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Michael Beekes

Publications and source records attributed to Michael Beekes.

At least 19 recordsLinked to original sources
PubMed2006

Propagation of scrapie in peripheral nerves after footpad infection in normal and neurotoxin exposed hamsters.

As is known from various animal models, the spread of agents causing transmissible spongiform encephalopathies (TSE) after peripheral infection affects peripheral nerves before reaching the central nervous system (CNS) and leading to a fatal end of the disease. The lack of therapeutic approaches for TSE is partially due to the limited amount of information available on the involvement of host biological compartments and processes in the propagation of the infectious agent. The in vivo model presented here can provide information on the spread of the scrapie agent via the peripheral nerves of hamsters under normal and altered axonal conditions. Syrian hamsters were unilaterally footpad (f.p.) infected with scrapie. The results of the spatiotemporal ultrasensitive immunoblot-detection of scrapie-associated prion protein (PrP(Sc)) in serial nerve segments of both distal sciatic nerves could be interpreted as a centripetal and subsequent centrifugal neural spread of PrP(Sc) for this route of infection. In order to determine whether this propagation is dependent on main components in the axonal cytoskeleton (e.g. neurofilaments, also relevant for the component ;a' of slow axonal transport mechanisms), hamsters were treated -in an additional experiment- with the neurotoxin beta,beta-iminodiproprionitrile (IDPN) around the beginning of the scrapie infection. A comparison of the Western blot signals of PrP(Sc) in the ipsilateral and in the subsequently affected contralateral sciatic nerve segments with the results revealed from IDPN-untreated animals at preclinical and clinical stages of the TSE disease, indicated similar amounts of PrP(Sc). Furthermore, the mean survival time was unchanged in both groups. This in vivo model, therefore, suggests that the propagation of PrP(Sc) along peripheral nerves is not dependent on an intact neurofilament component of the axonal cytoskeleton. Additionally, the model indicates that the spread of PrP(Sc) is not mediated by the slow component ;a' of the axonal transport mechanism.

Animals↗
PubMed2006

Detection of preclinical scrapie from serum by infrared spectroscopy and chemometrics.

In this study we describe a methodology for diagnosing preclinical scrapie infection in hamsters from serum by a combination of Fourier-transform infrared (FT-IR) spectroscopy and chemometrics. Syrian hamsters (Mesocricetus auratus) were orally inoculated with the 263K scrapie agent, or mock-infected, and sera were obtained at 70, 100 and 130 days post infection (dpi) and at the terminal stage of scrapie (160 +/- 10 dpi). The analysis of hamster sera by FT-IR spectroscopy and artificial neural networks (ANN) confirmed results from earlier studies which had indicated the existence of disease-related structural and compositional alterations in the sera of infected donors in the terminal stage of scrapie [Schmitt et al. (2002) Anal Chem 74:3865-3868]. Experimental data from sera of animals in the preclinical stages of scrapie revealed subtle but reproducible spectral variations that permitted the identification of a preclinical scrapie infection at 100 dpi and later, but not at 70 dpi. The IR spectral features that were discriminatory for the preclinical stages differed from those of the terminally ill individuals. In order to reliably identify scrapie-negative as well as preclinical (100 and 130 dpi) and terminal scrapie-positive animals, a hierarchical classification system of independent artificial neural networks (ANN) was established. A "toplevel" ANN was designed which discriminates between animals in the terminal stage of scrapie and preclinical scrapie-positive or control animals. Spectra identified by the "toplevel" ANN as preclinical or controls were then further analyzed by a second classifier, the "sublevel" ANN. Using independent external validation procedures, the toplevel classifier produced an overall classification accuracy of 98%, while the sublevel classifier yielded an accuracy of 93%, indicating that scrapie-specific serum markers were also present for preclinical disease stages. Possible spectral markers responsible for the discrimination capacity of the two different ANNs are discussed.

Animals↗
PubMed2006

Cell-free formation of misfolded prion protein with authentic prion infectivity.

Prion propagation has been modeled in vitro; however, the low infectious titer of PrP(Sc) thus generated has cast doubt on the "protein-only" hypothesis. Here we show that prion delivery on suitable nitrocellulose carrier particles abrogates the apparent dissociation of PrP(Sc) and infectivity. Misfolded prion protein generated by protein misfolding cyclic amplification is as infectious as authentic brain-derived PrP(Sc) provided that confounding effects related to differences in the size distribution of prion protein aggregates generated in vitro and consecutive differences in regard to biological clearance are abolished.

