Prion-infected Brain Homogenate Research Articles

Efficacy of Wex-cide 128 disinfectant against multiple prion strains.

Prion diseases are transmissible, fatal neurologic diseases that include Creutzfeldt-Jakob Disease (CJD) in humans, chronic wasting disease (CWD) in cervids, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep. Prions are extremely difficult to inactivate and established methods to reduce prion infectivity are often dangerous, caustic, expensive, or impractical. Identifying viable and safe methods for treating prion contaminated materials is important for hospitals, research facilities, biologists, hunters, and meat-processors. For three decades, some prion researchers have used a phenolic product called Environ LpH (eLpH) to inactivate prions. ELpH has been discontinued, but a similar product, Wex-cide 128, containing the similar phenolic chemicals as eLpH is now available. In the current study, we directly compared the anti-prion efficacy of eLpH and Wex-cide 128 against prions from four different species (hamster 263K, cervid CWD, mouse 22L and human CJD). Decontamination was performed on either prion infected brain homogenates or prion contaminated steel wires and mouse bioassay was used to quantify the remaining prion infectivity. Our data show that both eLpH and Wex-cide 128 removed 4.0-5.5 logs of prion infectivity from 22L, CWD and 263K prion homogenates, but only about 1.25-1.50 logs of prion infectivity from human sporadic CJD. Wex-cide 128 is a viable substitute for inactivation of most prions from most species, but the resistance of CJD to phenolic inactivation is a concern and emphasizes the fact that inactivation methods should be confirmed for each target prion strain.

Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation.

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth factors. These cells show an ability to regulate neuroinflammation in the context of neurodegenerative diseases such as prion disease and other protein misfolding disorders. Prion diseases can be sporadic, acquired, or genetic; they can result from the misfolding and aggregation of the prion protein in the brain. These diseases are invariably fatal, with no available treatments. One of the earliest signs of disease is the activation of astrocytes and microglia and associated inflammation, which occurs prior to detectable prion aggregation and neuronal loss; thus, the anti-inflammatory and regulatory properties of MSCs can be harvested to treat astrogliosis in prion disease. Recently, we showed that adipose-derived MSCs (AdMSCs) co-cultured with BV2 cells or primary mixed glia reduce prion-induced inflammation through paracrine signaling. This paper describes a reliable treatment using stimulated AdMSCs to decrease prion-induced inflammation. A heterozygous population of AdMSCs can easily be isolated from murine adipose tissue and expanded in culture. Stimulating these cells with inflammatory cytokines enhances their ability to both migrate toward prion-infected brain homogenate and produce anti-inflammatory modulators in response. Together, these techniques can be used to investigate the therapeutic potential of MSCs on prion infection and can be adapted for other protein misfolding and neuroinflammatory diseases.

Intranasally delivered mesenchymal stromal cells decrease glial inflammation early in prion disease.

Mesenchymal stromal cells (MSCs) are an intriguing avenue for the treatment of neurological disorders due to their ability to migrate to sites of neuroinflammation and respond to paracrine signaling in those sites by secreting cytokines, growth factors, and other neuromodulators. We potentiated this ability by stimulating MSCs with inflammatory molecules, improving their migratory and secretory properties. We investigated the use of intranasally delivered adipose-derived MSCs (AdMSCs) in combating prion disease in a mouse model. Prion disease is a rare, lethal neurodegenerative disease that results from the misfolding and aggregation of the prion protein. Early signs of this disease include neuroinflammation, activation of microglia, and development of reactive astrocytes. Later stages of disease include development of vacuoles, neuronal loss, abundant aggregated prions, and astrogliosis. We demonstrate the ability of AdMSCs to upregulate anti-inflammatory genes and growth factors when stimulated with tumor necrosis factor alpha (TNFα) or prion-infected brain homogenates. We stimulated AdMSCs with TNFα and performed biweekly intranasal deliveries of AdMSCs on mice that had been intracranially inoculated with mouse-adapted prions. At early stages in disease, animals treated with AdMSCs showed decreased vacuolization throughout the brain. Expression of genes associated with Nuclear Factor-kappa B (NF-κB) and Nod-Like Receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling were decreased in the hippocampus. AdMSC treatment promoted a quiescent state in hippocampal microglia by inducing changes in both number and morphology. Animals that received AdMSCs showed a decrease in both overall and reactive astrocyte number, and morphological changes indicative of homeostatic astrocytes. Although this treatment did not prolong survival or rescue neurons, it demonstrates the benefits of MSCs in combatting neuroinflammation and astrogliosis.

