Monday, August 21, 2017

Protective Effect of Val129-PrP against Bovine Spongiform Encephalopathy but not Variant Creutzfeldt-Jakob Disease

Protective Effect of Val129-PrP against Bovine Spongiform Encephalopathy but not Variant Creutzfeldt-Jakob Disease


Natalia Fernández-Borges,1 Juan Carlos Espinosa,1 Alba Marín-Moreno, Patricia Aguilar-Calvo, Emmanuel A. Asante, Tetsuyuki Kitamoto, Shirou Mohri, Olivier Andréoletti, Juan María Torres


Bovine spongiform encephalopathy (BSE) is the only known zoonotic prion that causes variant Creutzfeldt-Jakob disease (vCJD) in humans. The major risk determinant for this disease is the polymorphic codon 129 of the human prion protein (HuPrP), where either methionine (Met129) or valine (Val129) can be encoded. To date, all clinical and neuropathologically confirmed vCJD cases have been Met129 homozygous, with the exception of 1 recently reported Met/Val heterozygous case. Here, we found that transgenic mice homozygous for Val129 Hu-PrP show severely restricted propagation of the BSE prion strain, but this constraint can be partially overcome by adaptation of the BSE agent to the Met129 Hu-PrP. In addition, the transmission of vCJD to transgenic mice homozygous for Val129 Hu-PrP resulted in a prion with distinct strain features. These observations may indicate increased risk for vCJD secondary transmission in Val129 Hu-PrP–positive humans with the emergence of new strain features. 


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Discussion


We report a detailed comparison of the transmission properties of BSE and vCJD prions among humanized transgenic mice with different PRNP codon 129 genotypes. Because a high expression level of PrP in transgenic mice directly influences prion disease susceptibility and incubation time, these transgenic mice have an advantage over knock-in mice for evaluating these features in the different human PrP genotypes. In addition, the 3 mouse models used in our study have equivalent PrP expression levels, making them suitable for studying comparative susceptibilities across the different PRNP codon 129 genotypes.


In previous reports, we demonstrated that Met129 homozygous individuals might be susceptible to a sheep or goat BSE agent to a higher degree than to cattle BSE and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD (31). In this study, we wanted to extend these results to the other human PRNP genotypes: Met/Val129 and Val/ Val129. We gained a different perspective when several BSE isolates adapted to different species inoculated in TgVal129 mice showed an apparent lack of transmission. In addition, almost all inoculated TgMet/Val129 mice did not transmit BSE; this finding supports the interpretation by Wadsworth et al. that human PrP Val129 severely restricts propagation of the BSE prion strain (27).


An unexpected result of this study was the finding that 1 BSE isolate from a goat (Ca-BSE/Go) was clinically transmitted to 1 of 10 TgMet/Val129 mice and subclinically transmitted to TgVal129 mice. This particular isolate is characterized by a high infectious titer (35) that could explain the potential for this inoculum to overcome the restriction on BSE prions to propagate in TgVal129 mice.


Although cattle BSE did not transmit to TgMet/Val129 mice directly, adaptation of the BSE agent to human PrP Met129 sequence and subsequent inoculation of the resultant vCJD prions to TgMet/Val129 mice produced a 100% attack rate. However, we did not detect clinical prion disease, supporting a slower rate of vCJD conversion compared with that among TgMet129 mice. This slow but potential conversion rate in TgMet/Val129 mice correlates well with the single vCJD case of a human carrying the PrP Met/Val129 genotype (22) and with the description of subclinical secondary transmissions through human vCJD–infected tissues (4–7,47). 


TgVal129 mice challenged with Hu-vCJD2 did not produce detectable brain PrPres and clinical signs, in spite of the overexpression of HuPrP-Val129 and the use of the more efficient intracerebral route of infection. However, subclinical infection in these TgVal129 mice was demonstrated in BoPrP-Tg110 mice. These data suggest that PrP Val129 could sustain a very slow and limited vCJD conversion rate that is consistent with the detection of PrPres in tonsils and appendixes of asymptomatic PrP Val129 persons (23–25). Previous studies of other transgenic mice expressing PrP Val129 have also shown a low transmission efficiency of vCJD (2,27,30).


The fluctuating subclinical transmissibility of both vCJD inocula in TgVal129 mice (negative for Hu-vCJD1 and positive for Hu-vCJD2 ) might be caused by differences in prion titer between inocula. This assessment was strengthened after the transmission of both vCJDs to TgMet129 mice, in which a shorter incubation period was observed in animals inoculated with Hu-vCJD2 . A certain variability in subclinical transmissibility and incubation time between different vCJD isolates is not uncommon, as has been previously reported (2,27,30), suggesting that a Val129 transmission barrier can only be overcome with highly infectious vCJD isolates.


The dramatic changes in the susceptibility of TgVal129 mice (Table 3) challenged with vCJD isolates first passaged in TgMet129 mice suggest an apparent increase in titer of both vCJD prion isolates; however, adaptation of the inocula to the new host mouse cannot be disregarded as being partly responsible for this increased susceptibility. We observed a 100% infection rate, but without clinical signs of prion disease. We observed similar transmission features when we passaged vCJD in TgMet/Val129 mice. In addition, the apparent PrPVal129 restricted propagation of cattle BSE and BSE from other species was completely abolished after its adaptation to human PrPMet129.


Although PrP overexpression and the inoculation route can affect transmission efficiency, our results and those previously reported in both overexpressing and knock-in transgenic mice (2,27,30) suggest that the Val129 PrP variant could sustain a very slow and limited vCJD conversion rate, and is unable to completely prevent vCJD transmission. Biochemical and neuropathological features of vCJD transmission to TgVal129 mice showed substantial differences compared to TgMet129 or TgMet/ Val129 mice. Similar to previous reports (2,27,28,48), a type 5 PrPSc associated with very weak and diffuse PrP plaques without a florid morphology was the hallmark among these mice. In addition, our demonstration of previously unreported type 5 PrPSc in brain samples of vCJDchallenged knock-in Ki-Hu129V/V mice (30) establishes that the evolution of type 5 PrPSc associated with the transmission of vCJD prions to the Val129 genotype is not an artifact of PrP overexpression. This finding further reinforces the specific biochemical features of vCJD when transmitted to the human-PrP Val129 sequence.


Extrapolation of results from prion transmission studies based on transgenic mice has to be done with caution, especially when human susceptibility to prions is analyzed. However, our results clearly indicate that PrPVal129 individuals are highly resistant to transmission of cattle BSE or BSE passaged in other species. Also, PrPVal129 individuals might be susceptible to infection with human-passaged BSE (vCJD) prions, and the propagated agents might transmit with molecular and neuropathological properties distinguishable from those of type 4 PrPres. Although the resultant type 5 PrPSc shares the same fragment sizes as those of type 2 PrPSc, the 2 PrPSc types can be distinguished by the predominance of the diglycosylated glycoform associated with type 5 PrPSc. Overall, our results indicate that human Val129-PrP polymorphic variant is a strong molecular protector against BSE zoonotic transmission but fails to prevent human-to-human vCJD transmission. Because potential late-onset vCJD cases could appear in the population (49,50) these findings underline the need for continued investigation of all forms of human prion disease.




''Overall, our results indicate that human Val129-PrP polymorphic variant is a strong molecular protector against BSE zoonotic transmission but fails to prevent human-to-human vCJD transmission. Because potential late-onset vCJD cases could appear in the population (49,50) these findings underline the need for continued investigation of all forms of human prion disease.''


THURSDAY, AUGUST 17, 2017 

JAVMA NEWS Atypical BSE found in Alabama cow September 01, 2017



MONDAY, AUGUST 21, 2017

Chronic Wasting Disease Prion Strain Emergence and Host Range Expansion



THURSDAY, AUGUST 17, 2017 

Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob disease in the United States revisited 2017




Terry S. Singeltary Sr.

Thursday, July 6, 2017

PRION 2017 CONFERENCE ABSTRACTS HUMAN TSE PRION DISEASE

Subject: PRION 2017 CONFERENCE ABSTRACTS HUMAN TSE PRION DISEASE


P26 Familial prion diseases evaluated at the US National Prion Disease Pathology Surveillance Center, 2005-2015

Dr. Ryan Maddox1, Ms. Elizabeth Harker1, Ms. Janis Blevins2, Dr. Jiri Safar2, Dr. Lawrence Schonberger1, Dr. Ermias Belay1

1Centers for Disease Control and Prevention (CDC), Atlanta, United States, 2National Prion Disease Pathology Surveillance Center (NPDPSC), Cleveland, United States

Aim: To describe characteristics of familial prion disease cases evaluated at the National Prion Disease Pathology Surveillance Center (NPDPSC).

Methods: Specimens from suspected prion disease cases from across the United States are submitted to NPDPSC for detailed diagnostic neuropathology, molecular classification of prions with Western blots, and PRNP gene sequencing. Cases are classified as having familial prion disease (familial CJD, Gerstmann-Straüssler-Scheinker syndrome (GSS), or fatal familial insomnia (FFI)) when a pathogenic mutation is identified or when other test results indicate prion disease in conjunction with a positive family history. NPDPSC data for 2005-2015 were analyzed for frequency and subtype distribution, and when available, medical records for familial prion disease cases <55 years of age were also reviewed.

Results: Out of 2309 neuropathology-confirmed prion disease cases submitted to NPDPSC with genetic test results available, 242 (10.5%) were classified as having a familial prion disease; an additional 16 familial cases were identified through blood analysis with no neuropathology (n=14) or through a family history for the disease (n=2). Familial CJD was found in 219 (85%) of the 258 total familial prion disease cases; 23 (9%) had GSS, and the remaining 16 (6%) had FFI. Over half of the familial CJD cases (117, 53%) had the E200K mutation, followed by V210I (26, 12%), D178N (12, 5%), and T188R (12, 5%). The mean duration of illness for cases with E200K and V210I mutations was under 6 months, while cases with D178N and T188R mutations had mean durations exceeding a year. Six mutations were identified with a diagnosis of GSS, with P102L accounting for almost half of the cases.

Conclusions: These data indicate that genetic prion disease accounts for approximately 11% of human prion disease cases in the United States. The category of familial prion diseases is remarkably heterogeneous, with broad frequency distribution of more than 25 pathogenic mutations identified among cases referred to NPDPSC during the 11-year period.

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Stem cell transplantation in the investigation and treatment of genetic Prion Diseases

Miss Kati Frid1, Miss Orli Binyamin1, Mr Guy Keller1, Prof Tamir Ben Hur1, Prof Ruth Gabizon1
1 Hadassah University Hospital, Jerusalem, Israel , Jerusalem, Israel

Aims: Recent studies have shown that transplantation of neural progenitor cell (NPCs) or mesenchymal stem cells (MSCs) to mice models of neurodegenerative diseases induce an immunomodulatory and neuroprotective affect which results in milder disease presentation. In this project, we plan to look whether NPCs transplantation into TgMHu2ME199K mice, a model for genetic prion disease, can delay disease onset. In parallel we will test whether NPCs from wt mice can be infected with prions if transplanted into TgMHu2ME199K brains and also establish if transplantation of TgMHu2ME199K mice NPCs into wt mice can transmit prion disease.

Methods: New born TgMHu2ME199K mice and wt mice were transplanted with embryonic NPCs from wt or TgMHu2ME199K mice. Mice from all groups were followed up until the age of 10 months for disease signs and subsequently sacrificed. Brain slices were immunostained for Nestin and disease related PrP in several areas and in particular in endogenous and transplanted NPCs.

Results: We show that transplantation of NPCs from either wt or TgMHu2ME199K embryos into 2 days old TgMHu2ME199K mice significantly delayed the progression of prion disease. Pathological examinations indicates that delay of disease aggravation was consistent with increased numbers of Nestin positive cells in sub ventricular zone (SVZ) and reduced neuronal death in hippocampus, but independent from levels of disease related PrP, indicating a non-prion related neuroprotective effect. In fact, there was no co-localization between Nestin and disease related PrP in both transplanted and endogenous NPCs, even 10 months after transplantation. In addition, we found no disease related PrP in wt NPCs or wt brains, indicating there were no infection from TgMHu2ME199K NPCs to wt mice.

Conclusions: We conclude that transplantation of NPCs may be considered an optional treatment in the prevention of genetic CJD and other neurodegenerative conditions. In addition, these experiments may elucidate the mechanism of de-novo generation of prion infectivity in genetically affected CJD patients.

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Widening the phenotypic spectrum of sporadic fatal insomnia: clinical findings, results of diagnostic investigations and neuropathological features of new cases.

Dr. Samir Abu Rumeileh1, Dr. Maria Chiara Casadio1, Prof. Pietro Cortelli1,2, Prof. Annemieke J.M. Rozemuller3, Prof. Sandro Sorbi4, Dr. Giorgio Giaccone5, Dr. Sabina Capellari1,2, Prof. Piero Parchi1,2
1Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy, 2IRCCS, Institute of Neurological Sciences of Bologna, Bologna, Italy, 3University Medical Center Utrecht, Utrecht, the Netherlands, 4Department of Neuroscience, Psychology, Drug Research and Child Health, University of Florence, Florence, Italy, 5IRCCS, Foundation Carlo Besta Neurological Institute, Milan, Italy

Aims: Fatal insomnia (FI) is a rare prion disease comprising both a genetic (FFI) and a sporadic form (sFI). At variance with FFI, whose distinctive phenotype has been reported in several affected members belonging to more than 30 families worldwide, only very few well-characterized cases of sFI are on record to date. Here we describe the phenotypic features of a series of new cases of sFI.

Methods: Four patients who received a definite diagnosis of sFI at death were investigated using an extensive protocol which included neurological evaluation, CSF biomarkers and genetic analyses, and neurophysiological and neuroimaging assessments.

Results: Mean age at onset was 41 years (range 27-55) and mean disease duration 30.5 months (range 8-54). All patients presented with visual disturbances and/or cognitive-psychiatric impairment and developed dysarthria, myoclonus, extrapyramidal signs, and akinetic mutism during clinical course. Moreover, three out of 4 also had sleep disruption, cerebellar and pyramidal signs, while autonomic hyperactivation was present in two. CSF analysis revealed a negative 14-3-3 protein assay in all patients, t-tau levels above 1250 pg/ml in one out of three patients and a positive prion RT-QuIC in one out of two. EEG periodic sharp-waves complexes were only seen in one patient. In all three patients who underwent polysomnographic evaluation or long-term EEG monitoring, there was a severe reduction of total sleep time, a poorly organized sleep and the absence of non-REM sleep figures. Brain MRI variably showed signal abnormalities in medial thalamic nuclei, inferior olives, and cerebral cortex. FDG-PET studies revealed a pronounced bilateral thalamic hypometabolism and scattered hypometabolic cortical areas in two out of three cases, while SPECT scans showed abnormal cortical perfusion in two patients. Neuropathological assessment revealed severe neuronal loss of the anterior medial thalamus and inferior olives; mild to moderate neuronal loss in the cerebellum and a variable degree of cortical spongiform changes reflecting disease duration. Western blot confirmed the presence of type 2 PrPSc, while analyses of PRNP gene revealed methionine homozygosity (MM) at codon 129 but no mutations.

Conclusions: At variance with FFI patients carrying MM at codon 129, those with sFI have cognitive and motor disturbances predominating over sleep and autonomic abnormalities and a longer disease duration. The young age at onset, the absence of pathognomonic clinical signs and the relatively low sensitivity of diagnostic investigations such as EEG, CSF biomarker assays and brain MRI makes the clinical diagnosis of sFI quite challenging.

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P145 Therapeutic effect of autologous compact bone-derived mesenchymal stem cell transplantation on prion disease

Dr Zhifu Shan1, Mr Yuji Hirai1, Mr Roy Hayashi1, Dr Takeshi Yamasaki1, Dr Rie Hasebe1, Dr Chang-Hyun Song2, Dr Motohiro Horiuchi1
1Graduate School Of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, Japan, 2College of Korean Medicine, Daegu Haany University, Gyeongsan, Republic of Korea

Aims: Prion diseases are fatal neurodegenerative disorders of humans and animals and no effective treatments are available to date. Allogenic transplantation of immortalized human mesenchymal stem cells (MSCs) can prolong the survival of mice infected with prions. However, autologous transplantation is an appropriate model for evaluating the effects of MSCs on prion diseases. Therefore, we analyzed the effect of mouse MSCs on prion-infected mice.

Methods: We isolated and purified MSCs from the femur and tibia of mice as compact bone-derived MSCs (CB-MSCs). Expressions surface markers were analyzed by flow cytometer. CB-MSCs were stereotaxically transplanted into hippocampus at 120 days post inoculation.

Results: Flow cytometric analysis showed that CB-MSCs were negative for myeloid stem cell-derived cell markers CD11b and CD45, but positive for molecules, such as Sca-1, CD105, and CD90.2, which are reported to be expressed on MSCs. The ability of CB-MSCs to migrate to brain extracts from prion-infected mice was confirmed by an in vitro migration assay. Intra-hippocampus transplantation of CB-MSCs at 120 days post inoculation marginally but significantly prolonged the survival of mice infected with the Chandler prion strain. The transplantation of CB-MSCs did not influence the accumulation of disease-specific prion protein. However, the CB-MSCs transplantation enhanced microglial activation, which appeared to be polarized to the M2-type activation state.

Conclusions: These results suggest that autologous MSC transplantation is a possible treatment for prion diseases and the modification of microglial activation may be a therapeutic target for neurodegenerative diseases.

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P85 Inherited human prion disease modelled in PrP transgenic Drosophila

Dr. Alana M. Thackray1, Ms Alzbeta Cardova1, Hanna Wolf2, Lydia Pradl2, Ina Vorberg2, Dr. Walker S. Jackson2, Dr. Raymond Bujdoso1 

1Cambridge University, Cambridge, United Kingdom, 2German Center For Neurodegenerative Disease (DZNE), Bonn, Germany

Inherited human prion diseases, such as FFI and familial CJD (fCJD), are associated with autosomal dominant mutations in the human prion protein gene PRNP characterised by the accumulation of PrPSc, an abnormal isomer of the normal host protein PrPC, in the brain of affected individuals. PrPSc is considered to be the principal component of the transmissible, neurotoxic prion agent. Mutations in PRNP may promote PrPC mis-folding, enhance mis-folded PrP to aggregate, or increase the stability of PrPSc. It is important to identify the molecular pathways and cellular processes that regulate prion formation and prion-induced neurotoxicity. This will allow identification of possible therapeutic interventions for individuals with, or at risk from, genetic human prion disease. Genetic forms of human prion diseases are difficult to study in the natural host as they are relatively rare and are characterised by a long asymptomatic phase prior to clinical disease. Attempts have been made to model inherited human prion diseases in mice transgenic for human or murine PrP carrying mutations associated with these conditions, or other modified forms of mouse PrP. Increasingly, Drosophila melanogaster has been used to model human neurodegenerative disease.

We have demonstrated that transmissible mammalian prion disease can be modelled in the fly by studies that show PrP transgenic Drosophila develop a neurotoxic phenotype after exposure to exogenous prions. The prion-induced fly phenotype was associated with Proteinase-K resistant PrPSc and was transmissible to PrP transgenic hosts, including mice (Thackray et al 2016 Biochem. J. 473(23):4399-4412). An important unanswered question is whether genetic prion disease with concomitant spontaneous formation of transmissible prions can be modelled in Drosophila.

We have generated a Drosophila model of genetic human prion disease. We used pUAST / PhiC31-mediated site-directed mutagenesis to generate Drosophila transgenic for murine or hamster PrP that carry single codon mutations associated with the genetic forms of human prion disease. Drosophila transgenic for humanised mouse or hamster PrP harbouring an FFI (D178N) or fCJD (E200K) mutation displayed a spontaneous decline in locomotor ability at adulthood that increased in severity as the flies aged. Significantly, this mutant PrP-mediated neurotoxic fly phenotype was transmissible to recipient Drosophila that expressed the wild type form of the transgene. Collectively, our novel data are indicative of spontaneous formation of a transmissible PrP-dependent moiety in FFI- or CJD-PrP transgenic Drosophila and show that inherited human prion disease can be modelled in this invertebrate host.

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P21 Clinicopathological features of Gerstmann-Sträussler-Scheinker syndrome with P105L mutation.

Fumiko Furukawa1, Tetsuyuki Kitamoto2, Yoshikazu Nakamura3, Masahito Yamada4, Tadashi Tsukamoto5, Hidehiro Mizusawa5, Takanori Yokota1, Nobuo Sanjo1

1 Department of Neurology and Neurological Science, Tokyo Medical and Dental University, Tokyo, Japan, 2Department of Prion Protein Research, Division of CJD Science and Technology, Tohoku University Graduate School of Medicine, Sendai, Japan, 3Department of Public Health, Jichi Medical University, Tochigi, Japan, 4Department of Neurology and Neurobiology of Aging, Kanazawa, Japan, 5National Center of Neurology and Psychiatry, Tokyo, Japan

Aims:
Although the common clinical features of Gerstmann-Sträussler-Scheinker syndrome (GSS) with P105L mutation in the prion protein gene (PRNP; GSS105) have been reported to be spastic paraparesis and slowly progressive dementia, the recent report by Mano et al. (2016) revealed that parkinsonism was also a noteworthy symptom in GSS105. There have been reported a few patients with this mutation and their clinical and pathological features have not been fully clarified yet. We reviewed the clinical records of the patients with GSS105, and evaluated the relationship between neuropathological findings and clinical features.