Animals↗
PubMed2006

FTIR-microspectroscopy of prion-infected nervous tissue.

The family of transmissible spongiform encephalopathies (TSE), also termed prion diseases, is a group of fatal, neurodegenerative diseases characterized by the accumulation of a misfolded protein, the disease-associated prion protein PrPSc. This glycoprotein differs in secondary structure from its normal, cellular isoform PrPC, which is physiologically expressed mostly by neurons. Scrapie is a prion disease first described in the 18th century in sheep and goats, and has been established as a model in rodents to study the pathogenesis and pathology of prion diseases. Assuming a multitude of molecular parameters change in the tissue in the course of the disease, FTIR microspectroscopy has been proposed as a valuable new method to study and identify prion-affected tissues due to its ability to detect a variety of changes in molecular structure and composition simultaneously. This paper reviews and discusses results from previous FTIR microspectroscopic studies on nervous tissue of scrapie-infected hamsters in the context of histological and molecular alterations known from conventional pathogenesis studies. In particular, data from studies reporting on disease-specific changes of protein structure characteristics, and also results of a recent study on hamster dorsal root ganglia (DRG) are discussed. These data include an illustration on how the application of a brilliant IR synchrotron light source enables the in situ investigation of localized changes in protein structure and composition in nervous cells or tissue due to PrPSc deposition, and a demonstration on how the IR spectral information can be correlated with results of complementary studies using immunohistochemistry and x-ray fluorescence techniques. Using IR microspectroscopy, some neurons exhibited a high accumulation of disease-associated prion protein evidenced by an increased amount of beta-sheet at narrow regions in or around the infected nervous cells. However, not all neurons from terminally diseased hamsters showed PrPSc deposition. Generally, the average spectral differences between all control and diseased DRG spectra are small but consistent as demonstrated by independent experiments. Along with studies on the purified misfolded prion protein, these data suggest that synchrotron FTIR microspectroscopy is capable of detecting the misfolded prion protein in situ without the necessity of immunostaining or purification procedures.

Animals↗
PubMed2006

Structural differences between TSEs strains investigated by FT-IR spectroscopy.

Strain diversity in transmissible spongiform encephalopathies (TSEs) has been suggested to be "enciphered" in the structure of the misfolded prion protein isoform PrP(Sc). We have recently demonstrated the strain typing potential of the FT-IR spectroscopy technique, analyzing four different TSE agents adapted to Syrian hamsters [A. Thomzig, S. Spassov, M. Friedrich, D. Naumann and M. Beekes, Discriminating scrapie and BSE isolates by infrared spectroscopy of pathological prion protein J. Biol. Chem. 279 (2004) 33847-33854.] [1]. In the present paper, we have extended the FT-IR study, exploring the secondary structure, temperature stability, and hydrogen-deuterium exchange characteristics of PrP27-30, from the TSE agents 263K, ME7-H, 22A-H, and BSE-H. The strain differentiation capacity of the FT-IR approach was objectively proven for the first time by multivariate cluster analysis. The second derivative FT-IR spectra obtained from dried protein films or samples hydrated in H(2)O or D(2)O consistently exhibited strain-specific infrared characteristics in the secondary structure sensitive amide I region, complemented by strain dependent spectral traits in the amide II and amide A absorption regions, and the different H/D-exchange behaviour of the various PrP27-30 samples. FT-IR spectra of PrP27-30 samples from 263K, ME7-H and 22A-H exposed to increasing temperature (up to 90 degrees C) showed that a strain-specific response to heat treatment is associated with strain specific thermostability of distinct secondary structure elements, providing additional means for TSEs strain discrimination.

Animals↗
PubMed2006

Photo-induced crosslinking of prion protein oligomers and prions.

Prion diseases are caused by a unique type of infectious agent, which is thought to consist of a misfolded beta-sheeted form of the alpha-helical cellular prion protein (PrPC). This misfolded isoform (PrPSc) tends to form insoluble amyloid-like aggregates, impeding classical structural analysis by X-ray crystallography or NMR. Intermolecular crosslinking may provide a means of stabilizing notoriously elusive oligomers for further analysis and may be used for analyzing aggregate architecture by characterising intermolecular contact sites. Using a photo-induced crosslinking method (PICUP), aggregates of recombinant PrP (rPrP) and PrPSc were linked at interacting surfaces via amino acid side chains. The degree of crosslinking within PrP aggregates was adjustable using varying light intensities and could efficiently be monitored by fluorescence correlation spectroscopy. Specific intermolecular crosslinking of PrPSc molecules was achieved even in crude brain homogenate. Functional studies showed that stabilized aggregates of rPrP did not loose their capacity to induce further protein aggregation and crosslinking of PrPSc did not alter significantly the level of infectivity, indicating that photo-induced covalent linkage of PrPSc does not destruct surfaces important for prion propagation.

