Prion Preparations Research Articles

Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies.

The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions.

Ex vivo mammalian prions are formed of paired double helical prion protein fibrils.

Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity and the three-dimensional structure of infectious prions has remained obscure. Recently, we developed novel methods to obtain exceptionally pure preparations of prions from mouse brain and showed that pathogenic PrP in these high-titre preparations is assembled into rod-like assemblies. Here, we have used precise cell culture-based prion infectivity assays to define the physical relationship between the PrP rods and prion infectivity and have used electron tomography to define their architecture. We show that infectious PrP rods isolated from multiple prion strains have a common hierarchical assembly comprising twisted pairs of short fibres with repeating substructure. The architecture of the PrP rods provides a new structural basis for understanding prion infectivity and can explain the inability to systematically generate high-titre synthetic prions from recombinant PrP.

Pathogen safety of human C1 esterase inhibitor concentrate

Human plasma-derived products--such as C1 esterase inhibitor (C1-INH) concentrate, used to treat hereditary angioedema--carry with them the risk of transmitting blood-borne viruses and, theoretically, prion proteins. To minimize this risk, three complementary approaches are implemented: selection and testing of plasma donations for the absence of pathogenic blood-borne viruses, similarly testing and releasing the plasma pool for fractionation, and ensuring that the manufacturing process includes validated steps for pathogen inactivation and removal. This article describes the selection of plasma for the production of C1-INH and the studies used to confirm the pathogen reduction capacity of the manufacturing process: three independent virus reduction steps--pasteurization, hydrophobic interaction chromatography (HIC), and virus filtration--and two prion reduction steps. Samples of product intermediates from the manufacturing steps were spiked with a panel of enveloped and nonenveloped viruses and two prion preparations and subjected to a valid scaled-down version of the respective manufacturing steps resulting in the quantification of the pathogen reduction factors. Validation studies demonstrated overall virus reduction factors for all viruses of more than 15 log, considerably exceeding the potential amount of virus present in a plasma pool for fractionation. Prion proteins were also efficiently removed by the manufacturing process, as currently determined in evaluating the prion removal capacity of the ammonium sulfate precipitation and HIC steps. The pathogen reduction capacity demonstrated here indicates that the manufacturing process of the C1-INH Berinert is highly effective for reducing enveloped and nonenveloped viruses and prion proteins.

High Margin of Pathogen Safety for a Plasma-Derived FXIII Concentrate for the Treatment of Congenital FXIII Deficiency