Methods:
We obtained clinical symptoms of GSS105 from the data collected by Japanese Prion Disease Surveillance between April 1999 and September 2014. In addition, we reviewed clinicopathological findings of several previous case reports of this mutation since 1993, and analyzed our recent autopsied case.

Results:
Parkinsonism was the most common initial symptom (44%), and the second was dementia (16%). In clinical features, slowly progressive dementia, pyramidal tract sign, and extrapyramidal tract signs were observed in 26/27 cases (96%), 20/26 cases (77%) and 15/19 cases (79%) of GSS105 respectively. The 12/28 cases (43%) of GSS105 had all of three features. The average number of months at the triad completed in a patient was 51 from the time at onset.
In the previous reports, pathological evaluation was performed in 7 cases out of 20 cases. Neuropathogically, there was remarkable neuronal loss and gliosis in deep layers of cerebral cortices without spongiform change, and degeneration of corticospinal tract was also observed in all cases without PrPSc deposition. Two autopsied cases with extrapyramidal sings showed degeneration of the substantia nigra and striatum, and one of these 2 cases had a few mesh and unicentric core type PrPSc deposition in substantia nigra. In 3 autopsied cases without extrapyramidal sign, 1 case showed multicentric type PrPSc deposition in striatum, and other 2 cases had single core or multicentric type PrPSc deposition in caudate and putamen. Tiny PrPSc depositions were found in the spinal posterior horns of two patients, but the corresponding clinical symptoms were not described.

Conclusions:
This study revealed slowly progressive dementia, pyramidal signs, and extrapyramidal signs can be a major clinical triad of GSS105. Although corresponding pathological features were observed in some reported cases, the area of neuronal degeneration was different from that of PrPSc deposition. The triad can become completed over 4 years in most cases from the time at onset.

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P82 Utilizing the Bank Vole Prion Protein to Characterize Mutations Associated with Gerstmann-Sträussler-Scheinker Disease

Mr Hamza Arshad1,2, Mr Matthew Bourkas1,2, Dr. Joel Watts1,2
1Department of Biochemistry, University of Toronto, Toronto , Canada, 2Tanz Centre for Research in Neurodegenerative Diseases, Toronto, Canada

Aims:
Gerstmann-Sträussler-Scheinker (GSS) disease is a familial form of prion disease that is inherited in an autosomal dominant manner. Mutations associated with GSS are scattered throughout the sequence of the PRNP gene that codes for the prion protein (PrP). With atypical molecular properties, GSS has only been efficiently propagated in bank voles, and little is known about how GSS-associated mutations lead to prion formation. Given that the bank vole prion protein (BVPrP) shows a unique promiscuity for adopting misfolded conformations, we hypothesized that it would represent an ideal substrate for studying the biochemical effects of GSS-associated mutations.

Methods:
A diverse range of GSS-causing mutations were introduced into the sequence of BVPrP, and then the mutant proteins were expressed in N2a cells in which all copies of the endogenous mouse PrP gene had been ablated using CRISPR/Cas9 (N2a-PrP-/- cells). Total PrP expression levels in cells expressing wild-type and mutant BVPrP were compared by immunoblotting and ELISA. Detergent insolubility experiments were carried out on lysates from cells stably expressing wild-type or mutant BVPrP to check for the presence of aggregated PrP. BVPrP turnover rates in stably transfected cells, used as a measure of protein stability, were determined using cycloheximide chase experiments in which residual BVPrP levels were measured at various time points following arrest of protein translation.

Results:
The presence of GSS-causing mutations in the globular domain (such as F198S and D202N) significantly decreased steady-state BVPrP levels, reduced BVPrP solubility, and increased the rate of BVPrP turnover. Octapeptide repeat insertion mutations also significantly destabilized the protein, and this effect was magnified with increasing repeat number. Other mutations located outside of the globular domain, including A117V and P102L, did not appreciably influence the solubility or turnover rate of BVPrP.

Conclusions:
GSS-causing mutations located within the globular domain of PrP destabilize the structure of PrP, resulting in increased turnover rates and lower steady-state protein expression levels. This destabilization may also increase the likelihood of protein misfolding and aggregation. However, not all GSS-associated mutations had this effect, suggesting that there are multiple molecular pathways that can lead to GSS pathogenesis.

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P88 Structural stability of a novel GSS-causing PrP mutant with a partial duplication of the hydrophobic region

Miss Ze-Lin Fu1,2, Dr. Robert C. C. Mercer1, Dr. Brian D. Sykes2, Dr. David Westaway1,2
1Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada, 2Department of Biochemistry, University of Alberta, Edmonton, Canada

Aims: We aim to understand the molecular basis of a novel prion protein (PrP) mutant that misfolds into a pathogenic form and causes Gerstmann-Straussler-Scheinker (GSS) disease. This mutant designated "HRdup" was defined in a 34-year-old GSS patient and contains an 8-amino-acid duplication (LGGLGGYV) after Val129. This allele encompasses both part of the glycine-rich portion of the hydrophobic region (HR) and all of the first beta strand and is thus distinct from other GSS mutations that comprise missense mutations or insertions of extra octarepeats. Transgenic (Tg) mice expressing HRdup PrP develop a spontaneous neurological syndrome and proteinase K digestion of brain homogenates from these mice shows a pathognomonic GSS band at ~8kDa.

Methods: For structural analysis, we purified recombinant mouse PrP with the HRdup insert (118-231) along with control mouse PrP (mPrP) alleles, M128V (118-231) and WT mPrP (118-231) These proteins were subjected to increasing concentrations of urea while monitoring the denaturation by use of 1D ¹H NMR. We plotted the integral values of five chosen resonances (I182 Hγ2, F198 Hα, Y162 Hα, Y218 Hδ, Y163 Hε) against urea concentrations and obtained the value of urea concentration where half of the proteins were denatured, this being an important measurement of protein stability. 2D ¹H NOESY NMR experiments were performed to determine if the insertion was disrupting the β-sheet structure of PrP, while ¹³C, ¹⁵N double-labeling experiments were performed to define the rigidity of the 8 extra residues.

Results: Mouse HRdup was found to have similar sensitivity towards urea denaturation compared with M128V and WT mPrP. Moreover, 2D ¹H NOESY NMR experiments demonstrated that the β-sheet of mouse HRdup is formed in the same manner as in mPrP M128V.

Conclusions: The novel HRdup insert in PrP does not disrupt the stability of the C-terminal globular domain, nor the short β-sheet therein. Along with limited proteolysis experiments to assess brain expressed HRdup in Tg mice, our data define a type of misfolding that is distinct from C-terminal PrP alterations in other prion diseases such as Creutzfeldt-Jakob Disease and scrapie as it encompasses sequences N-terminal to the HRdup insert. Further NMR experiments aim at a complete structural determination for this highly pathogenic allele.

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P79 Atypical PrPSc retains high infectivity after protease K treatment

Dr Ilaria Vanni1, DVM Laura Pirisinu1, Mr Stefano Marcon1, Dr Michele Angelo Di Bari1, DVM Claudia D'Agostino1, Dr Joaquin Castilla2,3, MD Pierluigi Gambetti4, DVM Romolo Nonno1, DVM Umberto Agrimi1 1Istituto Superiore Di Sanità, Department of Veterinary Public Health and Food Safety, Rome, Italy, 2CIC bioGUNE, Parque Tecnológico de Bizkaia, Derio, Spain, 3IKERBASQUE, Bilbao, Spain, 4Case Western Reserve University, Department of Pathology, Cleveland, United States of America

Aims: Latest studies reported that prion diseases associated with PrPSc characterized by an unusual protease resistant core (PrPres) cleaved at both N- and C-termini, are transmissible in rodent models (1,2). Although these studies suggested that atypical prions are associated with true infectivity similarly to classical prions, definite proof of the infectious role of these unusual PrPres aggregates is still missing.
To pursue this issue, we focused on human and experimental prion diseases associated with atypical PrPSc, respectively the Gerstmann-Sträussler-Scheinker (GSS) disease associated with the A117V PrP mutation and the neurological disease spontaneously developed by transgenic mice expressing wt bank vole PrP (TgL1).

Methods: Frozen brain tissues were obtained from human or experimental prion diseases associated with classical PrPSc (sCJD-MM1 and vole-adapted CWD) or atypical PrPSc (GSS with the A117V mutation and spontaneously sick TgL1 mice). Brain homogenates were either treated with PK (20 μg/ml, 37°C for 1 hour) or left untreated. PK-treated or untreated homogenates were then endpoint titrated by bioassay in bank voles. Infectivity titers were calculated as ID₅₀ U/g by the Spearman and Karber method.

Results: Overall, atypical prion diseases showed infectious titers as high, or even higher, than classical prions. Notably, GSS-A117V showed the highest infectious titer (10<9.3>), followed by vole-adapted CWD (10<8.4>), spontaneously sick TgL1 (10<7.6>) and sCJD MM1 (10<5.6>). After PK treatment, ID₅₀ U/g were 10<9.0> for GSS-A117V, 10<8.1> for CWD, 10<8.4> for TgL1 and 10<5.9> for sCJD MM1. The variation of the infectious titres after PK treatment was within the -0.3-to-+0.8 log range. Thus, in both classical and atypical prions PK treatment did not substantially affected the infectious titer.

Conclusions: Our study reveals that prion diseases characterized by atypical PrPSc exhibit high infectious titers and that such infectivity is retained upon PK treatment, as it happens for classical PrPSc. Notably, these data argue against the hypothesis of “protease sensitive” forms of PrPSc as being the misfolded conformers responsible for the abovementioned infectivity. Furthermore, our results highlight that the C-terminal region of PrPSc is dispensable for infectivity, thus suggesting that PrPres aggregates containing exclusively the central PrP domain, i.e. composed of PrP peptides spanning aa ̴90-150, could be considered fully competent “miniprions”. Efforts towards the purification of these aggregates are underway.

REFERENCES:

1. Pirisinu et al. Sci Rep. 2016; 6:20443.

2. Asante et al. PLoS Pathog. 2013; 9(9):e1003643.

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P177 Transmission study of human prion diseases in PrP glycan-KO transgenic mice

Phd Silvio Notari1, PhD Laura Cracco1, PhD Satish Kumar Nemani1, MD Bernardino Ghetti2, PhD Qingzhong Kong1, MD Pierluigi Gambetti1
1Department of Pathology, Case Western Reserve University, Cleveland, United States, 2Department of Pathology and Laboratory Medicine, Indiana University, Indianapolis, United States

Aims:
The role of PrP glycosylation in various transmission barriers including species, mutation and 129 polymorphism barriers has been studied in several in vitro and in vivo models. We report an update of our studies on the propensity to transmit and reproduce the histopathological phenotype human prion diseases, including familial and sporadic forms historically difficult to transmit to humanized transgenic mice [Tg(HuPrP)]. Hamster Hyperactive (Hyper) and Drowsy prion strains have also been examined.

Methods:
We generated a new transgenic mouse line expressing unglycosylated PrP-129M [Tg(HuPrP-129MGlycKO)], here after referred to as TgNN6h, by substituting asparagine with glutamine at both residue 181 and 197 glycosylation sites (N181Q/N197Q). TgNN6h were inoculated with different human prion strains: Variably protease sensitive prionopathy (VPSPr), Gerstmann-Sträussler-Scheinker (GSS) mutations (A117V, P102L8kDa, P102L21kDa, F198S), sporadic Creutzfeldt-Jakob disease (sCJD, subtypes MM2, VV2, MM1, VV1, MV2K, MV2C), familial CJD V180I (fCJDv180I), sporadic fatal insomnia (sFI) as well as hamster Hyper and Drowsy strains.

Results:
TgNN6h efficiently transmitted VPSPr with attack rates (ARs) and incubation periods (IPs) changed as a function of the VPSPr 129 genotype (highest in 129MM/MV and lowest in 129VV). To note that the transmission to TgNN6h was relatively efficient even with VPSPr-VV despite the 129 genotypic barrier, which is totally divergent from the lack of transmission of VPSPr-VV to Tg mice expressing wild type human PrP [Tg(HuWTPrP)].
Among GSS mutations subtypes, P102L21kDa, F198S, P102L8kDa and A117V were transmitted with various efficiencies. Despite limited neuropathology and medium-long average IP (380-700 days), a high rate of GSS-inoculated animals suffered of typical prion-related clinical signs before death, a finding hinting to the presence of PrPSc (investigation in progress).
Familial CJD V180I was easily transmitted with AR, IP and phenotype comparable to those of VPSPr, confirming the conformation similarity of the PrPSc species in these two diseases.
Within the sCJD group, sCJDMM2 and sFI were the only conditions that transmitted to TgNN6h much more efficiently than to [Tg(HuWTPrP)] with IP reductions of approximately 50% and 90% at first and second passage, respectively.
Hamster Hyper and Drowsy strains did not transmit efficiently to TgNN6h.

Conclusions:
Our data indicate that glycan-free TgNN6h may play a role in testing experimental transmission and studying transmission barriers of human prion diseases, especially those difficult to transmit to Tg mice expressing wild type human PrPC.
Compared to previous studies, our data underscore the variability of the response to transmission between different models of glycan-free Tg mice.

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P23 Enteral feeding has strong effects on survival in prion diseases

Kristy McNiven1, Akin Nihat1, TzeHow Mok1, Michele Gorham1, Peter Rudge1, Prof John Collinge1, Prof Simon Mead1

1National Prion Clinic, National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust, London, United Kingdom

Aims:
Most if not all patients diagnosed with Creutzfeldt-Jakob disease (CJD) will develop dysphagia during the course of their illness. A minority of patients are managed with enteral feeding via percutaneous endoscopic gastrostomy (PEG) or radiologically inserted gastrostomy (RIG); however there is no clear evidence about the risks and benefits, both of which are important in helping clinicians and relatives in making the decision to pursue enteral feeding. This study aims provide evidence to support decision making based on the experiences of the National Prion Clinic.

Methods:
Case - matched control study of 25 patients with CJD (12 sporadic, 7 acquired and 6 inherited) treated with enteral feeding via PEG or RIG tube and 84 patients not fed in this way. All were enrolled in longitudinal observational studies. Up to four controls per patient were matched by diagnosis, age +/-5 years, codon 129 genotype and disease severity measured by the MRC Prion Disease Rating Scale (score 0-20). Survival was analysed using a log-rank (Mantel-Cox) test and function was measured using the MRC Scale.

Results:
A total of 664 symptomatic patients from the PRION-1 Study and the National Prion Monitoring Cohort Study were considered, 25 (3.76%) had enteral feeding.
Cases treated with enteral feeding showed a marked increase in survival compared with controls, in sporadic CJD by a mean of 132 days (P<0.001), in acquired CJD by 813 days (P<0.001) and in inherited prion disease by 1006 days (P<0.01). The MRC Scale score showed evidence against sustained improvement in function in any patient post RIG/PEG insertion, in fact, patients continued to deteriorate. We discuss some of the motivations for and complications of the treatment.

Conclusions:
This study provides evidence that enteral feeding via RIG or PEG tubes is associated with longer survival in patients with prion disease; but does not improve overall function. This effect may be directly related to the intervention, or may relate to patient selection. Supportive treatments should be considered as a confounding factor in clinical trials that use survival as an outcome measure.

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P24 Enhanced Creutzfeldt-Jakob Disease surveillance in patients with atypical dementia: challenges to recruitment in the clinical setting.

Mrs Gemma Logan1, Dr Briony Waddell1,2, Dr Guy Holloway2, Dr Susan D Shenkin2,4, Professor Richard S G Knight1, Dr Suvankar Pal1,3, Dr Anna M Molesworth1

1National CJD Research and Surveillance Unit, University of Edinburgh, United Kingdom , 2NHS Lothian, , United Kingdom, 3Anne Rowling Regenerative Neurology Clinic, University of Edinburgh, United Kingdom, 4University of Edinburgh, , United Kingdom

Aims: This research is part of a larger study that aims to find out if prion disease, such as Creutzfeldt-Jakob Disease (CJD) is being missed in the older population in Lothian, Scotland and, if so, why. Here, we aim to assess some of the challenges faced by the study team in the first year of patient recruitment by considering factors that affect the response rates to the study.

Methods: The study involves patients aged sixty-five or older with atypical clinical features not suggestive of the commonly recognised forms of dementia, who are accessing local Old Age Psychiatry, Medicine of the Elderly and Neurology services. Patients are recruited into the study following their referral by clinicians to the dedicated study clinical research team. Between January and December 2016, 61 eligible patients were referred to the study of whom only 16 (26%) were subsequently recruited. We sought feedback from healthcare professionals, patients and their relatives to understand some of the reasons for the relatively low participation rates.

Results: We have identified a number of reasons that affect both the willingness and ability of clinicians and patients to participate:

Patient/caregiver factors incorporate factors relating to the the patient or their caregiver that have impacted on their willingness to participate in the study or their ability to complete the study components, such as fear, time and the appropriateness of the approach taken.

Provider Factors incorporate factors relating to the referring clinicians and healthcare workers, such as the provider’s knowledge of the different forms of dementia and the suitability of case definitions for referral, their confidence in introducing research to patients and other reasons contributing to their willingness to participate in research.

Operational Factors incorporate factors relating to the study methodology or the organisation from which patients are identified and referred, such as the time available to care providers to engage with patients in research, or the timing of this following a dementia diagnosis.

Conclusion: It is important that the methods and approach are continuously evaluated and improvements made where necessary. Gaining feedback from those involved can provide relevant, first-hand experiences of the challenges faced, enabling changes to be incorporated that have significance. Reasons for relatively low participation in this study is multi-factorial. We have have identified a number of areas in which efforts should be focused to overcome the barriers to success and help improve study participation rates.

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P134 A clinical description of atypical dementia patients: are we missing Creutzfeldt-Jakob Disease in older patients?

Dr Briony Waddell1, Ms Gemma Logan2, Dr Suvankar Pal1,3, Dr Guy Holloway1,3, Dr Susan Shenkin1, Dr David Summers1,2, Professor Richard Knight1,2, Professor Colin Smith1,4, Dr Anna Molesworth2
1NHS Lothian, Edinburgh, United Kingdom, 2NCJDRSU, Edinburgh, United Kingdom, 3Anne Rowling Regenerative Neurology Clinic, Edinburgh, United Kingdom, 4Edinburgh Brain and Tissue Bank, Edinburgh, United Kingdom

Aims: Ageing is the greatest risk factor for most forms of dementia, however in the UK over half of all dementia cases are not diagnosed and even amongst those who are, particularly in older patients the underlying cause of dementia may be missed. Ascertaining the correct diagnosis is important not only to the patient and their carers but for appropriate clinical care. In Creutzfeldt-Jakob Disease (CJD) case under-ascertainment may also have public health implications, due to the potential for person-to-person transmission through medical procedures, including certain types of surgery or transfusion of blood. We aim to describe the clinic-pathological features of older patients with atypical dementia, who were recruited into a study to determine if there are otherwise unrecognized cases of CJD that may have been missed.

Methods: Since January 2016, patients aged ≥65 years seen by neurology, old-age psychiatry or medicine for the elderly specialties in NHS Lothian with a diagnosis of non-CJD dementia but with atypical features (for example unusual speed of progression, focal neurology, or psychiatric features) have been invited to participate in the study. For each participant, a symptom questionnaire and epidemiological review were undertaken by a member of the study’s clinical research team. The clinical examination included the Addenbrooke’s Cognitive Examination – III, the frontal assessment battery, the hospital anxiety and depression scale, the Barthel’s Index, and the Edinburgh Motor Assessment Scale. In addition, MRI was undertaken with consent (including DWI and FLAIR sequences), a blood sample was taken for codon-129 molecular subtyping and patients were also invited to donate their brain tissue for research in the event of their death. The guardian or next of kin was approached if a patient lacked the capacity to consent for his- or herself.

Results: Recruitment is ongoing; the clinical, radiological, and pathological characteristics of participants referred in the first 18 months of the study will be presented.

Conclusion: There may be more people affected by CJD but the diagnosis missed, perhaps because the symptoms are atypical of CJD and more similar to other forms of dementia

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P28 Enhanced Creutzfeldt-Jakob Disease Surveillance in the Older Population in the UK using Biochemical Testing: Logistical Aspects.

Dr Alex Peden1, Ms Adriana Libori1, Ms Helen Yull1, Professor Colin Smith2, Dr Anna Molesworth1
1National CJD Research & Surveillance Unit, Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom, 2Edinburgh Brain & Tissue Bank, Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom

Aims: Our aim is to determine whether there is otherwise unrecognised Creutzfeldt-Jakob disease (CJD) or other prionopathies in individuals who are 65 or more years of age in the UK. We are developing a systematic protocol for the biochemical screening of locally banked brain tissue donations for evidence of the disease-associated misfolded forms of the prion protein (PrPSc) found in variant CJD, sporadic CJD and variably protease sensitive prionopathy. Our aim is to maximise both the sensitivity of this protocol for detecting prion disease and the range of prion disease subtypes that can be detected.

