Wednesday, January 16, 2019

Early preclinical detection of prions in the skin of prion-infected animals

Early preclinical detection of prions in the skin of prion-infected animals

Zerui Wang1,2, Matteo Manca3, Aaron Foutz4, Manuel V. Camacho1 , Gregory J. Raymond3, Brent Race3, Christina D. Orru3, Jue Yuan1 , Pingping Shen1,2, Baiya Li1,5, Yue Lang1,2, Johnny Dang1 , Alise Adornato1 , Katie Williams3, Nicholas R. Maurer1 , Pierluigi Gambetti1 , Bin Xu6, Witold Surewicz7, Robert B. Petersen1,8, Xiaoping Dong9, Brian S. Appleby1,4,10, Byron Caughey 3, Li Cui2, Qingzhong Kong1,4,10,11 & Wen-Quan Zou1,2,4,9,10,11

A definitive pre-mortem diagnosis of prion disease depends on brain biopsy for prion detection currently and no validated alternative preclinical diagnostic tests have been reported to date. To determine the feasibility of using skin for preclinical diagnosis, here we report ultrasensitive serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays of skin samples from hamsters and humanized transgenic mice (Tg40h) at different time points after intracerebral inoculation with 263K and sCJDMM1 prions, respectively. sPMCA detects skin PrPSc as early as 2 weeks post inoculation (wpi) in hamsters and 4 wpi in Tg40h mice; RT-QuIC assay reveals earliest skin prion-seeding activity at 3 wpi in hamsters and 20 wpi in Tg40h mice. Unlike 263Kinoculated animals, mock-inoculated animals show detectable skin/brain PrPSc only after long cohabitation periods with scrapie-infected animals. Our study provides the proof-of-concept evidence that skin prions could be a biomarker for preclinical diagnosis of prion disease.

snip...

Discussion

Several lines of evidence have recently suggested that skin is the place where misfolded proteins often stay, which may play a role in the pathogenesis and early detection of neurodegenerative diseases25. While it has been known for a long time that sheep and goats with scrapie often have skin lesions26, prions had not been detected in skin until prion infectivity was first found in skin of prion-infected greater kudu using an animal-based bioassay27. Subsequently, skin PrPSc was detected directly by western blotting after the enrichment of PrPSc in experimentally or naturally scrapie-infected hamsters and sheep, as well as in a single cadaver with vCJD28,29. We recently observed both prion-seeding activity and prion infectivity in the skin of patients with sCJD and vCJD at the terminal stage of the diseases using RT-QuIC assay and a bioassay humanized Tg mouse-based, respectively13. In the current study, we further demonstrated that skin PrPSc is preclinically detectable not only by RT-QuIC, but also by sPMCA before brain damage occurs in two animal models of prion diseases, 263K-inoculated hamsters and sCJDMM1-inoculated humanized Tg40h mice, with parallel RT-QuIC findings in an independent set of scrapie-infected hamsters done in an independent laboratory.

In terms of the earliest time point at which skin PrPSc becomes detectable in animals infected by the intracerebral inoculation of prions, sPMCA showed detection at 2 wpi for hamsters and 4 wpi for Tg40h mice, while RT-QuIC detection was at 3 wpi for hamsters and 20 wpi for Tg40h (Fig. 8a). These findings indicate that skin PrPSc is detectable at least 5 weeks earlier in scrapieinfected animals before brain pathology is observed. Of the five body areas examined, the ear pinna and back skin were the areas that showed earliest prion-seeding activity (3 wpi), while the thigh skin was the latest (9 wpi). The latter was also confirmed by two sets of hamsters examined in two-independent laboratories in this study. In contrast to the prion-seeding activity found in the skin of infected hamsters at the early stage of infection, the earliest time point showing skin prion-seeding activity was at 20 wpi in humanized Tg mice by RT-QuIC (Fig. 8b). This time point was similar whether recombinant hamster or human PrP substrate was used despite our expectation that human PrP might provide a more sensitive RT-QuIC for human PrPSc based on better sequence homology between the seeds and substrate.

Although the reasons for early and widespread presence of PrPSc in the skin remain unclear, possibilities include the spread of the prion inoculum itself, or endogenously replicating prions, from the brain through the peripheral nerves to the skin within the 2–3 weeks required for the first detection by our ultrasensitive

sPMCA and RT-QuIC assays. PrP seeding activity has been detected in the blood in the prion-infected hamsters and deer immediately after peripheral inoculation including oral, nasal, or blood route30. However, no reports have shown that PrPSc is consistently detectable in the blood of prion-infected hamsters within 2 weeks post intracerebral inoculation. Thus, the early spread of PrPSc from the brain-to-the skin in the intracerebrally 263K-inoculated hamsters is likely either not through the blood or, if initially from the blood, requires time-dependent concentration or replication in the skin to become detectable.

It is unclear why, according to RT-QuIC, the back skin more consistently accumulates PrPSc than the other skin areas tested. It may depend on the dermatomes of nerves and their distance from the CNS. Between the back and thigh areas examined, the back dermatome is more proximate to the CNS. Similarly, we found prion-seeding activity much earlier in the ear area than the thigh (3 wpi vs 9 wpi). Analogously, misfolded α-synuclein deposition in Parkinson’s disease patients is more frequently detected in proximate (100% in the cervical C7 site) compared to distal (35% in the thoracic T12 region) skin areas by immunofluorescence microscopy31–33. In future studies, it would be interesting to determine whether PrPSc in the skin of sCJD has a similar distribution, and whether factors besides dermatome distance from the brain are involved.