Abstract 4655Congenital Factor XIII (FXIII) deficiency is an extremely rare, serious, and life-threatening condition, especially due to intracranial haemorrhage, which can be treated by a plasma-derived FXIII concentrate. The pathogen safety of this plasma-derived protein is achieved by the complementary measures (i) selecting and testing the starting material, human plasma, (ii) releasing the plasma pool for fractionation when non-reactive for viral markers and genomic material of blood-borne viruses, and (iii) ensuring adequate capacity of the production process to reduce a wide range of viruses as well as prions. Virus validation and prion evaluation studies were performed to assess the reduction capacity of the manufacturing process of this FXIII concentrate.A minimal load of infectious virus, if any, in human plasma, the starting material for the production of a FXIII concentrate, is achieved by selecting donor centers and donors, by testing donations for viral markers and genomic material of HAV, HBV, HCV, HIV-1 and high titers of parvovirus B19 (B19V), and by releasing plasma pools for fractionation when non-reactive for these blood-borne viruses. Two dedicated virus reduction steps and further steps of the manufacturing process of the FXIII concentrate were investigated: heat treatment in aqueous, stabilized solution (“pasteurization”), virus filtration (two filters in series with mean pore sizes of approx. 19 nm (20N/20N)), and partitioning steps. The virus and prion reduction capacity of the manufacturing process of this FXIII concentrate was evaluated in in vitro studies.Product intermediates from selected steps of the manufacturing process for the FXIII concentrate, derived from different production lots, were spiked with enveloped and non-enveloped viruses of diverse physico-chemical characteristics and processed according to a scaled-down, validated manufacturing process. The virus panel employed in these studies consisted of HIV, BVDV (bovine viral diarrhea virus, a specific model virus for HCV and WNV), WNV, HAV, parvoviruses (B19V and CPV (canine parvovirus)) and PRV (pseudorabies virus, a non-specific enveloped DNA virus). Prion reduction capacity was currently assessed by studying one manufacturing process step with two different spike preparations and based on data for other products from CSL Behring or published data.Pasteurization (heat treatment) inactivates all blood-borne viruses or their specific model viruses effectively, i.e. HIV, BVDV, WNV, HAV and B19V were inactivated in the order of 4 log10 or more. Virus filtration removed effectively all viruses studied. Further manufacturing steps reliably contribute to the overall virus reduction capacity of the manufacturing process of this FXIII concentrate.Two different prion preparations prepared from brain homogenate of 263K infected hamsters were used in the prion evaluation study: a membrane-associated preparation of prion material (microsomes) and a PrPSc preparation without membranes (purified PrPSc). Both spike preparations were removed equally by the manufacturing step Al(OH)3 adsorption / defibrination. Prion material in the different fractions was quantified using a biochemical/serological method, the Conformation-Dependent Immunoassay (CDI).The pathogen reduction capacity for this FXIII concentrate is shown in the following table:Pathogen Reduction Factor [log10]Manufacturing stepsHIVBVDVWNVPRVHAVCPVB19VPrionsMicrosomespurified PrPScpublished dataCryoprecipitation8% ethanol pptAl(OH)3 adsorption/ defibrination7.02.62.7Ion exchange chromatography5.03.4≥6.53.43.73.0*Heat treatment (Pasteurization)≥7.7≥8.1≥7.44.31.0≥4.0Ammonium sulphate precipitation20N/20N virus filtration≥6.1≥5.0≥6.4≥5.66.04.0*Formulation/sterile filtration/ lyophilizationOverall Reduction≥18.8≥16.5N/A≥19.913.310.7N/A9.69.7*based on preliminary data (for other plasma proteins from CSL Behring) or published data, included in the overall prion reduction factor.Based on the epidemiology in the donor population and donation frequency, the sensitivity of the NAT assay, the amount of plasma needed to produce one vial of FXIII concentrate and the virus reduction factors demonstrated, it can be concluded that the measures taken result in a FXIII concentrate with a very high margin of safety for a wide range of viruses and other pathogens. Disclosures:Groener:CSL Behring: Employment. Nowak:CSL Behring: Employment. Popp:CSL Behring: Employment. Schäfer:CSL Behring: Employment.

Multiple Dedicated Measures Result in a Plasma-Derived FVIII Concentrate with High Margin of Safety Regarding Pathogen Transmission

Abstract 4355A minimal load of infectious virus, if any, in human plasma, the starting material for the production of a FVIII concentrate, is achieved by selecting donor centers and donors, by testing donations for viral markers and genomic material of HAV, HBV, HCV, HIV-1 and high titers of parvovirus B19 (B19V), and by releasing plasma pools for fractionation when non-reactive for these blood-borne viruses. Two dedicated virus reduction steps and further steps of the manufacturing process of this FVIII concentrate [Beriate] were investigated: heat treatment in aqueous, stabilized solution (“pasteurization”), virus filtration (filter with a mean pore size of approx. 19 nm (Planova 20N)), and a chromatography step. The virus and prion reduction capacity of the manufacturing process of this FVIII concentrate was evaluated in in vitro studies.Product intermediates from selected steps of the manufacturing process for the FVIII concentrate, derived from different production lots, were spiked with enveloped and non-enveloped viruses of diverse physico-chemical characteristics and processed according to a scaled-down, validated manufacturing process. The virus panel employed in these studies consisted of HIV, BVDV (bovine viral diarrhea virus, a specific model virus for HCV and WNV), HAV, parvoviruses (B19V and CPV (canine parvovirus)) and PRV (pseudorabies virus, a non-specific enveloped DNA virus). The prion reduction capacity was studied by spiking cryoprecipitate with two different spike preparations and performing the whole manufacturing process including the virus filtration according to a valid down scaled process.Pasteurization (heat treatment) inactivates all blood-borne viruses or their specific model viruses effectively, i.e. HIV, BVDV, HAV and B19V were inactivated in the order of 4 log10 or more. Virus filtration removed effectively all enveloped viruses and HAV and removed CPV, a small non-enveloped virus, to a very high degree. Further manufacturing steps reliably contribute to the overall virus reduction capacity of the manufacturing process of this FVIII concentrate.Two different prion preparations prepared from brain homogenate of 263K infected hamsters were used in prion evaluation studies: a membrane-associated preparation of prion material (microsomes) and a PrPSc preparation without membranes (purified PrPSc) were used to evaluate the prion reduction capacity of the FVIII production process. Studying the whole manufacturing process in a combined step approach documented the removal of both spike preparations below the limit of detection. Prion material in the different fractions was quantified using a biochemical/serological method, the Conformation-Dependent Immunoassay (CDI). Spiking different manufacturing steps independently and adding the individual reduction factors to the overall prion reduction factor was considered not appropriate as conditioning of the spiked prion material by the production process may impact the reduction capacity.The pathogen reduction capacity for the FVIII concentrate Beriate is shown in the following table:Based on the epidemiology in the donor population and donation frequency, the sensitivity of the NAT assay, the amount of plasma needed to produce one vial of FVIII concentrate and the virus reduction factors demonstrated, it can be concluded that the measures taken result in a FVIII concentrate [Beriate] with a very high margin of safety for a wide range of viruses and other pathogens. Disclosures:Groener:CSL Behring: Employment. Nowak:CSL Behring: Employment. Popp:CSL Behring: Employment. Schäfer:CSL Behring: Employment.Manufacturing stepsPathogen Reduction Factor [log10]PrionsHIVBVDVPRVHAVParvoviruses*Microsomespurified PrPScCryoprecipitationAdsorption to Al(OH)3/IEX resin≥ 3.6≥ 4.1Heat treatment (Pasteurization)≥6.8≥9.34.73.9≥3.81IEX chromatography3.33.02.11.33.4220N virus filtration≥6.0≥5.8≥7.2≥5.53.42Formulation/sterile filtration/lyophilizationOverall Reduction≥16.1≥18.1≥14.0≥10.7≥10.6≥3.6≥4.1*parvoviruses used in virus validation studies1B19V (human parvovirus B19),2CPV (animal parvovirus: canine parvovirus)