Methods: To achieve these aims, five biochemical tests are employed based on different principles for detecting PrPSc. Frozen samples from six defined brain regions from each donated brain are analysed. The five methods are: Western blotting for protease resistant PrPSc, sodium phosphotungstate precipitation of PrPSc prior to Western blotting, conformation dependent immunoassay and the in vitro amplification techniques protein misfolding cyclic amplification, and real time quaking induced conversion (RT-QuIC).

Results: Based on two years’ experience, we will review how that protocol has worked in practice. For each method in turn we will present (1) the rationale for using the method for enhanced surveillance for prion disease, (2) the algorithmic process that we have used to further investigate initial results that are indeterminate and (3) the potential for the method to be made high-throughput and automated for wider population surveillance.

Conclusions: It is important to establish the feasibility of employing in depth biochemical analysis as part of a programme of enhanced CJD surveillance for evidence of prion disease in the older population. The panel of biochemical methods used in this study is designed to maximise the potential for detecting low levels of prion accumulation, or novel prionopathies in the screening of locally banked brain tissue. Western blotting is a well-established diagnostic method in prion disease research and surveillance, but has limited sensitivity and is limited to detection of protease resistant PrPSc. The four other techniques used in this study have higher sensitives or use a different principle for detecting PrPSc. However, these other methods remain to be evaluated as tools for screening large numbers of presumed negative samples in a surveillance setting. Therefore, when using this panel of methods careful consideration must be given to the process used for assigning positive results and assessing any anomalous findings.

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P30 The clinical and referral characteristics of older patients with Creutzfeldt-Jakob Disease: a retrospective review of cases occurring in the UK

Dr Briony Waddell1, Ms Jan Mackenzie2, Professor Richard Knight1,2, Dr Suvankar Pal1,2, Dr Anna Molesworth2 1NHS Lothian, Edinburgh, United Kingdom, 2NCJDRSU, Edinburgh, United Kingdom

Aims: Ageing is the greatest identified risk factor for most forms of dementia. However, variant Creutzfeldt-Jakob Disease (vCJD) is predominantly a disease of younger people and sporadic CJD (sCJD) mainly affects those under 80 years of age. The very low age-specific incidence of both vCJD and sCJD in the oldest age group may, in part, be due to case under ascertainment, perhaps due to under investigation, atypical clinical presentation or similarity with other forms of dementia. Here we describe the clinical and referral characteristics of older patients who have previously been diagnosed with CJD, and determine if they differ from younger cases.

Methods: In the UK, suspect cases of CJD are referred by clinicians to the National CJD Research & Surveillance Unit (NCJDRSU) for clinical assessment and epidemiological review. We undertook a retrospective review of CJD cases referred to the NCJDRSU, for vCJD between 1995 and 2015 (n = 177) and for sCJD between 2010 and 2015 (n = 584). Older patients were defined as those 45 years of age and above for vCJD and 80 years or older for sCJD. Their clinico-pathological features and referral characteristics (such as the referrer’s specialty and time of diagnosis in relation to death) were then compared between age groups.

Results: For both vCJD and sCJD older patients were more likely to present with rapid cognitive decline compared to younger patients, and were less likely to present with sensory or psychiatric features. In sCJD there was an overall lower burden of other neurological features in older patients. The sensitivity of MRI for the changes characteristic of both vCJD and sCJD was significantly lower in older patients. Older patients with sCJD were more likely to be referred to the NCJDRSU by a specialty other than neurology (e.g. general medicine and psychiatry). Older age was associated with later diagnosis in both vCJD and sCJD (p <0.001).

Conclusion: This study indicates that older patients with CJD were more likely than younger patients to be referred later in the disease process compared to younger patients. These findings may reflect less rigorous diagnostic pursuit in the elderly and are consistent with the possibility that there may be more people affected by CJD but the diagnosis missed, perhaps because presentation may be atypical of CJD and more in keeping with other forms of dementia.

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P120 Early preclinical detection of prions in blood of macaques peripherally infected with the variant CJD agent.

Luis Concha1,2, Claudio Soto1
1University Of Texas, Houston, United States, 2Universidad de los Andes, Santiago, Chile

Aim: Preclinical detection of prions in blood of experimentally infected non-human primates.
The detection of prions in blood of patients affected by variant Creutzfeldt-Jakob disease (vCJD) has been recently achieved, by means of the protein misfolding cyclic amplification (PMCA) technique (Concha-Marambio et. al., 2016). Moreover, a few blood samples were shown to contain prions before disease onset (Bougard et. al. 2016). However, the unknown time of infection makes impossible to determine when in the incubation period prions can be detected in blood. Thus, we studied blood samples longitudinally collected from 3 macaques infected with the macaque adapted vCJD agent (m-vCJD).

Methods: Three macaques were peripherally infected with m-vCJD (McDowell et. al., 2016). Blood was collected longitudinally, starting 2 months post inoculation (mpi) until the endpoint of the disease. The samples were divided in three panels: early preclinical (2 to 12 mpi), late preclinical (12 mpi to onset) and clinical (onset to final bleed). These samples were kindly provided by Dr Luisa Gregory as de-identified samples.
The PMCA protocol previously used was optimized to detect prions in blood of vCJD patients, for the detection of m-vCJD prions in macaque blood, using human PrP from transgenic mice as substrate. The substrate was supplemented with 100 μg/ml heparin and few modifications were introduced into the PMCA protocol.

Results: m-vCJD prions from macaque brain homogenate (BH) were amplified at similar efficiencies vCJD prions (10-10 to 10-11 dilutions of BH). Prions were readily detected in whole blood, buffy coat and plasma during the clinical phase of the disease. Preclinical samples were more challenging to amplify. However, after PMCA optimization, we could detect with high sensitivity and specificity all the early and late preclinical samples. Our results show that m-vCJD prions from macaque blood can be detected at least ~800 days before the onset of disease.

Conclusions: PMCA was adapted for the efficient amplification of m-vCJD prions present in blood of macaques peripherally challenged with the vCJD agent. Our results suggest that PMCA can detect prions in blood more than 800 days before onset with high sensitivity and specificity. Since the first sample was collected 2 mpi and it was positive, PMCA can probably detect prions in blood weeks after inoculation. Overall, our results show the consistent and reproducible preclinical detection of prions in macaques, which suggest that this protocol could be used in humans for pre-symptomatic detection of carriers infected with vCJD prions.

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P146 Earliest Detection Of Prion Seeding Activity In Blood Of Human Prp Transgenic Mice Infected With Vcjd

Christelle Jas-Duval1,2, Dr Mohammed Moudjou2, Lilian Bruyere-Ostells1, Maxime Belondrade1, Charly Mayran1, Laetitia Herzog2, Fabienne Reine2, Pr Stéphane Haïk3, Dr Daisy Bougard1, Dr Vincent Béringue2 1UMR 1058 Pathogénèse et Contrôle des Infections Chroniques (Inserm-EFS-Université de Montpellier), Montpellier, France, 2VIM, INRA, Université Paris-Saclay, Jouy-en-Josas, France, 3Sorbonne Universités, UPMC Univ Paris 06, Inserm, CNRS, Institut du cerveau et la moelle (ICM) Hôpital Pitié-Salpêtrière, Paris, France

Aims: Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease, linked to dietary exposure to Bovine Spongiform Encephalopathy prions. It is now evident that vCJD can be transmitted from person to person through blood transfusion and this has raised concerns that a reservoir of infectious asymptomatic people could exist in the blood donor population. Three secondary cases of vCJD linked to transfusion of non-leukodepleted blood cells from asymptomatic donors have been described in United Kingdom. While the number of clinical cases of vCJD remains limited, there is evidence that as many as 1 in 2000 individuals exposed to BSE may carry clinically silent vCJD prions in the lymphoid tissue. Very low levels of disease-specific prion protein (PrPSc) can be amplified and detected in tissue and body fluids of vCJD-affected individuals by a technique designated protein misfolding cyclic amplification (PMCA). A sensitive version of this assay allowed detection of minute amounts of PrPSc in blood samples of patients between 1.3 and 2.6 years before they develop vCJD. To further substantiate the use of PMCA as screening test for silent vCJD infection, we sought to define, using human PrP transgenic mice experimentally challenged with vCJD, the time-course of vCJD prion seeding activity in blood.

Methods: Human PrP transgenic mice (Met 129 allele; tg650 line) and, as controls, PrP knock-out mice (Zurich I) were inoculated by intracerebral route with vCJD brain extract. At regular time points until the terminal stage of disease, blood was sampled and submitted to independent runs of PMCA. PrPSc was detected in the amplified products by immunoblot. As controls for prion replication in mice, brain and spleens were collected for PrPSc analysis.

Results and Conclusions: PrPSc seeding activity of the inoculum was detected in the blood of both human PrP and PrP knock-out mice at day 2 post-inoculation. From day 10 onwards, no seeding activity was evidenced in the blood from PrP knock-out mice. In human PrP mice, the earliest seeding activity was detected from day 35, and persisted until the disease terminal stage at day 500. Partitioning of the activity in the different blood fractions and comparison of seeding activity with that observed with brain and spleen material will be presented.
These data indicate that vCJD seeding activity can be detected in blood nearly throughout the incubation period and reinforces the use of PMCA as preclinical screening test for the presence of vCJD prions.

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P148 Comparison of Protein Misfolding Cyclic Amplification (PMCA) Assays for Analytical and Diagnostic Sensitivity

Mr Kaetan Ladhani1, Dr Sandra McCutcheon2, Mr Boon Chin-Tan2, Dr Fiona Houston2, Dr Jillian Cooper1
1CJD Research and Resource Centre, NIBSC, South Mimms, United Kingdom, 2Roslin Institute, University of Edinburgh, Easter Bush, United Kingdom

Aims: Assessment of PMCA for analytical and diagnostic sensitivity
Identical sets of samples were generated to allow direct comparison of assays intended for the diagnosis of variant Creutzfeld-Jakob disease (vCJD).

1. Analytical sensitivity using spikes of vCJD tissue homogenate in pooled human plasma

2. Diagnostic sensitivity using blood components from sheep infected with BSE through oral ingestion or by blood transfusion (Roslin Insitute)

Methods: Protein misfolding cyclic amplification methods were used in the assessment of blinded sets of samples for both the analytical and diagnostic sensitivity. A range of substrates were used for amplification and included brain homogenates prepared from transgenic mice or cell lines overexpressing human prion protein. Initial enrichment steps included precipitation and centrifugation or affinity capture.

1. Analytical sensitivity. Tissue spiked samples were provided in triplicate with normal brain/ plasma controls. The range of vCJD tissue spikes were between 10-6 and 10-14 of 10% W/V vCJD brain homogenate.

2. Diagnostic sensitivity assessment. Blood components, plasma or buffy coat enriched fractions were provided from animals confirmed to be incubating disease at the clinical and preclinical phase of disease. Control samples were from animals challenged with control brain or samples derived from a scrapie-free flock.

Results: For analytical sensitivity 3 different groups have been assessed using vCJD and control tissues spiked into pooled human plasma. The limits of detection were consistent and comparable between laboratories with the last dilution of vCJD brain spikes detected being 10-8 of the 10% W/V tissue homogenate. The analysis of the spiked plasma panels highlighted that PMCA assay can incorrectly score control samples positive.

The assessment of BSE infected sheep blood components demonstrated that the sensitivity of PMCA was dependent on the component tested. For known clinical samples all plasma samples tested were positive, none of the preclinical samples were scored positive. In contrast when buffy coat enriched fractions were tested the majority of clinical and all preclinical samples were scored positive.

Conclusions: PMCA is a very sensitive method for detecting markers of both BSE and vCJD infection. The analysis undertaken here has further demonstrated the capacity of this assay to detect preclinical samples. PMCA has the sensitivity needed to detect blood components positive ahead of clinical onset of disease although when compared to other detection methods the analytical sensitivity is several logs lower with vCJD brain. A full assessment of assay specificity is needed to ensure performance criteria are suitably met.

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P127 Prion surveillance in primary immunodeficiency patients exposed to UK-sourced immunoglobulin in the UK, 1996-2000

Mrs Kudzai Karekwaivanane1, Ms Catherine Bangs2, Mr Kaetan Ladhani3, Dr Jillian Cooper3, Ms Suzanne Lowrie1, Professor James Ironside1, Dr Matthew Helbert, Dr Anna Molesworth1
1National CJD Research & Surveillance Unit, University of Edinburgh, Edinburgh, United Kingdom, 2Central Manchester University Hospitals NHS Foundation Trust , Manchester, United Kingdom, 3National Institute for Biological Standards and Control , South Mimms, United Kingdom

Aims:
Variant Creutzfeldt-Jakob disease (vCJD) is a very rare disease associated with an abnormal form of a naturally occurring protein, the prion protein, the presence of which can be detected in certain body tissues. Most cases of vCJD can be attributed to eating contaminated meat products, however infection may be spread through blood transfusion or treatment with certain blood products. For patients with primary immunodeficiency treated with these products there is a very low risk of their having been infected with vCJD. We aim to find out whether any evidence of abnormal prion protein and/or prion disease can be found in this patient group.

Methods:
Patients with primary immunodeficiency who received UK-sourced immunoglobulin products between December 1996 and December 2000 are eligible for inclusion in this study, with informed consent. By following these patients over several years and testing any available tissue, including biopsies, post-mortem material and when a suitable test becomes available, their blood, this study can look for evidence of prionopathy.

Results:
Since 2006 when the study began and the time of abstract submission, a total of 77 patients have been recruited from 16 centres across Britain. All participants had received UK sourced immunoglobulin for all or part of the period between 1996 and 2000, with 9 known to have been treated with batches derived from the plasma of donors who subsequently developed vCJD. The participants have now been followed up for approximately 1250 person-years following first exposure and a total of 43 participants have donated tissue specimens, including 4 post-mortem donations. In this time no patients have shown any clinical or pathological features of vCJD or evidence of abnormal prion protein.

Conclusion:
The results are reassuring but it is important to continue to monitor patients over the longer term, to look for evidence of vCJD, assess if infection was acquired through the use of blood products and consider the wider implications to patients and the public health.

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P131 Pathologically Confirmed Variant Creutzfeldt - Jakob disease in A Patient Heterozygous at Codon 129 of Prion Protein Gene

Dr Tze How Mok1, Dr Zane Jaunmuktane1, Susan Joiner1, Tracy Campbell1, Dr Catherine Morgan1, Dr Benjamin Wakerley1, Dr Farhad Golestani1, Dr Peter Rudge1, Prof Simon Mead1, Dr. Jonathan DF Wadsworth1, Prof Sebastian Brandner1, Prof John Collinge1
1NHS National Prion Clinic, London, United Kingdom

Background:
The human transmission of bovine spongiform encephalopathy (BSE) as variant Creutzfeldt-Jakob disease (vCJD) provoked an international public health crisis. The incidence of vCJD has declined since its peak in the early 2000s but caution continues to be exercised in view of the long incubation periods observed in acquired prion disease, seen for example in individuals who are methionine-valine heterozygous (MV) in their codon 129 of the prion protein gene (PRNP) during the kuru epidemic. All cases of definite vCJD thus far have been in individuals who are methionine homozygous (MM) at codon 129 of the PRNP.

Results:
A 36-year-old Caucasian male was referred to the NHS National Prion Clinic in 2015 with a progressive frontal behavioural syndrome, cognitive impairment, gait ataxia, myoclonus, visual hallucinations, dysphagia and eventually severe agitation prior to death over 16 months. His cerebrospinal fluid protein S100B level was normal while protein 14.3.3 and real-time quaking-induced conversion assay were both negative; a diffusely slow rhythm was seen on his electroencephalogram but not periodic complexes. Magnetic Resonance Imaging (MRI) of his brain showed restricted diffusion in the caudate heads, putamen, medial thalami and hypothalamus. The brain pathology showed classical appearances of vCJD, supported by the immunoblot findings of PrPSc type 4 (London Classification), seen uniquely in vCJD.

Conclusions:
This first pathologically confirmed case of vCJD in a PRNP codon 129 MV individual signifies a potential change in clinic-radiological phenotype where although the neuropathology and immunoblot studies were typical, the clinical phenotype and MRI findings are characteristic for sporadic CJD, rather than vCJD. Whether this marks the start of a “second wave” in vCJD cases remains unclear, but it emphasises the importance of autopsy surveillance in determining the true prevalence of BSE-related deaths in humans.

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P32 Enhanced Creutzfeldt-Jakob Disease Surveillance in the Older Population in the UK using Neuropathology and Biochemical Testing: interim findings

Ms Helen.M. Yull1, Miss Adriana Libori1, Dr Diane Ritchie1, Dr Alexander Peden1, Professor Colin Smith1,2, Dr Anna Molesworth1

1National CJD Research & Surveillance Unit, University of Edinburgh, Edinburgh, United Kingdom, 2Edinburgh Brain & Tissue Bank, University of Edinburgh, Edinburgh, United Kingdom

Aims: Our aim is to determine whether there is otherwise unrecognised Creutzfeldt-Jakob disease (CJD) or other prionopathy in individuals who are 65 or more years of age by undertaking neuropathological and biochemical screening of locally banked brain tissue donations for evidence of abnormal prion protein and/or disease. In so doing we will assess the feasibility of deploying these surveillance methods in the UK general population.

Methods: Since April 2015, we have undertaken in-depth biochemical and histopathological screening of brain tissue donations to the MRC Edinburgh Brain & Tissue Bank for any evidence of unrecognised prionopathy, using the full range of techniques available. Each tissue donation is subjected to histopathological screening for changes attributable to prionopathy. In addition, formalin fixed tissue from two brain regions from each case are analysed by immunocytochemistry using two anti-PrP antibodies. Frozen samples from six defined brain regions from each donated brain are analysed using a range of approaches that are designed to maximise detection of the differing forms of PrPSc. The methods used are Western blotting for protease resistant PrPSc (WB), sodium phosphotungstate (NaPTA) precipitation of PrPSc prior to Western blotting (NaPTA-WB), conformation dependent immunoassay (CDI) and the in vitro amplification techniques of protein misfolding cyclic amplification (PMCA) and real time quaking induced conversion (RT-QuIC). In addition, the PRNP codon-129 genotype is also determined for each donation received. Any anomalous findings from the use of any one of these techniques are rigorously investigated.

Results: At the time of abstract submission, we had received brain tissue from 55 post mortem donors. The full histopathological, biochemical and molecular genetic analysis has been completed for 43 of these cases and a total of 1570 fixed and frozen tissue sample analyses have been performed. No evidence of prionopathy has been detected so far in any of these tissue samples. The experience and challenges of the first two years of this study will be presented.

Conclusions: Enhanced CJD surveillance in the 65+ age group remains a priority because of the possibility of the under-ascertainment of variant CJD, sporadic CJD and variably protease sensitive prionopathy in the older population. In-depth investigation of brain tissue donated for research can inform this process, but the logistics of wider population surveillance are less clear. Continuing evaluation will help assess the feasibility of deploying these methods throughout the rest of the UK.

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P27 Developing a motor rating scale for sporadic CJD

Dr Akin Nihat1, Dr Andrew Thompson1, Dr TH Mok1, Dr Peter Rudge1, Professor John Collinge1, Professor Simon Mead1

1MRC Prion Unit, National Hospital for Neurology and Neurosurgery, Queen Square, London, QC1N 3BG, United Kingdom

Aims
Sensitive and objective outcome measures are crucial in evaluating and quantifying clinical change in Creutzfeldt-Jakob disease (CJD); particularly in advance of clinical trials, where greater accuracy in detecting change will inform the design of appropriately powered studies. An important milestone was the validation of a robust, global functional scale (the Medical Research Council Prion Disease Rating Scale, or MRC Scale), now widely used in patients with sporadic CJD. However, sporadic CJD is a multifaceted disease, and further outcome measures identifying change in specific domains are required. We sought to construct a rating scale targeted to objective and functional motor features in sporadic CJD.

Methods
A subset of patients enrolled to the National Prion Monitoring Cohort underwent individual assessments (n=145) with a battery of 23 bedside tests designed to reflect the scope of motor features in sporadic CJD. These included tests validated to measure specific motor function in other conditions, such as the Scale for the Assessment and Rating of Ataxia (SARA). The group included assessment of cerebellar, pyramidal and extrapyramidal function, myoclonus, and functional measures such as mobility; these were further pared based on data completeness and relative spread of responses, to leave 17 tests, or items.

These items underwent Rasch analysis using the RUMM2030™ statistical package. Rasch analysis is a form of statistical modelling commonly used in educational and healthcare test design. It allows combination of multiple ordinal tests (items) into a single interval scale that reflects a “latent trait”, or outcome measure, of interest, and allows meaningful separation of participants based on this single measure.