Both sPMCA and RT-QuIC assays detected skin PrPSc early in scrapie-infected hamsters. However, sPMCA amplified PrPSc in skin samples from CJD-infected Tg40 mice at 4 wpi, while RTQuIC assay detected prion-seeding activity only at 20 wpi (Fig. 8). The reason for the difference in Tg40 mice is not clear, but may be due in part to differences between the assays and the prion strains involved. sPMCA is performed in brain homogenates, which provide naturally post-translationally modified (glycosylated and GPI-anchored) PrPC as the substrate, and other potential brain-derived co-factors. RT-QuIC reactions include only unmodified recombinant PrPC as substrate, and no natural co-factors. sPMCA reactions are accelerated by sonication, whereas RT-QuIC reactions are shaken. Also, in successive rounds of sPMCA, the substrate and other brain components are refreshed, but our RT-QuIC reactions were performed in one round, with no refreshment. To exclude the effect of mismatch between seeds and substrates on the sensitivity of RT-QuIC reactions, we tested two recombinant PrP molecules as substrates from two different species including hamster and human and they all showed the similar sensitivity with the same earliest time point at 20 wpi. Finally, 263K scrapie and MM1 sCJD prions undoubtedly differ in conformation, and therefore, perhaps, their interactions with co-factors, various PrPC substrates, and/or skinderived inhibitors of RT-QuIC reactions. These factors might differentially affect the sensitivity of detection of MM1 sCJD in the skin of Tg40 mice by sPMCA and RT-QuIC. It is also possible that the RT-QuIC assay may become as sensitive as sPMCA for skin prion detection in the Tg40h mice after further optimization of RT-QuIC’s experimental conditions.

Our early detection of PrPSc in the skin of sCJD- and scrapieinfected rodents suggests that it may be possible to do the same with the skin of humans who carry PrP mutations associated with genetic prion diseases such as familial CJD, Gerstmann–Sträussler–Scheinker syndrome, or fatal familial insomnia because it is expected that their mutant PrPC spontaneously converts into PrPSc and accumulates later in life. Skinbased RT-QuIC may reveal early prion-seeding activity in PrP mutation-carriers, or people with suspected exposures to prion infections, while they are still asymptomatic. Even for suspected sCJD cases, who are only identified in the symptomatic phase, skin-based RT-QuIC might be useful for monitoring disease progression, defining severity and diversity, and evaluating the treatment efficacy when potential drugs become available.

Although neither clinical signs nor brain PrPSc were observed in control animals cohabitating with 263K-inoculated hamsters within 12 weeks, the mock-inoculated animals that were housed with scrapie-infected animals had amplifiable PrPSc in the brain and skin via amplification techniques. Moreover, in contrast to the skin PrPSc amplified from the 263K-inoculated hamsters as early as 2 wpi, the control animals that co-habitated with infected hamsters were found to have amplified skin PrPSc after cohabitation for 11 weeks. This finding implies that the skin PrPSc detected early in the scrapie-infected hamsters is not the result of environmental contamination; otherwise, the control animals would exhibit skin PrPSc at 2 wpi as well. The finding of skin PrPSc in the cohabitating control animals may be relevant to the environmental transmission of prions observed in natural animal prion diseases, such as scrapie and CWD. Interestingly, prion transmission has been observed in hamsters by contact with prion-contaminated surfaces through rubbing and bedding34, in which cases skin is expected to be involved. The role that skin may plays in the environmental transmission of prions warrants future investigation.

Skin PrPSc may derive from urine or fecal prion contamination in addition to possible skin shedding due to scratching or biting each other. Indeed, scrapie infectivity was reported in the urine of prion-infected mice coincident with lymphocytic nephritis during their preclinical and clinical stages of prion infection35,36. It was also observed in their urine in intracerebrally inoculated hamsters even without any apparent inflammation21. In addition, deer with clinical CWD and mild to moderate nephritis were found to have sPMCA-detectable PrPSc and CWD-infectivity in urine22. Using sPMCA, PrPSc was detected in urine of ~80% of the hamsters intraperitoneally inoculated with 263K prions at the symptomatic stage23. Notably, PrPSc was detected in urine, but only at the terminal stage of disease in intracerebrally inoculated hamsters, except for a few days immediately after oral administration24. Similar to the observations by Gonzalez-Romero et al.23, Murayama et al. also found that not all infected hamsters had detectable urine PrPSc even at the terminal stage24. The skin PrPSc detected early in the intracerebrally infected hamsters, but not in the co-habitated-negative controls, at 2 wpi suggests that skin prions may not result from urine at the early stage of infection.

