Transmissible Spongiform Encephalopathy Diseases Research Articles

Ultra-efficient Replication of Infectious Prions by Automated Protein Misfolding Cyclic Amplification

Prions are the unconventional infectious agents responsible for transmissible spongiform encephalopathies, which appear to be composed mainly or exclusively of the misfolded prion protein (PrPSc). Prion replication involves the conversion of the normal prion protein (PrPC) into the misfolded isoform, catalyzed by tiny quantities of PrPSc present in the infectious material. We have recently developed the protein misfolding cyclic amplification (PMCA) technology to sustain the autocatalytic replication of infectious prions in vitro. Here we show that PMCA enables the specific and reproducible amplification of exceptionally minute quantities of PrPSc. Indeed, after seven rounds of PMCA, we were able to generate large amounts of PrPSc starting from a 1x10(-12) dilution of scrapie hamster brain, which contains the equivalent of approximately 26 molecules of protein monomers. According to recent data, this quantity is similar to the minimum number of molecules present in a single particle of infectious PrPSc, indicating that PMCA may enable detection of as little as one oligomeric PrPSc infectious particle. Interestingly, the in vitro generated PrPSc was infectious when injected in wild-type hamsters, producing a disease identical to the one generated by inoculation of the brain infectious material. The unprecedented amplification efficiency of PMCA leads to a several billion-fold increase of sensitivity for PrPSc detection as compared with standard tests used to screen prion-infected cattle and at least 4000 times more sensitivity than the animal bioassay. Therefore, PMCA offers great promise for the development of highly sensitive, specific, and early diagnosis of transmissible spongiform encephalopathy and to further understand the molecular basis of prion propagation.

Linkage disequilibrium across six prion gene regions spanning 20 kbp in U.S. sheep

Single nucleotide polymorphisms (SNPs) and haplotype alleles within the prion gene (PRNP) coding sequence of domestic sheep (Ovis aries) are associated with genetic predisposition to scrapie, a transmissible spongiform encephalopathy disease of sheep. This report describes regions of linkage disequilibrium (LD) throughout the PRNP gene region in U.S. sheep and provides a genetic framework for identifying additional PRNP determinants associated with scrapie resistance. Four sequence tagged sites (i.e., STS or amplicons) totaling 3869 bp and spanning 20 kbp of genomic PRNP sequence were sequenced in a diverse panel of 90 sires representing ten popular U.S. breeds of sheep. Analysis of these sequences identified 36 previously unreported polymorphisms. In combination with two previously characterized STS, 62 polymorphisms were analyzed in a 20-kbp PRNP region in this panel of U.S. sheep. Two regions of strong LD and ten common haplotypes were identified. The haplotype encoding amino acid residues A, R, and Q at codons 136, 154, and 171, respectively, was observed on nine larger haplotypes spanning PRNP from the promoter region to the 3' untranslated region. The haplotype encoding VRQ was observed on two larger haplotypes, whereas ARR, ARH, and AHQ were each present on a single haplotype. The existence of multiple haplotypes encoding ARQ raises the question of whether sheep bearing these different haplotypes are equally susceptible to scrapie. The haplotype structure within the 20-kbp region of PRNP identified in this study is important for higher-resolution analysis of genetics contributions to scrapie susceptibility.

P29 Macopharma Prion Capture (P‐CAPT™) Filter Removes Brain‐Derived Prion In Leucoreduced Human RBC