Chemical Induction of Misfolded Prion Protein Conformers in Cell Culture

Prion-infected cells accumulate a heterogeneous population of aberrantly folded PrP conformers, including the disease-causing isoform (PrP(Sc)). We found that specific chemicals can modulate the levels of various PrP conformers in cultured cells. Positively charged polyamidoamines (dendrimers) eliminated protease-resistant (r) PrP(Sc) from prion-infected cells and induced the formation of insoluble, protease-sensitive PrP aggregates (designated PrP(A)). Larger, positively charged polyamidoamines more efficaciously induced the formation of PrP(A) and cleared rPrP(Sc), whereas negatively charged polyamidoamines neither induced PrP(A) nor cleared rPrP(Sc). Although the biochemical properties of PrP(A) were shown to be similar to protease-sensitive (s) PrP(Sc), bioassays of PrP(A) indicated that it is not infectious. Our studies argue that PrP(A) represents an aggregated PrP species that is off-pathway relative to the formation of rPrP(Sc). It remains to be established whether the formation of PrP(A) inhibits the formation of rPrP(Sc) by sequestering PrP(C) in the form of benign, insoluble aggregates.

Prion detection by an amyloid seeding assay

Polymerization of recombinant prion protein (recPrP), which was produced in bacteria, into amyloid fibers was accompanied by the acquisition of prion infectivity. We report here that partially purified preparations of prions seed the polymerization of recPrP into amyloid as detected by a fluorescence shift in the dye Thioflavin T. Our amyloid seeding assay (ASA) detected PrP(Sc), the sole component of the prion, in brain samples from humans with sporadic Creutzfeldt-Jakob disease, as well as in rodents with experimental prion disease. The ASA detected a variety of prion strains passaged in both mice and hamsters. The sensitivity of the ASA varied with strain type; for hamster Sc237 prions, the limit of detection was approximately 1 fg. Some prion strains consist largely of protease-sensitive PrP(Sc) (sPrP(Sc)), and these strains were readily detected by ASA. Our studies show that the ASA provides an alternative methodology for detecting both sPrP(Sc) and protease-resistant PrP(Sc) that does not rely on protease digestion or immunodetection.

Removal of the glycosylphosphatidylinositol anchor from PrPSc by cathepsin D does not reduce prion infectivity

According to the protein-only hypothesis of prion propagation, prions are composed principally of PrP(Sc), an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrP(C)), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrP(Sc) to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrP(C). Removal of the GPI anchor from PrP(Sc) requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrP(Sc) in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrP(Sc), (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrP(Sc) or on prion infectivity.

Removal of Prions by the Manufacturing Process of Haemate® P/Humate-P®.