Adipose-derived mesenchymal stromal cells decrease prion-induced glial inflammation in vitro

Prion diseases are characterized by the cellular prion protein, PrPC, misfolding and aggregating into the infectious prion protein, PrPSc, which leads to neurodegeneration and death. An early sign of disease is inflammation in the brain and the shift of resting glial cells to reactive astrocytes and activated microglia. Few therapeutics target this stage of disease. Mesenchymal stromal cells produce anti-inflammatory molecules when exposed to inflammatory signals and damaged tissue. Here, we show that adipose-derived mesenchymal stromal cells (AdMSCs) migrate toward prion-infected brain homogenate and produce the anti-inflammatory molecules transforming growth factor β (TGFβ) and tumor necrosis factor-stimulated gene 6 (TSG-6). In an in vitro model of prion exposure of both primary mixed glia and BV2 microglial cell line, co-culturing with AdMSCs led to a significant decrease in inflammatory cytokine mRNA and markers of reactive astrocytes and activated microglia. This protection against in vitro prion-associated inflammatory responses is independent of PrPSc replication. These data support a role for AdMSCs as a beneficial therapeutic for decreasing the early onset of glial inflammation and reprogramming glial cells to a protective phenotype.

Hydrogen Peroxide Gas Plasma Sterilizer Combined with Dielectric Barrier Discharge and Corona Discharge Inactivates Prions

Prions are highly resistant to physical or chemical damage, although previous studies have shown that STERRAD®, a hydrogen gas plasma sterilizer using radiofrequency (RF) discharge, has an inactivation effect. Here, the effect of hydrogen peroxide gas combined with dielectric barrier discharge (DBD) plasma and corona discharge plasma using a RENO-S130 sterilizer on scrapie prions was examined. Scrapie prion-infected mouse brain homogenate was air-dried on a cover glass, sealed in a Tyvek pouch, and subjected to RENO-S130 treatment using either non-lumen mode (28 min) or Eco mode (45 min) with hydrogen peroxide gas derived from 50% hydrogen peroxide. Control (untreated) samples were prepared on a cover glass using the same procedure but without exposure to RENO-S130. PrPres (proteinase K (PK)-resistant prion protein), an index of the conformational variant of prion protein (PrPSc), was decreased by treatment with RENO-S130 under both modes of operation. Specifically, PrPres was identified after the 1st and 2nd cycles of protein misfolding cyclic amplification (PMCA) in control samples but was below the detection limit in RENO-S130-treated samples. A bioassay showed that treatment of prions with RENO-S130 (non-lumen or Eco mode) significantly prolonged mouse survival time. Taken together, these findings show hydrogen peroxide gas combined with DBD/corona discharge plasma can inactivate prions by reducing prion propagation and prion infectivity. This treatment is potentially applicable to the sterilization of prion-contaminated heat-sensitive medical devices.

A cold water, ultrasonically activated stream efficiently removes proteins and prion-associated amyloid from surgical stainless steel

