Monday, February 15, 2016

Distinctive properties of plaque-type dura mater graft-associated Creutzfeldt–Jakob disease in cell-protein misfolding cyclic amplification

Distinctive properties of plaque-type dura mater graft-associated Creutzfeldt–Jakob disease in cell-protein misfolding cyclic amplification


Atsuko Takeuchi, Atsushi Kobayashi, Piero Parchi, Masahito Yamada, Masanori Morita, Shusei Uno and Tetsuyuki Kitamoto




There are two distinct subtypes of dura mater graft-associated Creutzfeldt–Jakob disease (dCJD) with methionine homozygosity at codon 129 of the PRNP gene. The majority of cases is represented by a non-plaque-type (np-dCJD) resembling sporadic CJD (sCJD)-MM1 or -MV1, while the minority by a plaque-type (p-dCJD). p-dCJD shows distinctive phenotypic features, namely numerous kuru plaques and an abnormal isoform of prion protein (PrPSc) intermediate in size between types 1 and 2. Transmission studies have shown that the unusual phenotypic features of p-dCJD are linked to the V2 prion strain that is associated with sCJD subtypes VV2 or -MV2. In this study, we applied protein misfolding cyclic amplification (PMCA) using recombinant human prion protein as a substrate and demonstrated that p-dCJD prions show amplification features that are distinct from those of np-dCJD. Although no amplification of np-dCJD prions was observed with either 129 M or 129 V substrate, p-dCJD prions were drastically amplified with the 129 V substrates, despite the PRNP codon 129 incompatibility between seed and substrate. Moreover, by using a type 2 PrPSc-specific antibody not recognizing PrPSc in p-dCJD, we found that type 2 products are generated de novo from p-dCJD prions during PMCA with the 129 V substrates. These findings suggest that our cell-PMCA is a useful tool for easily and rapidly identifying acquired CJD associated with the transmission of the V2 CJD strain to codon 129 methionine homozygotes, based on the preference for the 129 V substrate and the type of the amplified products.



all iatrogenic cjd is, is sporadic cjd, until the iatrogenic event is traced back, discovered, documented, put into the academic domain and finally public domain. ...tss


These findings support the view of the co-occurrence of multiple PrPres fragments within MM1 prions rather than the co-occurrence of multiple prion strains within the same individual.


In conclusion, the present study, together with evidence from other groups,11,12 suggests that the co-occurrence of multiple PrPres fragments within a single sCJD patient is a universal phenomenon. These findings show that the conventional typing of PrPres merely represents the predominant PrPres subpopulation among multiple co-existing PrPres fragments. Besides the general co-occurrence of multiple PrPres fragments, the condition of PK digestion easily affects the size of PrPres.27,28 Indeed, insufficient PK digestion can generate type 1 PrPres-specific antibody–reactive fragments in the sCJD patients classified as type 2.29 Furthermore, it is possible that the conventional Western blot analysis fails to detect type 2 PrPres in sCJD-MM1+2 cases showing very focal perivacuolar PrP deposition in the brain.9 These confusing aspects of PrPres typing question the validity of the conventional molecular typing system. For a precise classification, it may be appropriate that the neuropathological phenotyping [synaptic (SY), perivacuolar (PV), plaque (PL), or patchy plaque (PP)] be combined with the molecular typing [eg, sCJD-MM1(+2)/SY+PV] the sCJD-MM patient showing synaptic + perivacuolar PrP deposition, but not type 2 PrPres in the conventional Western blot analysis.



Even though experiments in rodents performed some years ago demonstrated the phenomenon of multiple PrPSc types within one animal [21], incongruity regarding the frequency of co-occurrence of different PrPSc types in sCJD patients still endures. Initial studies suggested that this is a relatively rare event, occurring in less than 5% of patients [8]. This low incidence may, at least partially, be attributed to the fact that these analyses are routinely performed on a limited range of distinct brain regions per patient [8,13]. The fact that region-specific presence of distinct PrPSc types may occur in sCJD was highlighted by a publication investigating ten defined regions within the central nervous system in 14 patients with sCJD [16]. The authors found more than one PrPSc type in five individuals and hypothesized that the co-occurrence of more than one PrPSc type could be the rule, rather than the exception, if the entire central nervous system was investigated. In our analysis of nine distinct central-nervous-system regions in 50 patients with sCJD using a standardized protocol [22], we detected more than one PrPSc type in nine individuals. The observation that most patients harboring two PrPSc types are codon-129 methionine/valine heterozygotes stresses the significance of this polymorphism in the replicative cycle of PrPSc.