Amyloid↗
PubMed2006

Pathological prion protein in muscles of hamsters and mice infected with rodent-adapted BSE or vCJD.

Recently, pathological prion protein (PrP(TSE)) was detected in muscle from sheep infected with scrapie, the archetype of transmissible spongiform encephalopathies (TSEs). This finding has highlighted the question of whether mammalian muscle may potentially also provide a reservoir for TSE agents related to bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob Disease (vCJD). Here, results are reported from studies in hamsters and mice that provide direct experimental evidence, for the first time, of BSE- and vCJD-associated PrP(TSE) deposition in muscles. Our findings emphasize the need for further assessment of possible public-health risks from TSE involvement of skeletal muscle.

Animals↗
PubMed2006

Acid inactivation of prions: efficient at elevated temperature or high acid concentration.

Scrapie prion rods isolated from hamster and non-infectious aggregates of the corresponding recombinant protein rPrP(90-231) were incubated with hydrochloric acid. The amount of PrP and of infectivity that survived incubation in HCl at varying times, acid concentrations and temperatures was quantified by Western blot densitometry and bioassays, respectively. Prion rods and rPrP aggregates showed similar HCl hydrolysis kinetics of PrP, indicating structural homology. For 1 M HCl and 25 degrees C, the rate of PrP hydrolysis follows first-order kinetics at 0.014 h(-1); the rate of infectivity inactivation is 0.54 h(-1). Hydrolysis for 1 h at 25 degrees C was only slightly proportional to HCl concentration up to 5 M, but complete loss of infectivity and PrP reduction to <2 % was observed at 8 M HCl. The temperature dependence of unhydrolysed PrP, as well as infectivity at 1 M HCl for 1 h, showed a slight decrease up to 45 degrees C, but a sigmoidal decrease by several orders of magnitude at higher temperatures. The slow hydrolysis of PrP and inactivation of infectivity by acid treatment at room temperature are attributed to solvent inaccessibility of prion rods and rPrP aggregates, respectively. The more effective hydrolysis and inactivation at temperatures above 45 degrees C are interpreted as thermally induced disaggregation with an activation energy of 50-60 kJ mol(-1). Most importantly, infectivity was always inactivated faster or to a higher extent than PrP was hydrolysed at several incubation times, HCl concentrations and temperatures.

Animals↗
PubMed2006

Detection of prion particles in samples of BSE and scrapie by fluorescence correlation spectroscopy without proteinase K digestion.

A characteristic feature of prion diseases such as bovine spongiform encephalopathy (BSE) is the accumulation of a pathological isoform of the host-encoded prion protein, PrP. In contrast to its cellular isoform PrP(C), the pathological isoform PrP(Sc) forms insoluble aggregates. All commercial BSE tests currently used for routine testing are based on the proteinase K (PK) resistance of PrP, but not all pathological PrP is PK-resistant. In the present study, single prion particles were counted by fluorescence correlation spectroscopy (FCS). The property of PK resistance is not required, i.e., both the PK-resistant and the PK-sensitive parts of the prion particles are detectable. PrP aggregates were prepared from the brains of BSE-infected cattle, as well as from scrapie-infected hamsters, by the NaPTA precipitation method without PK digestion. They were labeled using two different PrP-specific antibodies for FCS measurements in the dual-color mode (2D-FIDA). Within the limited number of samples tested, BSE-infected cattle and scrapie-infected hamsters in the clinical stage of the disease could be distinguished with 100% specificity from a control group. Thus, a diagnostic tool for BSE detection with complete avoidance of PK treatment is presented, which should have particular advantages for testing animals in the preclinical stage.

Animals↗
PubMed2005

The polysaccharide scaffold of PrP 27-30 is a common compound of natural prions and consists of alpha-linked polyglucose.