Background Transmissible spongiform encephalopathy (TSE) diseases (prion) can be transmitted by blood transfusion. Precautionary measures against the spreading of TSE through the blood supply have been implemented, but alone those measures are not sufficient. Pathogen Removal and Diagnostic Technologies (PRDT) developed a resin that reduced brain‐derived TSE infectivity by approximately 4 log10 while endogenous TSE infectivity from hamster blood was reduced to below the limit of detection of the bioassay. The resin was incorporated into membrane layers that were developed by MacoPharma into a prion capture filter termed P‐CAPTTM. In the present study we investigated the performance of the P‐CAPTTM filter to capture PrPres from TSE infected hamster brain homogenate mixed in leucoreduced human RBC.Methods Hamster brain homogenate infected with the 263K strain of scrapie was added to a unit of leucoreduced (LST1 MacoPharma whole blood filter) human RBC. The unit bag was sterile docked to a P‐CAPTTM kit containing an in‐line P‐CAPTTM filter and a transfer bag to collect the treated RBC. The spiked RBC was applied to the P‐CAPTTM filter by gravity at ambient temperature according to the manufacturer's instructions. At the end of the run, the filter was extensively washed and the captured proteins were analyzed by Western blot. In some studies, two P‐CAPTTM filters and two columns in series were tested to investigate the level of PrPres removal provided by the filter. Other parameters such as reproducibility of binding and filtration time were also analyzed.Results Western blot analysis of the filter‐bound proteins indicated that the P‐CAPTTM filter captured brain PrPres from a unit of human leucoreduced RBC. The binding was reproducible within the experimental variability of the Western blot signal and the filtration time was also reproducible. The results also indicated that all input PrPres (pathogenic prion protein) was adsorbed by the first filter and no Western blot signal was captured by the second filter in series.Conclusions P‐CAPTTM filters contain a resin that, when tested in column format, captured brain PrPres, brain‐derived infectivity and endogenous hamster blood infectivity. We showed here that the same resin incorporated into a filter was still capable of capturing brain PrPres.

Molecular aspects of disease pathogenesis in the transmissible spongiform encephalopathies

The transmissible spongiform encephalopathies (TSE), or prion diseases, are a group of rare, fatal, and transmissible neurodegenerative diseases of mammals for which there are no known viral or bacterial etiological agents. The bovine form of these diseases, bovine spongiform encephalopathy (BSE), has crossed over into humans to cause variant Creutzfeldt-Jakob disease. As a result, BSE and the TSE diseases are now considered a significant threat to human health. Understanding the basic mechanisms of TSE pathogenesis is essential for the development of effective TSE diagnostic tests and anti-TSE therapeutic regimens. This review provides an overview of the molecular mechanisms that underlie this enigmatic group of diseases.

Absence of Spiroplasma or Other Bacterial 16S rRNA Genes in Brain Tissue of Hamsters with Scrapie

Spiroplasma spp. have been proposed to be the etiological agents of the transmissible spongiform encephalopathies (TSEs). In a blind study, a panel of 20 DNA samples was prepared from the brains of uninfected hamsters or hamsters infected with the 263K strain of scrapie. The brains of the infected hamsters contained > or =10(10) infectious doses/g. The coded panel was searched for bacterial 16S rRNA gene sequences, using primers selective for spiroplasma sequences, primers selective for mollicutes in general, and universal bacterial primers. After 35 PCR cycles, no samples were positive for spiroplasma or any other bacterial DNA, while control Spiroplasma mirum genomic DNA, spiked at 1% of the concentration required to account for the scrapie infectivity present, was readily detected. After 70 PCR cycles, nearly all samples yielded amplified products which were homologous to various bacterial 16S rRNA gene sequences, including those of frequent environmental contaminants. These sequences were seen in uninfected as well as infected samples. Because the concentration of scrapie infectivity was at a known high level, it is very unlikely that a bacterial infection at the same concentration could have escaped detection. We conclude that the infectious agent responsible for TSE disease cannot be a spiroplasma or any other eubacterial species.

The use of non-prion biomarkers for the diagnosis of Transmissible Spongiform Encephalopathies in the live animal

Scrapie and bovine spongiform encephalopathy (BSE) are major global concerns and the emergence of variant Creutzfeldt-Jakob disease (vCJD) has caused turmoil for blood transfusion services and hospitals worldwide. Recent reports of iatrogenic CJD (iCJD) cases following blood transfusions from Transmissible Spongiform Encephalopathies (TSE)-infected donors have fuelled this concern. Major diagnostic tests for BSE and scrapie are conducted post-mortem from animals in late stages of the disease. Although the lymphoreticular system is involved in the earlier pathogenesis of some forms of sheep scrapie and vCJD, which presents great opportunity for diagnostic development, other TSE diseases (some strains of scrapie, sporadic CJD (sCJD) and bovine BSE) do not present such a diagnostic opportunity. Thus, there is an urgent need for pre-mortem tests that differentiate between healthy and diseased individuals at early stages of illness, in accessible samples such as blood and urine using less invasive procedures. This review reports on the current state of progress in the development and use of prion and non-prion biomarkers in the diagnosis of TSE diseases. Some of these efforts have concentrated on improving the sensitivity of PrPSc detection to allow in vivo diagnosis at low abundances of PrPSc whilst others have sought to identify non-prion protein biomarkers of TSE disease, many of which are still at early stages of development. In this review we comment upon the limitations of prion based tests and review current research on the development of tests for TSE that rely on non-prion disease markers in body fluids that may allow preclinical disease diagnosis.