BackgroundSterile service department decontamination procedures for surgical instruments struggle to demonstrate efficient removal of the hardiest infectious contaminants, such as prion proteins. A recently designed novel system, which uses a low pressure ultrasonically activated, cold water stream, has previously demonstrated efficient hard surface cleaning of several biological contaminants.AimTo test the efficacy of an ultrasonically activated stream for the removal of tissue proteins, including prion-associated amyloid, from surgical stainless steel surfaces.MethodsTest surfaces were contaminated with 22L, ME7 or 263K prion-infected brain homogenates. The surfaces were treated with the ultrasonically activated water stream for contact times of 5 and 10 s. Residual proteinaceous and amyloid contamination were quantified using sensitive microscopic analysis, and immunoblotting was used to characterize the eluted prion residues before and after treatment with the ultrasonically activated stream.FindingsEfficient removal of the different prion strains from the surgical stainless steel surfaces was observed, and reduced levels of protease-susceptible and -resistant prion protein was detected in recovered supernatant.ConclusionThis study demonstrated that an ultrasonically activated stream has the potential to be a cost-effective solution to improve current decontamination practices and has the potential to reduce hospital-acquired infections.

Altered distribution, aggregation, and protease resistance of cellular prion protein following intracranial inoculation.

Prion protein (PrPC) is a protease-sensitive and soluble cell surface glycoprotein expressed in almost all mammalian cell types. PrPSc, a protease-resistant and insoluble form of PrPC, is the causative agent of prion diseases, fatal and transmissible neurogenerative diseases of mammals. Prion infection is initiated via either ingestion or inoculation of PrPSc or when host PrPC stochastically refolds into PrPSc. In either instance, the early events that occur during prion infection remain poorly understood. We have used transgenic mice expressing mouse PrPC tagged with a unique antibody epitope to monitor the response of host PrPC to prion inoculation. Following intracranial inoculation of either prion-infected or uninfected brain homogenate, we show that host PrPC can accumulate both intra-axonally and within the myelin membrane of axons suggesting that it may play a role in axonal loss following brain injury. Moreover, in response to the inoculation host PrPC exhibits an increased insolubility and protease resistance similar to that of PrPSc, even in the absence of infectious prions. Thus, our results raise the possibility that damage to the brain may be one trigger by which PrPC stochastically refolds into pathogenic PrPSc leading to productive prion infection.

Lithium as a disease-modifying agent for prion diseases

Prion diseases still remain incurable despite multiple efforts to develop a treatment. Therefore, it is important to find strategies to at least reduce the symptoms. Lithium has been considered as a neuroprotective agent for years, and the objective of this preclinical study was to evaluate the efficacy of lithium delivered as a water-in-oil microemulsion (Aonys®). This delivery system allows using low doses of lithium and to avoid the toxicity observed in chronic treatments. C57BL/6J mice were intracranially inoculated with ME7 prion-infected brain homogenates and then were treated with lithium from day 90 post inoculation until their death. Lithium was administered at traditional doses (16 mg/kg/day) by the gavage route and at lower doses (40 or 160 µg/kg/day; Aonys®) by the rectal mucosa route. Low doses of lithium (Aonys®) improved the survival of prion-inoculated mice, and also decreased vacuolization, astrogliosis, and neuronal loss compared with controls (vehicle alone). The extent of the protective effects in mice treated with low-dose lithium was comparable or even higher than what was observed in mice that received lithium at the traditional dose. These results indicate that lithium administered using this innovative delivery system could represent a potential therapeutic approach not only for prion diseases but also for other neurodegenerative diseases.

Generation of Infectious Prions and Detection with the Prion-Infected Cell Assay.

Cell lines propagating prions are an efficient and useful means for studying the cellular and molecular mechanisms implicated in prion disease. Utilization of cell-based models has led to the finding that PrPC and PrPSc are released from cells in association with extracellular vesicles known as exosomes. Exosomes have been shown to act as vehicles for infectivity, transferringinfectivity between cell lines and providing a mechanism for prion spread between tissues. Here, we describe the methods for generating a prion-propagating cell line with prion-infected brain homogenate, cell lysate, conditioned media, and exosomes and also detection of protease-resistant PrP with the prion-infected cell assay.

Age and Environment Influences on Mouse Prion Disease Progression: Behavioral Changes and Morphometry and Stereology of Hippocampal Astrocytes.