An inert polysaccharide scaffold identified as a 5-15% component of prion rods (PrP 27-30) is unambiguously distinguishable from the N-glycosyl groups and the GPI anchor of PrP, and consists predominantly of 1,4-linked glucose with some branching via 1,4,6-linked glucose. We show that this polysaccharide scaffold is a common secondary component of prions found in hamster full-length PrP(Sc), prion rods and in mouse ScN2a prions from cell culture. The preparation from prion rods was improved, resulting in a polysaccharide scaffold free of remaining infectivity. Furthermore, we determined the stereochemistry of the glycoside linkages as pre-dominantly if not entirely alpha-glycosidic. The origin of the polysaccharide, its interaction with PrP and its potential relation to glycogen and corpora amylacea are discussed.

Animals↗
PubMed2004

Autocatalytic self-propagation of misfolded prion protein.

Prions are thought to replicate in an autocatalytic process that converts cellular prion protein (PrP(C)) to the disease-associated misfolded PrP isoform (PrP(Sc)). Our study scrutinizes this hypothesis by in vitro protein misfolding cyclic amplification (PMCA). In serial transmission PMCA experiments, PrP(Sc) was inoculated into healthy hamster brain homogenate containing PrP(C). Misfolded PrP was amplified by rounds of sonication and incubation and reinoculated into fresh brain homogenate every 10 PMCA rounds. The amplification depended on PrP(C) substrate and could be inhibited by recombinant hamster PrP. In serial dilution experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalyzed the structural conversion of PrP(C) as efficiently as PrP(Sc) from brain of scrapie (263K)-infected hamsters, yielding an approximately 300-fold total amplification of PrPres after 100 rounds, which confirms an autocatalytic PrP-misfolding cascade as postulated by the prion hypothesis. PrPres formation was not paralleled by replication of biological infectivity, which appears to require factors additional to PrP-misfolding autocatalysis.

Animals↗
PubMed2004

Discriminating scrapie and bovine spongiform encephalopathy isolates by infrared spectroscopy of pathological prion protein.

For the surveillance of transmissible spongiform encephalopathies (TSEs) in animals and humans, the discrimination of different TSE strains causing scrapie, BSE, or Creutzfeldt-Jakob disease constitutes a substantial challenge. We addressed this problem by Fourier transform-infrared (FT-IR) spectroscopy of pathological prion protein PrP27-30. Different isolates of hamster-adapted scrapie (263K, 22A-H, and ME7-H) and BSE (BSE-H) were passaged in Syrian hamsters. Two of these agents, 22A-H and ME7-H, caused TSEs with indistinguishable clinical symptoms, neuropathological changes, and electrophoretic mobilities and glycosylation patterns of PrP27-30. However, FT-IR spectroscopy revealed that PrP27-30 of all four isolates featured different characteristics in the secondary structure, allowing a clear distinction between the passaged TSE agents. FT-IR analysis showed that phenotypic information is mirrored in beta-sheet and other secondary structure elements of PrP27-30, also in cases where immunobiochemical typing failed to detect structural differences. If the findings of this study hold true for nonexperimental TSEs in animals and humans, FT-IR characterization of PrP27-30 may provide a versatile tool for molecular strain typing without antibodies and without restrictions to specific TSEs or mammalian species.

Animals↗
PubMed2004

Decontamination of surgical instruments from prion proteins: in vitro studies on the detachment, destabilization and degradation of PrPSc bound to steel surfaces.

Effective reprocessing of surgical instruments ensuring elimination of inadvertent contamination with infectious agents causing transmissible spongiform encephalopathies (TSEs) is essential for the prevention of iatrogenic transmission of Creutzfeldt-Jakob disease (CJD) or its new variant (vCJD) from asymptomatic carriers. In a search for effective yet instrument-friendly and routinely applicable reprocessing procedures, we used an in vitro carrier assay to assess the decontamination activity exerted by different reagents on pathological prion protein (PrP(Sc)), the biochemical marker for TSE infectivity, attached to steel surfaces. In this assay, steel wires were contaminated with 263K scrapie brain homogenate and reprocessed for decontamination by exposure to several different test reagents. Residual contamination with PrP(Sc) and its protease-resistant core PrP27-30, still present after reprocessing on the wire surface or in the cleaning solution, was monitored by sensitive Western blot detection without or after proteinase K digestion. Using this approach, various reagents and processing conditions were screened for both their efficacy of decontamination and their active principles, such as detachment, destabilization or degradation of surface-bound prion protein. This revealed that, under appropriate conditions, relatively mild reagents such as 0.2 % SDS/0.3 % NaOH (pH 12.8), a commercially available alkaline cleaner (pH 11.9-12.2), a disinfectant containing 0.2 % peracetic acid and low concentrations of NaOH (pH 8.9) or 5 % SDS (pH 7.1) exert potent decontaminating activities on PrP(Sc)/PrP27-30 attached to steel surfaces. For in vivo validation, wires reprocessed in these reagents have been implanted into reporter animals in ongoing experiments.

