Amplification techniques and diagnosis of prion diseases.

Protein misfolding cyclic amplification assay (PMCA) and real-time quaking-induced conversion (RT-QuIC) are two amplification techniques based on the ability of PrPsc to induce a conformational change in PrP allowing the detection of minute amounts of PrPsc in body fluids or tissues. PMCA and RT-QuIC have different ability to amplify PrPsc from sporadic, variant and genetic forms of Creutzfeldt-Jakob disease (CJD). PMCA readily amplifies PrPsc from variant CJD (vCJD) tissue while RT-QuIC easily amplifies PrPsc from sporadic CJD (sCJD) patient tissues. In terms of diagnosis, this implies the possibility of distinguishing vCJD from sCJD and explains the wider use of RT-QuIC given the respective frequencies of vCJD and sCJD. The sensitivity values of RT-QuIC for the diagnosis of sCJD are comparable or higher than those of the other tests (EEG, MRI, detection of 14-3-3 or tau proteins in cerebrospinal fluid) but with a specificity close to 100%. These new diagnostic methods could also be useful for the diagnosis of other neurodegenerative diseases.

Combined Creutzfeldt-Jakob/ Alzheimer's Disease Cases are Important in Search for Microbes in Alzheimer's Disease.

The question whether Alzheimer's disease is infectious as brought up in the recent editorial published in the Journal of Alzheimer's Disease is complicated by the controversy whether the causal agent is a microbe or a misfolded host protein (amyloid). The replicating amyloid (prion) theory, based upon data from studies of Creutzfeldt-Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs), has been challenged since the prion can be separated from TSE infectivity, and spiroplasma, a wall-less bacterium, has been shown to be involved in the pathogenesis of CJD. Further support for a microbial cause for AD comes from occurrence of mixed CJD/AD cases involving up to 15% of AD brains submitted to brain banks. The association of CJD with AD suggests a common etiology rather than simply being a medical curiosity. A co-infection with the transmissible agent of CJD, which we propose to be a Spiroplasma sp., would explain the diversity of bacteria shown to be associated with cases of AD.

An alarming presentation of Creutzfeldt-Jakob disease following a self-inflicted gunshot wound to the head.

Transmissible spongiform encephalopathies (TSE), also known as prion diseases, are characterized by rapid and fatal neurological decline. They not only detrimentally affect the patient, but also present additional challenges to healthcare systems due to the infectivity of the tissues and the difficulty of inactivating the prion. The most common TSE is Creutzfeldt-Jakob disease (CJD), which can occur after familial, spontaneous or acquired transmission. TSEs received more attention after the development of variant CJD (vCJD), also known as Mad Cow Disease, in the UK during the mid-1990s. Unlike familial or spontaneous CJD, this variant was connected to consumption of cattle contaminated with the prion disease, bovine spongiform encephalopathy.This development increased interest in the etiology of CJD and other TSEs and the risk it presents as an infectious disease. The following details the case of a 59-year-old male infected with CJD presented to our level II trauma center for treatment following a self-inflicted gunshot wound to the head.

Creutzfeldt-Jakob disease and mad cows: lessons learnt from yeast cells.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that affect mammals including humans. The proteinaceous nature of the infectious agent, the prion, and its propagation, challenge established dogmas in biology. It is now widely accepted that prion diseases are caused by unconventional agents principally composed of a misfolded host-encoded protein, PrP. Surprisingly, major break-throughs in prion research came from studies on functionally unrelated proteins in yeast and filamentous fungi. Aggregates composed of these proteins act as epigenetic elements of inheritance that can propagate their alternative states by a conformational switch into an ordered ß-sheet rich polymer just like mammalian prions. Since their discovery prions of lower eukaryotes have provided invaluable insights into all aspects of prion biogenesis. Importantly, yeast prions provide proof-of-principle that distinct protein conformers can be infectious and can serve as genetic elements that have the capacity to encipher strain specific information. As a powerful and tractable model system, yeast prions will continue to increase our understanding of prion-host cell interaction and potential mechanisms of protein-based epigenetic inheritance.

Linking chronic wasting disease to scrapie by comparison of Spiroplasma mirum ribosomal DNA sequences.

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative diseases of man and animals and are transmitted by a filterable pathogen whose identity is currently unresolved. Our data indicates that Spiroplasma, a wall-less bacterium, is involved in the pathogenesis of TSE. We searched for Spiroplasma ribosomal gene sequences in 10 scrapie-infected sheep brains and 10 normal sheep brains, 7 cervid samples infected with chronic wasting disease (CWD), and 7 normal cervid brains. DNA was extracted from these tissue samples and amplified by polymerase chain reaction (PCR) using primers specific for Spiroplasma-specific 16S rDNA. Specificity of the amplicon was determined by Southern blotting and DNA sequence analyses. Spiroplasma 16S rDNA was found in 8 of 10 scrapie-infected sheep brains and 6 of 7 CWD-infected tissue samples. All normal animal brain samples were negative. Spiroplasma 16S rDNA was also found in two human Creutzfeldt-Jakob diseased (CJD) brains but not in two age-matched normal human brains. DNA sequence analyses of the amplified PCR products from human and animal TSE cases revealed greater than 99% nucleotide sequence homology with Spiroplasma mirum. The presence of Spiroplasma DNA in TSE-infected tissues supports our hypothesis that Spiroplasma may be involved in the pathogenesis of these diseases.