Prions, the causative agents of Creutzfeldt-Jakob Disease (CJD) and related neurodegenerative disorders in humans including variant CJD (vCJD) have up to now never been demonstrated in human blood although if in UK two probable transmissions of vCJD by blood transfusion were reported (Llewelyn et al., Lancet 2004; 363:417–421 and Peden et al., Lancet 2004; 364:527–529). In order to minimize a theoretical risk of human prion contamination of plasma pools, potential donors at risk of incubating CJD are excluded from donation permanently. Due to the implementation of these precautionary measures, the risk to collect plasma from a donor developing vCJD subsequent to a donation is very low. In addition, the amount of prions present in plasma of a healthy donor (i.e., a donor in the incubation period of (v)CJD) can be considered very low (Comer Risk Assessment of Exposure to vCJD Infectivity in Blood and Blood Products for Department of Health [UK], Revision DE, Det Nordske Veritas, February 2003). Even with this very low risk related to plasma for fractionation, the capacity of selected steps of the manufacturing process of the VWF/FVIII product Haemate® P/Humate-P® to remove prions has been evaluated in prion removal studies. As it has never been possible to isolate infectious prion protein, termed PrPSc, from human blood or plasma, the model prion strain 263K (isolated from brain of deliberately infected hamsters) was spiked to intermediates of scaled-down plasma protein purification processes. As there is no knowledge of the biophysicochemical nature of potential prion contaminants in plasma, we employed two different prion spike preparations in these studies: membrane associated prions in so-called microsomes and purified prion protein PrPSc, an almost native, molecular, non-membrane associated form of prion infectivity. Laboratory studies were performed by spiking either experimental plasma pools or an intermediate at a production stage further downstream with prion preparations and investigating the prion reduction by consecutive scaled-down process steps. These investigational studies resulted in a mean overall prion reduction factor of 5.3 log10 and 6.4 log10, respectively, for the two hamster derived spike preparations.Reduction of prions by selected manufacturing steps for Haemate® P/Humate-P®Manufacturing stagesPrion Reduction Factor [log10]MicrosomesPrPScCombined steps cryoprecipitation, Al(OH)3, glycine and NaCl precipitation3.53.9Combined steps ultracentrifugation/sterile filtration1.82.5Mean Overall Prion Reduction5.36.4Additionally, confirmative data were obtained in studies employing human brain-derived prion preparations (sCJD and vCJD) as spiking material in selected studies of a single manufacturing step. Therefore, it can be concluded that the risk of prion transmission by the VWF/FVIII product Haemate® P/Humate-P® is extremely remote based on complementary safety procedures, i.e., collection of plasma by stringent donor selection and the overall prion reduction factor which clearly exceeds a potential prion load in the manufacturing pool. This high safety margin is confirmed in a probabilistic risk calculation, demonstrating at a probability of 95% a safety factor for each vial of the product of 6.4 log10.

Two Virus Reduction Steps Are Inherent in the Manufacturing Process of Berinert P, a C1-Esterase-Inhibitor Concentrate.

