Biochem Biophys Rep. 2023 Jul; 34: 101446.
Published online 2023 Mar 3. doi: 10.1016/j.bbrep.2023.101446
PMCID: PMC10009011
PMID: 36923008
Autoclave treatment fails to completely inactivate DLB alpha-synuclein seeding activity
Jung-Youn Han,a,1 Kyung-Je Park,b,1 Hoo-Chang Park,b Yu-Ran Lee,b Roger A. Moore,c Hyun-Joo Sohn,b,∗∗ and Young Pyo Choia,∗
Abstract
Synucleinopathies are characterized by the deposition of alpha-synuclein (α-syn) aggregates in brain tissue. Pathological α-syn aggregates propagate in a prion-like manner and display prion-like biochemical properties. Using RT-QuIC, we measured α-syn seeding activity from brains of Dementia with Lewy body (DLB) patients post autoclave. Here, we show that autoclaving at 121 °C removes one to two log10 of α-syn seeding activity but the remaining 50% seeding dose (SD50) is more than 107/mg tissue. DLB brain samples autoclaved at 132 °C still revealed an SD50 of approximately 106/mg tissue. Our data suggest that DLB α-syn seeds are incompletely inactivated by standard autoclave, thus highlighting the need for evaluating laboratory procedures that fully inactivate them.
Keywords: Alpha-synuclein, Seeds, Inactivation, Autoclave, RT-QuIC, Prion
snip...
3. Results and discussion
We first measured α-syn concentrations by ELISA in the brain homogenates used for seeding. Concentrations of α-syn were measured in the range of 0.9 μg/ml to 2.1 μg/ml in the 10% homogenates, showing some variation between samples (Table 1). Similarly, immunoblot band intensities of α-syn migrating approximately at 18–19 KDa also displayed minor variation between samples (Fig. 1). We then examined α-syn seeding activity in non-autoclaved DLB brain homogenates with serial dilutions up to 10–11. Flat responses were seen in control (Con1) reactions and positive responses rising above baseline were detected in reactions with 10–4 to 10-8 DLB1 BA9 tissue dilutions (Fig. 2A) or 10-4 to 10-9 DLB1 BA39 tissue dilutions (Fig. 2E). The SD50 values reached 8.78 log10 SD50/mg tissue (BA9) or 9.7 log10 SD50/mg tissue (BA39), respectively. We then analyzed α-syn seeding activity in the BA9 tissue of three more DLB cases. Positive RT-QuIC responses were detectable in reactions seeded with up to 10-7 to 10-8 dilutions, with relative concentrations of α-syn seeding activity in the range of 8–9 log10 SD50/mg tissue (Table 1).
Next, brain homogenates from DLB1 patient were autoclaved either at 121 °C or 132 °C for 20 min prior to RT-QuIC. At 121 °C, positive responses were identified in reactions seeded with up to 10-7 dilution of BA9 tissue but were not detectable with tissue dilutions of 10–8 to 10-9 (Fig. 2B). The remaining α-syn seeding activity was 107.12/mg tissue. At 132 °C, α-syn seeding activity was identified only in reactions seeded with 10–4 to 10-5 dilutions of BA9 tissue with the SD50 value of 105.95/mg tissue (Fig. 2C). When compared to non-autoclaved BA9 homogenate, SD50 values were reduced by 1.67 log10 following autoclave at 121 °C or by 2.83 log10 following 132 °C. Protein aggregation rates (PARs) were calculated as the inverse of the lag phase. Temperature-dependent reductions in PAR were observed at each dilution following autoclave treatment (Fig. 2D). Similar RT-QuIC kinetics were observed for the DLB1 BA39 homogenates (Fig. 2F, G and H). Following autoclave of the DLB1 BA39 homogenates, seeding activity titers decreased by 2.42 log10 (at 121 °C) or 3.13 log10 (at 132 °C) when compared to the corresponding non-autoclaved sample. The analysis of BA9 tissue from three additional DLB cases autoclaved at 121 °C showed similar results to DLB1, with the reduction of SD50 values in the range of 0.88–1.25 log10 (Table 1).