Because enriched environment (EE) and exercise increase and aging decreases immune response, we hypothesized that environmental enrichment and aging will, respectively, delay and increase prion disease progression. Mice dorsal striatum received bilateral stereotaxic intracerebral injections of normal or ME7 prion infected mouse brain homogenates. After behavior analysis, animals were euthanized and their brains processed for astrocyte GFAP immunolabeling. Our analysis related to the environmental influence are limited to young adult mice, whereas age influence refers to aged mice raised on standard cages. Burrowing activity began to reduce in ME7-SE two weeks before ME7-EE, while no changes were apparent in ME7 aged mice (ME7-A). Object placement recognition was impaired in ME7-SE, NBH-A, and ME7-A but normal in all other groups. Object identity recognition was impaired in ME7-A. Cluster analysis revealed two morphological families of astrocytes in NBH-SE animals, three in NBH-A and ME7-A, and four in NBH-EE, ME7-SE, and ME7-EE. As compared with control groups, astrocytes from DG and CA3 prion-diseased animals show significant numerical and morphological differences and environmental enrichment did not reverse these changes but induced different morphological changes in GFAP+ hippocampal astroglia. We suggest that environmental enrichment and aging delayed hippocampal-dependent behavioral and neuropathological signs of disease progression.

Specificity, Size, and Frequency of Spaces That Characterize the Mechanism of Bulk Transepithelial Transport of Prions in the Nasal Cavities of Hamsters and Mice.

Inhalation of infected brain homogenate results in transepithelial transport of prions across the nasal mucosa of hamsters, some of which occurs rapidly in relatively large amounts between cells (A. E. Kincaid, K. F. Hudson, M. W. Richey, and J. C. Bartz, J. Virol 86:12731-12740, 2012, doi:http://dx.doi.org/10.1128/JVI.01930-12). Bulk transepithelial transport in the nasal cavity has not been studied to date. In the present study, we characterized the frequency, size, and specificity of the intercellular spaces that mediate the bulk transport of inhaled prions between cells of mice or hamsters following extranasal inoculation with mock-infected brain homogenate, different strains of prion-infected brain homogenate, or brain homogenate mixed with India ink. Infected or mock-infected inoculum was identified within lymphatic vessels of the lamina propria and in spaces of >5 μm between a small number of cells of the nasal mucosa in >90% of animals from 5 to 60 min after inhalation. The width of the spaces between cells, the amount of the inoculum within the lumen of lymphatic vessels, and the timing of the transport indicate that this type of transport was taking place through preexisting spaces in the nasal cavity that were orders of magnitude wider than what is normally reported for paracellular transport. The indiscriminate rapid bulk transport of brain homogenate in the nasal cavity results in immediate entry into nasal cavity lymphatics following inhalation. This novel mechanism may underlie the recent report of the early detection of prions in blood following inhalation and has implications for horizontal prion transmission. The results of these studies demonstrate that the nasal mucosa of mice and hamsters is not an absolute anatomical barrier to inhaled prion-infected or uninfected brain homogenate. Relatively large amounts of infected and uninfected brain homogenate rapidly cross the nasal mucosa and enter the lumen of lymphatic vessels following inhalation. These bulk transepithelial transport events were relatively rare but present in >90% of animals 5 to 60 min following inhalation. This novel mechanism of bulk transepithelial transport was seen in experimental and control hamsters and mice, indicating that it was not species specific or in response to prion exposure. The indiscriminate bulk intercellular transport of inhaled pathogens across the nasal mucosa followed by entry into the lymphatic system may be a mechanism that underlies the entry and spread of other toxins and pathogens in olfactory system-driven animals.

A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases.