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The most infectious prion protein particles

Neurodegenerative diseases such as Alzheimer's, Parkinson's and the transmissible spongiform encephalopathies (TSEs) are characterized by abnormal protein deposits, often with large amyloid fibrils. However, questions have arisen as to whether such fibrils or smaller subfibrillar oligomers are the prime causes of disease. Abnormal deposits in TSEs are rich in PrP(res), a protease-resistant form of the PrP protein with the ability to convert the normal, protease-sensitive form of the protein (PrP(sen)) into PrP(res) (ref. 3). TSEs can be transmitted between organisms by an enigmatic agent (prion) that contains PrP(res) (refs 4 and 5). To evaluate systematically the relationship between infectivity, converting activity and the size of various PrP(res)-containing aggregates, PrP(res) was partially disaggregated, fractionated by size and analysed by light scattering and non-denaturing gel electrophoresis. Our analyses revealed that with respect to PrP content, infectivity and converting activity peaked markedly in 17-27-nm (300-600 kDa) particles, whereas these activities were substantially lower in large fibrils and virtually absent in oligomers of < or =5 PrP molecules. These results suggest that non-fibrillar particles, with masses equivalent to 14-28 PrP molecules, are the most efficient initiators of TSE disease.

High incidence of genetic human transmissible spongiform encephalopathies in Italy

To assess the incidence and mortality rates of genetic transmissible spongiform encephalopathy (TSE) diseases in Italy. The authors have sequenced the prion protein gene (PRNP) in 643 patients referred to the Italian Registry of Creutzfeldt-Jakob disease (CJD) and related disorders between 1993 and 2002. Crude age- and sex-specific incidence and mortality rates were calculated. Differences in morbidity from genetic TSE diseases in the 20 Italian regions were assessed by the standardized morbidity ratio (SMR). A total of 130 cases were classified as genetic TSE diseases with a mean yearly incidence rate of 0.28 cases per million people. Genetic TSE diseases represent 17.7% of all TSE diseases, including sporadic, iatrogenic, and variant CJD. The most frequent mutation was the V210I (n = 54), and the second most common the E200K (n = 42). Mortality rates for genetic TSE diseases did not increase in any of the age groups under examination over the 10 years of surveillance. The analysis of regional distribution of genetic cases by place of birth revealed that in Campania and Calabria regions the number of genetic TSE cases was higher than in other regions. In Italy the incidence of genetic transmissible spongiform encephalopathy (TSE) diseases is the second highest among European countries. Genetic analysis is important for a correct classification of patients with TSE.

Rapid presymptomatic detection of PrP Sc via conformationally responsive palindromic PrP peptides

Structurally unique, synthetic prion peptides provide the basis of a simple assay to serve as both a detection and signal amplification system that distinguishes the normal prion protein, PrP C, from the misfolded prion protein, PrP Sc, that is associated with the occurrence of transmissible spongiform encephalopathies (TSE). Proof-of-principle has been shown on brain samples from an experimental scrapie hamster model. The assay demonstrates very sensitive detection of PrP Sc in animal brain tissue with potential application for early presymptomatic detection in animal screening. Furthermore, the sensitivity of the assay could enable blood tests for this TSE disease as well as other amyloid and/or misfolded protein diseases.