The clinical picture of hereditary angio-edema (HAE) is characterized by acute attacks of circumscribed swellings of the skin and sub-mucosa which recur after symptomless intervals of days to years. Episodes of swelling may involve the upper respiratory tract, including the tongue, pharynx, and larynx. This contributes to the mortality of up to 30% (or more in some families) due to suffocation as complication of laryngeal edema. This increased vascular permeability and massive local uncontrolled edema is most probably caused by uncontrolled activation of the complement system and by increased formation of bradykinin and C2 kinin due to deficiency or complete absence of C1-esterase inhibitor (C1-INH). The first line therapy for treatment of an acute attack and for short-term prophylaxis is intravenous administration of a plasma-derived C1-INH concentrate (where licensed). Such a C1-INH product is Berinert® P, licensed in e.g., Germany, Switzerland, France, and Japan. As, in general, plasma-derived products may potentially transmit viruses, special care is taken to minimize this risk by careful selection of plasma collection centers and donors. Furthermore, each donation is tested thoroughly for the absence of hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency viruses (HIV), and high titers of parvovirus B19 (B19V) by serological and/or NAT/PCR methods. In addition, the plasma pool for fractionation is tested and only released for further processing if the pool is non-reactive (negative) for HBsAg and non-reactive for antibodies against HCV and HIV-1/-2 as well as non-reactive for genomic sequences of HAV, HBV, HCV, HIV-1, and high titers of B19V (not exceeding 105 IU/ml). Selecting and testing the starting material for absence of blood borne viruses is a very important prerequisite to the production of the C1-INH concentrate; however, the manufacturing process of this product with its inherent capacity to inactivate and/or remove potentially present viruses in the plasma pool for fractionation complements the effort for a final product with a high margin of safety with regard to viruses. Two manufacturing steps - pasteurization (heat treatment of stabilized aqueous solution at 60°C for 10 hours) and hydrophobic interaction chromatography (HIC) - were validated for their capacity to inactivate and/or remove viruses. In these laboratory studies, the relevant blood borne viruses or specific and non-specific model viruses were employed: HIV and HAV as viruses of risk, bovine viral diarrhea virus (BVDV) as model virus for HCV and related viruses as West Nile virus (WNV), and canine parvovirus (CPV) as model virus for parvovirus B19 (B19V) demonstrating a very effective reduction of the infectivity by both manufacturing steps independently. Based on these virus validation studies it is evident that two complementary virus reduction steps are inherent in the manufacturing process of Berinert® P.Mean overall virus reduction factors for Berinert® PVirusVirus Reduction by Two Manufacturing Steps [log10]HI ChromatographyPasteurizationOverall Virus ReductionHIV≥ 4.5≥ 6.6≥ 11.1BVDV≥ 5.1≥ 9.2≥ 14.3PRV≥ 6.76.6≥ 13.3HAV≥ 3.3≥6.4≥9.7CPV6.71.48.1Furthermore, investigational studies demonstrate (i) an effective inactivation of B19V by pasteurization resulting in a virus reduction factor of ≥ 4.3 log10 and (ii) the removal of two different prion preparations by a single manufacturing step (microsomes and purified PrPSc by 3.1 log10 and 3.2 log10, respectively). Therefore, a high safety margin for pathogens can be attributed to Berinert® P.

Search for a Prion-Specific Nucleic Acid

Diversity of prion strains was attributed to an elusive nucleic acid, yet a search spanning nearly two decades has failed to identify a prion-specific polynucleotide. In our search for a prion-specific nucleic acid, we analyzed nucleic acids in purified fractions from the brains of Syrian hamsters infected with Sc237 prions. Purification of Sc237 prions removed nucleic acids larger than 50 nucleotides as measured by return refocusing electrophoresis (RRGE). To determine the size of the largest polynucleotide present in purified fractions at an abundance of one molecule per infectious (ID50) unit, we measured prions present after inoculation. In order to account for the rapid clearance of prions after intracerebral inoculation, we determined the number of PrP(Sc) molecules and ID50 units of prions that were retained in brain. Factoring in clearance after inoculation, we estimate that the largest polynucleotide present in our purified fractions at one molecule per ID50 unit is approximately 25 nucleotides in length. In the same fractions, there were approximately 3,000 protease-resistant PrP(Sc) molecules per ID50 unit after accounting for clearance of PrP(Sc) following inoculation. We compared the resistance of Sc237 and 139H prions to inactivation by UV irradiation at 254 nm. Irradiation of homogenates and microsomes diminished prion infectivity by a factor of approximately 1,000 but did not alter the strain-specified properties of the Sc237 and 139H prions. The data reported here combined with the production of synthetic prions argue that the 25-mer polynucleotides found in purified prion preparations are likely to be host encoded and of variable sequence; additionally, these 25-mers are unlikely to be prion specific.

Heparan Sulfate Is a Cellular Receptor for Purified Infectious Prions

Prions replicate in the host cell by the self-propagating refolding of the normal cell surface protein, PrP(C), into a beta-sheet-rich conformer, PrP(Sc). Exposure of cells to prion-infected material and subsequent endocytosis can sometimes result in the establishment of an infected culture. However, the relevant cell surface receptors have remained unknown. We have previously shown that cellular heparan sulfates (HS) are involved in the ongoing formation of scrapie prion protein (PrP(Sc)) in chronically infected cells. Here we studied the initial steps in the internalization of prions and in the infection of cells. Purified prion "rods" are arguably the purest prion preparation available. The only proteinaceous component of rods is PrP(Sc). Mouse neuroblastoma N2a, hypothalamus GT1-1, and Chinese hamster ovary cells efficiently bound both hamster and mouse prion rods (at 4 degrees C) and internalized them (at 37 degrees C). Treating cells with bacterial heparinase III or chlorate (a general inhibitor of sulfation) strongly reduced both binding and uptake of rods, whereas chondroitinase ABC was inactive. These results suggested that the cell surface receptor of prion rods involves sulfated HS chains. Sulfated glycans inhibited both binding and uptake of rods, probably by competing with the binding of rods to cellular HS. Treatments that prevented endocytosis of rods also prevented the de novo infection of GT1-1 cells when applied during their initial exposure to prions. These results indicate that HS are an essential part of the cellular receptor used both for prion uptake and for cell infection. Cellular HS thus play a dual role in prion propagation, both as a cofactor for PrP(Sc) synthesis and as a receptor for productive prion uptake.