BackgroundPrion diseases are characterized by the accumulation in the central nervous system of an abnormally folded isoform of the prion protein, named PrPSc. Aggregation of PrPSc into oligomers and fibrils is critically involved in the pathogenesis of prion diseases. Oligomers are supposed to be the key neurotoxic agents in prion disease, so modulation of prion aggregation pathways with small molecules can be a valuable strategy for studying prion pathogenicity and for developing new diagnostic and therapeutic approaches. We previously identified thienyl pyrimidine compounds that induce SDS-resistant PrPSc (rSDS-PrPSc) oligomers in prion-infected samples.ResultsDue to the low effective doses of the thienyl pyrimidine hits, we synthesized a quaterthiophene-bis-triazine compound, called MR100 to better evaluate their diagnostic and therapeutic potentials. This molecule exhibits a powerful activity inducing rSDS-PrPSc oligomers at nanomolar concentrations in prion-infected cells. Fluorescence interaction studies of MR100 with mouse PrP fibrils showed substantial modification of the spectrum, and the interaction was confirmed in vitro by production of rSDS-oligomer species upon incubation of MR100 with fibrils in SDS-PAGE gel. We further explored whether MR100 compound has a potential to be used in the diagnosis of prion diseases. Our results showed that: (i) MR100 can detect rSDS-oligomers in prion-infected brain homogenates of various species, including human samples from CJD patients; (ii) A protocol, called “Rapid Centrifugation Assay” (RCA), was developed based on MR100 property of inducing rSDS-PrPSc oligomers only in prion-infected samples, and avoiding the protease digestion step. RCA allows the detection of both PK-sensitive and PK-resistant PrPSc species in rodents samples but also from patients with different CJD forms (sporadic and new variant); (iii) A correlation could be established between the amount of rSDS-PrPSc oligomers revealed by MR100 and the duration of the symptomatic phase of the disease in CJD patients; and (iv) Bioassay experiments showed that MR100 can trap prion infectivity more efficiently than P30 drug.ConclusionsMR100 is a powerful tool not only for studying the prion aggregation pathways regarding oligomeric and sPrPSc species, but also for developing alternative methods for the detection of prion-infected samples. Considering our bioassay results, MR100 is a promising molecule for the development of prion decontamination approaches.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0074-7) contains supplementary material, which is available to authorized users.

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

During prion infection, the normal, protease-sensitive conformation of prion protein (PrPC) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrPSc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrPSc in prion-infected brain homogenates as an initiating seed to convert PrPC and trigger the self-propagation of PrPSc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrPSc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrPSc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrPSc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.

Rapid Transepithelial Transport of Prions following Inhalation

Prion infection and pathogenesis are dependent on the agent crossing an epithelial barrier to gain access to the recipient nervous system. Several routes of infection have been identified, but the mechanism(s) and timing of in vivo prion transport across an epithelium have not been determined. The hamster model of nasal cavity infection was used to determine the temporal and spatial parameters of prion-infected brain homogenate uptake following inhalation and to test the hypothesis that prions cross the nasal mucosa via M cells. A small drop of infected or uninfected brain homogenate was placed below each nostril, where it was immediately inhaled into the nasal cavity. Regularly spaced tissue sections through the entire extent of the nasal cavity were processed immunohistochemically to identify brain homogenate and the disease-associated isoform of the prion protein (PrP(d)). Infected or uninfected brain homogenate was identified adhering to M cells, passing between cells of the nasal mucosa, and within lymphatic vessels of the nasal cavity at all time points examined. PrP(d) was identified within a limited number of M cells 15 to 180 min following inoculation, but not in the adjacent nasal mucosa-associated lymphoid tissue (NALT). While these results support M cell transport of prions, larger amounts of infected brain homogenate were transported paracellularly across the respiratory, olfactory, and follicle-associated epithelia of the nasal cavity. These results indicate that prions can immediately cross the nasal mucosa via multiple routes and quickly enter lymphatics, where they can spread systemically via lymph draining the nasal cavity.