Results
A final 9-item scale showed good fit to the Rasch model (χ2 < 30, p > 0.05) for both items (fit residual -0.40, SD = 1.1) and persons (fit residual -0.36, SD = 0.9), and included cerebellar, extrapyramidal, pyramidal and functional items. There was no significant multidimensionality, nor differential item functioning to age, gender or cognition – indicating that the majority of scale variation was explained by a single latent trait, and its performance did not alter based on variables listed. There was an acceptable correlation between the motor scale and MRC Scale (r2 > 0.5).

Conclusions
We present a newly developed, Rasch-derived outcome measure to assess functional and objective motor domains in sporadic CJD. This may form part of a future assessment battery in clinical treatment trials, and a marker of disease progression. Further development will include robust external validation.

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P133 CJD with M232R: Its clinicoepidemiological features.

Dr. Tadashi Tsukamoto1,6, Dr. Nobuo Sanjo2, Dr. Tsuyoshi Hamaguchi3, Dr. Yoshikazu Nakamura4, Dr. Tetsuyuki Kitamoto5, Dr. Masahito Yamada3, Dr. Hidehiro Mizusawa1,6
1National Center Hospital, National Center Of Neurolgy And Psychiatry, Kodaira-City, Japan, 2Department of Neurology and Neurological Science, Tokyo Medical and Dental University, Graduate School of Medical and Dental Sciences, Bunkyo-ku, Japan, 3Department of Neurology and Neurobiology of Aging, Kanazawa University Graduate School of Medical Science, Kanazawa city, Tokyo, 4Department of Public Health, Jichi Medical University, Shimotsuke, Japan, 5Department of Prion Protein Research, Division of CJD Science and Technology, Tohoku University, Graduate School of Medicine, Sendai City, Japan, 6Prion Disease Surveillance Committee, , Japan

Aims:
CJD with M232R subtype has been reported almost exclusively from Japan. But, the entity of M232R as a genetic CJD has been challenged mainly by European and American researchers. Using the data from the database of Prion Disease Surveillance Committee, Japan, we try to discuss the importance of M232R substitution.

Methods:
We collected information of 95 cases of M232R(codon 129 Met/Met, 219 Glu/Glu: abbreviated to MM/EE), 2 cases of M232R(MV/EE), 2 cases of M232R(MM/EK), 3 cases of R232R(MM/EE), 3 cases of M232R + V180I(MM/EE), and a case of M232R + V203F(MM/EE). We compared the differences of mean value of duration time from disease onset to death, age at onset, and duration time from onset to the appearance of myoclonus, dementia, visual/cerebellar signs, pyramidal/extrapyramidal signs and akinetic mutism. We compared those values of 95 cases of M232R(MM/EE) and 1191 cases of possible/probable sCJD(MM/EE). Statistical analysis was done by SPSS Statistics® ver.20.

Results:
Ages(years) at onset of M232R(MM,EE), M232R(MV/EE), M232R(MM/EK), R232R(MM/EE), M232R+V180I(MM/EE) , M232R+V203F(MM/EE)) and probable/possible sCJD were respectively 65.7±9.9, 64.0±9.9, 71.0±8.5, 55.5±3.5, 70.7±4.9 ,38.0 and 68.9±9.6 (mean value ± SD). V203F+M232R and R232R had the tendency to have onset young. Total disease duration time of M232R(MV/EE) was longer than M232R(MM/EE), 89.5 years±12.0 and 19.8 months±27.3. Using independent t test of mean value of total duration time, onset age, duration from onset to appearance of signs, we found the significant difference of onset time between M232R(MM/EE) and sCJD(MM/EE), t value -2.923, significance probability 0.003. We also revealed the difference of mean value of duration time from onset to appearance of pyramidal sign between those two groups, t value 2.072, significance probability 0.039.

Conclusions:
From the statistical analysis, methionine to arginine substitution seems to exert an influence on sCJD natural history to hasten the disease onset but slow down the appearance of pyramidal sign.
M232R may not be a mere polymorphism.

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P29 Is sporadic CJD an acquired disease? Exploring the UK sCJD cases for evidence of iatrogenic transmission through transplantation and surgery.

Dr Patrick Urwin1, Ms Jan Mackenzie1, Prof Richard Knight1, Prof Robert Will1, Dr Anna Molesworth1
1National CJD Research and Surveillance Unit, Edinburgh, United Kingdom

Background:
While sporadic CJD is believed to occur as a result of a spontaneous protein misfolding event, the possibility of person-to-person transmission cannot be ruled out. Iatrogenic transmission of CJD has been recognised in recipients of cadaveric dura mater or growth hormone, and via contaminated blood from variant CJD donors. Prior epidemiological studies have suggested surgery as a risk factor for the development of sporadic CJD. It is possible that at least a small number of sporadic CJD cases may in fact represent unrecognised iatrogenic transmission through tissue or organ transplantation, or by contamination of surgical instruments.

Aims:
We have looked for epidemiological evidence to indicate transplant or surgical transmission of sCJD between patients in the UK seen by the NCJDRSU between 1st January 2010 and 31st December 2015.

Methods:
All patients in the UK suspected to have CJD are referred to the NCJDRSU for assessment by our clinical neurologists, as well as for CSF biochemical and tissue neuropathological review. All cases seen are classified according to the WHO diagnostic criteria. A detailed, standardised questionnaire about the current illness, and background medical and surgical history is collected from the family, supplemented with data obtained from the general practitioner (family doctor) medical records when available. Details of any tissue or organ transplantation, or any surgery is collected from all available data sources.
For the 579 cases categorised as definite or probable sporadic CJD, we identified all procedures where tissue or organ transplantation occurred, and attempted to explore the possibility of transplant transmission of CJD among this group. We scrutinised the medical histories of these cases to look for any overlap between different patients undergoing related surgical procedures in the same hospital within +/- 1 year. These potential “linkage events” have been assessed for clinical relevance.

Results:
Data on 4437 operations were collated from both sources. 30 non-autologous transplant procedures were identified. 106 surgical “linkage events” were identified. Further details of both the transplant procedures and the “linkage events” will be presented.

Conclusion:
We have found no evidence of transplant related transmission of sCJD. The number of identified surgical “linkage events” is small compared to the number of cases in the study, and many are not regarded as clinically plausible for potential transmission. Interpretation of these events is difficult, given significant limitations in the data, but there is no clear evidence of transmission of sCJD through surgical intervention.

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P33 Rapid testing for Creutzfeldt-Jakob disease in cadaveric donors of human tissues

PhD Luisa Gregori1, MS Arthur Serer1, PhD Kristy McDowell1, MD Juraj Cervenak1, MD David Asher1
1United States Food & Drug Administration, Silver Spring,, United States

Aims
At least six iatrogenic transmissions of Creutzfeldt-Jakob disease (CJD) attributed to corneal transplantation have been confirmed or suspected worldwide. Over 40,000 corneas are transplanted yearly in the US. In many countries, including the US, eye banks screen donors by history to identify risk factors and test for several communicable diseases. Medical review should exclude demented donors and others at increased CJD risk. But cadaveric donors are not tested for CJD for several reasons: no marketed human CJD screening test, low prevalence of CJD and low overall autopsy rate. We aimed to show that existing technology might overcome some logistical difficulties to allow screening.

Methods
Optic nerves and frontal lobes of persons with CJD generally contain both infectivity and the abnormal prion protein (PrPTSE) that accumulates in transmissible spongiform encephalopathies (TSEs). We hypothesized that the small amounts of tissue accessible after enucleation using a disposable biopsy punch through a retro-orbital trocar hole into the frontal lobe might be sufficient to test for PrPTSE. We applied the procedure to collect brain samples and optic nerves from two macaques experimentally infected with variant CJD agent. We also tested, in triplicate, small tissue samples from frozen brains of six confirmed human CJD cases. Each of the 18 specimens was serially diluted in half-log increments using 10% normal human brain homogenate as diluent. We assayed control samples (10% w/v brain homogenates) from 28 individuals with several non-TSE neurodegenerative diseases and ten normal individuals. All samples were tested with the IDEXX HerdChek® BSE-Scrapie Ag Kit to detect PrPTSE.

Results
Our collection procedure obtained sufficient macaque brains and optic nerves to detect PrPTSE; all macaque specimens were positive. All 18 human CJD brain samples were positive at 1% and 0.3% dilutions. (One sample of a triplicate gave a low positive result, while the other two replicates gave high positive results). We found no reactive results with any control brain suspension (100% specificity). IDEXX detected PrPTSE ≥30-fold more sensitively than Western blot.

Conclusions
We showed that a commercial test of retro-orbital tissues might offer a practical solution to screen brains of cornea donors for evidence of CJD. A rapid sensitive postmortem screening test for CJD appears feasible without conducting a full autopsy and could enhance the safety of transplanted tissues. While results of this study suggest proof of principle, we do not endorse routine off-label use of commercial veterinary tests with human tissues.

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P29 Is sporadic CJD an acquired disease? Exploring the UK sCJD cases for evidence of iatrogenic transmission through transplantation and surgery.

Dr Patrick Urwin1, Ms Jan Mackenzie1, Prof Richard Knight1, Prof Robert Will1, Dr Anna Molesworth1
1National CJD Research and Surveillance Unit, Edinburgh, United Kingdom

Background:
While sporadic CJD is believed to occur as a result of a spontaneous protein misfolding event, the possibility of person-to-person transmission cannot be ruled out. Iatrogenic transmission of CJD has been recognised in recipients of cadaveric dura mater or growth hormone, and via contaminated blood from variant CJD donors. Prior epidemiological studies have suggested surgery as a risk factor for the development of sporadic CJD. It is possible that at least a small number of sporadic CJD cases may in fact represent unrecognised iatrogenic transmission through tissue or organ transplantation, or by contamination of surgical instruments.

Aims:
We have looked for epidemiological evidence to indicate transplant or surgical transmission of sCJD between patients in the UK seen by the NCJDRSU between 1st January 2010 and 31st December 2015.

Methods:
All patients in the UK suspected to have CJD are referred to the NCJDRSU for assessment by our clinical neurologists, as well as for CSF biochemical and tissue neuropathological review. All cases seen are classified according to the WHO diagnostic criteria. A detailed, standardised questionnaire about the current illness, and background medical and surgical history is collected from the family, supplemented with data obtained from the general practitioner (family doctor) medical records when available. Details of any tissue or organ transplantation, or any surgery is collected from all available data sources.
For the 579 cases categorised as definite or probable sporadic CJD, we identified all procedures where tissue or organ transplantation occurred, and attempted to explore the possibility of transplant transmission of CJD among this group. We scrutinised the medical histories of these cases to look for any overlap between different patients undergoing related surgical procedures in the same hospital within +/- 1 year. These potential “linkage events” have been assessed for clinical relevance.

Results:
Data on 4437 operations were collated from both sources. 30 non-autologous transplant procedures were identified. 106 surgical “linkage events” were identified. Further details of both the transplant procedures and the “linkage events” will be presented.

Conclusion:
We have found no evidence of transplant related transmission of sCJD. The number of identified surgical “linkage events” is small compared to the number of cases in the study, and many are not regarded as clinically plausible for potential transmission. Interpretation of these events is difficult, given significant limitations in the data, but there is no clear evidence of transmission of sCJD through surgical intervention.

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P81 Iatrogenic Creutzfeldt-Jakob disease related to dura mater grafts

Professor Masahito Yamada1, Dr. Tsuyoshi Hamaguchi1, Dr. Kenji Sakai1, Dr. Moeko Noguchi-Shinohara1, Dr. Ichiro Nozaki1, Yu Taniguchi1, Dr. Atushi Kobayashi2, Dr. Atsuko Takeuchi3, Prof. Tetsuyuki Kitamoto3, Prof. Yosikazu Nakamura4, Prof. Nobuo Sanjo5, Dr. Tadashi Tsukamoto6, Prof. Masaki Takao7, Dr. Shigeo Murayama8, Dr. Yasushi Iwasaki9, Prof. Mari Yoshida9, Dr. Hiroshi Shimizu10, Prof. Akiyoshi Kakita10, Prof. Hitoshi Takahashi10, Dr. Hiroyoshi Suzuki11, Prof. Hironobu Naiki12, Prof. Hidehiro MIzusawa6

1Kanazawa University, Kanazawa, Japan, 2Hokkaido University, Sapporo, Japan, 3Tohoku University, Sendai, Japan, 4Jichi Medical University, Shimotsuke, Japan, 5Tokyo Medical and Dental University, Tokyo, Japan, 6National Center of Neurology and Psychiatry, Tokyo, Japan, 7Saitama Medical University International Medical Center, Hidaka, Japan, 8Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan, 9Aichi Medical University, Nagakute, Japan, 10University of Niigata,, Niigata, Japan, 11National Hospital Organization Sendai Medical Center, Sendai, Japan, 12University of Fukui, Fukui, Japan

Aims: To elucidate epidemiological, clinicopathological, and molecular features of dura mater graft-associated Creutzfeldt-Jakob disease (dCJD), and deposition of pathogenic proteins of neurodegenerative diseases in dCJD.

Methods: We investigated dCJD patients identified in Japan, including epidemiological and clinicopathological features, genetic and molecular characteristics of prion protein (PrP), and transmission properties to mice expressing human PrP (Ki-HuPrP). Deposition of amyloid β protein (Aβ) and other pathogenic proteins in dCJD patients were analysed in comparison with age-matched patients with sporadic CJD (sCJD).

Results: Until September 2016, 152 patients with dCJD have been identified in Japan, comprising about two-thirds of the world cases. They received dura mater grafts between 1975 and 1993 at the ages of 1-70 (av. 43) years for the surgery of brain tumor, hemorrhage, hemifacial spasm, etc. After the incubation periods of 1-30 (av. 13) years, they had dCJD onset at the ages of 15-80 (av. 56) years in 1985-2015. The higher incidence of dCJD in Japan may be related to more frequent use of cadaveric dura mater for non-life-threatening conditions than in other countries. dCJD was classified to plaque and non-plaque types. Non-plaque type had clinicopathological features of classical CJD, while, plaque type was characterized by relatively slow progression, lack or late occurrence of periodic encephalogram, unique MRI findings, methionine homozygosity (M/M) at codon 129 of the PrP gene, intermediate type PrPres (type i), and kuru plaques in the brain (MMiK). Transmission studies with the plaque type dCJD to Ki-HuPrP mice showed properties identical to sCJD-VV2 and MV2, indicating that the origin of the plaque type would be VV2 or MV2 sCJD (V2 prion strain). Furthermore, we identified two atypical cases of MMiK type among patients with “sCJD”; the two MMiK cases showed same properties as sCJD-VV2 or MV2 in transmission and protein misfolding cyclic amplification (PMCA) studies, indicating transmission of the V2 prion prion to these cases in the absence of dura mater or other grafts. Compared with sCJD, dCJD showed significantly severe cerebrovascular Aβ deposition (CAA), especially meningeal CAA, and subpial Aβ deposition; the severity was correlated with incubation period from dura mater grafting to death.

Conclusions: We revealed epidemiological, clinicopathological, and molecular features of dCJD. Plaque type dCJD was characterized by MMiK, indicating cross-sequence transmission of the V2 prion strain to methionine homozygotes. MMiK could be a marker to identify acquired prion diseases. Cerebral β-amyloidosis could be transmitted from humans to humans via cadaveric dura mater grafting.

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P129 Occupations of cases of variant Creutzfeldt-Jakob Disease (vCJD) in the UK, 1995-2016

Miss Jan Mackenzie1, Dr Patrick Urwin1, Dr Graeme Mackenzie1, Professor Richard Knight1, Professor Robert Will1, Dr Anna Molesworth1
1National CJD Research & Surveillance Unit, Edinburgh Eh4 2xu, United Kingdom

Aims: Our aims are to review the occupational histories of UK vCJD cases to date, with emphasis on occupations where possible exposure to a transmissible spongiform encephalopathy (TSE) may occur.

Methods: Since 1990, cases of suspected CJD, including vCJD, have been referred by local clinicians to the National CJD Research & Surveillance Unit in Edinburgh for further clinical assessment and epidemiological review. Wherever possible a close relative is interviewed about a wide range of potential risk factors, including the patient’s lifetime occupational history prior to developing vCJD. The occupational history is grouped into several categories: medical/paramedical/nursing/dentistry; animal/pharmaceutical/other research laboratories; animal farming/veterinary medicine; meat industry and other occupations involving animal products. Because people can have several occupations, cases can appear in more than one occupational category.
The occupational health records of healthcare workers who have overlapped with inpatient stays of other patients, and the records of laboratory workers working in research laboratories are then investigated further to identify any possible exposures involving TSE-infected animals or tissues.
Results: A total of 178 cases of definite or probable vCJD have been reported in the UK to date. Information on occupational history was available in 175. Six cases were identified as ever having worked in a medical/paramedical/nursing/dentistry capacity. A further 6 could be considered as support staff in healthcare settings: one GP receptionist, 2 hospital-based clerical staff, 2 hospital-based domestic workers and one case who worked in a dental surgery. Two cases were reported to have worked in laboratories. Ten cases were employed in the animal farming/veterinary industry, 12 in the meat industry and 6 in other occupations involving animal products.

Conclusion: A previous investigation lead by Public Health England failed to identify any occupational exposure of healthcare workers to patients who later developed vCJD, or any exposure to TSE-infected animals or tissue in the laboratory. A case-control comparison of lifetime occupational histories has also concluded that it was unlikely that a high proportion of UK vCJD cases had been infected through occupational exposure. We have no evidence, based on our recent review of UK cases, to indicate the occurrence of occupational exposure to TSEs in cases of vCJD. However the possibility that this may occur cannot be ruled out and the monitoring of occupation should continue to be integral to national surveillance initiatives.

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P136 16 year follow-up of surgically exposed contacts of patients with CJD in the UK

Dr Sarah Wallace2, Marta Checchi1, Dr Hester Ward3, Dr Anna Molesworth4, Jan Mackenzie4, Professor Robert Will4, Dr Katy Sinka1
1Public Health England, London, United Kingdom, 2North London Public Health Training Scheme, London, United Kingdom, 3Health Protection Scotland, Glasgow, United Kingdom, 4National CJD Research and Surveillance Unit, Edinburgh, United Kingdom

Aims: Past Creutzfeldt-Jakob disease (CJD) transmission by surgical instruments has been documented. In the UK, to reduce the likelihood of further occurrences there is detailed guidance for the use and management of surgical instruments. In addition, the surgical histories of patients newly diagnosed with CJD are investigated to identify past procedures that could have exposed other patients to a risk of prion transmission. If any are identified, individuals exposed to potentially contaminated surgical instruments are traced. The number of surgical contacts to trace is guided by a risk assessment considering the nature of the surgery, the type of CJD and the time between the operation and diagnosis of the index patient. Surgical contacts are informed and asked to take public health precautions. Long term follow-up of this group, to detect any transmissions of CJD is ongoing. Here, we present the findings after 16 years of this public health surveillance in the UK.

Methods: A surveillance system was set up in 2007 and retrospectively included all patients identified through CJD surgical lookbacks since 2000. Records are flagged to identify date and cause of death. Periodic cross-referencing with the national CJD surveillance data and a post mortem review of medical notes by a neurology specialist is done. Person years of follow-up, years of survival post operation and causes of death among those patients who have died were summarised.

Results: At June 2016, 231 CJD surgical contacts, associated with 18 surgical incidents had been followed up for a total of 1,828 person years. No clinical cases of CJD had been identified. Five incidents involved neurosurgery, three were ophthalmology procedures, one invasive nasal surgery and the remainder involved gut associated lymphoid tissues in patients with or at risk of variant CJD. Of the total, 82% had survived for five years post exposure. Sixty-nine patients had died, 16 of whom had neurological causes of death, mainly related to the diagnosis for which their original surgery was done. The median (range) follow-up time since operation for the remaining patients is 8.8 years (2.6 to 20.8 years).

Conclusion: CJD surgical incidents are rare but continue to occur. Systematic follow-up has not identified any associated development of CJD to date. Public health follow-up will continue long term and add to the body of evidence informing the transmissible risks of CJD through modern surgery.

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P173 Biosafety risk assessment: managing prion diseases in veterinary laboratories

Miss Luna Montesion1, Mrs Elena Bozzetta1, Mrs Cristina Casalone1, Mrs Elsa Manzardo1, Mrs Maria Gabriella Perotta2, Mrs Maria Caramelli1, Mrs Daniela Meloni1
1Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Turin, Italy, 2Ministry of Health, Rome, Italy

Aims
Laboratories handling biological agents need to effectively manage occupational health and safety, other than protect the environment in the surrounding areas. Although laboratory-acquired prion infections are not documented in humans, possible ways of contamination are thought to be the accidental inoculation or the ingestion of infected material. The possibility of infection by aerosol exposure could not also be discarded. The determination of which mitigation measures should be used to address the specific laboratory risks should be designed upon a risk assessment. Ideally, a risk assessment should be conducted in a manner which is standardized and systematic, which allows it to be repeatable and comparable. Because the infectious nature of prions is not well characterized and destruction of these agents goes beyond the procedures typically required for biohazard inactivation, working with prions requires special considerations for biocontainment to minimize risks. The Italian Reference Laboratory for Animal Transmissible Spongiform Encephalopathies (CEA) developed a biorisk assessment methodology addressed to strengthen risk governance in the laboratories managing prion diseases.