We then investigated prion seeding activity in CWD-positive elk brains before and after autoclave treatment. The SD50 values in non-autoclaved brain homogenates from CWD-positive elk were in the range of 6–7 log10 SD50/mg tissue (Table 1). Following autoclave treatment at 121 °C for 20 min, SD50 values were reduced by 1.25–1.38 log10 when compared to the corresponding non-autoclaved samples and the remaining prion seeding titers reached 5 to 5.5 log10 SD50/mg tissue.
In this study, we quantitated the decrease of seeding activity in autoclaved DLB α-syn aggregates or CWD prions using end-point RT-QuIC [8]. Our results show that autoclaving at 121 °C removes one to two log10 of α-syn seeding activity but the remaining seeding activity titer is more than 107/mg tissue. Consistent with our data, α-syn seeding activity from other synucleinopathies such as PD or multiple system atrophy (MSA) has also been reported as highly resistant to physiochemical inactivation. Tarutani and colleagues showed that MSA α-syn seeding activity measured using a cellular system was shown to be reduced by approximately 1 log10 SD50 following autoclaving at 134 °C for 20 min [6]. Another study performed by Pinder and colleagues reported that autoclaving PD brain homogenates at 134 °C removed 2.2 to 2.4 log10 of α-syn seeding titers irrespective of autoclave time [13]. Thus, our data suggest that α-syn seeding activity in DLB brains is more susceptible to inactivation by autoclave than that of PD or MSA brains. Notably, pathogenic α-syn aggregates derived from DLB, PD or MSA brains have been reported to differ in their physiochemical and biological properties [[14], [15], [16], [17]]. While further investigations are needed to more precisely quantify the levels of resistance of α-syn seeds from various synucleinopathies, remaining α-syn seeding activity after standard autoclave might be a public health problem given the structural, biochemical and mechanistic similarities between PrPSc and other proteopathic seeds [18,19]. There are growing concerns that neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease (AD) might be transmissible between humans [20]. In fact, multiple cases of iatrogenic amyloid β transmission have been reported in recent years [21,22]. To the best of our knowledge, there is so far no evidence of α-syn transmission between humans. Nonetheless, incomplete inactivation of α-syn aggregates after standard autoclave procedures could be a concern in hospital environments. Therefore, more effective protocols for pathological α-syn aggregate inactivation are needed to fully remove any risk associated with human-to-human transmission. These inactivation methods could include treatments with 1–2 N sodium hydroxide, 20,000 ppm sodium hypochlorite, vaporized hydrogen peroxide, a mildly acidic formulation of hypochlorous acid or acidic SDS, and prolonged autoclaving at higher temperature [5,[23], [24], [25], [26], [27]], all of which were found to be highly effective in decontaminating prions.
***> Our data suggest that DLB α-syn seeds are incompletely inactivated by standard autoclave, thus highlighting the need for evaluating laboratory procedures that fully inactivate them.
Wednesday, September 7, 2022
Multiple system atrophy MSA, Prion, TSE, iatrogenic, What If? ANOTHER CRITICAL REVIEW
Friday, February 4, 2022
Different α-synuclein prion strains cause dementia with Lewy bodies and multiple system atrophy, iatrogenic transmission, what if?
SUNDAY, MARCH 4, 2018
Can Aβ Seeds Be Transferred During Neurosurgery?