Detection of prion infection in variant Creutzfeldt-Jakob disease: a blood-based assay

Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative disorder originating from exposure to bovine-spongiform-encephalopathy-like prions. Prion infections are associated with long and clinically silent incubations. The number of asymptomatic individuals with vCJD prion infection is unknown, posing risk to others via blood transfusion, blood products, organ or tissue grafts, and contaminated medical instruments. We aimed to establish the sensitivity and specificity of a blood-based assay for detection of vCJD prion infection. We developed a solid-state binding matrix to capture and concentrate disease-associated prion proteins and coupled this method to direct immunodetection of surface-bound material. Quantitative assay sensitivity was assessed with a serial dilution series of 10⁻⁷ to 10⁻¹⁰ of vCJD prion-infected brain homogenate into whole human blood, with a baseline control of normal human brain homogenate in whole blood (10⁻⁶). To establish the sensitivity and specificity of the assay for detection of endogenous vCJD, we analysed a masked panel of 190 whole blood samples from 21 patients with vCJD, 27 with sporadic CJD, 42 with other neurological diseases, and 100 normal controls. Samples were masked and numbered by individuals independent of the assay and analysis. Each sample was tested twice in independent assay runs; only samples that were reactive in both runs were scored as positive overall. We were able to distinguish a 10⁻¹⁰ dilution of exogenous vCJD prion-infected brain from a 10⁻⁶ dilution of normal brain (mean chemiluminescent signal, 1·3×10⁵ [SD 1·1×10⁴] for vCJD vs 9·9×10⁴ [4·5×10³] for normal brain; p<0·0001)—an assay sensitivity that was orders of magnitude higher than any previously reported. 15 samples in the masked panel were scored as positive. All 15 samples were from patients with vCJD, showing an assay sensitivity for vCJD of 71·4% (95% CI 47·8–88·7) and a specificity of 100% (95% CIs between 97·8% and 100%). These initial studies provide a prototype blood test for diagnosis of vCJD in symptomatic individuals, which could allow development of large-scale screening tests for asymptomatic vCJD prion infection. UK Medical Research Council.

Current risk of iatrogenic Creutzfeld–Jakob disease in the UK: efficacy of available cleaning chemistries and reusability of neurosurgical instruments

The initial cleaning of reusable surgical devices is critical to ensure the efficacy of the subsequent sterilisation process. Transmissible spongiform encephalopathies (TSEs) are incurable and fatal neurodegenerative diseases apparently transmitted simply by the absorption or ingestion of self-aggregating protease-resistant prions (PrP(Sc)), which are very resilient to most standard cleaning chemistries and heat-based decontamination techniques. Therefore there is a risk of iatrogenic transmission from reusable surgical devices if these are allowed to retain potentially infectious material after standard reprocessing through sterile service departments (SSDs). We aimed to assess the current state of surgical instrument decontamination with the collaboration of anonymous SSDs. Surgical stainless steel surfaces were spiked with prion-infected brain homogenates, and episcopic differential interference contrast/epifluorescence (EDIC/EF) microscopy was applied to quantify the amount of residual prion amyloid and other proteins remaining after decontamination with enzymatic cleaners currently employed by SSDs. Reusable instruments deemed 'clean and ready to use' were also stained for comparison with our findings in the laboratory. All cleaning chemistries were only partially effective under the recommended conditions. More importantly, PrP(Sc) constituted the main fraction of the remaining contamination left on these surfaces. The neurosurgery instruments also harboured amyloid and general protein contamination. This study shows that currently marketed cleaning chemistries and recent decontamination protocols do not completely suppress the threat from iatrogenic CJD. These findings should be taken into account for risk assessment purposes and re-evaluating instrument handling and decontamination practices.

In vitro infectivity assay for prion titration for application to the evaluation of the prion removal capacity of biological products manufacturing processes

Prions can be detected and quantified currently by using either immunoassays such as Western-blot, ELISA or conformation dependent immunoassay, or an infectivity assay in laboratory animals (bioassay). While immunoassays are inexpensive and rapid, they are based on the detection of PrP Sc, the abnormal isoform of the prion protein, a surrogate marker for prion infectivity. The bioassay is considered the gold-standard analytical method for measuring prion infectivity, but it is very costly and time-consuming, involving the destruction of large numbers of animals. The use of the transgenic MovS6 cell line is described for the development of an in vitro tissue culture infectivity assay (TCIA) for prion detection and quantitation. Compared to a bioassay, the TCIA is rapid (∼8 weeks), easy to implement, much less expensive, and requires far fewer animals. After titrating concomitantly a prion-infected brain homogenate sample by Western-blot, TCIA and bioassay, data show that the sensitivity of the TCIA is close to that of the bioassay, since 1 TCID 50 corresponds to 4 ID 50, and 80-fold more sensitive than the Western-blot. The application of the TCIA to the evaluation of prion removal in biological products manufacturing processes is described using a 15 nm-nanofiltration step of human albumin as a model.