Unlike the situation with urine, it has not been very clear whether PrPSc is present in feces of intracerebrally inoculated hamsters at the early stage of prion infection. High titers of prion infectivity were detected in feces throughout the disease incubation in orally inoculated hamsters while low levels of infectivity were occasionally observed in intracerebrally- or intraperitoneally-inoculated animals18. For instance, no prion infectivity was detected in feces of hamsters within 3 wpi, including at 1, 2, and 22 days post inoculation (dpi), except for 8 dpi when 17% transmission rate was detected in feces18. However, fecal PrPSc was only detected during the clinical stage of disease by sPMCA in hamsters with lower doses of oral inoculum19. Western blotting of fecal extracts showed shedding of PrPSc in the excrement at 24–72 h post inoculation, but not at 0–24 h post inoculation, or at later preclinical or clinical time points19. Consistent with this observation, prion infectivity was not detected in feces of mule deer after oral challenge with CWD prions within the first 12–16 wpi, but feces contained infectivity after 36 wpi through to clinical disease stages at 64–80 wpi20. It is likely that PrPSc is present in feces of infected animals at the late stage of prion infection, which may contaminate the skin of cohabitating control animals.

Although prion contamination of skin by excrement may not be a major concern in human prion diseases, it is an important issue for prion transmission in animals, such as cattle, sheep, goats, and cervids. It is worth noting that the high incidence of scrapie in sheep and goats as well as CWD in cervids is believed to be attributable to contamination of the environment due to high prion shedding. The detection of PrPSc in excretions including saliva, urine, and feces clearly indicates this shedding. Oral ingestion due to the coprophagic behavior of animals has been believed to cause wide horizontal transmission of scrapie and CWD. However, our current finding of skin PrPSc in cohabitating prion-inoculated and PBS-inoculated control animals, as well as the occurrence of brain PrPSc in the PBS-inoculated animals at the late stage suggests that prion contamination of skin may be a potential route of transmission of prion diseases.

In conclusion, our study indicates that skin PrPSc may be a useful biomarker not only for the preclinical diagnosis of prion diseases, but also for monitoring disease progression following infection and treatment. Since the chance that PrPSc can be consistently detected in blood and urine of sCJD patients by sPMCA and RT-QuIC assays has been virtually very low10,11,37, it is possible that detection of PrPSc in the skin, a highly accessible tissue, could be developed for evaluating therapeutic efficiency and drug screening. As mentioned earlier, RT-QuIC analysis of CSF and nasal brushing specimens to date has been used for diagnosis of human prion diseases only at the clinical stage. Moreover, it is much less practical in live animals to collect CSF and nasal brushing specimens. In cervids, at least, there has been more focus on RT-QuIC analyses of RAMALT biopsies and various excreta38. Although these analyses are currently the most accurate tests available for chronic wasting disease in live cervids, they do not yet provide 100% diagnostic sensitivity and specificity38. Thus, it may be helpful to have additional or alternative diagnostic specimens, such as skin or ear pinna punches, for RT-QuIC and sPMCA testing 


''In cervids, at least, there has been more focus on RT-QuIC analyses of RAMALT biopsies and various excreta38. Although these analyses are currently the most accurate tests available for chronic wasting disease in live cervids, they do not yet provide 100% diagnostic sensitivity and specificity38.''

***> they do not yet provide 100% diagnostic sensitivity and specificity38.''

FRIDAY, MARCH 30, 2018

Detection of Creutzfeldt-Jakob disease prions in skin: implications for healthcare


WEDNESDAY, NOVEMBER 22, 2017 

NIH scientists and collaborators find infectious prion protein in skin of CJD patients


WEDNESDAY, NOVEMBER 22, 2017 

Prion seeding activity and infectivity in skin samples from patients with sporadic Creutzfeldt-Jakob disease


TUESDAY, JUNE 20, 2017 

Prion 2017 Conference 

Transmissible prions in the skin of Creutzfeldt-Jakob disease patients

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

Dr. Wenguan 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, 4Universitv 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- QulC) 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 ClD 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) 

DISORDERS PRION 2017 DECIPHERING NEURODEGENERATIVE 


*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. 

Oral Session14：45～15：00O-12 Wenquan Zou

*** PrPSc in the skin of CJD patients


Accessing transmissibility and diagnostic marker of skin prions.

Kong, Qingzhong Safar, Jiri G. Zou, Wen-Quan

Case Western Reserve University, Cleveland, OH, United States

Abstract The fatal, transmissible animal and human prion diseases are characterized by the deposition in the brain of a proteinase K (PK)-resistant infectious prion protein (PrPSc), an isoform derived from the cellular protein (PrPC) through misfolding. A definitive antemortem diagnosis is virtually impossible for most patients because of the difficulty in obtaining the brain tissues by biopsy. Recently, PrPSc has been reported to be detected in the skin of experimentally or naturally scrapie-infected animals (Thomzig et al., 2007). Consistent with this finding, we have observed PK-resistant PrP in the skin of a patient with variant Creutzfeldt-Jakob disease (vCJD), an acquired form of human prion disease caused by bovine prion (Notari et al., 2010). Unexpectedly, our latest preliminary study identified two types of PK-resistant PrP molecules [with gel mobility similar to the PrPSc types 1 and 2 from the brain of sporadic CJD (sCJD)] in the fibroblast cells extracted from the skin of clinical sCJD patients and asymptomatic subjects carrying PrP mutations linked to familial CJD (fCJD). We also detected PrPSc in the skin of humanized transgenic (Tg) mice inoculated intracerebrally with a human prion. Moreover, prion infectivity has been observed in the skin of infected greater kudu (Cunningham et al., 2004) and a murine prion inoculated to mice via skin scarification can not only propagate in the skin, but also spread to the brain to cause prion disease (Wathne et al., 2012). We hypothesize that the skin of patients with prion disease harbors prion infectivity and the presence of PK-resistant PrP in the skin is a novel diagnostic marker for preclinical CJD patients. To test the hypotheses, we propose to (1) determine prion infectivity of the skin- derived fibroblasts and skin of sCJD patients and asymptomatic PrP-mutation carriers using humanized Tg mouse bioassay, (2) to pinpoint the earliest stage at which PrPSc becomes detectable in the skin of prion- infected Tg mice, and (3) to detect PrPSc in the skin of various human prion diseases, using conventional as well as highly sensitive RT-QuIC assays for both (2) and (3). If successful, our proposal may not only help prevent potential transmission of human prion diseases but also enable definitive and less intrusive antemortem diagnosis of prion diseases. Finally, knowledge generated from this study may also enhance our understanding of other neurodegenerative diseases such as Alzheimer's disease.