Methods
CEA biorisk assessment methodology was drawn up on the basis of the algorithm proposed by INAL (Italian authority for occupational health and safety protection). This tool produces a robust and useful risk estimation according to the World Health Organization indications or guidelines.It provides a visualization of risks by a quantitative approach, by balancing risks as a function of likelihood of infection and likelihood of exposure through a particular infectious route based on procedures and work practices and the consequences of disease assuming infection. Risks are ranked on the basis of a 5 point scale of unacceptable, high, medium, low and extremely low.

Results:
The CEA biorisk assessment model has been shown to provide useful data from which to make an informed decision about whether the risk situation is well characterized. The technique leaded to laboratory risk estimations being judged tolerable (medium).

Conclusion:
The use of exposure information enables these assessments to be prioritised based on the relative reliability required of the risk controls. The methodology can provide a framework for discussing biosafety risk assessment broadly and also complement the assessment of laboratory security in a biorisk management system. This scheme aids program managers in allocating resources and may help future laboratories in better defining the mitigation strategies.

=====PLEASE NOTE, OCCUPATIONAL RISK FACTORS FOR IATROGENIC TSE PRION...TSS=====

There has been limited systematic research targeted at identifying occupational risk factors for sCJD in healthcare settings. This paper reports on the data supplied to the Spanish CJD registry in response to the request, and on the results of two literature reviews of sCJD – one on case reports involving health professionals and the other on epidemiologically assessed healthcare-related occupational risk of sCJD.

snip...

REFERENCES

Terry S. Singeltary Sr. Doctor Antonio Ruiz Villaespesa, pathologist and CJD researcher deceased because of Creutzfeldt-Jakob Disease SPAIN. 21 Apr 2009. [Accessed 11 Apr 2012]. In: Monitoring the occurrence of emerging forms of CJD [blog]. Available from: http://cjdusa.blogspot.com.es/2009/04/doctor-antonio-ruiz-villaespesa.html

snip...see full text ;













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PRION 2017 CONFERENCE ABSTRACTS HUMAN TSE PRION DISEASE CONTINUED

P119 Type-dependant diverse extension patterns of hyperintensity on diffusion-weighted MR images in dura mater graft-associated Creutzfeldt-Jakob disease

Dr Kenji Sakai1, Dr Tsuyoshi Hamaguchi1, Dr Nobuo Sanjo2, Dr Hiroyuki Murai3, Dr Yasushi Iwasaki4, Dr Tadanori Hamano5, Dr Mari Honma6, Dr Moeko Noguchi-Shinohara1, Dr Ichiro Nozaki1, Prof Yosikazu Nakamura7, Prof Tetsuyuki Kitamoto8, Dr Hidehiro Mizusawa9, Prof Masahito Yamada1
1Kanazawa University, Kanazawa, Japan, 2Tokyo Medical and Dental University, Tokyo, Japan, 3Kyushu University, Fukuoka, Japan, 4Aichi Medical University, Nagakute, Japan, 5University of Fukui, Fukui, Japan, 6Masu Memorial Hospital, Nihonmatsu, Japan, 7Jichi Medical University, Shimtsuke, Japan, 8Tohoku University, Sendai, Japan, 9National Center of Neurology and Psychiatry, Kodaira, Japan

Aims: Studies of dura mater graft-associated Creutzfeldt-Jakob disease (dCJD) cases have proposed that abnormal prion protein may propagate directly from the contaminated dura mater graft to the adjacent brain regions and spread to other brain areas. We aimed to elucidate extension patterns of the hyperintense areas on diffusion-weighted images (DWI) in patients with dCJD.

Methods: We collected the MR images and the medical records of dCJD cases identified by CJD Surveillance Committee in Japan between April 1999 and September 2016. The dCJD cases were classified into two clinicopathological subtypes (non-plaque type and plaque type). We analyzed a relationship among the abnormal signals on DWI, the pathological classification, and the sites of grafting. Sequential images were also assessed.

Results: We collected MR images of 11 patients with dCJD. Lyodura® was transplanted in all proven cases. Four cases were female. The median with range of the age at onset, the age at dural grafting, and the incubation period were 41 (26–76) years, 19 (10–53) years, and 22 (16–29) years, respectively. The dCJD cases were classified into 8 cases of the non-plaque type and 3 cases of the plaque type. Four of the 8 non-plaque-type cases and all the plaque-type cases were pathologically confirmed. Brain MRI was performed 3 (1–22) months, reported as median (range), after the onset. Initial brain MRI was taken significantly earlier in cases with non-plaque type (non-plaque 2.5 months vs plaque 10 months; P = 0.012).
Regarding non-plaque type, hyperintense cerebral cortex and basal ganglia (BG) were obvious in all cases. Abnormal signals were much brighter on the side of dural grafting. Subsequent MRI performed in 5 cases showed widespread hyperintense lesions in the brain. In contrast, the plaque type cases showed diverse patterns of hyperintensity on DWI. In one case, initial hyperintense areas were shown in the BG, frontal cortex, and thalamus. The other case demonstrated the lesion confined to the BG and thalamus. Sequential images presented with frontal lesions in addition to BG and thalamus. The third case showed no apparent abnormalities in the cerebral cortex or BG on DWI 7 months after the onset; however, serial images showed hyperintensity in the cerebral cortex, thalamus, and cerebellum.

Conclusions: In cases with non-plaque type dCJD, there could be a close relationship between the hyperintense signals on DWI and the sites of dural grafting. Plaque-type prions could have different patterns of propagation in human CNS.

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P126 Clinical and pathological characterization of “sporadic Creutzfeldt-Jakob disease” with histories of neurosurgery to identify iatrogenic cases

Dr. Tsuyoshi Hamaguchi1, Dr. Kenji Sakai1, Dr. Atsushi Kobayashi2, Professor Tetsuyuki Kitamoto3, Dr. Ryusuke Ae4, Professor Yosikazu Nakamura4, Professor Nobuo Sanjo5, Dr. Tadashi Tsukamoto6, Professor Hidehiro Mizusawa6, Professor Masahito Yamada1
1Department Of Neurology, Kanazawa University Hospital, Kanazawa, Japan, 2Laboratory of Comparative Pathology, Hokkaido University, Sapporo, Japan, 3Department of Neurological Science, Tohoku University, Sendai, Japan, 4Division of Public Health, Center for Community Medicine, Jichi Medical University, Shimotsuke, Japan, 5Department of Neurology and Neurological Science, Tokyo Medical and Dental University, Tokyo, Japan, 6Department of Neurology, National Center of Neurology and Psychiatry, Kodaira, Japan

Aims: Recently, we revealed that patients with plaque-type dura mater graft-associated Creutzfeldt-Jakob disease (dCJD) showed atypical molecular features characterized by methionine homozygote at codon 129 of the prion protein gene (PrP), intermediate type PrPSc, and Kuru plaques, called MMiK. Surprisingly, we discovered that 2 patients who had been diagnosed as having sporadic Creutzfeldt-Jakob disease (sCJD) showed clinical, pathological, and molecular features identical to MMiK type, suggesting possible iatrogenic transmission. In this study, the objective is to investigate the possibility of iatrogenic transmission of prion disease in patients who have been diagnosed as having sCJD.

Methods: We investigated 1836 patients who were diagnosed as having sCJD and 5 patients who were diagnosed as having unclassified type CJD because of insufficient information of dura mater grafting in the nationwide surveillance of CJD in Japan, and compared between those with and without histories of neurosurgeries.

Results: Among 1841 patients with sCJD, 51 had histories of neurosurgery, but 13 patients were excluded from the study because they underwent neurosurgeries within 1 year before or after the onset of CJD. Between the patients with and without histories of neurosurgery, positive rate (84.2%, 32/38 patients) of periodic sharp-wave complexes (PSWCs) on electroencephalogram (EEG) in the patients with histories of neurosurgery were significantly less than that (94.4%, 1677/1776 patients) in the patients without histories of neurosurgery, although age at onset of CJD, sex distribution, distribution of polymorphism at codon 129 of PrP, disease duration of CJD (duration between the onset of CJD and the appearance of the akinetic mutism or death in the patients who died without akintic mutism), and positive rate of 14-3-3 protein and total tau (cut off 1200 pg/ml) in cerebrospinal fluid were not significantly different. Among 38 sCJD patients with histories of neurosurgery, 9 (23.7%) had atypical CJD features that showed no PSWCs on EEG or had disease duration longer than 1 year. Three of the 9 atypical patients with histories of neurosurgery were autopsied, and one patient was MM2-thalamic form, one patient was MM2-cortical form, and another one patient was MMiK.

Conclusions: Among the patients who were diagnosed as sCJD or unclassified type CJD, some patients with histories of neurosurgery, but without dura mater grafting, had atypical clinical and neuropathological features similar to plaque type dCJD. Neuropathological study with PrP typing is essential to identify iatrogenic cases in patients with prion disease and history of neurosurgery.

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Iatrogenic CJD after human cadaver-sourced growth hormone treatment in France: identifying risk factors associated with susceptibility

Ms Laurene Peckeu1, PhD Dominique Costagliola4, MD Jean-Philippe Brandel1,2, MD, PhD Stephane Haïk1,2,3
1Institut Du Cerveau Et De La Moelle Épinière (ICM) - Team "alzheimer's And Prion Diseases "inserm Umr-1127/cnrs Umr 7225, Paris, France, 2AP-HP, Cellule Nationale de Référence des maladies de Creutzfeldt-Jakob, Groupe Hospitalier Pitié-Salpêtrière, Paris, France, 3AP-HP, Laboratoire de Neuropathologie R Escourolle, Groupe Hospitalier Pitié-Salpêtrière, Paris, France, 4Institut Pierre Louis d’Epidémiologie et de Santé Publique, Sorbonne Universités, Inserm et UPMC Univ Paris 06 (UMR_S 1136), Paris, France

Background: Human growth hormone (hGH) treatment is the main cause of iatrogenic CJD (iCJD-hGH) in France. Among the 1443 patients who received cadaver-sourced hGH during the high-risk period from December 1983 to July 1985, 119 patients developed Creutzfeldt-Jakob disease. In this study, risk factors associated with the susceptibility of developing iCJD-hGH were analysed.

Methods: Two multivariate cox analyses were performed on the 1443 subjects. The first one (Bonferroni correction, level of significance for univariate analysis at 0.0008) aimed to determine, among all the 63 batches dispensed during this period, those significantly associated with the risk of developing CJD. The second model aimed to assess the association of sex, age at beginning of treatment, treatment duration and number of doses received from each batch with susceptibility. In this model all variables individually associated with susceptibility at a significant level of p<0.1 were then included in the multivariate analysis (level of significance: p<0.05).

Results: Nineteen batches were identified after univariate analysis. Five of them were significantly associated with susceptibility after multivariate analysis. The number of doses delivered from batches identified at risk was statistically different (mean=55.3 for hGH-CJD group and 16.1 for non hGH-CJD group). iCJD-hGH was significantly more frequent in males (10%) compared to females (5%). Median age at the beginning of treatment was 12.0 years in non hGH-CJD group, and 12.1 in hGH-CJD group. Treatment duration was significantly higher in hGH-CJD group. Risk factors associated with the susceptibility identified by the second model were gender (HR, 1.70; 95%CI, 1.08-2.67) and the number of doses from the batches identified at risk by the first analysis (HR, 2.90; 95%CI, 2.22-3.79).

Discussion: In the final model, among the five batches that were significantly associated with susceptibility, four were previously reported as at risk (Huillard d’Aignaux et al., 1998). Of the 1443 patients, 550 were exposed to at least one of these five batches and only 18% (n=100) of them developed iCJD-hGH suggesting that additional risk factors were involved. The second model showed a significant association between sex, doses and susceptibility of developing iCJD-hGH. While the relationship between infectious dosis and attack rate is a well known phenomenon in experimental infectious models of prion diseases, this is the first evidence, to our knowledge, that it also applies in humans. Surprinsingly, we observed an effect of gender with a two-fold higher proportion in male that could not been explained by a difference in exposure.

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Lessons learned from the systematic public health follow-up of all diagnoses of CJD for surgical risk, in England

Rachel Glass1, Marta Checchi1, Dr Katy Sinka1
1Public Health England, London, United Kingdom

Aims: On average 100 new diagnoses of CJD are made each year in England. Around 80% of these will have had surgery in the past, some including tissues considered infective for CJD. Public health follow-up aims to identify these incidents and take action to reduce the risk of transmission through re-usable surgical instruments.

Methods: Since 2000, the surgical history of patients diagnosed with CJD has been reviewed and the transmission risk of invasive procedures assessed. This considers the type of CJD, type of procedure, tissues involved and time elapsed between the surgery and point at which CJD is suspected or diagnosed. The follow-up identifies 1) potentially contaminated instruments and 2) exposed surgical contacts. The risk assessment determines whether instruments should be quarantined or destroyed and whether informing exposed surgical contacts is advised. We used data collected by the CJD incidents panel (CJDIP) before 2013 and from the national Health Protection software system (HPZone) from 2013 to take stock of 16 years of public health follow-up for CJD.

Results: Between 2000 and 2013, 320 reports of surgery following clinical suspicion or diagnosis of CJD were reviewed by the CJDIP, approximately 30% of all CJD diagnoses during the period. Most reports (67%) concerned sporadic CJD, followed by variant CJD (13%) and familial CJD (6%). The remainder were non-CJD diagnoses after further investigation. The median number of surgical procedures per patient was two (range 1 to 17), almost all of which involved low infectivity tissues for CJD. In 19 instances surgery involved high or medium infectivity tissues and traceable patients, who were notified. Of these 12 concerned cataract operations and were later reclassified as low risk. From 2013 to 2016, 419 new reports of CJD were logged on HPZONE, aligning to 90% of CJD diagnoses during the period. Over 90% of these diagnoses were sporadic CJD, reflecting both the decline in variant CJD and the continuing increase in sporadic CJD diagnoses. Three incidents, involved high infectivity tissue and notification of surgical contacts. The median time from logging to closing the public health investigation on HPZONE was 1.7 months.

Conclusion: Systematic follow-up of new CJD diagnoses provides a cornerstone of the public health response to reduce the risk of healthcare related transmission of CJD. The process has evolved alongside changes in decontamination practice, infection control and surgical instrument traceability.

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P138 Rapid and highly sensitive detection of Variant Creutzfeldt - Jakob Disease prions binded on steel surfaces by PMCA: Application to Prion decontamination studies

Engineer Maxime Belondrade1, Dr Simon Nicot1, Dr Vincent Béringue2, Dr Joliette Coste1, Pr Sylvain Lehmann3, Dr Daisy Bougard1
1UMR 1058 Pathogénèse et Contrôle des Infections Chroniques (Inserm-EFS-Université de Montpellier) , Montpellier, France, 2Institut National de la Recherche Agronomique, UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France, 3CHRU de Montpellier and Université de Montpellier, IRMB, INSERM U1183, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France

Aims:
Among human prions, variant Creutzfeldt-Jakob (vCJD) is considered at risk of iatrogenic transmission due to its widespread tissue distribution, the existence of asymptomatic infected individuals, and the uncertainties surrounding the prevalence of the disease in the general population.
Due to the exceptional resistance to conventional hospital sterilization methods, prions remain a serious challenge for healthcare facilities especially regarding inactivation of re-usable medical devices, like endoscopes. Decontamination procedures were validated on animal-intensive, time-consuming and expensive bioassay infectivity studies using non-human prion strains. However, studies showed that inactivation procedures validated on rodent prions cannot be fully extrapolated to inactivation of human prions.
Our aim was to develop a rapid, sensitive and cost-effective assay for the detection of human vCJD prions adsorbed onto a surface for the evaluation of prion decontamination.

Methods:
Stainless Steel wires, used as carrier models of prions for inactivation studies by mimicking the surface of surgical instruments, were contaminated by contact with vCJD infected brain homogenate. The presence of residual prions after several decontamination methods were evaluated using the in vitro prion amplification technique called Protein Misfolding Cyclic Amplification (PMCA).

Results:
Using PMCA directly on steel-wires (Surf-PMCA) we were able to detect vCJD prions on a single wire infected with a 10-8 brain dilution. This technique has enabled us to evaluate on this human strain the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD prions was lower than expected.

Conclusions:
Our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of vCJD prions adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.

=====PLEASE NOTE, ENDOSCOPY AND TSE PRION...TSS=====

WEDNESDAY, MARCH 02, 2016 

Endoscope Maker Olympus Agrees To $646 Million Settlement Over Kickbacks, while still ignoring the elephant in the room, CJD TSE PRIONS

I told Olympus 15 years ago about these risk factors from endoscopy equipment, disinfection, even spoke with the Doctor at Olympus, this was back in 1999. I tried to tell them that they were exposing patients to dangerous pathogens such as the CJD TSE prion, because they could not properly clean them. even presented my concern to a peer review journal GUT, that was going to publish, but then it was pulled by Professor Michael Farthing et al... see history ;

Subject: CJD * Olympus Endoscope

Date: Sun, 10 Oct 1999 16:41:49 –0500

From: "Terry S. Singeltary Sr."

To: GOLDSS@...


=====END...TSS=====

PRION 2017 CONFERENCE ABSTRACTS HUMAN TSE PRION DISEASE CONTINUED

P61 vCJD strain properties in a Spanish mother and son replicate as those of a young UK case

Dr Abigail Diack1, Mrs Aileen Boyle, Dr Diane Ritchie2, Jesus de Pedro-Cuesta3, Alberto Rábano4, Prof Robert Will2, Prof Jean Manson1

1The Roslin Institute, Easter Bush, United Kingdom, 2National CJD Research & Surveillance Centre, Edinburgh, United Kingdom, 3National Center of Epidemiology and CIBERNED, Madrid, Spain, 4CIEN Tissue Bank, CIEN Foundation, and CIBERNED, Madrid, Spain

Aims
Since the first report in 1996, there have been 228 cases of vCJD worldwide. Comparative studies of vCJD cases have demonstrated similar clinical symptoms, patterns of neuropathology and biochemical phenotype between cases originating from different countries. Here we have investigated transmission characteristics of the first cases of vCJD in first-degree relatives, a mother and son who resided in Spain. Both individuals are in the older age range of reported vCJD cases and had relatively atypical early clinical symptoms when compared to UK cases.

Methods
A strain typing panel of wild-type mice was inoculated with brain homogenate prepared from both the Spanish mother (aged 64) and son (aged 41) and compared with a UK case of vCJD. Mice were assessed for clinical signs, neuropathology and PrPres glycoform profile.

Results
All three vCJD brain isolates transmitted successfully to the wild-type mouse panel with the appearance of clinical and pathological signs associated with TSE transmission to mice. Inocula from the Spanish mother and son showed the same temporal order of clinical endpoint in each mouse strain when compared with the UK case. The distribution of TSE vacuolation was consistent for each vCJD inocula however variability in the intensity of vacuolation distribution was apparent. This was most evident in the VM mice challenged with the Spanish mother and may be indicative of low titre. PrP deposition patterns were similar between inocula with variability in the intensity of PrP accumulation between mice observed both within and between groups. PrPres was detected in the brains of RIII and VM mice challenged with all three isolates with no differences detected.

Conclusions
This study of two first degree relatives with vCJD confirms that the same infectious TSE agent was responsible for both cases and is consistent with that of a UK case of vCJD and historical vCJD transmission data. This is the first strain characterisation study of an older individual with vCJD and highlights the need for awareness of vCJD in older age groups particularly in those presenting with atypical dementias. These findings add additional supporting evidence to the hypothesis that a single strain of TSE agent is responsible for vCJD cases regardless of geographical origin or age at infection and indirectly support the hypothesis of a dietary origin for primary cases of vCJD.

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P75 Strain characterisation of a PRNP codon 129MV individual reveals similar strain properties to those of codon 129MM individuals

Dr Abigail Diack1, Mr Chris Plinston1, Mrs Aileen Boyle1, Dr Diane Ritchie2, Miss Emma Hunt1, Dr Matthew Bishop2, Professor Robert Will2, Professor Jean Manson1

1The Roslin Institute and R(D)SVS, University of Edinburgh, Edinburgh, United Kingdom, 2National CJD Research and Surveillance Unit, Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom

Aims
The identification of vCJD in a PRNP 129 heterozygous individual in 2016 has raised concerns over the potential for the properties of the vCJD agent to change after transmission in a different genetic background. In 2004, a sub-clinical case of vCJD in a PRNP heterozygous individual infected via blood transfusion was reported. We now report the results of a full strain characterisation study of the PrP positive spleen tissue from this individual.

Methods
Spleen material from a PRNP 129MV individual was inoculated into a panel of wild-type mice. Serial passage was then performed by inoculating brain homogenate from a TSE positive mouse from each mouse strain into the wild-type mouse panel. This was then repeated for a second mouse passage. In each passage, a combination of clinical signs, neuropathology (TSE vacuolation and PrP deposition) and biochemical analysis were carried out.