Role of glycosylphosphatidylinositols in the activation of phospholipase A2 and the neurotoxicity of prions

Prion-induced neuronal injury in vivo is associated with prostaglandin E(2) production, a process that can be reproduced in tissue-culture models of prion disease. In the present study, neuronal phospholipase A(2) was activated by glycosylphosphatidylinositols (GPIs) isolated from the cellular prion protein (PrP(c)) or from disease-associated isoforms (PrP(Sc)), resulting in prostaglandin E(2) production, but not by GPIs isolated from Thy-1. The ability of GPIs to activate neuronal phospholipase A(2) was lost following the removal of acyl chains or cleavage of the phosphatidylinositol-glycan linkage, and was inhibited by a mAb that recognized phosphatidylinositol. In competition assays, pretreatment of neurons with partial GPIs, inositol monophosphate or sialic acid reduced the production of prostaglandin E(2) in response to a synthetic miniprion (sPrP106), a synthetic correlate of a PrP(Sc) species found in Gerstmann-Straussler-Scheinker disease (HuPrP82-146), prion preparations or high concentrations of PrP-GPIs. In addition, neurons treated with inositol monophosphate or sialic acid were resistant to the otherwise toxic effects of sPrP106, HuPrP82-146 or prion preparations. This protective effect was selective, as inositol monophosphate- or sialic acid-treated neurons remained susceptible to the toxicity of arachidonic acid or platelet-activating factor. Addition of PrP-GPIs to cortical neuronal cultures increased caspase-3 activity, a marker of apoptosis that is elevated in prion diseases. In contrast, treatment of such cultures with inositol monophosphate or sialic acid greatly reduced sPrP106-induced caspase-3 activity and, in co-cultures, reduced the killing of sPrP106-treated neurons by microglia. These results implicate phospholipase A(2) activation by PrP-GPIs as an early event in prion-induced neurodegeneration.

Hamster Prions Are a Suitable Model for Partitioning of Human CJD Prions during Plasma Processing Steps.

Prion removal evaluation of plasma processing procedures is one important basis to assess the margin of safety of plasma protein therapeutics. Currently, in these evaluation studies to assess the removal capacity of selected manufacturing steps for human prions mainly prions derived from scrapie-infected hamsters or mice are used in spiking studies. In order to test the validity of hamster prions instead of different human prion strains as spiking reagents, we compared the partitioning of these prion preparations at two purification steps common to the manufacturing of several human plasma-derived therapeutic proteins at ZLB Behring. The glycine precipitation (inherent in the manufacture of e.g., vWF/Factor VIII) and the 25% ethanol precipitation step (inherent in the manufacture of e.g., alpha-1-proteinase inhibitor and albumin) were down scaled to mimic the production process. A microsomal membrane preparation as well as purified full-length pathogenic prion (PrPSc; without membrane association) prepared (a) from hamster brains infected with the model scrapie strain Sc237, (b) from brains of transgenic mice infected with human sporadic CJD prions, and (c) from the brain of a patient who died of vCJD were used for spiking experiments. The different prion preparations were spiked to the starting material of the manufacturing steps studied and the manufacturing step was performed in a dedicated laboratory. The amount of PrPSc in all fractions was determined quantitatively utilizing an ELISA-formatted Conformation Dependent Immunoassay [Safar et al., Nat Med 1998; 41157–1165]. The 25% ethanol precipitation removed very effectively all prion preparations equally regardless of their origin (reduction factor ≥ 3 log10). The glycine precipitation removed the microsomal as well as the purified prion preparations from all species equally whereby prions of the purified prion preparation were removed to a considerably higher degree than the membrane preparation (reduction factor ≥ 3 log10 vs. 1.6 to 1.8 log10). In parallel, prion infectivity of each sample is currently tested in animal bioassays involving transgenic mice susceptible for the tested prion strain. It can be concluded, up to now based on immunological data, that spiked hamster prions are a reliable model to confirm the safety of human plasma-derived products also for human derived prions.