Public Health Relevance Currently it is unclear whether or not the skin of patients with prion diseases is infectious and, moreover, there is no alternative preclinical definitive testing or the brain biopsy in the prion diseases. The aim of our proposal is to address the issues by detection of the infectivity of patients' skin samples using animal bioassay and a new highly sensitive RT-QuIC assay. We believe that our study will not only provide insights into the pathogenesis and transmissibility of prion disease but also will develop preclinical definitive testing for prion disease.

Funding Agency Agency National Institute of Health (NIH)

Institute National Institute of Neurological Disorders and Stroke (NINDS)

Type Exploratory/Developmental Grants (R21)

Project # 1R21NS096626-01

Application # 9092119

Study Section Special Emphasis Panel (ZRG1)

Program Officer Wong, May Project Start 2016-02-01

Project End 2018-01-31

Budget Start 2016-02-01

Budget End 2017-01-31

Support Year 1

Fiscal Year 2016

Total Cost

Indirect Cost Institution Name Case Western Reserve University

Department Pathology

Type Schools of Medicine

DUNS # 077758407

City Cleveland

State OH

Country United States

Zip Code 44106



TUESDAY, MAY 10, 2016 

Accessing transmissibility and diagnostic marker of skin prions


2019

***>  Rapid recontamination of a farm building occurs after attempted prion removal 

http://dx.doi.org/10.1136/vr.105054 

Here, an efficient scrapie decontamination protocol was applied to a farm with high levels of environmental contamination with the scrapie agent. 

snip... 

However, the introduction into this decontaminated barn of 25 VRQ/VRQ sheep (a genotype highly susceptible to classical scrapie) demonstrated that all animals, with the exception of 1 lamb that died at 122 dpe, had detectable PrPSc in lymphoid tissue, indicating infection with the scrapie agent. This included 14 animals (54 per cent) that were PrPSc-positive on the first RAMALT analysis at 372 dpe or 419 dpe. 

This study clearly demonstrates the difficulty in removing scrapie infectivity from the farm environment. Practical and effective prion decontamination methods are still urgently required for decontamination of scrapie infectivity from farms that have had cases of scrapie and this is particularly relevant for scrapiepositive goatherds, which currently have limited genetic resistance to scrapie within commercial breeds.24 

***> This is very likely to have parallels with control efforts for CWD in cervids. 

Funding This study was funded by DEFRA within project SE1865. 

Competing interests None declared. 

https://veterinaryrecord.bmj.com/content/early/2019/01/02/vr.105054.long 

***> see more of study here; 

https://prionprp.blogspot.com/2019/01/rapid-recontamination-of-farm-building.html

 FRIDAY, DECEMBER 28, 2018 

***> Chronic Wasting Disease CWD TSE Prion 2019 Where The Rubber Meets The Road 


Saturday, December 15, 2018 

***> ADRD Summit RFI Singeltary COMMENT SUBMISSION BSE, SCRAPIE, CWD, AND HUMAN TSE PRION DISEASE 

December 14, 2018 


TUESDAY, JANUARY 1, 2019 

CHILDHOOD EXPOSURE TO CADAVERIC DURA 


SATURDAY, JANUARY 5, 2019 

Low levels of classical BSE infectivity in rendered fat tissue 

 
FRIDAY, DECEMBER 14, 2018 MAD COW USA FLASHBACK 

FRIDAY DECEMBER 14, 2018 

 
THURSDAY, JANUARY 3, 2019 

MAD COW USDA DISEASE BSE TSE Prion 


NSLP DEAD STOCK DOWNER COW SCHOOL LUNCH PROGRAM


THURSDAY, AUGUST 30, 2018 

TRACKING HERD MATES USDA MAD COW DISEASE, TRACE FORWARD, TRACE BACK RECORDS, WHO CARES, NOT THE OIE


USDA ONLY TESTING 20k HEAD OF CATTLE A YEAR FOR MAD COW DISEASE ...LOL!