Coexistence of multiple PrPSc types in individuals with Creutzfeldt-Jakob disease


Magdalini Polymenidou, Katharina Stoeck, Markus Glatzel, Martin Vey, Anne Bellon, and Adriano Aguzzi




Background The molecular typing of sporadic Creutzfeldt-Jakob disease (CJD) is based on the size and glycoform ratio of protease-resistant prion protein (PrPSc), and on PRNP haplotype. On digestion with proteinase K, type 1 and type 2 PrPSc display unglycosylated core fragments of 21 kDa and 19 kDa, resulting from cleavage around amino acids 82 and 97, respectively.


Methods We generated anti-PrP monoclonal antibodies to epitopes immediately preceding the differential proteinase K cleavage sites. These antibodies, which were designated POM2 and POM12, recognise type 1, but not type 2, PrPSc.


Findings We studied 114 brain samples from 70 patients with sporadic CJD and three patients with variant CJD. Every patient classified as CJD type 2, and all variant CJD patients, showed POM2/POM12 reactivity in the cerebellum and other PrPSc-rich brain areas, with a typical PrPSc type 1 migration pattern.


Interpretation The regular coexistence of multiple PrPSc types in patients with CJD casts doubts on the validity of electrophoretic PrPSc mobilities as surrogates for prion strains, and questions the rational basis of current CJD classifications.






The discovery of heritable polymorphic PK cleavage sites in PrPSc has been used for the molecular classification of CJD cases.11,13,14,17 In concert with the codon 129 PRNP haplotype, the different PrPSc types correlate with distinct disease phenotypes. Most patients with the MM1 or MV1 subtype present with so-called classic CJD, and show rapid progressive dementia, early myoclonus, visual disturbances including cortical blindness, and a disease duration of approximately 4 months. By contrast, patients with the MV2 or VV2 subtype show an atypical disease course, with a longer disease duration (6–18 months), early ataxia, predominant extrapyramidal symptoms, and late-onset dementia.18


Because of the limited resolution of conventional western blot systems, the coexistence of PK-digested PrPSc bands at 19 kDa and 21 kDa is not recognisable by conventional antibodies binding to both PrPSc types, including POM1. By mixing brain homogenates that contain solely type 1 or type 2 PrPSc, we have found that 3F4-based or POM1-based western blots consistently fail to detect type 1 PrPSc whenever it represents less than 30–40% of total PrPSc. We addressed these problems by developing a set of antibodies binding Nproximal epitopes of PrP, which recognise PK-digested type 1 PrPSc (cleaved at amino acid 82 or upstream), but not type 2 PrPSc (cleaved at amino acids 97 or 86).


On investigation of 70 sporadic CJD cases, we determined that 50% of the patients formerly classified as CJD type 2 had low but detectable amounts of PrPSc type 1 in their cerebral cortex. Investigations of additional brain areas revealed that significant amounts of PrPSc type 1 coexist with type 2 in at least some areas of all patients classified as having type 2 disease. Co-occurrence of CJD types in the same brain has been previously reported in approximately 30% of the tested cases.24 Using unambiguous analytical tools, we have established that type 2 PrPSc does not exist independently from type 1 PrPSc, at least not in the Swiss CJD patients included in our study.


Furthermore, using our type 1-specific antibodies, we found type 1 PrPSc content in three cortical samples of vCJD patients.


Glycotyping of blots incubated with 3F4/POM1 versus POM2/POM12 showed no significant differences in glycoform ratios, neither for the same groups incubated with different antibodies, nor for the two PrPSc groups, in accordance with previously published data.33 The homogeneity of glycoforms suggests that our collective is comparable to those investigated in other countries, despite the peculiarities of Swiss CJD epidemiology.34


The results presented here do not question the validity of the established correlations between PrPSc types and clinical findings. The coincident presence of PrPSc type 1 may not alter the expected clinical outcome of patients with predominant PrPSc type 2 deposition. However, the existence of heritable PrPSc types implies that strainspecific characteristics of prions are enshrined in the conformation of PrPSc, and that the different core fragment sizes of PK-digested PrPSc may be used as surrogates of such conformational variations. In the light of the data presented here, the strength of these arguments becomes somewhat questionable.