In Vitro Generation of Infectious Scrapie Prions

Prions are unconventional infectious agents responsible for transmissible spongiform encephalopathy (TSE) diseases. They are thought to be composed exclusively of the protease-resistant prion protein (PrPres) that replicates in the body by inducing the misfolding of the cellular prion protein (PrPC). Although compelling evidence supports this hypothesis, generation of infectious prion particles in vitro has not been convincingly demonstrated. Here we show that PrPC --> PrPres conversion can be mimicked in vitro by cyclic amplification of protein misfolding, resulting in indefinite amplification of PrPres. The in vitro-generated forms of PrPres share similar biochemical and structural properties with PrPres derived from sick brains. Inoculation of wild-type hamsters with in vitro-produced PrPres led to a scrapie disease identical to the illness produced by brain infectious material. These findings demonstrate that prions can be generated in vitro and provide strong evidence in support of the protein-only hypothesis of prion transmission.

Transmissible spongiform encephalopathy strain, PrP genotype and brain region all affect the degree of glycosylation of PrPSc

Transmissible spongiform encephalopathies (TSEs), sometimes known as prion diseases, are caused by an infectious agent whose molecular properties have not been determined. Traditionally, different strains of TSE diseases are characterized by a series of phenotypic properties after passage in experimental animals. More recently it has been recognized that diversity in the degree to which an abnormal form of the host protein PrP, denoted PrP(Sc), is glycosylated and the migration of aglycosyl forms of PrP(Sc) on immunoblots may have some differential diagnostic potential. It has been recognized that these factors are affected by the strain of TSE agent but also by other factors, e.g. location within the brain. This study shows in some cases, but not others, that host PrP genotype has a major influence on the degree of PrP(Sc) glycosylation and migration on gels and provides further evidence of the effect of brain location. Accordingly both the degree of glycosylation and the apparent molecular mass of PrP(Sc) may be of some value for differential diagnosis between TSE strains, but only when host effects are taken into account. Furthermore, the data inform the debate about how these differences arise, and favour hypotheses proposing that TSE agents affect glycosylation of PrP during its biosynthesis.

Cre-loxP mediated control of PrP to study transmissible spongiform encephalopathy diseases.

Expression of the PrP glycoprotein is essential for the development of the transmissible spongiform encephalopathy (TSE) or prion diseases. Although PrP is widely expressed in the mouse, the precise relevance of different PrP-expressing cell types to disease remains unclear. To address this, we generated two lines of floxed PrP gene-targeted transgenic mice using the Cre recombinase-loxP system. These floxed mice allow a functional PrP allele to be either switched "on" or "off." We demonstrate control of PrP expression for both alleles following Cre-mediated recombination, as determined by PrP mRNA and protein expression in the brain. Moreover, we show that Cre-mediated alteration of PrP expression in these mice has a major influence on the development of TSE disease. These floxed PrP mice will allow the involvement of PrP expression in specific cell types following TSE infection to be defined, which may identify potential sites for therapeutic intervention.

Flexible N-terminal Region of Prion Protein Influences Conformation of Protease-resistant Prion Protein Isoforms Associated with Cross-species Scrapie Infection in Vivo and in Vitro

Transmissible spongiform encephalopathy (TSE) diseases are characterized by the accumulation in brain of an abnormal protease-resistant form of the host-encoded prion protein (PrP), PrP-res. PrP-res conformation differs among TSE agents derived from various sources, and these conformational differences are thought to influence the biological characteristics of these agents. In this study, we introduced deletions into the flexible N-terminal region of PrP (residues 34-124) and investigated the effect of this region on the conformation of PrP-res generated in an in vitro cell-free conversion assay. PrP deleted from residues 34 to 99 generated 12-16-kDa protease-resistant bands with intact C termini but variable N termini. The variable N termini were the result of exposure of new protease cleavage sites in PrP-res between residues 130 and 157, suggesting that these new cleavage sites were caused by alterations in the conformation of the PrP-res generated. Similarly truncated 12-16-kDa PrP bands were also identified in brain homogenates from mice infected with mouse-passaged hamster scrapie as well as in the cell-free conversion assay using conditions that mimicked the hamster/mouse species barrier to infection. Thus, by its effects on PrP-res conformation, the flexible N-terminal region of PrP seemed to influence TSE pathogenesis and cross-species TSE transmission.

Inactivation of transmissible spongiform encephalopathy (prion) agents by environ LpH.