Results
Primary passage of the spleen material into wild-type mice demonstrated that the spleen could propagate infectivity however clinical signs and both TSE vacuolation and PrP deposition were only observed in RIII mice whereas VM and C57BL/6 mice showed no or very limited vacuolation with positive transmission determined by PrP deposition. Upon sub- passage, transmission rates increased in all mouse lines but a lengthening in incubation periods and a change in incubation period rankings were observed. Minor differences in TSE vacuolation distribution were also apparent. A second sub-passage has now been completed; strain characteristics have stabilised and appear to be consistent to that of codon 129MM individuals and historical vCJD transmissions.

Conclusions
This is the first strain characterisation study of both a subclinical and PRNP heterozygous individual. Unlike previously published studies, transmission properties on primary passage demonstrated low transmission rates and a lack of both clinical and TSE vacuolation pathology. As the BSE/vCJD strain is unique in its stable properties at primary passage, serial passage of this individual was required in order to fully ascertain the strain. Following serial passage, strain characteristics have stabilised and are consistent with those of 129MM individuals. This study is the first to indicate that strain characterisation can be carried out during the subclinical or asymptomatic phase of disease and that strain properties of vCJD are not affected by the PRNP codon 129 genotype of an individual.

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P39 Identification of biomarkers for prion infection in blood.

Ms Ciara Farren1, Mr Boon Chin Tan1, Dr Dominic Kurian1, Dr Andrew Gill1, Dr Fiona Houston1
1Roslin Institute, Univeristy Of Edinburgh, Edinburgh, United Kingdom

Aims:
Surveys of archived human appendix tissue for PrPd deposition have suggested that up to 1/2000 of the UK population may be silent carriers of variant Creutzfeld-Jakob disease (vCJD) and thus could potentially transmit infection to others e.g. via blood transfusion. There is a lack of diagnostic/ screening tests that can be performed on easily accessible samples (e.g. blood, urine) to identify individuals with preclinical or subclinical infection. As an alternative to PrPSc detection, the aim of this project was to identify novel protein biomarkers in blood that could be used for diagnosis of prion diseases, using an archive of blood samples from sheep experimentally infected with bovine spongiform encephalopathy (BSE).

Methods:
Plasma samples collected from nine sheep before infection and 10 months after oral infection with BSE (when their blood was demonstrated to be infectious but they were showing no clinical signs of disease) were pooled to control for individual variation, giving two samples (uninfected and infected) for comparison. The samples were treated to selectively deplete highly abundant plasma proteins and enrich for less abundant proteins using ProteoMiner™ columns. Using isotopic labelling to distinguish between the two samples, differentially expressed proteins in the infected sample compared with the uninfected sample were evaluated by mass spectrometry. Proteins were quantified by comparing the peak ratio of the intensities on the mass spectrum to derive relative abundance.

Results:
Originally 153 proteins were identified. Identification of proteins based on more than one peptide match was ensured by removing those identified using fewer than 2 peptides. The coefficient variation (calculated by dividing the standard deviation of peptide profiles by the mean) was set to less than 100% to confirm variation between peptides was not too high. The extent of differential expression was assessed by filtering the median H/L (heavy/light isotopes) and proteins between >1.5 and <0.666 were retained, leaving a dataset of 44 proteins. The most common plasma proteins were eliminated and we now have a shortlist of 8 candidate proteins to follow up on.

Conclusions:
In order to validate a protein biomarker panel for the purpose of efficient blood-based diagnostics, western blots will be performed on the pooled sheep plasma samples to confirm up or down expression of proteins. This will be followed by further validation on plasma samples from individual sheep infected with BSE and scrapie, and at different time points during disease.

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P135 Accuracy of a history of blood donation from surrogate witnesses: data from the UK TMER study

Professor Robert Will1, Ms Jan MacKenzie1, Mr Vince Divine2, Dr Anna Molesworth1, Dr Suvankar Pal1, Dr Charlotte Llewelyn3, Dr Patricia Hewitt2
1NCJDRSU, Edinburgh, United Kingdom, 2NHS Blood and Transplant, London, UK, 3NHS Blood and Transplant, Cambridge, UK

Aims
Look-back studies of blood transfusion in Creutzfeldt-Jakob disease (CJD) commonly rely on reported history from surrogate witnesses. Data from the UK Transfusion Medicine Epidemiology Review (TMER) has been analysed to determine the accuracy of a history of blood donation provided by the relatives of cases.

Methods
Details of blood donation history are routinely sought through interview with family members, and have been obtained in the great majority of cases of CJD. Identifiers in all variant CJD cases eligible to donate (168/178) are circulated to the UK blood transfusion services (UKBS) to check whether the individual had donated. For other CJD cases, identifiers are normally forwarded only from those with a reported history of blood donation. In this analysis, all sporadic CJD cases in England for the period 2010-2016, regardless of reported history, were also checked against the national donor database, which was introduced in the late 1990s.
The actual blood donation data were compared with the information provided by relatives on donation history.

Results
In 32 variant CJD cases reported by relatives to have donated blood, 20 (63%) were traced as donors and in 17 blood components were issued. In 9 of 12 cases not traced, donation was reported prior to 1990, but 3 reported to have donated blood after 1990 could not be confirmed. In addition, 4 of 135 (3%) variant CJD cases reported to have not donated blood were registered as donors, with one having components issued.
Details of donation history were available in 500 of 554 sporadic CJD cases identified in England and 49 of 138 (36%) cases reported to have donated were traced as donors. The majority of those not traced had a date for donation predating available records. 362 cases were reported not to have donated blood and 11 (3%) of these had donated.

Conclusion
Reported history of blood donation from relatives is fairly accurate overall, but clearly a small proportion of cases reported to have been blood donors have not actually donated blood and vice versa. Look-back studies in CJD are unlikely to obtain complete information on blood donation history from family members and checking donation history through other sources may improve the accuracy of these studies.

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P46 Investigation of the effect of sCJD subtypes on the sensitivity of CSF RT-QuIC.

Doctor Neil McKenzie1, Doctor Lynne McGuire1, Mr Graham Fairfoul1, Ms Helen Yull1, Ms Laura Banks1, Ms Suzanne Lowrie1, Professor James Ironside1, Professor Robert Wills1, Professor Richard Knight1, Doctor Alison Green1

1National CJD Research and Surveillance Unit (NCJDRSU), Edinburgh, United Kingdom

Aims:
CSF RT-QuIC analysis has proven to be a sensitive and specific diagnostic test for the investigation of patients with suspected sporadic Creutzfeldt-Jakob disease (sCJD). There is increasing evidence that the sensitivity of CSF RT-QuIC may vary between sCJD subtypes. In this study we investigate the effect of sCJD subtypes on the sensitivity of CSF RT-QuIC.

Methods:
CSF samples from 117 neuropathologically confirmed sCJD patients with known codon 129 genotype and PrPSc isotypes were analysed using RT-QuIC using full-length Hamster PrP (Ha PrP FL) as previously described. Of these 117 sCJD patients: 64 (25M:39F; mean age ± SD: 69.1 ±9.6 years) were MM1; 9 (7M:2F; mean age ± SD: 69.7 ± 8.4 years) were MM2, 8 (3M:5F; mean age ± SD: 61.8 ± 12.0 years) were MV1; 14 (7M:7F; mean age ± SD: 66.9 ± 7.4 years) were MV2; 3 (2M:1F; mean age ± SD: 51.3 ± 24.5 years) were VV1 and 19 (7M:12F; mean age ± SD: 65.8 ± 9.6 years) were VV2. For each patient the positivity of the RT-QuIC was noted, alongside the final relative fluorescence units (RFU) at 90 hours was recorded.

Results:
The highest RT-QuIC sensitivity was seen with the MM1 and VV2 sCJD subtypes (92% and 100% respectively) and the lowest sensitivity was seen with the VV1 sCJD subtypes (33%). The MM2, MV1 and MV2 subtypes had intermediate sensitivities of 78%, 87% and 82%. The mean ± SD RFU for each of the subtypes was MM1: 28540 ± 15688; MM2: 26982 ± 15016; MV1: 22060 ± 11928; MV2: 28613 ± 15768; VV1: 13559 ± 6463 and VV2:34838 ± 15945. The mean RFU for the VV1 subtypes was significantly lower than those of the other sCJD subtypes, p <0.005.

Conclusions:
RT-QuIC has been shown to be a sensitive and specific test for diagnosis of sCJD which gives reproducible results in multi-centre trials. However the data described here show that the RPNP-codon 129 and PrPSc isotype may influence the positivity of RT-QuIC. The significance of this study is limited by the small numbers in each of the sCJD subtypes and further work is in progress to investigate these findings further.

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P73 Biological characterization of sporadic Creutzfeldt Jakob disease subtypes in bank voles and comparison with scrapie strains

DVM Romolo Nonno1, Michele Di Bari1, Laura Pirisinu1, Claudia D'Agostino1, Daniela Saverioni2, Ilaria Vanni1, Geraldina Riccardi1, Stefano Marcon1, Gabriele Vaccari1, Sabina Capellari2, Maurizio Pocchiari3, Piero Parchi2, Umberto Agrimi1

1Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità , Rome, Italy, 2IRCCS, Intitute of Neurological Sciences, Bologna, Italy, 3Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy

Aims:
Sporadic Creutzfeldt-Jakob disease (sCJD) represents around 90% of human prion cases. Although the precise cause of sCJD is unknown, it is supposed to derive from spontaneous PrP misfolding. Alternatively, sCJD might derive from exposure to animal or human prion sources. sCJD encompasses six subtypes, namely MM/MV1, VV2, MV2K, MM/MV2C, MM2T and VV1, which largely represent distinct prion strains. However, a comprehensive understanding of CJD strains and of their relationships with animal prion strain diversity is still lacking. Here we investigated the biological properties of sCJD in bank voles and compared them with scrapie strains.

Methods:
Sixteen sCJD isolates were inoculated in two genetic lines of bank voles, carrying isoleucine or methionine at PRNP codon 109 (Bv109I and Bv109M). Vole-adapted strains, defined as a set of disease phenotypes, were obtained upon second passages in congenic bank voles.

Results:
The transmission efficiency was highly variable, with the main source of variation being the sCJD subtype and codon 129 genotype. MM/MV1, MM2T and MM/MV2C cases transmitted with the highest efficiency, with MM/MV1 associated with survival times shorter in Bv109M than in Bv109I, while MM2T and MM/MV2C showed the opposite behavior. MV2K, VV1 and VV2 cases gave partial attack rates, with MV2K associated with survival times shorter in Bv109I than in Bv109M.

Upon second passages, six different vole-adapted strains were isolated. Two vole-adapted strains were, sometimes, isolated from some single isolates, suggesting the presence of different sub-strains or a strain mutation after passage in voles. MM/MV1, MM2T and VV2 were associated with three distinct vole-adapted strains. The MM/MV1 strain was also isolated from VV1 and one out of three MV2K. A minor strain component was isolated from single VV1 and MM2C cases. Two further strains were mostly isolated from MM/MV2C and MV2K, and frequently co-occurred in the same isolate.

Conclusions:
Overall, the relative transmission efficiency in Bv109M and Bv109I and the vole-adapted strain(s) isolated upon second passages confirms that sCJD subtypes are associated with distinct biological properties. However, a distinctive subtype/strain association was evident only for MM/MV1 and MM2T, and partially for VV2. VV1 mainly led to the isolation of the MM1 strain. A more complex scenario was associated with MM/MV2C and MV2K, which showed partially overlapping features.
The comparison with small ruminant strains suggest that classical scrapie and sCJD are associated with different prion strains. In contrast, some cases of the MM/MV2C subtype showed biological properties similar to small ruminant CH1641 isolates.

=====PLEASE NOTE, VERY IMPORTANT...TSS=====

***Our transmission study demonstrates that CH 1641-like scrapie is likely to be more virulent than classical scrapie in cattle. 

In the US, scrapie is reported primarily in sheep homozygous for 136A/171Q (AAQQ) and the disease phenotype is similar to that seen with experimental strain CH1641.


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P76 Atypical Creutzfeldt Jakob disease with PrP-amyloid plaques in white matter: Molecular characterization and transmission to bank voles show the M1 strain signature

Mr Marcello Rossi1, Ms Daniela Saverioni1, Mr Michele Di Bari2, Ms Sabina Capellari1, Ms Laura Pirisinu2, Mr Simone Baiardi3, Ms Annemieke Rozemuller4, Mr Romolo Nonno2, Mr Piero Parchi1,3
1IRCCS Istituto Delle Scienze Neurologiche di Bologna, Bologna, Italy, 2Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy, 3Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy, 4Department of Pathology, University Medical Center Utrecht, Utrecht, Utrecht, The Netherlands

Aims: PrP-amyloid plaques represent a pathological hallmark in CJD and show a strong correlation with both prion strain and PRNP genotype. Florid plaques, for example, are a distinctive feature of vCJD (BSE strain), while kuru-type plaques are the pathological hallmark of the CJD V2 strain in subjects carrying MV or MM at PRNP codon 129. Besides these well-defined phenotypes, initial evidence indicate that amyloid plaques (limited to the white matter) can be rarely seen in sCJD MM1. However, the factor/s determining such a distinctive histopathology in a subgroup of MM1 patients remain/s unknown.

Methods: We compared clinical features, type and regional distribution of histopathological changes and PrPSc phisico-chemical properties between 5 MM1 CJD cases showing PrP-amyloid plaques in white matter and a larger group of typical sCJD MM1 subjects. Furthermore, we compared the transmission properties of the index case with those of typical MM1 inocula by bioassay in two genetic lines of bank voles carrying either isoleucine or methionine at PRNP codon 109 (Bv109I and Bv109M, respectively).

Results: All five atypical CJD cases showed several PrP-amyloid plaques in subcortical white matter and a disease duration longer than 12 months. Three cases were classified as sCJD MM1, one as sCJD MM1+2C and one as gCJD E200K-MM1. Extensive immunoblot analyses did not reveal any significant differences in the electrophoretic mobility, proteinase-K sensitivity and thermo-stability of PrPSc between atypical CJD MM1 with plaques and classical CJD MM1. Likewise, transmission properties such as incubation time, lesion profile and PrPSc electrophoretic profile in bank voles did not show any remarkable differences between the two groups.

Conclusions: According to our data, the occurrence of PrP-amyloid plaques in the subcortical white matter of CJD MM1 (or MM1+2C) is not a strain-specific feature. Since cases lacking amyloid plaques may also manifest a prolonged disease course, unidentified, host-specific factors must also play a role, in addition to the long disease duration, in generating white matter PrP-amyloid plaques in CJD MM1.

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P221 Genome wide differential DNA methylation profiles in Creutzfeldt-Jakob disease peripheral blood

Luke Dabin1, Holger Hummerich1, Rachel Raybould2, John Collinge1, Emmanuelle Viré1, Simon Mead1
1 MRC Prion Unit, University College London, London, United Kingdom 2 Institute of Psychological Medicine & Clinical Neurosciences, Cardiff University, Cardiff, Wales

Aims: Epigenetic modifications can occur at the level of DNA, chromatin or RNA, and regulate the action of genes temporally and contextually. As well as organising cell differentiation and development, such modifications are known to play crucial roles in the mechanisms of complex diseases such as cancer, and can also serve as useful biomarkers. Of these modifications, DNA methylation has recently been implicated in neurodegenerative diseases such as Alzheimer’s disease, frontotemporal dementia and amyotrophic-lateral sclerosis. Such diseases share with prion diseases a trend towards sporadic, late-onset cases characterised by protein dysmetabolism and progressive loss of neurons.

In order to better understand the pathophysiology of prion diseases, we are combining knowledge gained from GWAS and gene expression studies with epigenetic analyses. Here we investigate genome-wide methylation in the blood of sporadic and variant prion disease patients compared to methylation in healthy control subjects. Our findings suggest the potential for novel and accessible peripheral biomarkers for prion neurodegeneration and further insight into the mechanisms of prion and prion-like diseases.

Methods: Pre-mortem blood samples from 116 sCJD patients and 20 vCJD patients were matched by sex and age with 111 cognitively normal controls. Genomic DNA was extracted and bisulphite treated (Zymo EZ Methylation-Gold kit). Bisulphite treated DNA was hybridised to the Illumina Infinium 450k array. Raw array intensity files were imported into R and normalised in ChAMP. ComBat was used to correct for batch effects and a reference-based correction method was employed to account for cell-type heterogeneity. A linear regression model including age and sex as covariates was used to calculate percentage change in methylation. Methylation was correlated with a range of clinical parameters from the National Prion Monitoring Cohort study.

Results: After correction for multiple testing in sCJD, significant differences to DNA methylation profiles were found at 25 sites between cases and controls. Decreased methylation at some of these sites also correlated with disease severity measured by the MRC Prion Disease Rating Scale. A separate analysis of cases only suggested sites at which altered methylation predicted the rate of disease progression. There were fewer significant findings in vCJD possibly because of small sample size.

Conclusions: By defining changes in the blood methylome of sCJD we aim to contribute to differential diagnosis, prognosis and understanding pathophysiology of the diseases. These results may be relevant to other neurodegenerative disorders involving propagation of proteopathic seeds.

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Transmissible prions in the skin of Creutzfeldt-Jakob disease patients

Dr. Wenquan Zou1, Dr. Christina Orru2, Jue Yuan1, Brian Appleby1, Baiya Li1, Dane Winner1, Yian Zhan1,3, Mark Rodgers1, Jason Rarick1, Robert Wyza1, Tripti Joshi1, Gongxian Wang3, Mark Cohen1, Shulin Zhang1, Bradley Groveman2, Robert Petersen1, James Ironside4, Miguel Quinones-Mateu1, Jiri Safar1, Qingzhong Kong1, Byron Caughey2

1Case Western Reserve University, Cleveland, United States, 2Rocky Mountain Laboratories, National Institutes of Health, Hamilton, United States, 3Nanchang University, Nanchang, China, 4University of Edinburgh, Edinburgh, United Kingdom

Aims: Sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is transmissible by neuroinvasive iatrogenic routes due to abundant prion infectivity in the central nervous system (CNS). The disease-associated prion protein (PrPSc) and its infectivity have never been detected in skin from sCJD patients; however, some epidemiological studies have associated sCJD risk with skin-involved non-CNS surgeries. The aims of our study were to explore potential prion seeding activity and infectivity of skin and the feasibility of skin-based CJD diagnosis.

Methods: Skin samples were collected at autopsy or biopsy from twenty-one sCJD, two variant CJD, and fifteen non-CJD patients and analysed by Western blotting and real-time quaking-induced conversion (RT-QuIC) for PrPSc. Infectivity of skin from two sCJD patients was determined by bioassay using two lines of humanized transgenic (Tg) mice.

Results: Western blotting demonstrated PrPSc in the skin of one of five deceased sCJD patients examined. However, the more sensitive RT-QuIC assay detected prion-seeding activity in skin from all 23 CJD decedents but not in non-CJD controls, indicating preliminary CJD diagnostic sensitivities and specificities of 100% (95% confidence intervals of 85-100%, and 78-100%, respectively). Although sCJD skins contained ~102-105-fold lower RT-QuIC seeding activity than sCJD brains, ten out of twelve mice from two Tg mouse lines inoculated with skin homogenates of two patients with two different subtypes of sCJD succumbed to prion disease within 450 days after inoculation.

Conclusions: sCJD patients’ skin may contain both detectable prion seeding activity and transmissible prions. Our findings not only suggest a new basis for diagnostic sCJD testing, but also raise concerns about the potential for iatrogenic sCJD transmission via skin. 

(Funded by the CJD Foundation, the National Institute of Neurological Disorders and Stroke, the Centers for Disease Control and Prevention, as well as others)

***Conclusions: sCJD patients’ skin may contain both detectable prion seeding activity and transmissible prions. Our findings not only suggest a new basis for diagnostic sCJD testing, 

***but also raise concerns about the potential for iatrogenic sCJD transmission via skin. 

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P31 CJD International Support Alliance (CJDISA) – The voice, the face, the human story behind this horrific disease.

Mrs. Deana Simpson1,2, Mrs. Suzanne Solvyns1,3

1CJD International Support Alliance, , , 2CJD Insight, Macomb, United States, 3CJD Support Group Network Australia, Glenahven, Australia

Aims:
The CJD International Support Alliance (CJDISA) was formed by a group of grassroots nonprofit organizations that work together as an international coalition on behalf of patients and families affected by prion disease and those at risk the world over. CJDISA was founded to fill the gap that exists on an international level and to assure excellence in the service to individuals affected/at risk of prion disease, their families, and caregivers. The participating organizations are dedicated to work together in meeting the educational, social, emotional, spiritual and practical needs of those they represent. Under the CJDISA umbrella, these organizations collaborate on educational initiatives, information dissemination, resource allocation, program design and implementation, and advocacy.

Methods:
The CJDISA was founded on the belief that by raising awareness of prion disease and educating healthcare professionals and the public at large, we could:

• Help remove the stigma surrounding CJD and other prion
diseases

• Promote research activities around early detection, prevention,
treatment opportunities, improved quality of life and ultimately
a cure

• Increase proper utilization of resources

• Promote continued access to care for those in active disease
or those at risk for prion disease

• Assist in ensuring safe blood and food supply.