Squalestatin Cures Prion-infected Neurons and Protects Against Prion Neurotoxicity

A key feature of prion diseases is the conversion of the normal, cellular prion protein (PrP(C)) into beta-sheet-rich disease-related isoforms (PrP(Sc)), the deposition of which is thought to lead to neurodegeneration. In the present study, the squalene synthase inhibitor squalestatin reduced the cholesterol content of cells and prevented the accumulation of PrP(Sc) in three prion-infected cell lines (ScN2a, SMB, and ScGT1 cells). ScN2a cells treated with squalestatin were also protected against microglia-mediated killing. Treatment of neurons with squalestatin resulted in a redistribution of PrP(C) away from Triton X-100 insoluble lipid rafts. These effects of squalestatin were dose-dependent, were evident at nanomolar concentrations, and were partially reversed by cholesterol. In addition, uninfected neurons treated with squalestatin became resistant to the otherwise toxic effect of PrP peptides, a synthetic miniprion (sPrP106) or partially purified prion preparations. The protective effect of squalestatin, which was reversed by the addition of water-soluble cholesterol, correlated with a reduction in prostaglandin E(2) production that is associated with neuronal injury in prion disease. These studies indicate a pivotal role for cholesterol-sensitive processes in controlling PrP(Sc) formation, and in the activation of signaling pathways associated with PrP-induced neuronal death.

Is mad cow disease caused by a bacteria?

Transmissible spongioform enchephalopathies (TSE's), include bovine spongiform encephalopathy (also called BSE or “mad cow disease”), Creutzfeldt–Jakob disease (CJD) in humans, and scrapie in sheep. They remain a mystery, their cause hotly debated. But between 1994 and 1996, 12 people in England came down with CJD, the human form of mad cow, and all had eaten beef from suspect cows. Current mad cow diagnosis lies solely in the detection of late appearing “prions”, an acronym for hypothesized, gene-less, misfolded proteins, somehow claimed to cause the disease. Yet laboratory preparations of prions contain other things, which could include unidentified bacteria or viruses. Furthermore, the rigors of prion purification alone, might, in and of themselves, have killed the causative virus or bacteria. Therefore, even if samples appear to infect animals, it is impossible to prove that prions are causative. Manuelidis found viral-like particles, which even when separated from prions, were responsible for spongiform STE's. Subsequently, Lasmezas's study showed that 55% of mice injected with cattle BSE, and who came down with disease, had no detectable prions. Still, incredibly, prions, are held as existing TSE dogma and Heino Dringer, who did pioneer work on their nature, candidly predicts “it will turn out that the prion concept is wrong.” Many animals that die of spongiform TSE's never show evidence of misfolded proteins, and Dr. Frank Bastian, of Tulane, an authority, thinks the disorder is caused by the bacterial DNA he found in this group of diseases. Recently, Roels and Walravens isolated Mycobacterium bovis it from the brain of a cow with the clinical and histopathological signs of mad cow. Moreover, epidemiologic maps of the origins and peak incidence of BSE in the UK, suggestively match those of England's areas of highest bovine tuberculosis, the Southwest, where Britain's mad cow epidemic began. The neurotaxic potential for cow tuberculosis was shown in pre-1960 England, where one quarter of all tuberculous meningitis victims suffered from Mycobacterium bovis infection. And Harley's study showed pathology identical to “mad cow” from systemic M. bovis in cattle, causing a tuberculous spongiform encephalitis. In addition to M. bovis, Mycobacterium avium subspecies paratuberculosis (fowl tuberculosis) causes Johne's disease, a problem known and neglected in cattle and sheep for almost a century, and rapidly emerging as the disease of the new millennium. Not only has M. paratuberculosis been found in human Crohn's disease, but both Crohn's and Johne's both cross-react with the antigens of cattle paratuberculosis. Furthermore, central neurologic manifestations of Crohn's disease are not unknown. There is no known disease which better fits into what is occurring in Mad Cow and the spongiform enchephalopathies than bovine tuberculosis and its blood–brain barrier penetrating, virus-like, cell-wall-deficient forms. It is for these reasons that future research needs to be aimed in this direction.