WEDNESDAY, AUGUST 29, 2018 

USDA Announces Atypical Bovine Spongiform Encephalopathy Detection USDA 08/29/2018 10:00 AM EDT





WEDNESDAY, AUGUST 29, 2018 

***> USDA DROPS MAD COW TESTING FROM 40K A YEAR TO JUST 20K A YEAR, IMPOSSIBLE TO FIND BSE, BUT THEY DID, IN FLORIDA!


Singeltary submission


Friday, December 14, 2018

FSIS Recalling 10,828 pounds raw intact bone-in beef quarters cattle Products may contain Specified Risk Materials (SRM) MOST HIGH RISK FOR BSE MAD COW DISEASE





 

Terry S. Singeltary Sr., Bacliff, Texas USA 77518, Galveston Bay, on the bottom...
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Monday, January 14, 2019

Two novel PRNP truncating mutations broaden the spectrum of prion amyloidosis with chronic diarrhea

Ann Clin Transl Neurol. 2018 Jun; 5(6): 777–783.

Published online 2018 Apr 26. doi: 10.1002/acn3.568 PMCID: PMC5989776 PMID: 29928661

Two novel PRNP truncating mutations broaden the spectrum of prion amyloidosis

Sabina Capellari,corresponding author 1 , 2 , † Simone Baiardi, 1 , † Rita Rinaldi, 3 Anna Bartoletti‐Stella, 1 , 2 Claudio Graziano, 4Silvia Piras, 2 Giovanna Calandra‐Buonaura, 1 , 2 Roberto D'Angelo, 3 Camilla Terziotti, 3 Raffaele Lodi, 1 , 5Vincenzo Donadio, 2 Loris Pironi, 6 Pietro Cortelli, 1 , 2 and Piero Parchicorresponding author 2 , 7 Author information Article notes Copyright and License information Disclaimer

Introduction Human prion diseases are neurodegenerative disorders characterized by tissue accumulation of a misfolded form (PrPSc) of the cellular prion protein (PrPC). They comprise three major phenotypes, namely Creutzfeldt‐Jakob disease (CJD), by far the most common, fatal insomnia (FI) and Gerstmann‐Straüssler‐Scheinker syndrome (GSS).1, 2 At variance with CJD and FI, GSS manifests a much slower clinical course and is mainly characterized by amyloid deposition rather than spongiform change in the affected brain tissue.2, 3 This major phenotypic difference directly reflects the composition and topology of PrPScaggregates. While in CJD and FI PrPSc accumulates as a full‐length or N‐terminally truncated fragments retaining the glycophosphatidylinositol (GPI) membrane anchor attaching the protein to the cell membrane,4 in GSS PrPSc mainly deposits in the extracellular space as unglycosylated anchorless fragments.2, 3 GSS was originally described as a parenchymal brain amyloidosis linked to missense or insertional mutations in the prion protein gene (PRNP).2 More recently, however, the discovery of premature truncating mutations in PRNP has expanded the phenotypic spectrum of anchorless PrP amyloidosis. The description of a PRNP stop‐codon mutation (p.Y145X) in a Japanese with dementia and both parenchymal and vascular PrP‐amyloid,5, 6 was followed by the findings of similar mutations at codons 160 (p.Q160X),7, 8 226 (p.Y226X),9 and 227 (p.Y227X)9 in patients with progressive dementia and pure PrP‐cerebral amyloid angiopathy (PrP‐CAA), GSS or mixed GSS/PrP‐CAA pathology.7, 9, 10Most significantly, PRNP truncating mutations at codons 163 (p.Y163X) and 203 (p.D203X), have been recently found in patients with chronic diarrhea, progressive autonomic failure, and peripheral polyneuropathy in association with widespread abnormal PrP deposition in peripheral organs and the CNS.11, 12 Based on the multiple organ system PrP deposition and the amyloid properties of the protein aggregates in the brain, the term PrP systemic amyloidosis was introduced, irrespectively of the demonstration of the classical pathological hallmarks of systemic amyloidosis.11, 13, 14

Here, we describe two Italian families with several affected members carrying novel truncating mutations in PRNP associated with progressive autonomic failure and variable cognitive impairment.


 A Novel Prion Disease Associated with Diarrhea and Autonomic Neuropathy 

 Simon Mead, M.D., Sonia Gandhi, M.D., Jon Beck, B.Sc., Diana Caine, Ph.D., Dillip Gallujipali, M.D., Christopher Carswell, M.D., Harpreet Hyare, M.D., Susan Joiner, M.Sc., Hilary Ayling, B.Sc., Tammaryn Lashley, Ph.D., Jacqueline M. Linehan, B.Sc., Huda Al-Doujaily, M.Sc., Bernadette Sharps, B.Sc., Tamas Revesz, M.D., Malin K. Sandberg, Ph.D., Mary M. Reilly, M.D., Martin Koltzenburg, M.D., Alastair Forbes, M.D., Peter Rudge, M.D., Sebastian Brandner, M.D., Jason D. Warren, M.D., Jonathan D.F. Wadsworth, Ph.D., Nicholas W. Wood, M.D., Janice L. Holton, M.D., and John Collinge, M.D. 

 Background Human prion diseases, although variable in clinicopathological phenotype, generally present as neurologic or neuropsychiatric conditions associated with rapid multifocal central nervous system degeneration that is usually dominated by dementia and cerebellar ataxia. Approximately 15% of cases of recognized prion disease are inherited and associated with coding mutations in the gene encoding prion protein (PRNP). The availability of genetic diagnosis has led to a progressive broadening of the recognized spectrum of disease. 