Agents causing transmissible spongiform encephalopathy (TSE) diseases are resistant to inactivation by several conventional decontamination methods. Using an animal bioassay, we compared the TSE agent disinfectant efficacy of a commercially available product referred to alternatively as LpH-SE, LpH-AG, or LpH-st to that of a similarly named but differently formulated product, Environ LpH, which was found to be an effective TSE agent disinfectant in a previous study. Here, we found LpH-SE to be at least 10(4)-fold to 10(5)-fold less effective than Environ LpH.

Genetics of transmissible spongiform encephalopathy susceptibility and the search for surrogate markers for infection

The transmissible spongiform encephalopathy diseases are unusual in that they can be sporadic or infectious, and that the infectious agent does not contain nucleic acids. Instead, infectivity is associated with a modified host-encoded protein referred to as a prion. During the course of disease, host encoded prion protein (PrP) is converted from the normal cellular form, PrPC, to a disease form, PrPSC/BSE, which is highly resistant to degradation by heat or proteinases. The occurrence of the sporadic form of transmissible spongiform encephalopathy disease in humans, as well as susceptibility to infection in humans and some animal species has been correlated with particular alleles of the host PrP gene. Scrapie has been endemic in sheep populations in many countries for over 100 years, with no apparent adverse effects on human health, however the occurrence of bovine spongiform encephalopathy in the mid 1980s in the United Kingdom and subsequently other countries has stimulated interest in the transmissible spongiform encephalopathy diseases. Bovine spongiform encephalopathy seems to have arisen from the recycling of ruminant offals, although it is not clear whether bovine spongiform encephalopathy was already endemic in the cattle population and this practice simply increased the incidence, or if the disease was newly introduced to cattle by transmission of scrapie from sheep. A new form of Creutzfeldt-Jacob disease that bares all the hallmarks of bovine spongiform encephalopathy was recently described in humans, suggesting that it arose from consumption of bovine spongiform encephalopathy-infected beef. Subsequent studies have demonstrated that transmissible spongiform encephalopathy disease can be experimentally transmitted between species and has alerted us to the potential risk of allowing scrapie to remain in the sheep population. In the United Kingdom, the sheep population is being PrP-genotyped and breeding stock selected from animals with genotypes that have been shown to be more resistant to scrapie. However, the resistant sheep genotypes may still harbour disease. There is no convincing evidence for PrP genotypes in cattle that are resistant to bovine spongiform encephalopathy. Work in cattle and mice has shown that there are loci other than PrP that influence the host response to a transmissible spongiform encephalopathy challenge. In order to monitor, and ultimately control, transmissible spongiform encephalopathy diseases in livestock it is important to identify markers for infection that are robust, appear early in infection and can be measured in an easily obtained sample, such as blood. We have developed a bovine ‘non-redundant’ cDNA set from brain to allow expression profiling using macro- and micro array technology. The identities of genes represented the in the non-redundant cDNAs set were assigned by oligo-nucleotide fingerprinting and have been confirmed by sequencing. This non-redundant cDNA set has been used to create a bovine array that is being used to examine expression in tissues from cattle during a time course of experimental bovine spongiform encephalopathy infection in order to identify genes with expression profiles that are altered following infection. Such genes may provide surrogate markers to detect transmissible spongiform encephalopathy infection during the early stages of disease. Ultimately knowledge of such genes may suggest targets for pharmacological intervention to arrest the disease process before the onset of the major neurological damage associated with the terminal stage of the disease.

Molecular aspects of disease pathogenesis in the transmissible spongiform encephalopathies.