Spiroplasma sp. 16S rDNA in Creutzfeldt-Jakob disease and scrapie as shown by PCR and DNA sequence analysis.

The pathogenesis of the transmissible spongiform encephalopathies (TSE), which include Creutzfeldt-Jakob disease (CJD) in humans and scrapie in sheep, remains an enigma. In this paper we present evidence for the association of Spiroplasma sp., a wall-less prokaryote, with TSE. We have shown PCR amplification of Spiroplasma 16S rDNA in TSE-infected brain tissues (13 of 13 CJD cases and 5 of 9 scrapie cases) and not in control brains (0 of 50). Direct sequencing of the amplified PCR products has confirmed the presence of Spiroplasma-like DNA in all 5 of the TSE brains tested. Our evidence is not necessarily in conflict with involvement of a PrPres--a protease-resistant host-derived protein referred to as the prion--in the pathogenesis of TSE, since there is evidence that another factor is involved. We propose a bacterium, namely Spiroplasma, as this associated factor although the role of Spiroplasma in TSE cannot be determined from these experiments. The presence of the nucleic acid sequence of this microbe in all cases of TSE in our laboratory and not in controls provides direct evidence of the association of Spiroplasma sp. with TSE.

Medicinal and other products and human and animal transmissible spongiform encephalopathies: memorandum from a WHO meeting.

The report in March 1996 of 10 human cases of a novel from of Creutzfeldt-Jakob disease in the United Kingdom, and its possible link to the agent that causes bovine spongiform encephalopathy (BSE), raises many questions about the safety of animal-derived products and by-products entering the food chain or being used in medicine. This Memorandum updates the preventive measures put forward in 1991 to minimize the risks associated with the use of bovine-derived materials in medicinal products and medical devices.

[Creutzfeldt-Jakob disease]. / Die Creutzfeldt-Jakob-Erkrankung.

Creutzfeldt-Jakob's disease is a transmissible encephalopathy manifesting with dementia and motor disturbances, which usually progresses rapidly and is lethal within months. It occurs mainly sporadically, but it can also be transmitted by proteinaceous infective particles called prions. The diagnosis has to rely on clinical symptoms, EEG and brain biopsy being the most suitable additional examinations. No therapy is yet known. "Naturally" occurring transmission has not been observed: all transmitted cases reported so far have been iatrogenic and followed administration of cadaveric hypophyseal hormones, transplantation of tissue from CNS or related organs, or brain surgery with contaminated instruments. Remarkable discoveries in the past decades with respect to the molecular and genetic characterization of the transmissible pathogen have led to a new understanding of the disease. The infective agent appears to be an abnormal isoform of a physiologically occurring protein: the cellular prion (PrPc). The crucial pathogenetic event is the conformational conversion of PrPc into its pathological isoform (PrPsc), an event thought to be triggered autocatalytically by the infectious agent itself. The disease can be elicited in experimental animals by inoculation of PrPsc. In the sporadic cases of Creutzfeldt-Jakob's disease, PrPsc is thought to arise through spontaneous conversion of PrPc. A growing body of evidence indicates that specific alleles of the prion gene confer a genetic predisposition to Creutzfeldt-Jakob's disease and to related pathologies.

Dementias, neurodegeneration, and viral mechanisms of disease from the perspective of human transmissible encephalopathies.

Our transmission experiments with human CJD emphasize the centrality of an exogenous infectious pathogen that can exist in symbiosis with its host for extended periods. Many latent or persistent viruses can cause neurodegenerative disease and may have a role in late onset dementias. There are reasons to believe that CJD infections may share properties with some of these latent viruses in causing dementia, and several retroviral mechanisms may be operative in CJD. In order to clarify viral-like attributes of the CJD agent we have closely followed infectivity and find the following: 1) the CJD agent has a virus-like size and density, and is biochemically separable from most host-encoded prion protein (PrP); 2) Endogenous retroviral IAP RNA sequences of 5,000 bases, as well as several gag-like nucleic acid binding proteins, co-purify with infectivity in preparations treated with high concentrations of anionic detergents and exhaustive nuclease digestion. They signify the purification of true viral cores rather than aggregation artifacts, and diminish claims that there are no protected nucleic acids of > 50 bases in highly purified infectious preparations; 3) In established hamster CJD, temporal studies show the agent has an effective doubling time of approximately 7.5 days in brain, consistent with complex host-viral interactions common to slow viral infections; 4) PrP-res does not correspond to titered levels of infectivity either in a biochemical or an in vivo setting but may function as a viral receptor that can modulate disease expression. Interestingly, functional changes in glial cells occur earlier than PrP-res changes, and indicate an important role for glial cells in evolving infections; 5) Human-rodent transmission studies suggest that CJD, or a CJD-like variant can be a common but latent infection of humans, with relatively infrequent expression of neurological disease. Susceptibility to disease can rest on host attributes and possibly age-related co-factors. Nonetheless, fundamental viral principles are also operative. Agent strain variants, viral burden, and the routes of infection are critical parameters for latency and disease expression. The properties described above have led me to return to the inclusion of CJD (and scrapie) in the panorama of conventional slow viral infections of the brain, as originally proposed by Sigurdsson. Identification of virus-specific molecules are essential for elucidating the role of these agents in the spectrum of human dementias.