Results:
Our work is guided by 31 prion researchers and professionals and form the ‘Friends and Advisors Group’ of the CJDISA. These dedicated and compassionate individuals offer their expert guidance on various programs and services established by the CJDISA, provide lay interpretation of research articles so that families can understand how that research may or may not apply to them, and they act as knowledgeable resources for countries that don’t have a CJD support organization yet have patients and families in need of assistance.

A priority for the CJDISA is to assist countries in the establishment of CJD Support Organizations wherever possible. We currently have nine Member Organizations and five Associate Member Organizations in 13 Countries. Having support organizations in each country is critical in order to provide timely and accurate assistance to patients and families in their native country. During time of great stress and sadness families deserve the most knowledgeable resources guiding them through the tragedies of prion disease.

Conclusions:
Most member organizations are run by individuals directly impacted by prion disease. We are the voice, the face, the human story behind this horrific disease – representing those impacted by prion disease on a global level.

=====

P130 Human prion disease surveillance in Washington state, 2006-2015

Dr. Liliana Sanchez-Gonzalez1, Dr. Ryan Maddox2, Ms. Janis Blevins3, Ms. Elizabeth Harker2, Dr. Jiri Safar3, Dr. Lawrence Schonberger2, Dr. Ermias Belay2, Mr. Chas DeBolt1, Dr. Kathy Lofy1
1Washington State Department of Health, Shoreline, United States, 2Centers for Disease Control and Prevention (CDC), Atlanta, United States, 3National Prion Disease Pathology Surveillance Center (NPDPSC), Cleveland, United States

Aim: To report human prion disease surveillance findings from the U.S. state of Washington, the only state in the country where classic bovine spongiform encephalopathy (BSE), in a dairy cow in 2003, has been identified. Because a presumptive diagnosis of BSE was not made until two weeks after this animal was slaughtered, the carcass was released for use in food after routine removal of tissues considered to be at high-risk of transmission of the BSE agent (e.g., brain, spinal cord, small intestine). Although beef from cattle slaughtered in the same plant on the same day as the BSE-positive cow was recalled, some meat products were distributed to locations in Washington and several other states.

Methods: A state-wide surveillance system consisting of 35 local health jurisdictions has been in place in Washington since 2004. Suspected human prion disease cases reported from multiple surveillance sources are investigated, and information regarding clinical presentation, tests results, and confirmatory pathology testing is reviewed and analyzed. Cases are classified based on the World Health Organization Creutzfeldt-Jakob disease (CJD) case definition and Centers for Disease Control and Prevention diagnostic criteria for variant CJD (vCJD), with an emphasis on excluding the possibility of a vCJD diagnosis. Findings from 2006 to 2015 are presented.

Results: During 2006-2015, 111 human prion disease cases among Washington state residents were reported and investigated: 103 sporadic cases (73 confirmed through neuropathological analysis, 26 probable CJD, 4 possible CJD), 7 genetic prion disease cases (6 familial CJD, 1 Gerstmann-Straüssler-Scheinker syndrome), and 1 iatrogenic CJD case. Excluding the 4 possible CJD cases, the average annual age-adjusted incidence was 1.4 per million. The highest incidence of cases was in people aged 60–79; among the neuropathology-confirmed sporadic cases, 40 (55%) were methionine homozygous, 13 (18%) were valine homozygous, and 20 (27%) were heterozygous at codon 129 of the PRNP gene. The iatrogenic CJD case, attributed to contaminated human growth hormone received as a child, had an atypical neuropathological phenotype that likely represents a specific neuropathological finding for iatrogenic CJD versus sporadic CJD. None of the reported cases was found to meet the criteria for a vCJD diagnosis.

Conclusions: The incidence of prion diseases among Washington state residents during the 10-year period was consistent with previous national reports. No evidence for vCJD was found in the state; however, given the long incubation periods associated with the disease, continued active surveillance is recommended.

=====

P165 Transmission of macaque prion strains to transgenic mice expressing simian PrP

Dr Emmanuel Comoy1, Prof Jacqueline Mikol1, Jérome Delmotte1, Steve Cottin1, Dr Jean-Philippe Deslys1
1Commissariat à l'Energie Atomique, Fontenay-aux-roses, France

Aims: Cynomolgus macaques are considered as one of the utmost model of human situation towards prion infection, according to their phylogenetic and physiological proximities with human. This model provided the first experimental evidence of the transmission of Bovine Spongiform Encephalopathy (BSE) to human, and oral and intravenous exposures of macaques provided important clues for primary and secondary risk assessment of BSE for human health. Moreover, we recently described the transmission of BSE-L and classical scrapie to macaques, questioning the zoonotic potential of those animal prions.

However, this primate model with long incubation periods (2 to more than 10 years) is not appropriate to perform secondary transmission experiments that moreover need several animals. We thus developed, as alternative models to macaques, transgenic mice expressing the macaque PRNP gene (TgMac) for assessing the distribution of infectivity in organs of macaques and characterizing the prion strains transmitted to macaques.

Methods: TgMac mice were generated by direct microinjection of DNA coding for the macaque PrP gene into C57Bl/6 pronuclei, according to classical transgenetic techniques as previously described (Castilla et al., Arch. Virol. 2003, 148:677). Resulting mice were back-crossed with PrP knock-out mice (Manson et al. Mol. Neurobiol. 1994, 8:121) to eliminate endogenous murine PrP. TgMac mice were inoculated with CNS samples derived from macaques infected with c-BSE, L-BSE, scrapie, sporadic CJD and vCJD strains, and myelopathic primates. Spongiform changes were scored on sections of paraffin-embedded, formalin-fixed brains and PrPres was detected using rapid tests (Bio-Rad).

Results: The different prion strains issued from macaques induced neurological signs in TgMac mice after incubation periods of at least nine months. Clinically affected mice exhibited spongiform changes, and PrPres was detectable using TeSeE rapid tests, confirming the transmissibility of prions from macaques to TgMac. The susceptibility of these transgenic mice to those different prion strains is compared to other conventional or transgenic mice models.

Conclusions: We developed a new transgenic model of mice complementary to the macaque model. The combination of these macaque and transgenic mouse models should provide important information in the risk assessment for public health of animal prion diseases (primary risk) and inter-human transmission (secondary iatrogenic risks).

=====WOW=====


PLEASE NOTE, SPORADIC CJD HAS NOW BEEN LINKED TO TYPICAL AND ATYPICAL BSE, TYPICAL AND ATYPICAL SCRAPIE, AND NOW TO CWD TSE PRION, STILL GOING BY THE UKBSEnvCJD ONLY THEORY IS LIKE STILL LIVING IN THE STONE AGES...SEE SOURCE REFERENCES AT THE BOTTOM THAT CONFIRMS THIS...TSS

=====


SPONTANEOUS ATYPICAL BOVINE SPONGIFORM ENCEPHALOPATHY

***Moreover, sporadic disease has never been observed in breeding colonies or primate research laboratories, most notably among hundreds of animals over several decades of study at the National Institutes of Health25, and in nearly twenty older animals continuously housed in our own facility.***

Saturday, April 23, 2016 

PRION 2016 TOKYO Saturday, April 23, 2016 

SCRAPIE WS-01: Prion diseases in animals and zoonotic potential 2016 Prion. 10:S15-S21. 2016 ISSN: 1933-6896 printl 1933-690X online Taylor & Francis Prion 2016 Animal Prion Disease Workshop 

Abstracts 

WS-01: Prion diseases in animals and zoonotic potential 

Juan Maria Torres a, Olivier Andreoletti b, J uan-Carlos Espinosa a. Vincent Beringue c. Patricia Aguilar a, Natalia Fernandez-Borges a. and Alba Marin-Moreno a "Centro de Investigacion en Sanidad Animal ( CISA-INIA ). Valdeolmos, Madrid. Spain; b UMR INRA -ENVT 1225 Interactions Holes Agents Pathogenes. ENVT. Toulouse. France: "UR892. Virologie lmmunologie MolécuIaires, Jouy-en-Josas. France 

Dietary exposure to bovine spongiform encephalopathy (BSE) contaminated bovine tissues is considered as the origin of variant Creutzfeldt Jakob (vCJD) disease in human. To date, BSE agent is the only recognized zoonotic prion. Despite the variety of Transmissible Spongiform Encephalopathy (TSE) agents that have been circulating for centuries in farmed ruminants there is no apparent epidemiological link between exposure to ruminant products and the occurrence of other form of TSE in human like sporadic Creutzfeldt Jakob Disease (sCJD). However, the zoonotic potential of the diversity of circulating TSE agents has never been systematically assessed. The major issue in experimental assessment of TSEs zoonotic potential lies in the modeling of the ‘species barrier‘, the biological phenomenon that limits TSE agents’ propagation from a species to another. In the last decade, mice genetically engineered to express normal forms of the human prion protein has proved essential in studying human prions pathogenesis and modeling the capacity of TSEs to cross the human species barrier. To assess the zoonotic potential of prions circulating in farmed ruminants, we study their transmission ability in transgenic mice expressing human PrPC (HuPrP-Tg). Two lines of mice expressing different forms of the human PrPC (129Met or 129Val) are used to determine the role of the Met129Val dimorphism in susceptibility/resistance to the different agents. These transmission experiments confirm the ability of BSE prions to propagate in 129M- HuPrP-Tg mice and demonstrate that Met129 homozygotes may be susceptible to BSE in sheep or goat to a greater degree than the BSE agent in cattle and that these agents can convey molecular properties and neuropathological indistinguishable from vCJD. However homozygous 129V mice are resistant to all tested BSE derived prions independently of the originating species suggesting a higher transmission barrier for 129V-PrP variant. Transmission data also revealed that several scrapie prions propagate in HuPrP-Tg mice with efficiency comparable to that of cattle BSE. While the efficiency of transmission at primary passage was low, subsequent passages resulted in a highly virulent prion disease in both Met129 and Val129 mice. Transmission of the different scrapie isolates in these mice leads to the emergence of prion strain phenotypes that showed similar characteristics to those displayed by MM1 or VV2 sCJD prion. 

***These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.


Title: Transmission of scrapie prions to primate after an extended silent incubation period) *** 

In complement to the recent demonstration that humanized mice are susceptible to scrapie, we report here the first observation of direct transmission of a natural classical scrapie isolate to a macaque after a 10-year incubation period. Neuropathologic examination revealed all of the features of a prion disease: spongiform change, neuronal loss, and accumulation of PrPres throughout the CNS. 

*** This observation strengthens the questioning of the harmlessness of scrapie to humans, at a time when protective measures for human and animal health are being dismantled and reduced as c-BSE is considered controlled and being eradicated. 

*** Our results underscore the importance of precautionary and protective measures and the necessity for long-term experimental transmission studies to assess the zoonotic potential of other animal prion strains. 


SCRAPIE WS-01: Prion diseases in animals and zoonotic potential 2016 

Prion. 10:S15-S21. 2016 ISSN: 1933-6896 printl 1933-690X online 



O.05: Transmission of prions to primates after extended silent incubation periods: Implications for BSE and scrapie risk assessment in human populations 

Emmanuel Comoy, Jacqueline Mikol, Valerie Durand, Sophie Luccantoni, Evelyne Correia, Nathalie Lescoutra, Capucine Dehen, and Jean-Philippe Deslys Atomic Energy Commission; Fontenay-aux-Roses, France 

Prion diseases (PD) are the unique neurodegenerative proteinopathies reputed to be transmissible under field conditions since decades. The transmission of Bovine Spongiform Encephalopathy (BSE) to humans evidenced that an animal PD might be zoonotic under appropriate conditions. Contrarily, in the absence of obvious (epidemiological or experimental) elements supporting a transmission or genetic predispositions, PD, like the other proteinopathies, are reputed to occur spontaneously (atpical animal prion strains, sporadic CJD summing 80% of human prion cases). Non-human primate models provided the first evidences supporting the transmissibiity of human prion strains and the zoonotic potential of BSE. Among them, cynomolgus macaques brought major information for BSE risk assessment for human health (Chen, 2014), according to their phylogenetic proximity to humans and extended lifetime. We used this model to assess the zoonotic potential of other animal PD from bovine, ovine and cervid origins even after very long silent incubation periods. 

*** We recently observed the direct transmission of a natural classical scrapie isolate to macaque after a 10-year silent incubation period, 

***with features similar to some reported for human cases of sporadic CJD, albeit requiring fourfold long incubation than BSE. Scrapie, as recently evoked in humanized mice (Cassard, 2014), 

***is the third potentially zoonotic PD (with BSE and L-type BSE), 

***thus questioning the origin of human sporadic cases. We will present an updated panorama of our different transmission studies and discuss the implications of such extended incubation periods on risk assessment of animal PD for human health. 

=============== 

***thus questioning the origin of human sporadic cases*** 

***our findings suggest that possible transmission risk of H-type BSE to sheep and human. Bioassay will be required to determine whether the PMCA products are infectious to these animals. 


LOOKING FOR CWD IN HUMANS AS nvCJD or as an ATYPICAL CJD, LOOKING IN ALL THE WRONG PLACES $$$ 

*** These results would seem to suggest that CWD does indeed have zoonotic potential, at least as judged by the compatibility of CWD prions and their human PrPC target. Furthermore, extrapolation from this simple in vitro assay suggests that if zoonotic CWD occurred, it would most likely effect those of the PRNP codon 129-MM genotype and that the PrPres type would be similar to that found in the most common subtype of sCJD (MM1).*** 




*** The potential impact of prion diseases on human health was greatly magnified by the recognition that interspecies transfer of BSE to humans by beef ingestion resulted in vCJD. While changes in animal feed constituents and slaughter practices appear to have curtailed vCJD, there is concern that CWD of free-ranging deer and elk in the U.S. might also cross the species barrier. Thus, consuming venison could be a source of human prion disease. Whether BSE and CWD represent interspecies scrapie transfer or are newly arisen prion diseases is unknown. Therefore, the possibility of transmission of prion disease through other food animals cannot be ruled out. There is evidence that vCJD can be transmitted through blood transfusion. There is likely a pool of unknown size of asymptomatic individuals infected with vCJD, ***

*** and there may be asymptomatic individuals infected with the CWD equivalent. 

*** These circumstances represent a potential threat to blood, blood products, and plasma supplies. 


SCRAPIE WS-01: Prion diseases in animals and zoonotic potential 2016

Prion. 10:S15-S21. 2016 ISSN: 1933-6896 printl 1933-690X online




why do we not want to do TSE transmission studies on chimpanzees $ 

5. A positive result from a chimpanzee challenged severly would likely create alarm in some circles even if the result could not be interpreted for man. I have a view that all these agents could be transmitted provided a large enough dose by appropriate routes was given and the animals kept long enough. Until the mechanisms of the species barrier are more clearly understood it might be best to retain that hypothesis. 

snip... 

R. BRADLEY 


Subject: PRION 2017 CONFERENCE ABSTRACT First evidence of intracranial and peroral transmission of Chronic Wasting Disease (CWD) into Cynomolgus macaques: a work in progress

First evidence of intracranial and peroral transmission of Chronic Wasting Disease (CWD) into Cynomolgus macaques: a work in progress
Stefanie Czub1, Walter Schulz-Schaeffer2, Christiane Stahl-Hennig3, Michael Beekes4, Hermann Schaetzl5 and Dirk Motzkus6 1 

University of Calgary Faculty of Veterinary Medicine/Canadian Food Inspection Agency; 2Universitatsklinikum des Saarlandes und Medizinische Fakultat der Universitat des Saarlandes; 3 Deutsches Primaten Zentrum/Goettingen; 4 Robert-Koch-Institut Berlin; 5 University of Calgary Faculty of Veterinary Medicine; 6 presently: Boehringer Ingelheim Veterinary Research Center; previously: Deutsches Primaten Zentrum/Goettingen 

This is a progress report of a project which started in 2009. 21 cynomolgus macaques were challenged with characterized CWD material from white-tailed deer (WTD) or elk by intracerebral (ic), oral, and skin exposure routes. Additional blood transfusion experiments are supposed to assess the CWD contamination risk of human blood product. Challenge materials originated from symptomatic cervids for ic, skin scarification and partially per oral routes (WTD brain). Challenge material for feeding of muscle derived from preclinical WTD and from preclinical macaques for blood transfusion experiments. We have confirmed that the CWD challenge material contained at least two different CWD agents (brain material) as well as CWD prions in muscle-associated nerves. 

Here we present first data on a group of animals either challenged ic with steel wires or per orally and sacrificed with incubation times ranging from 4.5 to 6.9 years at postmortem. Three animals displayed signs of mild clinical disease, including anxiety, apathy, ataxia and/or tremor. In four animals wasting was observed, two of those had confirmed diabetes. All animals have variable signs of prion neuropathology in spinal cords and brains and by supersensitive IHC, reaction was detected in spinal cord segments of all animals. Protein misfolding cyclic amplification (PMCA), real-time quaking-induced conversion (RT-QuiC) and PET-blot assays to further substantiate these findings are on the way, as well as bioassays in bank voles and transgenic mice. 

At present, a total of 10 animals are sacrificed and read-outs are ongoing. Preclinical incubation of the remaining macaques covers a range from 6.4 to 7.10 years. Based on the species barrier and an incubation time of > 5 years for BSE in macaques and about 10 years for scrapie in macaques, we expected an onset of clinical disease beyond 6 years post inoculation. 

PRION 2017 DECIPHERING NEURODEGENERATIVE DISORDERS

Chronic Wasting Disease CWD TSE Prion to Humans, who makes that final call, when, or, has it already happened?

IF human transmission studies are unethical and will never take place, how much evidence is enough, and how much exposure do we allow, before a call is made. 

HOW many humans do we expose before enough is enough?

How many body bags now are enough, for a very long incubating disease that the body bags will for sure mount later, if something is NOT finally done NOW.

the public must know.

Now, i will tell you all how this will be interpreted by our fine federal friends, and their lobbyist et al from corporate America, and Doctors there from, here is how this will still read, rubber stamped ;

''There is no direct evidence that CWD can transmit to humans, and CWD has never been identified in humans anywhere in the world, including in areas where CWD has been present in animal populations for decades.''

this is absurd, and fake news at it's finest.

what is 'direct evidence', if human transmission is not possible?

there is more than enough evidence to make that call now. 

with that, who will finally make that judgement call, knowing that if cwd transmits to humans, it will look like the most common human tse prion i.e. sporadic cjd?

who makes that final call, when, and how many more humans must die before that decision is made and put in the public domain so we can go on with this and try to implement rules and regulations that might finally turn the tide, or do just let corporate science run rampant? 

or, will they continue to run with the infamous UKBSEnvCJD only theory$

with cwd now being documented to transmit macaque, AND TO PIGS orally (lot of human medicine made from pigs), the price of continuing to play TSE Prion Poker with humans goes up drastically. 

This is criminal negligence now, imo...terry

*** Subject: CWD TRANSMITS TO MACAQUE ORALLY MUSCLE INTAKE ***

Notice to Members Regarding Chronic Wasting Disease (CWD)

Posted on: May 31st, 2017 

To: MNA Members From: Métis Nation of Alberta 

Date: Wednesday, May 31, 2017 

*** Métis Nation of Alberta (MNA) was made aware of a recent Canadian research study examining the transmission of Chronic Wasting Disease. The initial results of the study indicate that macaque monkeys (genetically similar to humans) can be infected with Chronic Wasting Disease (CWD) after eating deer that is infected with CWD. CWD is a prion disease, which are fatal, transmissible diseases characterized by abnormal proteins in the brain and nervous system. To date no research has shown that CWD can be passed on to humans, and no human cases of CWD have ever been identified. However, this new research indicates that it is a possibility. The Deputy Chief Medical Officer of Health has reached out to us to share with our Métis harvesters this important information. For more information you can visit:


Chronic Wasting Disease: CFIA Research Summary 

 Embargoed until May 23, 2017 

(OCR of a scanned original) 

Research Findings 

Chronic Wasting Disease (CWD) is a progressive, fatal disease of the nervous system of cervids including deer, elk, moose, and reindeer that is caused by abnormal proteins called prions. It is known as a transmissible spongiform encephalopathy (TSE). Other TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans.

A limited number of experimental studies have demonstrated that non-human primates, specifically squirrel monkeys, are susceptible to CWD prions. An ongoing research study has now shown that CWD can also be transmitted to macaques, which are genetically closer to humans. 

The study led by Dr. Stefanie Czub, a scientist at the Canadian Food Inspection Agency (CFIA), and funded by the Alberta Prion Research institute has demonstrated that by orally administering material under experimental conditions from cervids (deer and elk) naturally infected with CWD, the disease can be transmitted to macaques. 

in this project, which began in 2009, 18 macaques were exposed to CWD in a variety of ways: by injecting into the brain, through contact with skin, oral administration, and intravenously (into the bloodstream through veins). So far, results are available from 5 animals. At this point, two animals that were exposed to CWD by direct introduction into the brain, one that was administered infected brain material by oral administration and two that were given infected muscle by oral administration have become infected with CWD. The study is ongoing and testing continues in the remaining animals. The early results will be presented at PRlON 2017, the annual international conference on prion diseases, in Edinburgh, Scotland, May 23 to 26, 2017. 