Why did the co-existence of type 1 and type 2 PrPSc go often undetected in the past, despite its ubiquitous presence? Maybe the 21 kDa band characteristic of type 1 PrPSc is easily obfuscated by the simultaneous occurrence of the neighbouring 19 kDa type 2 band and of the monoglycosylated PrPSc band. We tested this hypothesis by ascertaining the detectability threshold of type 1 PrPSc in mixtures of pure type 1 and type 2 samples. Indeed, we found that type 1 PrPSc becomes undiscernible as soon as 40% or more type 2 PrPSc is present. This observation provides a plausible explanation for the failure to appreciate the invariable coexistence of PrPSc types in previous reports.


To determine whether a similar phenomenon occurs in patients classified as type 1 would be interesting. However, this issue is impossible to address at present, as there is no CJD type-2-specific antibody. The fact that the type 1 coexistence is also apparent in cortical samples from CJD patients, indicates that the currently described phenomenon might be a more general one. The above results set the existing CJD classifications into debate and introduce interesting questions about human CJD types. For example, do human prion types exist in a dynamic equilibrium in the brains of affected individuals? Do they coexist in most or even all CJD cases? Is the biochemically identified PrPSc type simply the dominant type, and not the only PrPSc species?



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


07 02:27 AM


Terry S. Singeltary Sr. said:


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


2015-12-07 02:27 AM


Terry S. Singeltary Sr. said: re-Evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy


Nature 525, 247?250 (10 September 2015) doi:10.1038/nature15369 Received 26 April 2015 Accepted 14 August 2015 Published online 09 September 2015 Updated online 11 September 2015 Erratum (October, 2015)



I would kindly like to comment on the Nature Paper, the Lancet reply, and the newspaper articles.


First, I applaud Nature, the Scientist and Authors of the Nature paper, for bringing this important finding to the attention of the public domain, and the media for printing said findings.


Secondly, it seems once again, politics is getting in the way possibly of more important Transmissible Spongiform Encephalopathy TSE Prion scientific findings. findings that could have great implications for human health, and great implications for the medical surgical arena. but apparently, the government peer review process, of the peer review science, tries to intervene again to water down said disturbing findings.


where have we all heard this before? it’s been well documented via the BSE Inquiry. have they not learned a lesson from the last time?


we have seen this time and time again in England (and other Country’s) with the BSE mad cow TSE Prion debacle.


snip...see full Singeltary Nature comment here;



see Singeltary comments to Plos ;




BSE101/1 0136






From: . Dr J S Metiers DCMO


4 November 1992




1. Thank you for showing me Diana Dunstan's letter. I am glad that MRC have recognised the public sensitivity of these findings and intend to report them in their proper context. 'This hopefully will avoid misunderstanding and possible distortion by the media to portray the results as having more greater significance than the findings so far justify.


2. Using a highly unusual route of transmission (intra-cerebral injection) the researchers have demonstrated the transmission of a pathological process from two cases one of severe Alzheimer's disease the other of Gerstmann-Straussler disease to marmosets. However they have not demonstrated the transmission of either clinical condition as the "animals were behaving normally when killed". As the report emphasises the unanswered question is whether the disease condition would have revealed itself if the marmosets had lived longer. They are planning further research to see if the conditions, as opposed to the partial pathological process, is transmissible.


what are the implications for public health?


3. The route 'of transmission is very specific and in the natural state of things highly unusual. However it could be argued that the results reveal a potential risk, in that brain tissue from these two patients has been shown to transmit a pathological process. Should therefore brain tissue from such cases be regarded as potentially infective? Pathologists, morticians, neuro surgeons and those assisting at neuro surgical procedures and others coming into contact with "raw" human brain tissue could in theory be at risk. However, on a priori grounds given the highly specific route of transmission in these experiments that risk must be negligible if the usual precautions for handling brain tissue are observed.






BSE101/1 0137


4. The other dimension to consider is the public reaction. To some extent the GSS case demonstrates little more than the transmission of BSE to a pig by intra-cerebral injection. If other prion diseases can be transmitted in this way it is little surprise that some pathological findings observed in GSS were also transmissible to a marmoset. But the transmission of features of Alzheimer's pathology is a different matter, given the much greater frequency of this disease and raises the unanswered question whether some cases are the result of a transmissible prion. The only tenable public line will be that "more research is required’’ before that hypothesis could be evaluated. The possibility on a transmissible prion remains open. In the meantime MRC needs carefully to consider the range and sequence of studies needed to follow through from the preliminary observations in these two cases. Not a particularly comfortable message, but until we know more about the causation of Alzheimer's disease the total reassurance is not practical.


J S METTERS Room 509 Richmond House Pager No: 081-884 3344 Callsign: DOH 832 llllYc!eS 2 92/11.4/1.2



>>> 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 ?