Endogenous viral complexes with long RNA cosediment with the agent of Creutzfeldt-Jakob disease.

A class of viruslike agents that induces Creutzfeldt-Jakob Disease (CJD) and scrapie remains undefined at the molecular level. Several investigators believe this infectious agent is constituted by a single host protein or 'prion', and have emphasized data that would seem to exclude the presence of any viral nucleic acids. However, more rigorous evaluations in scrapie have shown reasonably abundant nucleic acids. Additionally, in highly purified 120S CJD preparations that have been treated with nucleases, RNAs as long as 6,000 bases have been detected. Few nucleic acids have been characterized in either scrapie or CJD, but previous cloning experiments delineated relatively short LTR regions of the endogenous IAP retrovirus in 120S CJD preparations. We therefore used specific primers encompassing the entire IAP genome to test for the presence of long viral RNAs, and here show approximately 5,000 contiguous bases of the IAP RNA genome can be recovered from reasonable amounts of starting brain. The 3' env region of IAP is comparably truncated in CJD and normal preparations, and we find no evidence for IAP transduction of CJD-specific sequences. Because IAP cores can coencapsidate unrelated sequences, and are unusually resistant to physical and chemical treatments, it was relevant to find if cosedimenting cognate proteins of the IAP core, such as gag, could be detected. The predicted approximately 65 kd acidic gag protein, showing appropriate antigenic and nucleic acid binding features, was apparent in both one and 2-D Western blots. This data strongly indicates specific viral complexes cofractionate with the CJD agent. Interestingly, these nuclease resistant IAPs do not appear to be in morphologically recognizable 'R' particles. This cosedimenting viral assembly therefore provides a paradigm for non-particulate CJD complexes in infectious preparations. In developing strategies to identify a CJD specific sequence, cosedimenting IAPs can be used to assess the quality, length and recovery of RNAs extracted from highly resistant viral complexes.

Similar genetic susceptibility in iatrogenic and sporadic Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD) is one of the transmissible spongiform encephalopathies (TSEs). In all TSEs host susceptibility is an important factor in the development of clinical disease. The prion protein (PrP) gene appears to confer the main component of this susceptibility. The appearance of spontaneous neurodegeneration in PrP transgenic mice carrying a human mutation has raised the possibility that the origin of sporadic CJD is solely genetic. We studied PrP codon 129 polymorphism in 23 of the 25 CJD cases in France related to human growth hormone (hGH) therapy. They constitute the largest and most homogeneous hGH-related iatrogenic CJD population yet analysed. All these CJD cases were homozygous at codon 129, compared with only 50% in the healthy control group (P < 0.00002). These iatrogenic cases also displayed a genotype frequency distribution similar to that observed in sporadic CJD. These results underline the importance of the PrP gene and especially the homozygous codon 129 genotype in determining the risk of developing CJD after contamination by a TSE agent. They also suggest that highly susceptible individuals may exist and raise the possibility that sporadic CJD may have an environmental origin.

Kinetics of infectivity are dissociated from PrP accumulation in salivary glands of Creutzfeldt-Jakob disease agent-inoculated mice.

The protease-resistant isoform of prion protein (PrP) has been implicated in the pathogenesis and transmission of Creutzfeldt-Jakob disease (CJD), scrapie and other related diseases, but the relationship between the infectious agent and PrP awaits elucidation. In the present study, we have examined levels of infectivity together with accumulation of the protease-resistant form of PrP (PrPCJD) in various tissues of CJD agent-inoculated mice. Accumulation of PrPCJD occurred only in tissues, including brain, salivary gland and spleen, in which infectivity was readily detectable throughout the course of the experiment. The brain showed the highest levels of both infectivity and PrPCJD accumulation, with well correlated kinetics. On the other hand, the high titres of infectivity detected in salivary gland and spleen early after inoculation of the agent were obviously distinguishable from PrPCJD. Furthermore, in the salivary gland, the kinetics of infectivity and the accumulation of PrPCJD reversed; infectivity declined as PrPCJD accumulated in the tissue. Our findings indicate that PrPCJD accumulation is associated with replication of the agent; however, PrPCJD is unlikely to be the agent itself.