The transmissible spongiform encephalopathy (TSE) diseases are a group of rare, fatal, and transmissible neurodegenerative diseases that include kuru and Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, transmissible mink encephalopathy (TME), and chronic wasting disease (CWD) in mule deer and elk. Over the last 20 yr, they have gone from a fascinating but relatively obscure group of diseases to one that is a major agricultural and economic problem as well as a threat to human health. The shift in the relative impact of the TSE diseases began in the late 1970s when the United Kingdom altered the process by which animal carcasses were rendered to provide a protein supplement (i.e., meat and bone meal) to sheep, cattle, and other livestock. Several years later a new disease was recognized in the British cattle population. The pathological and immunohistochemical characteristics of the disease clearly placed it among the TSEs. The new disease was named bovine spongiform encephalopathy (BSE) by the scientific community and "mad cow disease" by the less-than-scientific press. At its peak in the UK, several thousand cattle a year were diagnosed with BSE, and millions of cattle were slaughtered. Introduction of the specified offals ban as well as banning the practice of feeding ruminants to other ruminants has led to a drastic decrease in the number of yearly BSE cases in the UK (less than 500 in 2003), and the epidemic is clearly on the wane. However, BSE has now spread throughout the rest of Europe, as well as to Japan, Russia, Canada, and Israel and thus remains a worldwide problem.A primary concern following the identification of BSE in 1985 was that it might cross species barriers to infect humans. Initially, it was thought that transmission of BSE to humans was unlikely, given that humans appeared to be resistant to scrapie, an animal TSE that had been endemic in British sheep for centuries. However, a few years after BSE was first recognized, a previously unknown form of CJD (variant CJD or vCJD) was identified in young people in Great Britain. The hypothesis that vCJD was the consequence of exposure of humans to BSE has now been supported by several different studies, and over 140 cases of vCJD have been confirmed.

Prion protein and the molecular features of transmissible spongiform encephalopathy agents.

Transmissible spongiform encephalopathy (TSE) diseases, or prion diseases, are neurodegenerative diseases found in a number of mammals, including man. Although they are generally rare, TSEs are always fatal, and as of yet there are no practical therapeutic avenues to slow the course of disease. The epidemic of bovine spongiform encephalopathy (BSE) in the UK greatly increased the awareness of TSE diseases. Although it appears that BSE has not spread to North America, chronic wasting disease (CWD), a TSE found in cervids, is causing significant concern. Despite decades of investigation, the exact nature of the infectious agent of the TSEs is still controversial. Although many questions remain, substantial efforts have been made to understand the molecular features of TSE agents, with the hope of enhancing diagnosis and treatment of disease, as well as understanding the fundamental nature of the infectious agent itself. This review summarizes the current understanding of these molecular features, focusing on the role of the prion protein (PrP(c)) and its relationship to the disease-associated isoform (PrP(Sc)).

Transcript level of erythroid differentiation-related factor, a candidate surrogate marker for transmissible spongiform encephalopathy diseases in blood, shows a broad range of variation in healthy individuals.

In 2001, it was demonstrated that the expression of the erythroid differentiation-related factor (EDRF) is reduced in lymphatic tissues of rodents and cattle as well as in whole blood of sheep that suffer from transmissible spongiform encephalopathy. To determine whether the normal range of EDRF expression varies in healthy individuals, mRNA levels were measured in whole blood samples from 106 healthy blood donors by quantitative real-time RT-PCR. Furthermore, the correlations of transcript levels with individual physical characteristics were analyzed. In addition, EDRF expression was examined in total RNA samples from a lymph node and the intestine. The data show that EDRF mRNA levels in healthy persons vary within a total range of 2 log units as well as they display a weak correlation with body height. Furthermore, it was found that EDRF is also expressed in lymph nodes and the intestine. Owing to its broad range of variation, measuring the EDRF expression does not seem to be a good surrogate marker, unless an altered expression is distinctively different from the varying level in healthy humans.

Prophylactic and therapeutic effects of phthalocyanine tetrasulfonate in scrapie-infected mice.

The transmissible spongiform encephalopathy (TSE) diseases are rare, neurodegenerative diseases that include scrapie in sheep, bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease in humans. There are no effective treatments available for clinical use in humans. We now demonstrate that, in 2 different rodent models of scrapie, multiple pretreatments with the cyclic tetrapyrrole phthalocyanine tetrasulfonate (PcTS) were as effective at delaying disease as multiple treatments starting at the time of infection. At low doses of scrapie infectivity, PcTS also protected some mice from peripheral scrapie infection, even if treatment was initiated several weeks after infection. Furthermore, PcTS completely inactivated low levels of scrapie infectivity when incubated with the infectious inoculum. Thus, PcTS has a broad range of antiscrapie activities. These findings suggest that cyclic tetrapyrroles may be useful both prophylactically and therapeutically against TSE diseases in vivo, as well as for inactivation of TSE infectivity suspended in solution.