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



Sunday, November 22, 2015


*** Effect of heating on the stability of amyloid A (AA) fibrils and the intra- and cross-species transmission of AA amyloidosis Abstract


Amyloid A (AA) amyloidosis is a protein misfolding disease characterized by extracellular deposition of AA fibrils. AA fibrils are found in several tissues from food animals with AA amyloidosis. For hygienic purposes, heating is widely used to inactivate microbes in food, but it is uncertain whether heating is sufficient to inactivate AA fibrils and prevent intra- or cross-species transmission. We examined the effect of heating (at 60 °C or 100 °C) and autoclaving (at 121 °C or 135 °C) on murine and bovine AA fibrils using Western blot analysis, transmission electron microscopy (TEM), and mouse model transmission experiments. TEM revealed that a mixture of AA fibrils and amorphous aggregates appeared after heating at 100 °C, whereas autoclaving at 135 °C produced large amorphous aggregates. AA fibrils retained antigen specificity in Western blot analysis when heated at 100 °C or autoclaved at 121 °C, but not when autoclaved at 135 °C. Transmissible pathogenicity of murine and bovine AA fibrils subjected to heating (at 60 °C or 100 °C) was significantly stimulated and resulted in amyloid deposition in mice. Autoclaving of murine AA fibrils at 121 °C or 135 °C significantly decreased amyloid deposition. Moreover, amyloid deposition in mice injected with murine AA fibrils was more severe than that in mice injected with bovine AA fibrils. Bovine AA fibrils autoclaved at 121 °C or 135 °C did not induce amyloid deposition in mice. These results suggest that AA fibrils are relatively heat stable and that similar to prions, autoclaving at 135 °C is required to destroy the pathogenicity of AA fibrils. These findings may contribute to the prevention of AA fibril transmission through food materials to different animals and especially to humans.


Purchase options Price * Issue Purchase USD 511.00 Article Purchase USD 54.00




*** Transmission of Creutzfeldt-Jakob disease to a chimpanzee by electrodes contaminated during neurosurgery ***


Gibbs CJ Jr, Asher DM, Kobrine A, Amyx HL, Sulima MP, Gajdusek DC. Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.


Stereotactic multicontact electrodes used to probe the cerebral cortex of a middle aged woman with progressive dementia were previously implicated in the accidental transmission of Creutzfeldt-Jakob disease (CJD) to two younger patients. The diagnoses of CJD have been confirmed for all three cases. More than two years after their last use in humans, after three cleanings and repeated sterilisation in ethanol and formaldehyde vapour, the electrodes were implanted in the cortex of a chimpanzee. Eighteen months later the animal became ill with CJD. This finding serves to re-emphasise the potential danger posed by reuse of instruments contaminated with the agents of spongiform encephalopathies, even after scrupulous attempts to clean them.



the warning shots fired over the bow of the boat that were never heard ;




This was used to help cows super ovulate. This tissue was considered to be of greatest risk of containing BSE and consequently transmitting the disease...







I was quite prepared to believe in unofficial pituitary hormones, also in the 1970's, whether as described by Dr. Little, or in other circumstances, for animal use.




The fact that there were jars of pituitaries (or extract) around on shelves is attested by the still potent 1943 pituitaries, described in Stockell Hartree et al. (J/RF/17/291) which had come from the lab. at Mill Hill. Having taken the trouble to collect them, they were not lightly thrown out...



3. The extraction is from a pool of pituitary glands collected from abbatoirs and the process used is unlikely to have any effect on the BSE agent. Hormones extracted from human pituitary glands have been responsible for a small number of Creutzfeldt Jacob disease in man.










snip...see at bottom ;


Friday, January 10, 2014


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



Tuesday, August 4, 2015


*** FDA U.S. Measures to Protect Against BSE ***



*** now, from all the consumption and exposure above, now think iatrogenic cjd tse prion at a hospital near you, what if?


Thursday, August 13, 2015


Iatrogenic CJD due to pituitary-derived growth hormone with genetically determined incubation times of up to 40 years



Monday, August 17, 2015


*** FDA Says Endoscope Makers Failed to Report Superbug Problems OLYMPUS


*** 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 ;



Thursday, September 10, 2015


25th Meeting of the Transmissible Spongiform Encephalopathies Advisory Committee Food and Drug Administration Silver Spring, Maryland June 1, 2015



Sunday, January 17, 2016


Of Grave Concern Heidenhain Variant Creutzfeldt Jakob Disease



Saturday, February 13, 2016


The Risk of Prion Infection through Bovine Grafting Materials in dentistry



kind regards, terry