Potential impacts of the new finding

Since 2003 Canada has a policy that recommends that animals and materials known to be infected with prions be removed from the food chain and from health products. Although no direct evidence of CWD prion transmission to humans has ever been recorded, the policy advocates a precautionary approach to managing CWD and potential human exposure to prions. These initial findings do not change Health Canada’s Health Products and Food Branch (HPFB) position on food and health products. A precautionary approach is still recommended to manage the potential risks of exposure to prions through food and health products. Measures are in place at federal, provincial and territorial levels to reduce human exposure to products potentially contaminated by CWD by preventing known infected animals from entering the marketplace. 

While Federal and P/T government’s animal disease control policies continue to divert known CWD-infected animals away from entering the food and feed supply, research and development of sensitive detection methods including live-animal sampling techniques remain crucial for ensuring an accurate diagnosis. In addition, consistent federal, provincial and territorial communications of appropriate precautionary measures for hunters and indigenous communities are required. 

Next Steps

The CFlA will continue to collaborate with national and international partners to develop and validate new diagnostic techniques. The CFlA will also continue to offer confirmatory testing services and reference laboratory expertise to provincial and territorial partners on demand. 

Currently, CFlA laboratories are leading or collaborating on several research projects to understand the potential for CWD to infect humans. These projects use non‐human primates, genetically modified mice, and cell-free amplification approaches. Given the present findings, CFiA encourages continued research into TSEs. 

The results of this study reinforce the need to redesign the federal program to foster greater adoption of risk mitigation measures for farmed cervids. Federal and provincial government collaboration will continue as new program options are assessed. 

The results of Dr. Czub’s research into CWD will be of interest to scientists, governments, industry and people who consume cervid products. After the presentation at PRION 2017, the research will follow the normal steps of completion, peer review and publication. The Government of Canada will monitor the response to this research and determine whether further review of the science is required. Other studies underway by other researchers may also become public as a result of the presentation of Dr. Czub’s research. 

The Public Health Agency of Canada, Health Canada, CFlA and other Federal partners are working together to assess what policies or programs need further review as well as preparing other communications about the research and health policy and advice to Canadian. 2017/04/28 

===end...UNOFFICIAL...NO URL LINK...TSS===UPDATE, THE ABOVE INTERNAL DOCUMENT HAS NOW BEEN CONFIRMED, but still no link...TSS===

0:30 First evidence of intracranial and peroral transmission of Chronic Wasting Disease (CWD) into Cynomolgus macaques: a work in progress 

Dr Stefanie Czub University of Calgary Faculty of Veterinary Medicine/Canadian Food Inspection Agency Canada 


see science to date that the call should be made NOW, that cwd to humans is possible, and all precautions there fore, should be take will great urgency.

WEDNESDAY, MAY 03, 2017

*** First evidence of intracranial and peroral transmission of Chronic Wasting Disease (CWD) into Cynomolgus macaques


LOOKING FOR CWD IN HUMANS AS nvCJD or as an ATYPICAL CJD, LOOKING IN ALL THE WRONG PLACES $$$

*** These results would seem to suggest that CWD does indeed have zoonotic potential, at least as judged by the compatibility of CWD prions and their human PrPC target. Furthermore, extrapolation from this simple in vitro assay suggests that if zoonotic CWD occurred, it would most likely effect those of the PRNP codon 129-MM genotype and that the PrPres type would be similar to that found in the most common subtype of sCJD (MM1).***


Molecular Barriers to Zoonotic Transmission of Prions

 *** chronic wasting disease, there was no absolute barrier to conversion of the human prion protein.

 *** Furthermore, the form of human PrPres produced in this in vitro assay when seeded with CWD, resembles that found in the most common human prion disease, namely sCJD of the MM1 subtype.



*** The association between venison eating and risk of CJD shows similar pattern, with regular venison eating associated with a 9 FOLD INCREASE IN RISK OF CJD (p = 0.04). ***

*** The association between venison eating and risk of CJD shows similar pattern, with regular venison eating associated with a 9 FOLD INCREASE IN RISK OF CJD (p = 0.04). ***

*** The association between venison eating and risk of CJD shows similar pattern, with regular venison eating associated with a 9 FOLD INCREASE IN RISK OF CJD (p = 0.04). ***

There is some evidence that risk of CJD INCREASES WITH INCREASING FREQUENCY OF LAMB EATING (p = 0.02).

The evidence for such an association between beef eating and CJD is weaker (p = 0.14). When only controls for whom a relative was interviewed are included, this evidence becomes a little STRONGER (p = 0.08).

snip...

It was found that when veal was included in the model with another exposure, the association between veal and CJD remained statistically significant (p = < 0.05 for all exposures), while the other exposures ceased to be statistically significant (p = > 0.05).

snip...

In conclusion, an analysis of dietary histories revealed statistical associations between various meats/animal products and INCREASED RISK OF CJD. When some account was taken of possible confounding, the association between VEAL EATING AND RISK OF CJD EMERGED AS THE STRONGEST OF THESE ASSOCIATIONS STATISTICALLY. ...

snip...

In the study in the USA, a range of foodstuffs were associated with an increased risk of CJD, including liver consumption which was associated with an apparent SIX-FOLD INCREASE IN THE RISK OF CJD. By comparing the data from 3 studies in relation to this particular dietary factor, the risk of liver consumption became non-significant with an odds ratio of 1.2 (PERSONAL COMMUNICATION, PROFESSOR A. HOFMAN. ERASMUS UNIVERSITY, ROTTERDAM). (???...TSS)

snip...see full report ;

http://collections.europarchive.org/tna/20090505194948/http://bseinquiry.gov.uk/files/yb/1994/08/00004001.pdf

you can see more evidence here ;

http://chronic-wasting-disease.blogspot.com/2016/05/zoonotic-potential-of-cwd-prions-update.html

Wednesday, May 24, 2017 

PRION2017 CONFERENCE VIDEO UPDATE 23 – 26 May 2017 Edinburgh UPDATE 1


In Confidence - Perceptions of unconventional slow virus diseases of animals in the USA - APRIL-MAY 1989 - G A H Wells

3. Prof. A. Robertson gave a brief account of BSE. The US approach was to accord it a very low profile indeed. Dr. A Thiermann showed the picture in the ''Independent'' with cattle being incinerated and thought this was a fanatical incident to be avoided in the US at all costs. ...


snip...see full text ;


Evidence That Transmissible Mink Encephalopathy Results from Feeding Infected Cattle Over the next 8-10 weeks, approximately 40% of all the adult mink on the farm died from TME. snip... The rancher was a ''dead stock'' feeder using mostly (>95%) downer or dead dairy cattle... 

In Confidence - Perceptions of unconventional slow virus diseases of animals in the USA - APRIL-MAY 1989 - G A H Wells 3. Prof. A. Robertson gave a brief account of BSE. The US approach was to accord it a very low profile indeed. Dr. A Thiermann showed the picture in the ''Independent'' with cattle being incinerated and thought this was a fanatical incident to be avoided in the US at all costs. ... 


The occurrence of CWD must be viewed against the contest of the locations in which it occurred. It was an incidental and unwelcome complication of the respective wildlife research programmes. Despite its subsequent recognition as a new disease of cervids, therefore justifying direct investigation, no specific research funding was forthcoming. The USDA veiwed it as a wildlife problem and consequently not their province! ...page 26. 


*** Spraker suggested an interesting explanation for the occurrence of CWD. The deer pens at the Foot Hills Campus were built some 30-40 years ago by a Dr. Bob Davis. At or abut that time, allegedly, some scrapie work was conducted at this site. When deer were introduced to the pens they occupied ground that had previously been occupied by sheep. 


Wednesday, May 24, 2017 

PRION2017 CONFERENCE VIDEO UPDATE 23 – 26 May 2017 Edinburgh UPDATE 1 

Subject: PRION2017 CONFERENCE VIDEO UPDATE 23 – 26 May 2017 Edinburgh

*see archives of previous Prion Conferences, the ones that are still available, scroll down towards bottom in this link.


P120 Early preclinical detection of prions in blood of macaques peripherally infected with the variant CJD 
agent

Luis Concha1,2,Claudio Soto1

 1University Of Texas, Houston, United States, 2Universidad de los Andes, Santiago, Chile 

Aim: Preclinical detection of prions in blood of experimentally infected non-human primates. 
The detection of prions in blood of patients affected by variant Creutzfeldt-Jakob disease (vCJD) has been recently achieved, by means of the protein misfolding cyclic amplification (PMCA) technique (Concha- Marambio et. al., 2016). Moreover, a few blood samples were shown to contain prions before disease onset (Bougard et. al. 2016). However, the unknown time of infection makes impossible to determine when in the incubation period prions can be detected in blood. Thus, we studied blood samples longitudinally collected from 3 macaques infected with the macaque adapted vCJD agent (m-vCJD).  

Methods: Three macaques were peripherally infected with m-vCJD (McDowell et. al., 2016). Blood was collected longitudinally, starting 2 months post inoculation (mpi) until the endpoint of the disease. The samples were divided in three panels: early preclinical (2 to 12 mpi), late preclinical (12 mpi to onset) and clinical (onset to final bleed). These samples were kindly provided by Dr Luisa Gregory as de-identified samples. 
The PMCA protocol previously used was optimized to detect prions in blood of vCJD patients, for the detection of m-vCJD prions in macaque blood, using human PrP from transgenic mice as substrate. The substrate was supplemented with 100 ug/ml heparin and few modifications were introduced into the PMCA protocol. 

Results: m-vCJD prions from macaque brain homogenate (BH) were amplified at similar efficiencies vCJD prions (10-10 to 10-11 dilutions of BH). Prions were readily detected in whole blood, buffy coat and plasma during the clinical phase of the disease. Preclinical samples were more challenging to amplify. However, after PMCA optimization, we could detect with high sensitivity and specificity all the early and late preclinical samples. Our results show that m-vCJD prions from macaque blood can be detected at least ~800 days before the onset of disease. 

Conclusions: PMCA was adapted for the efficient amplification of m-vCJD prions present in blood of macaques peripherally challenged with the vCJD agent. Our results suggest that PMCA can detect prions in blood more than 800 days before onset with high sensitivity and specificity. Since the first sample was collected 2 mpi and it was positive, PMCA can probably detect prions in blood weeks after inoculation. Overall, our results show the consistent and reproducible preclinical detection of prions in macaques, which suggest that this protocol could be used in humans for pre-symptomatic detection of carriers infected with vCJD prions. 


DISORDERS PRION 2017  DECIPHERING NEURODEGENERATIVE



TUESDAY, JUNE 20, 2017 

Prion 2017 Conference Transmissible prions in the skin of Creutzfeldt-Jakob disease patients


WEDNESDAY, MAY 03, 2017

*** First evidence of intracranial and peroral transmission of Chronic Wasting Disease (CWD) into Cynomolgus macaques


FRIDAY, JUNE 16, 2017

PRION 2017 P55 Susceptibility of human prion protein to in vitro conversion by chronic wasting disease prions


MONDAY, JUNE 19, 2017 

PRION 2017 P20 Descriptive epidemiology of human prion diseases in Japan: a prospective 16-year surveillance study

Japan Prion Disease Increasing Annually to 2.3 patients per 1 million populations in 2014

http://creutzfeldt-jakob-disease.blogspot.com/2017/06/prion-2017-p20-descriptive-epidemiology.html

TUESDAY, JUNE 13, 2017

PRION 2017 CONFERENCE ABSTRACT Chronic Wasting Disease in European moose is associated with PrPSc features different from North American CWD


SATURDAY, JUNE 10, 2017

Chronic Wasting Disease CWD TSE Prion to Humans, who makes that final call, when, or, has it already happened?


MONDAY, JUNE 19, 2017 

PRION 2017 CONFERENCE ABSTRACT P61 vCJD strain properties in a Spanish mother and son replicate as those of a young UK case


WEDNESDAY, JUNE 14, 2017 

Amyloid-β accumulation in human growth hormone related iatrogenic CJD patients in the UK


Saturday, June 17, 2017

PRION 2017 P115 α- Synuclein prions from MSA patients exhibit similar transmission properties as PrPSc prions


THURSDAY, JUNE 22, 2017 

World Organisation for Animal Health (OIE) to establish liaison office in College Station, Texas


THURSDAY, JUNE 22, 2017 

National Prion Disease Pathology Surveillance Center Cases Examined(1) (May 18, 2017)


*** The potential impact of prion diseases on human health was greatly magnified by the recognition that interspecies transfer of BSE to humans by beef ingestion resulted in vCJD. While changes in animal feed constituents and slaughter practices appear to have curtailed vCJD, there is concern that CWD of free-ranging deer and elk in the U.S. might also cross the species barrier. Thus, consuming venison could be a source of human prion disease. Whether BSE and CWD represent interspecies scrapie transfer or are newly arisen prion diseases is unknown. Therefore, the possibility of transmission of prion disease through other food animals cannot be ruled out. There is evidence that vCJD can be transmitted through blood transfusion. There is likely a pool of unknown size of asymptomatic individuals infected with vCJD, ***

*** and there may be asymptomatic individuals infected with the CWD equivalent. 

*** These circumstances represent a potential threat to blood, blood products, and plasma supplies. 


Tracking spongiform encephalopathies in North America

Xavier Bosch

Published: August 2003


Summary;

“My name is Terry S Singeltary Sr, and I live in Bacliff, Texas. I lost my mom to hvCJD (Heidenhain variant CJD) and have been searching for answers ever since. What I have found is that we have not been told the truth. CWD in deer and elk is a small portion of a much bigger problem.”

49-year-old Singeltary is one of a number of people who have remained largely unsatisfied after being told that a close relative died from a rapidly progressive dementia compatible with spontaneous Creutzfeldt-Jakob disease (CJD). So he decided to gather hundreds of documents on transmissible spongiform encephalopathies (TSE) and realised that if Britons could get variant CJD from bovine spongiform encephalopathy (BSE), Americans might get a similar disorder from chronic wasting disease (CWD) the relative of mad cow disease seen among deer and elk in the USA. Although his feverish search did not lead him to the smoking gun linking CWD to a similar disease in North American people, it did uncover a largely disappointing situation.

Singeltary was greatly demoralised at the few attempts to monitor the occurrence of CJD and CWD in the USA.  Only a few states have made CJD reportable. Human and animal TSEs should be reportable nationwide and internationally, he complained in a letter to the Journal of the American Medical Association (JAMA 2003; 285: 733).  "I hope that the CDC does not continue to expect us to still believe that the 85% plus of all CJD cases which are sporadic are all spontaneous, without route or source."

Diagnosis and Reporting of Creutzfeldt-Jakob Disease
Singeltary, Sr et al. JAMA.2001; 285: 733-734. Vol. 285 No. 6, February 14, 2001 JAMA
Diagnosis and Reporting of Creutzfeldt-Jakob Disease
To the Editor: In their Research Letter, Dr Gibbons and colleagues1 reported that the annual US death rate due to Creutzfeldt-Jakob disease (CJD) has been stable since 1985. These estimates, however, are based only on reported cases, and do not include misdiagnosed or preclinical cases. It seems to me that misdiagnosis alone would drastically change these figures. An unknown number of persons with a diagnosis of Alzheimer disease in fact may have CJD, although only a small number of these patients receive the postmortem examination necessary to make this diagnosis. Furthermore, only a few states have made CJD reportable. Human and animal transmissible spongiform encephalopathies should be reportable nationwide and internationally.
Terry S. Singeltary, Sr Bacliff, Tex
1. Gibbons RV, Holman RC, Belay ED, Schonberger LB. Creutzfeldt-Jakob disease in the United States: 1979-1998. JAMA. 2000;284:2322-2323.

Until recently, CWD was thought to be confined to the wild in a small region in Colorado. But since early 2002, it has been reported in other areas, including Wisconsin, South Dakota, and the Canadian province of Saskatchewan. Indeed, the occurrence of CWD in states that were not endemic previously increased concern about a widespread outbreak and possible transmission to people and cattle.

To date, experimental studies have proven that the CWD agent can be transmitted to cattle by intracerebral inoculation and that it can cross the mucous membranes of the digestive tract to initiate infection in lymphoid tissue before invasion of the central nervous system. Yet the plausibility of CWD spreading to people has remained elusive.

Part of the problem seems to stem from the US surveillance system. CJD is only reported in those areas known to be endemic foci of CWD. Moreover, US authorities have been criticised for not having performed enough prionic tests in farm deer and elk.

Although in November last year the US Food and Drug Administration issued a directive to state public-health and agriculture officials prohibiting material from CWD-positive animals from being used as an ingredient in feed for any animal species, epidemiological control and research in the USA has been quite different from the situation in the UK and Europe regarding BSE.

"Getting data on TSEs in the USA from the government is like pulling teeth", Singeltary argues. "You get it when they want you to have it, and only what they want you to have."

Norman Foster, director of the Cognitive Disorders Clinic at the University of Michigan (Ann Arbor, MI, USA), says that  "current surveillance of prion disease in people in the USA is inadequate to detect whether CWD is occurring in human beings"; adding that, "the cases that we know about are reassuring, because they do not suggest the appearance of a new variant of CJD in the USA or atypical features in patients that might be exposed to CWD. However, until we establish a system that identifies and analyses a high proportion of suspected prion disease cases we will not know for sure". The USA should develop a system modelled on that established in the UK, he points out.

Ali Samii, a neurologist at Seattle VA Medical Center who recently reported the cases of three hunters "two of whom were friends" who died from pathologically confirmed CJD, says that "at present there are insufficient data to claim transmission of CWD into humans"; adding that "[only] by asking [the questions of venison consumption and deer/elk hunting] in every case can we collect suspect cases and look into the plausibility of transmission further". Samii argues that by making both doctors and hunters more aware of the possibility of prions spreading through eating venison, doctors treating hunters with dementia can consider a possible prion disease, and doctors treating CJD patients will know to ask whether they ate venison.

CDC spokesman Ermias Belay says that the CDC "will not be investigating the [Samii] cases because there is no evidence that the men ate CWD-infected meat". He notes that although "the likelihood of CWD jumping the species barrier to infect humans cannot be ruled out 100%" and that "[we] cannot be 100% sure that CWD does not exist in humans& the data seeking evidence of CWD transmission to humans have been very limited". 


http://www.thelancet.com/pdfs/journals/laninf/PIIS1473-3099(03)00715-1.pdf

15 November 1999
British Medical Journal
vCJD in the USA * BSE in U.S.
2 January 2000
British Medical Journal
U.S. Scientist should be concerned with a CJD epidemic in the U.S., as well
Diagnosis and Reporting of Creutzfeldt-Jakob Disease
Singeltary, Sr et al. JAMA.2001; 285: 733-734. Vol. 285 No. 6, February 14, 2001 JAMA
Diagnosis and Reporting of Creutzfeldt-Jakob Disease
To the Editor: In their Research Letter, Dr Gibbons and colleagues1 reported that the annual US death rate due to Creutzfeldt-Jakob disease (CJD) has been stable since 1985. These estimates, however, are based only on reported cases, and do not include misdiagnosed or preclinical cases. It seems to me that misdiagnosis alone would drastically change these figures. An unknown number of persons with a diagnosis of Alzheimer disease in fact may have CJD, although only a small number of these patients receive the postmortem examination necessary to make this diagnosis. Furthermore, only a few states have made CJD reportable. Human and animal transmissible spongiform encephalopathies should be reportable nationwide and internationally.
Terry S. Singeltary, Sr Bacliff, Tex
1. Gibbons RV, Holman RC, Belay ED, Schonberger LB. Creutzfeldt-Jakob disease in the United States: 1979-1998. JAMA. 2000;284:2322-2323.
26 March 2003
Terry S. Singeltary, retired (medically) CJD WATCH
I lost my mother to hvCJD (Heidenhain Variant CJD). I would like to comment on the CDC's attempts to monitor the occurrence of emerging forms of CJD. Asante, Collinge et al [1] have reported that BSE transmission to the 129-methionine genotype can lead to an alternate phenotype that is indistinguishable from type 2 PrPSc, the commonest sporadic CJD. However, CJD and all human TSEs are not reportable nationally. CJD and all human TSEs must be made reportable in every state and internationally. I hope that the CDC does not continue to expect us to still believe that the 85%+ of all CJD cases which are sporadic are all spontaneous, without route/source. We have many TSEs in the USA in both animal and man. CWD in deer/elk is spreading rapidly and CWD does transmit to mink, ferret, cattle, and squirrel monkey by intracerebral inoculation. With the known incubation periods in other TSEs, oral transmission studies of CWD may take much longer. Every victim/family of CJD/TSEs should be asked about route and source of this agent. To prolong this will only spread the agent and needlessly expose others. In light of the findings of Asante and Collinge et al, there should be drastic measures to safeguard the medical and surgical arena from sporadic CJDs and all human TSEs. I only ponder how many sporadic CJDs in the USA are type 2 PrPSc?
re-Human Prion Diseases in the United States
Posted by flounder on 01 Jan 2010 at 18:11 GMT

Terry S. Singeltary Sr.