 Methods We used longitudinal clinical assessments over a period of 20 years at one hospital combined with genealogical, neuropsychological, neurophysiological, neuroimaging, pathological, molecular genetic, and biochemical studies, as well as studies of animal transmission, to characterize a novel prion disease in a large British kindred. We studied 6 of 11 affected family members in detail, along with autopsy or biopsy samples obtained from 5 family members. 

 Results We identified a PRNP Y163X truncation mutation and describe a distinct and consistent phenotype of chronic diarrhea with autonomic failure and a length-dependent axonal, predominantly sensory, peripheral polyneuropathy with an onset in early adulthood. Cognitive decline and seizures occurred when the patients were in their 40s or 50s. The deposition of prion protein amyloid was seen throughout peripheral organs, including the bowel and peripheral nerves. Neuropathological examination during end-stage disease showed the deposition of prion protein in the form of frequent cortical amyloid plaques, cerebral amyloid angiopathy, and tauopathy. A unique pattern of abnormal prion protein fragments was seen in brain tissue. Transmission studies in laboratory mice were negative. 

 Conclusions Abnormal forms of prion protein that were found in multiple peripheral tissues were associated with diarrhea, autonomic failure, and neuropathy. (Funded by the U.K. Medical Research Council and others.) 

 snip... 

 Presentation with diarrhea led to invasive investigations or surgery in several patients, with concomitant potential for iatrogenic transmission of prions from the gut through contamination of medical or surgical instruments.28 It is reassuring, however, that murine studies did not show experimental transmissibility, although this finding does not completely rule out the presence of potentially infectious human prions. Although nonmutant prion protein was detected in the protein deposits in multiple tissues, Western blotting showed that the protein was not proteaseresistant. PRNP analysis should be considered in the investigation of unexplained chronic diarrhea associated with a neuropathy or an unexplained syndrome similar to familial amyloid polyneuropathy. The prevalence of systemic amyloidosis associated with prion protein is probably low but might be better characterized with more widespread testing of PRNP and histologic examination for prion protein in biopsy samples. 


***> Presentation with diarrhea led to invasive investigations or surgery in several patients, with concomitant potential for iatrogenic transmission of prions from the gut through contamination of medical or surgical instruments.28


''In particular the US data do not clearly exclude the possibility of human (sporadic or familial) TSE development due to consumption of venison. 

The Working Group thus recognizes a potential risk to consumers if a TSE would be present in European cervids.'' Scientific opinion on chronic wasting disease (II)

EFSA Panel on Biological Hazards (BIOHAZ) Antonia Ricci Ana Allende Declan Bolton Marianne Chemaly Robert Davies Pablo Salvador Fernández Escámez ... See all authors

First published: 17 January 2018 https://doi.org/10.2903/j.efsa.2018.5132 ;

also, see;

8. Even though human TSE‐exposure risk through consumption of game from European cervids can be assumed to be minor, if at all existing, no final conclusion can be drawn due to the overall lack of scientific data. In particular the US data do not clearly exclude the possibility of human (sporadic or familial) TSE development due to consumption of venison. The Working Group thus recognizes a potential risk to consumers if a TSE would be present in European cervids. It might be prudent considering appropriate measures to reduce such a risk, e.g. excluding tissues such as CNS and lymphoid tissues from the human food chain, which would greatly reduce any potential risk for consumers.. However, it is stressed that currently, no data regarding a risk of TSE infections from cervid products are available.

snip...

The tissue distribution of infectivity in CWD‐infected cervids is now known to extend beyond CNS and lymphoid tissues. While the removal of these specific tissues from the food chain would reduce human dietary exposure to infectivity, exclusion from the food chain of the whole carcass of any infected animal would be required to eliminate human dietary exposure.



ZOONOTIC CHRONIC WASTING DISEASE CWD TSE PRION UPDATE

here is the latest;

PRION 2018 CONFERENCE 

Oral transmission of CWD into Cynomolgus macaques: signs of atypical disease, prion conversion and infectivity in macaques and bio-assayed transgenic mice 

Hermann M. Schatzl, Samia Hannaoui, Yo-Ching Cheng, Sabine Gilch (Calgary Prion Research Unit, University of Calgary, Calgary, Canada) Michael Beekes (RKI Berlin), Walter Schulz-Schaeffer (University of Homburg/Saar, Germany), Christiane Stahl-Hennig (German Primate Center) & Stefanie Czub (CFIA Lethbridge). 

To date, BSE is the only example of interspecies transmission of an animal prion disease into humans. The potential zoonotic transmission of CWD is an alarming issue and was addressed by many groups using a variety of in vitro and in vivo experimental systems. Evidence from these studies indicated a substantial, if not absolute, species barrier, aligning with the absence of epidemiological evidence suggesting transmission into humans. Studies in non-human primates were not conclusive so far, with oral transmission into new-world monkeys and no transmission into old-world monkeys. Our consortium has challenged 18 Cynomolgus macaques with characterized CWD material, focusing on oral transmission with muscle tissue. Some macaques have orally received a total of 5 kg of muscle material over a period of 2 years. 