Transfer of Scrapie Prion Infectivity by Cell Contact in Culture

When a cell is infected with scrapie prions, newly synthesized molecules of the prion protein PrP(C) are expressed at the cell surface and may subsequently be converted to the abnormal form PrP(Sc). In an experimental scrapie infection of an animal, the initial innoculum of PrP(Sc) is cleared relatively rapidly, and the subsequent propagation of the infection depends on the ability of infected cells to convert uninfected target cells to stable production of PrP(Sc). The mechanism of such cell-based infection is not understood. We have established a system in dissociated cell culture in which scrapie-infected mouse SMB cells are able to stably convert genetically marked target cells by coculture. After coculture and rigorous removal of SMB cells, the target cells express PrP(Sc) and also incorporate [35S]methionine into PrP(Sc). The extent of conversion was sensitive to the ratio of the two cell types, and conversion by live SMB required 2500-fold less PrP(Sc) than conversion by a cell-free prion preparation. The conversion activity of SMB cells is not detectable in conditioned medium and apparently depends on close proximity or contact, as evidenced by culturing the SMB and target cells on neighboring but separate surfaces. SMB cells were killed by fixation in aldehydes, followed by washing, and were found to retain significant activity at conversion of target cells. Cell-mediated infection of target cells in this culture system is effective and requires significantly less PrP(Sc) than infection by a prion preparation. Several lines of evidence indicate that it depends on cell contact, in particular, the activity of aldehyde-fixed infected cells.

Killing of prion-damaged neurones by microglia.

The loss of neurones that occurs in the transmissible spongiform encephalopathies, or prion diseases, can be reproduced in vitro by incubating neuronal cultures with either peptides derived from the prion protein or with partially purified prion preparations. In the present studies, the extent of neuronal loss on exposure to these prions or prion peptides was increased by the addition of microglia, a process that was dependent upon the number of microglia added, the concentration of prions/peptides present and the degree of fibrillarity of the prion peptides. Microglia also killed scrapie-infected neuroblastoma cells expressing infectious PrP(SC). Microglia secreted low amounts of interleukin (IL)-6 when incubated with peptides alone but up to 10 times as much IL-6 when incubated with peptide-treated neurones, suggesting that microglia recognise peptide-induced changes in neurones.

Prion rods contain small amounts of two host sphingolipids as revealed by thin-layer chromatography and mass spectrometry.

Sphingolipids were detected in prions, the agents of transmissible spongiform encephalopathies. The analysis was carried out on highly purified, infectious prion rods, which are composed mainly of insoluble aggregates of the N-terminally truncated prion protein, so-called PrP 27-30. Lipid classes were quantified by high performance thin-layer chromatography with a detection limit of 25-50 ng per lipid class. Matrix-assisted laser desorption/ionization mass spectrometry was applied for the first time to lipid analysis in complex biological samples. A newly developed preparation technique improved the sensitivity to 1-20 pg per molecular species. Only the sphingolipids, galactosylceramide and sphingomyelin, were consistently observed in chloroform/methanol (2:1 v/v) extracts of prion rods. The molar ratio of PrP to the sphingolipids was between 2:1 and 40:1, depending on the purity of the prion preparation. The same lipids were also present in the low density fraction of a gradient centrifugation of prion-rods after sonication in 0.2% SDS. From the two alternatives, that the sphingolipids are either required for prion function or are relics from the cellular location of PrP in caveolae, the second alternative appears more plausible since the preparation of highest specific infectivity contained the lowest amount of sphingolipids.

Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.

The 'protein only' hypothesis postulates that the prion, the agent causing transmissible spongiform encephalopathies, is PrP(Sc), an isoform of the host protein PrP(C). Protease treatment of prion preparations cleaves off approximately 60 N-terminal residues of PrP(Sc) but does not abrogate infectivity. Disruption of the PrP gene in the mouse abolishes susceptibility to scrapie and prion replication. We have introduced into PrP knockout mice transgenes encoding wild-type PrP or PrP lacking 26 or 49 amino-proximal amino acids which are protease susceptible in PrP(Sc). Inoculation with prions led to fatal disease, prion propagation and accumulation of PrP(Sc) in mice expressing both wild-type and truncated PrPs. Within the framework of the 'protein only' hypothesis, this means that the amino-proximal segment of PrP(C) is not required either for its susceptibility to conversion into the pathogenic, infectious form of PrP or for the generation of PrP(Sc).