Decontamination↗
PubMed2004

Preclinical deposition of pathological prion protein PrPSc in muscles of hamsters orally exposed to scrapie.

Recently, pathological prion protein PrP(Sc), the putative key constituent of infectious agents causing transmissible spongiform encephalopathies (TSEs), was found in muscles of rodents experimentally infected with scrapie and in patients with Creutzfeldt-Jakob disease (CJD). For the assessment of risk scenarios originating from these findings (e.g., alimentary transmission of pathogens associated with bovine spongiform encephalopathy [BSE] and chronic wasting disease [CWD] via tainted beef and game or iatrogenic dissemination of CJD agent through contaminated surgical instruments) more detailed information about the time course of PrP(Sc) accumulation in muscles at preclinical and clinical stages of incubation is needed. Here we show that PrP(Sc) in muscles of hamsters fed with scrapie can be detected prior to the onset of clinical symptoms, but that the bulk of PrP(Sc) was deposited late in clinical disease. Additionally, regarding the question of how muscles become invaded, we report on the intramuscular location of PrP(Sc) and substantial indications for centrifugal spread of infection from spinal motor neurons to myofibers. Our findings in a well-established animal model for TSEs contribute to a better assessment of the risks for public health emanating from "Prions in skeletal muscle" and provide new insights into the pathophysiological spread of TSE agents through the body.

Administration, Oral↗
PubMed2003

Antemortem identification of bovine spongiform encephalopathy from serum using infrared spectroscopy.

Since 1986, more than 180 000 clinical cases of bovine spongiform encephalopathy (BSE) have been observed in the U.K. alone. Most of these cases were confirmed by postmortem examination of brain tissue. However, BSE-related risk assessment and risk management would greatly benefit from antemortem testing on living animals. A serum-based test could allow for screening of the cattle population; thus, even a BSE eradication program would be conceivable. Here we report on a novel method for antemortem BSE testing, which combines infrared spectroscopy of serum samples with multivariate pattern recognition analysis. A classification algorithm was trained using infrared spectra of bovine sera from more than 800 animals (including BSE-positive, healthy controls and animals suffering from classical viral or bacterial infections). In two validation studies, sensitivities of 85 and 84% and specificities of 86 and 91% were achieved, respectively. The combination of classification algorithms increased the sensitivity and specificity of BSE detection to 96 and 92%, respectively.

Animals↗
PubMed2003

In situ identification of protein structural changes in prion-infected tissue.

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders characterized by the conversion of the normal prion protein (PrP(C)) into aggregates of its pathological conformer (PrP(Sc)). The mechanism behind this structural conversion is unclear. We report the identification of disease-related protein structural differences directly within the tissue environment. Utilizing a synchrotron infrared (IR) light source, IR images of protein structure were obtained at a subcellular resolution, revealing regions of decreased alpha-helical content and elevated beta-sheet structure in and around infected neurons in the 263 K scrapie hamster model. PrP(Sc) immunostaining of the same tissue demonstrated that the elevated beta-sheet regions correspond to regions where the misfolded structure of PrP(Sc) is located. No evidence of these structural changes was observed in normal neurons.

Animals↗
PubMed2003

Widespread PrPSc accumulation in muscles of hamsters orally infected with scrapie.

Scrapie, bovine spongiform encephalopathy and chronic wasting disease are orally communicable, transmissible spongiform encephalopathies (TSEs). As zoonotic transmissions of TSE agents may pose a risk to human health, the identification of reservoirs for infectivity in animal tissues and their exclusion from human consumption has become a matter of great importance for consumer protection. In this study, a variety of muscles from hamsters that were orally challenged with scrapie was screened for the presence of a molecular marker for TSE infection, PrPSc (the pathological isoform of the prion protein PrP). Sensitive western blotting revealed consistent PrPSc accumulation in skeletal muscles from forelimb and hindlimb, head, back and shoulder, and in tongue. Previously, our animal model has provided substantial baseline information about the peripheral routing of infection in naturally occurring and orally acquired ruminant TSEs. Therefore, the findings described here highlight further the necessity to investigate thoroughly whether muscles of TSE-infected sheep, cattle, elk and deer contain infectious agents.

Animals↗