After 5-7 years of incubation time some animals showed clinical symptoms indicative of prion disease, and prion neuropathology and PrPSc deposition were detected in spinal cord and brain of some euthanized animals. PrPSc in immunoblot was weakly detected in some spinal cord materials and various tissues tested positive in RT-QuIC, including lymph node and spleen homogenates. To prove prion infectivity in the macaque tissues, we have intracerebrally inoculated 2 lines of transgenic mice, expressing either elk or human PrP. At least 3 TgElk mice, receiving tissues from 2 different macaques, showed clinical signs of a progressive prion disease and brains were positive in immunoblot and RT-QuIC. Tissues (brain, spinal cord and spleen) from these and pre-clinical mice are currently tested using various read-outs and by second passage in mice. Transgenic mice expressing human PrP were so far negative for clear clinical prion disease (some mice >300 days p.i.). In parallel, the same macaque materials are inoculated into bank voles. 

Taken together, there is strong evidence of transmissibility of CWD orally into macaques and from macaque tissues into transgenic mouse models, although with an incomplete attack rate. 

The clinical and pathological presentation in macaques was mostly atypical, with a strong emphasis on spinal cord pathology. 

Our ongoing studies will show whether the transmission of CWD into macaques and passage in transgenic mice represents a form of non-adaptive prion amplification, and whether macaque-adapted prions have the potential to infect mice expressing human PrP. 

The notion that CWD can be transmitted orally into both new-world and old-world non-human primates asks for a careful reevaluation of the zoonotic risk of CWD.. 

***> The notion that CWD can be transmitted orally into both new-world and old-world non-human primates asks for a careful reevaluation of the zoonotic risk of CWD. <*** 


READING OVER THE PRION 2018 ABSTRACT BOOK, LOOKS LIKE THEY FOUND THAT from this study ; 

P190 Human prion disease mortality rates by occurrence of chronic wasting disease in freeranging cervids, United States 

Abrams JY (1), Maddox RA (1), Schonberger LB (1), Person MK (1), Appleby BS (2), Belay ED (1) (1) Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic Infectious Diseases, Atlanta, GA, USA (2) Case Western Reserve University, National Prion Disease Pathology Surveillance Center (NPDPSC), Cleveland, OH, USA.. 

SEEMS THAT THEY FOUND Highly endemic states had a higher rate of prion disease mortality compared to non-CWD states. 

AND ANOTHER STUDY; 

P172 Peripheral Neuropathy in Patients with Prion Disease 

Wang H(1), Cohen M(1), Appleby BS(1,2) (1) University Hospitals Cleveland Medical Center, Cleveland, Ohio (2) National Prion Disease Pathology Surveillance Center, Cleveland, Ohio.. 

IN THIS STUDY, THERE WERE autopsy-proven prion cases from the National Prion Disease Pathology Surveillance Center that were diagnosed between September 2016 to March 2017, 

AND 

included 104 patients. SEEMS THEY FOUND THAT The most common sCJD subtype was MV1-2 (30%), followed by MM1-2 (20%), 

AND 

THAT The Majority of cases were male (60%), AND half of them had exposure to wild game. 

snip...see more on Prion 2017 Macaque study from Prion 2017 Conference and other updated science on cwd tse prion zoonosis below...terry 



Sunday, December 9, 2018 

***> Variable Protease-Sensitive Prionopathy Transmission to Bank Voles CDC December 14, 2018

https://vpspr.blogspot.com/2018/12/variable-protease-sensitive-prionopathy.html

FRIDAY, JANUARY 10, 2014

vpspr, sgss, sffi, TSE, an iatrogenic by-product of gss, ffi, familial type prion disease, what it ??? Greetings Friends, Neighbors, and Colleagues,

 vpspr, sgss, sffi, TSE, an iatrogenic by-product of gss, ffi, familial type prion disease, what it ???

 Confucius is confused again.

 I was just sitting and thinking about why there is no genetic link to some of these TSE prion sGSS, sFFi, and it’s really been working on my brain, and then it hit me today.

 what if, vpspr, sgss, sffi, TSE prion disease, was a by-product from iatrogenic gss, ffi, familial type prion disease ???

 it could explain the cases of no genetic link to the gss, ffi, familial type prion disease, to the family.

 sporadic and familial is a red herring, in my opinion, and underestimation is spot on, due to the crude prehistoric diagnostic procedures and criteria and definition of a prion disease.

 I say again, what if, iatrogenic, what if, with all these neurological disorders, with a common denominator that is increasingly showing up in the picture, called the prion.

 I urge all scientist to come together here, with this as the utmost of importance about all these neurological disease that are increasingly showing up as a prion mechanism, to put on the front burners, the IATROGENIC aspect and the potential of transmission there from, with diseases/disease??? in question.

 by definition, could they be a Transmissible Spongiform Encephalopathy TSE prion type disease, and if so, what are the iatrogenic chances of transmission?

 this is very important, and should be at the forefront of research, and if proven, could be a monumental breakthrough in science and battle against the spreading of these disease/diseases.

 the US National Library of Medicine National Institutes of Health pub-med site, a quick search of the word SPORADIC will give you a hit of 40,747. of those, there are a plethora of disease listed under sporadic. sporadic simply means (UNKNOWN).

 http://www.ncbi.nlm.nih.gov/pubmed/?term=sporadic

 

the US National Library of Medicine National Institutes of Health pub-med site, a quick search of the word FAMILIAL will give you a hit of 921,815. of those, there are a plethora of disease listed under familial.