Thursday, March 8, 2018
Familial human prion diseases associated with prion protein mutations Y226X and G131V are transmissible to transgenic mice expressing human prion protein
THURSDAY, FEBRUARY 15, 2018
Iatrogenic Creutzfeldt-Jakob disease with Amyloid-β pathology: an international study
MSA prions exhibit remarkable stability and resistance to inactivation
Authors
Authors and affiliations
Amanda L. WoermanSabeen A. KazmiSmita PatelYevgeniy FreymanAbby OehlerAtsushi AoyagiDaniel A. MordesGlenda M. HallidayLefkos T. MiddletonSteve M. GentlemanSteven H. OlsonStanley B. PrusinerEmail author
Original Paper
First Online: 28 August 2017
Abstract
In multiple system atrophy (MSA), progressive neurodegeneration results from the protein α-synuclein misfolding into a self-templating prion conformation that spreads throughout the brain. MSA prions are transmissible to transgenic (Tg) mice expressing mutated human α-synuclein (TgM83+/−), inducing neurological disease following intracranial inoculation with brain homogenate from deceased patient samples. Noting the similarities between α-synuclein prions and PrP scrapie (PrPSc) prions responsible for Creutzfeldt–Jakob disease (CJD), we investigated MSA transmission under conditions known to result in PrPSc transmission. When peripherally exposed to MSA via the peritoneal cavity, hind leg muscle, and tongue, TgM83+/− mice developed neurological signs accompanied by α-synuclein prions in the brain. Iatrogenic CJD, resulting from PrPSc prion adherence to surgical steel instruments, has been investigated by incubating steel sutures in contaminated brain homogenate before implantation into mouse brain. Mice studied using this model for MSA developed disease, whereas wire incubated in control homogenate had no effect on the animals. Notably, formalin fixation did not inactivate α-synuclein prions. Formalin-fixed MSA patient samples also transmitted disease to TgM83+/− mice, even after incubating in fixative for 244 months. Finally, at least 10% sarkosyl was found to be the concentration necessary to partially inactivate MSA prions. These results demonstrate the robustness of α-synuclein prions to denaturation. Moreover, they establish the parallel characteristics between PrPSc and α-synuclein prions, arguing that clinicians should exercise caution when working with materials that might contain α-synuclein prions to prevent disease.
Keywords
α-synuclein Neurodegeneration Propagation Proteinopathies Transmission models
***> These results demonstrate the robustness of α-synuclein prions to denaturation.
***> Moreover, they establish the parallel characteristics between PrPSc and α-synuclein prions, arguing that clinicians should exercise caution when working with materials that might contain α-synuclein prions to prevent disease.
THURSDAY, AUGUST 10, 2017
Minimise transmission risk of CJD and vCJD in healthcare settings Updated 10 August 2017
Friday, January 29, 2016
Synucleinopathies: Past, Present and Future, iatrogenic, what if?
snip...see full Singeltary Nature comment here;
re-Evidence for human transmission of amyloid-? 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)
http://www.nature.com/
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.
That 'anonymous' Lancet editorial was disgraceful. The editor, Dick Horton is not a scientist.
The pituitary cadavers were very likely elderly and among them some were on their way to CJD or Alzheimer's. Not a bit unusual. Then the recipients, who got pooled extracts injected from thousands of cadavers, were 100% certain to have been injected with both seeds. No surprise that they got both diseases going after thirty year incubations.
That the UK has a "system in place to assist science journalists" to squash embargoed science reports they find 'alarming' is pathetic.
Sounds like the journalists had it right in the first place: 'Alzheimer's may be a transmissible infection' in The Independent to 'You can catch Alzheimer's' in The Daily Mirror or 'Alzheimer's bombshell' in The Daily Express
if not for the journalist, the layperson would not know about these important findings.
where would we be today with sound science, from where we were 30 years ago, if not for the cloak of secrecy and save the industry at all cost mentality?
when you have a peer review system for science, from which a government constantly circumvents, then you have a problem with science, and humans die.
to date, as far as documented body bag count, with all TSE prion named to date, that count is still relatively low (one was too many in my case, Mom hvCJD), however that changes drastically once the TSE Prion link is made with Alzheimer's, the price of poker goes up drastically.
so, who makes that final decision, and how many more decades do we have to wait?
the iatrogenic mode of transmission of TSE prion, the many routes there from, load factor, threshold from said load factor to sub-clinical disease, to clinical disease, to death, much time is there to spread a TSE Prion to anywhere, but whom, by whom, and when, do we make that final decision to do something about it globally? how many documented body bags does it take? how many more decades do we wait? how many names can we make up for one disease, TSE prion?
Professor Collinge et al, and others, have had troubles in the past with the Government meddling in scientific findings, that might in some way involve industry, never mind human and or animal health.
