Our previous research had shown that human and bovine E. coli O157:H7 strains exhibit marked differences in EspD secretion levels. Here it is demonstrated that the proportion of the bacterial population expressing EspA filaments was associated with the level of EspD secretion. The ability of individual bacteria to express EspA filaments was not controlled at the level of LEE1-4 operon transcription, as demonstrated by using both beta-galactosidase and green fluorescent protein (GFP) promoter fusions. All bacteria, whether expressing EspA filaments or not, showed equivalent levels of GFP expression when LEE1-4 translational fusions were used. Despite this, the LEE4-espADB mRNA was more abundant from populations with a high proportion of nonsecreting bacteria (low secretors) than from populations with a high proportion of secreting and therefore filamented bacteria (high secretors). This research demonstrates that while specific environmental conditions are required to induce LEE1-4 expression, a further checkpoint exists before EspA filaments are produced on the bacterial surface and secretion of effector proteins occurs. This checkpoint in E. coli O157:H7 translocon expression is controlled by a posttranscriptional mechanism acting on LEE4-espADB mRNA. The heterogeneity in EspA filamentation could arise from phase-variable expression of regulators that control this posttranscriptional mechanism.