Prion Protein Adsorption to Soil in a Competitive Matrix Is Slow and Reduced

It is likely that the soil environment serves as a stable reservoir of infectious CWD and scrapie prions as well as a potential reservoir of BSE. Prion adsorption to soil could play an important role in prion mobility, proteolysis, and infectivity. We modified previously published methods to quantify adsorbed prions via direct detection and studied prion adsorption to soil and soil minerals as a function of time through 60 days. Prion-infected brain homogenate was used as a complex, relevant prion source. We determined that maximum PrP adsorption requires days or weeks, depending on the soil or mineral, and is 2-5 orders of magnitude lower than previous studies using purified PrP(Sc) or recPrP. Because PrP adsorption to soil is slow and reduced in tissue homogenate, the possibility of prion transport in soil environments cannot be excluded and requires further investigation. Our results indicate that binding to soil may protect prions from degradation, consistent with prions' longevity in the environment. Adsorption of PrP to sterilized soil did not differ significantly from adsorption to unsterilized soil, which suggests that active biological processes do not significantly affect prion adsorption or degradation in the soil environment.

The effects of prion protein proteolysis and disaggregation on the strain properties of hamster scrapie

Native mammalian prions exist in self-propagating strains that exhibit distinctive clinical, pathological and biochemical characteristics. Prion strain diversity is associated with variations in PrP(Sc) conformation, but it remains unknown precisely which physical properties of the PrP(Sc) molecules are required to encipher mammalian prion strain phenotypes. In this study, we subjected prion-infected brain homogenates derived from three different hamster scrapie strains to either (i) proteinase K digestion or (ii) sonication, and inoculated the modified samples into normal hamsters. The results show that the strain-specific clinical features and neuropathological profiles of inoculated animals were not affected by either treatment. Similarly, the strain-dependent biochemical characteristics of the PrP(Sc) molecules (including electrophoretic mobility, glycoform composition, conformational stability and susceptibility to protease digestion) in infected animals were unaffected by either proteolysis or sonication of the original inocula. These results indicate that the infectious strain properties of native prions do not appear to be altered by PrP(Sc) disaggregation, and that maintenance of such properties does not require the N-domain (approximately residues 23-90) of the protease-resistant PrP(Sc) molecules or protease-sensitive PrP(Sc) molecules.

Gene expression profile of quinacrine‐cured prion‐infected mouse neuronal cells

Prion diseases are transmissible fatal neurodegenerative diseases of humans and animals, characterised by the presence of an abnormal isoform (scrapie prion protein; PrP(Sc)) of the endogenous cellular prion protein (PrP(C)). The pathological mechanisms at the basis of prion diseases remain elusive, although the accumulation of PrP(Sc) has been linked to neurodegeneration. Different genomic approaches have been applied to carry out large-scale expression analysis in prion-infected brains and cell lines, in order to define factors potentially involved in pathogenesis. However, the general lack of overlap between the genes found in these studies prompted us to carry an analysis of gene expression using an alternative approach. Specifically, in order to avoid the complexities of shifting gene expression in a heterogeneous cell population, we used a single clone of GT1 cells that was de novo infected with mouse prion-infected brain homogenate and then treated with quinacrine to clear PrP(Sc). By comparing the gene expression profiles of about 15 000 genes in quinacrine-cured and not cured prion-infected GT1 cells, we investigated the influence of the presence or the absence of PrP(Sc). By real-time PCR, we confirmed that the gene encoding for laminin was down-regulated as a consequence of the elimination of PrP(Sc) by the quinacrine treatment. Thus, we speculate that this protein could be a specific candidate for further analysis of its role in prion infection and pathogenesis.