FOR any government to continue to circumvent science for monetary gain, fear factor, or any reason, shame, shame on you.
in my opinion, it's one of the reasons we are at where we are at to date, with regards to the TSE Prion disease science i.e. money, industry, politics, then comes science, in that order.
greed, corporate, lobbyist there from, and government, must be removed from the peer review process of sound science, it's bad enough having them in the pharmaceutical aspect of healthcare policy making, in my opinion.
my mother died from confirmed hvCJD, and her brother (my uncle) Alzheimer's of some type (no autopsy?). just made a promise, never forget, and never let them forget, before I do.
I kindly wish to remind the public of the past, and a possible future we all hopes never happens again. ...
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
Tuesday, September 8, 2015
Evidence for α-synuclein prions causing multiple system atrophy in humans with parkinsonism
FRIDAY, JANUARY 10, 2014
vpspr, sgss, sffi, TSE, an iatrogenic by-product of gss, ffi, familial type prion disease, what it ???
sporadic CJD, along with new TSE prion disease in humans, of which the young are dying, of which long duration of illness from onset of symptoms to death have been documented, only to have a new name added to the pot of prion disease i.e. sporadic GSS, sporadic FFI, and or VPSPR.
I only ponder how a familial type disease could be sporadic with no genetic link to any family member?
when the USA is the only documented Country in the world to have documented two different cases of atypical H-type BSE, with one case being called atypical H-G BSE with the G meaning Genetic, with new science now showing that indeed atypical H-type BSE is very possible transmitted to cattle via oral transmission (Prion2014). sporadic CJD and VPSPR have been rising in Canada, USA, and the UK, with the same old excuse, better surveillance.
You can only use that excuse for so many years, for so many decades, until one must conclude that CJD TSE prion cases are rising. a 48% increase in CJD in Canada is not just a blip or a reason of better surveillance, it is a mathematical rise in numbers.
More and more we are seeing more humans exposed in various circumstance in the Hospital, Medical, Surgical arenas to the TSE Prion disease, and at the same time in North America, more and more humans are becoming exposed to the TSE prion disease via consumption of the TSE prion via deer and elk, cattle, sheep and goats, and for those that are exposed via or consumption, go on to further expose many others via the iatrogenic modes of transmission of the TSE prion disease i.e. friendly fire.
I pondered this mode of transmission via the victims of sporadic FFI, sporadic GSS, could this be a iatrogenic event from someone sub-clinical with sFFI or sGSS ? what if?
Sunday, September 7, 2014
Twice as many cases of early dementia than was thought Alzheimer’s Society, the London School of Economics and the Institute of Psychiatry
FRIDAY, JANUARY 10, 2014
vpspr, sgss, sffi, TSE, an iatrogenic by-product of gss, ffi, familial type prion disease, what if ???
Greetings Friends, Neighbors, and Colleagues,
vpspr, sgss, sffi, TSE, an iatrogenic by-product of gss, ffi, familial type prion disease, what if ???
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.
sporadic CJD, along with new TSE prion disease in humans, of which the young are dying, of which long duration of illness from onset of symptoms to death have been documented, only to have a new name added to the pot of prion disease i.e. sporadic GSS, sporadic FFI, and or VPSPR.
I only ponder how a familial type disease could be sporadic with no genetic link to any family member?
when the USA is the only documented Country in the world to have documented two different cases of atypical H-type BSE, with one case being called atypical H-G BSE with the G meaning Genetic, with new science now showing that indeed atypical H-type BSE is very possible transmitted to cattle via oral transmission (Prion2014).
sporadic CJD and VPSPR have been rising in Canada, USA, and the UK, with the same old excuse, better surveillance.
You can only use that excuse for so many years, for so many decades, until one must conclude that CJD TSE prion cases are rising. a 48% incease in CJD in Canada is not just a blip or a reason of better surveillance, it is a mathematical rise in numbers.
More and more we are seeing more humans exposed in various circumstance in the Hospital, Medical, Surgical arenas to the TSE Prion disease, and at the same time in North America, more and more humans are becoming exposed to the TSE prion disease via consumption of the TSE prion via deer and elk, cattle, sheep and goats, and for those that are exposed via or consumption, go on to further expose many others via the iatrogenic modes of transmission of the TSE prion disease i.e. friendly fire.