 

http://www.ncbi.nlm.nih.gov/pubmed/?term=familial

 

again, sporadic and familial is a red herring, in my opinion. 

also, in my opinion, when you start have disease such as sporadic Fatal Familial Insomnia, (and or sporadic GSS, or the VPSPr type prion disease), and there is NO familial genetic linkage to the family of the diseased, I have serious questions there as to a familial type disease, and thus, being defined as such. 


http://transmissiblespongiformencephalopathy.blogspot.com/2014/01/vpspr-sgss-sffi-tse-iatrogenic-by.html

Alzheimer’s disease, iatrogenic, and Transmissible Spongiform Encephalopathy TSE Prion disease, that is the question ??? 

>>> The only tenable public line will be that "more research is required’’ <<< 

>>> possibility on a transmissible prion remains open<<< 

O.K., so it’s about 23 years later, so somebody please tell me, when is "more research is required’’ enough time for evaluation ?

[9. Whilst this matter is not at the moment directly concerned with the iatrogenic CJD cases from hgH, there remains a possibility of litigation here, and this presents an added complication. There are also results to be made available shortly (1) concerning a farmer with CJD who had BSE animals, (2) on the possible transmissibility of Alzheimer’s and (3) a CMO letter on prevention of iatrogenic CJD transmission in neurosurgery, all of which will serve to increase media interest.]

https://web.archive.org/web/2017012...einquiry.gov.uk/files/yb/1992/11/04001001.pdf

https://web.archive.org/web/2004031...einquiry.gov.uk/files/yb/1992/12/16005001.pdf

https://web.archive.org/web/20040315075058/www.bseinquiry.gov.uk/files/yb/1992/12/16005001.pdf

snip...see full Singeltary Nature comment here; 

http://www.nature.com/nature/journal/v525/n7568/full/nature15369.html#/comments

see Singeltary comments to Plos ; 


Alzheimer’s disease and Transmissible Spongiform Encephalopathy prion disease, Iatrogenic, what if ?

Posted by flounder on 05 Nov 2014 at 21:27 GMT


Alzheimer's disease

let's not forget the elephant in the room. curing Alzheimer's would be a great and wonderful thing, but for starters, why not start with the obvious, lets prove the cause or causes, and then start to stop that. think iatrogenic, friendly fire, or the pass it forward mode of transmission. think medical, surgical, dental, tissue, blood, related transmission. think transmissible spongiform encephalopathy aka tse prion disease aka mad cow type disease... 

Commentary: Evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy

http://journals.plos.org/plosone/article/comment?id=info:do I/10.1371/annotation/933cc83a-a384-45c3-b3b2-336882c30f9d
https://www.frontiersin.org/articles/10.3389/fnagi.2016.00005/full

Self-Propagative Replication of Ab Oligomers Suggests Potential Transmissibility in Alzheimer Disease 

*** Singeltary comment PLoS *** 

Alzheimer’s disease and Transmissible Spongiform Encephalopathy prion disease, Iatrogenic, what if ? 

Posted by flounder on 05 Nov 2014 at 21:27 GMT 

http://www.plosone.org/annotation/listThread.action?root=82860

Tuesday, December 12, 2017 

Neuropathology of iatrogenic Creutzfeldt–Jakob disease and immunoassay of French cadaver-sourced growth hormone batches suggest possible transmission of tauopathy and long incubation periods for the transmission of Abeta pathology

http://tauopathies.blogspot.com/2017/12/neuropathology-of-iatrogenic.html

PRION Conference Roundtable 2018

http://prionconference.blogspot.com/

FRIDAY, DECEMBER 14, 2018 

Transmission of amyloid-β protein pathology from cadaveric pituitary growth hormone December 14, 2018


TUESDAY, JANUARY 1, 2019 

CHILDHOOD EXPOSURE TO CADAVERIC DURA
 


MONDAY, JANUARY 14, 2019 

Evaluation of iatrogenic risk of CJD transmission associated with Chronic Wasting Disease TSE Prion in Texas TAHC TPWD Evaluation of iatrogenic risk of CJD transmission associated with Chronic Wasting Disease TSE Prion in Texas TAHC TPWD

It is a dimension as vast as space and as timeless as infinity. It is the middle ground between light and shadow, between science and superstition, and it lies between the pit of man's fears and the summit of his knowledge. This is the dimension of imagination. It is NOT, an area which we call the Twilight Zone, but an area that believes junk science, and the very industries and lobbyist some Texas Hunters, the cervid industry, that insist on shoving the fake news down their throats, we call this ted nugent junk science, and in TEXAS, sometimes you just can't fix stupid, this is where the rubber meets the road, here's your sign!

chronic wasting disease cwd tse prion aka mad deer elk disease, if you consume a cwd tse prion positive cervid, then months, years, decades later, go on to have surgery, dental, ophthalmology, endoscopy, donate tissue, blood, organs, you then expose those medical theaters and tissue, blood, organs, that are incubating the infectious cwd tse prion disease, to everyone that comes in contact.

these are not memes, these are actual statements from hunters/industry in Texas about CWD tse prion.

God help them, and us...terry

''Got a call today from TPWD, I’ve got a mule deer that tested early positive for CWD. I’m soon to turn into a zombie because I have already been eating it. They advised not to consume any of the meat...too late! They want to come confiscate what meat is left once they get more results back from another lab.''

snip...



Terry S. Singeltary Sr.
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