I pondered this mode of transmission via the victims of sporadic FFI, sporadic GSS, could this be a iatrogenic event from someone sub-clinical with sFFI or sGSS ? what if?
The occurrence of the disease in a patient who had contact with cases of familial C.J.D., but was not genetically related, has been described in Chile (Galvez et al., 1980) and in France (Brown et al., 1979b). In Chile the patient was related by marriage, but with no consanguinity, and had social contact with subsequently affected family members for 13 years before developing the disease. The contact case in France also married into a family in which C.J.D. was prevalent and had close contact with an affected member. In neither instance did the spouse of the non-familial case have the disease. The case described in this report was similarly related to affected family members and social contact had occurred for 20 years prior to developing C.J.D. If contact transmission had occurred, the minimum transmission period would be 11 years. Contact between sporadic cases has not been described and it is remarkable that possible contact transmissions have all been with familial cases. No method of transmission by casual social contact has been suggested. ***The occurrence of contact cases raises the possibility that transmission in families may be effected by an unusually virulent strain of the agent.
***The occurrence of contact cases raises the possibility that transmission in families may be effected by an unusually virulent strain of the agent.
see full text here;
Furthermore, GSS A117V infected vole brains were able to induce the same disease phenotype in recipient voles within 3–4 months after challenge, proving that a prion agent propagated in the brains of infected animals. These findings imply that brains of GSS patients harbor infectious prions with transmissibility features similar to those found in other human and animal TSEs.
https://www.nature.com/articles/srep20443
Wednesday, May 16, 2012
Alzheimer’s disease and Transmissible Spongiform Encephalopathy prion disease, Iatrogenic, what if ?
Terry S. Singeltary Sr. Proposal ID: 29403
*** 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.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8006664&dopt=Abstract
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
IN CONFIDENCE
5 NOVEMBER 1992
TRANSMISSION OF ALZHEIMER TYPE PLAQUES TO PRIMATES
[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.]
2012
Alzheimer’s disease and Transmissible Spongiform Encephalopathy prion disease, Iatrogenic, what if ?
Background
Alzheimer’s disease and Transmissible Spongiform Encephalopathy disease have both been around a long time, and was discovered in or around the same time frame, early 1900’s. Both diseases are incurable and debilitating brain disease, that are in the end, 100% fatal, with the incubation/clinical period of the Alzheimer’s disease being longer (most of the time) than the TSE prion disease. Symptoms are very similar, and pathology is very similar.
Methods
Through years of research, as a layperson, of peer review journals, transmission studies, and observations of loved ones and friends that have died from both Alzheimer’s and the TSE prion disease i.e. Heidenhain Variant Creutzfelt Jakob Disease CJD.
Results
I propose that Alzheimer’s is a TSE disease of low dose, slow, and long incubation disease, and that Alzheimer’s is Transmissible, and is a threat to the public via the many Iatrogenic routes and sources. It was said long ago that the only thing that disputes this, is Alzheimer’s disease transmissibility, or the lack of. The likelihood of many victims of Alzheimer’s disease from the many different Iatrogenic routes and modes of transmission as with the TSE prion disease.
Conclusions
There should be a Global Congressional Science round table event set up immediately to address these concerns from the many potential routes and sources of the TSE prion disease, including Alzheimer’s disease, and a emergency global doctrine put into effect to help combat the spread of Alzheimer’s disease via the medical, surgical, dental, tissue, and blood arena’s. All human and animal TSE prion disease, including Alzheimer’s should be made reportable in every state, and Internationally, WITH NO age restrictions. Until a proven method of decontamination and autoclaving is proven, and put forth in use universally, in all hospitals and medical, surgical arena’s, or the TSE prion agent will continue to spread. IF we wait until science and corporate politicians wait until politics lets science _prove_ this once and for all, and set forth regulations there from, we will all be exposed to the TSE Prion agents, if that has not happened already.
end...tss
Alzheimer’s disease and Transmissible Spongiform Encephalopathy prion disease, Iatrogenic, what if ?
source references ...end...tss
Terry S. Singeltary Sr.
