Diagnosis Of Prion Diseases Research Articles

Pathogenesis and Diagnosis of Prion Diseases

Pathogenesis and Diagnosis of Prion Diseases

Utilización y validez de la prueba de la proteína 14-3-3 en el diagnóstico de enfermedades por priones: estudio prospectivo de 4 años

The 14-3-3 test shows a high efficiency for the diagnosis of Creutzfeldt-Jakob disease (CJD), as long as an appropriate clinical setting exists. We analyze the evolution of the use and the validity of this test in Spain. From January 1997 to June 2001, 1,092 samples were tested in our laboratory; 674 samples were selected for the study. Diagnoses were obtained by the referring physicians and the national CJD surveillance system according to standard criteria (results of the test were not included). The number of samples analyzed (% of follow-up) was: 75 (96%) in 1997, 138 (82.3%) in 1998, 197 (73%) in 1999 and 264 (59%) in 2000. The negative predictive value, sensitivity and specificity for a diagnosis of prion disease revealed stable values (95, 86 and 86%, respectively). The positive predictive value (PPV) decreased from 77% in 1997 to 51% in 2000 because of an increase of false-positive (FP) results. Up to 25% of FP might have been excluded through neuroimaging or CSF characteristics. The observed increase of the demand suggests that the test is used as a screening technique. In this setting, the PPV of the test decreases due to an increase of false-positive results.

Profound Sex-Specific Effects on Incubation Times for Transmission of Bovine Spongiform Encephalopathy to Mice

Four strains of mice were inoculated intracerebrally with a primary isolate of bovine spongiform encephalopathy (BSE) and the cloned mouse-adapted scrapie strain ME7. Clinical prion disease diagnosis was made at the appearance of three or more neurological symptoms and their persistence for 3 consecutive weeks and confirmed by neuropathological criteria. For BSE, incubation periods were profoundly different between the sexes in all four mouse strains, being longer in the females. In contrast, ME7 scrapie incubation times were similar between the sexes. Our results indicate that sex-specific processes are involved in the course of primary BSE transmission. Research into this phenomenon may provide clues to the prophylaxis of BSE and have possible implications for new variant Creutzfeldt-Jakob disease in humans.

Protein-based PCR for prion diseases?

The development of a sensitive in vitro method to amplify the pathological form of the prion protein might provide a tool for the pre-clinical diagnosis of prion diseases.

Recent developments in the pathogenesis, diagnosis, and therapy of prion diseases

Prions continue to pose a formidable challenge to life sciences. While human prion diseases are still rare, the incidence of a new variant of Creutzfeldt-Jakob disease in the United Kingdom is increasing exponentially - raising fears that it might develop into a major epidemic. This disease is likely to represent the result of human infection with bovine prions. Therefore, understanding how prions replicate and damage the brain, and how their action may be possibly counteracted, has become a major public health issue. Here I examine some current hypotheses concerning the links between bovine and human prion diseases, and the mechanisms by which prions reach and damage the central nervous system after having entered the body at extracerebral sites.

High frequency of mutations in four different disease genes in early-onset dementia.

Heterozygous mutations in the genes for amyloid precursor protein (APP), the presenilins (PS1, PS2), prion protein (PrP), neuroserpin, and tau are associated with early-onset dementia (EOD) with or without neurological signs in the early disease stage. To investigate the proportion of EOD without early neurological signs attributable to known genes we prospectively (i.e., ante mortem) screened these six genes for mutations in 36 patients with EOD before age 60. Family history for dementia was positive (PFH) in 16, negative (NFH) in 17, and unknown (UFH) in 3 patients. In 12 patients, we found 5 novel mutations (PS1: F105L; PS2: T122P, M239I; PrP: Q160X, T188K) and 5 previously reported mutations (APP: in three most likely unrelated patients V717I; PS1: A79V, M139V; PrP: P102L, T183A) that all are considered disease causing. Of these 12 patients, 9 had PFH. This indicates a detection rate of 56% (9/16) in patients with PFH. We found 2 mutations (APP V717I) in 2 of the 3 the UFH-patients, and only 1 mutation (PrP T188K) in 1 of the 17 patients with NFH. No mutation was found in tau and neuroserpin genes. To date, three patients died and FAD, predicted by PS mutations in two patients, and prion disease, predicted by a PrP mutation in the third one, were histopathologically confirmed at autopsy. Up to now, mutation findings may be the most specific biomarkers for an ante mortem diagnosis of FAD or hereditary prion disease.

The Paraffin-Embedded Tissue Blot Detects PrP Sc Early in the Incubation Time in Prion Diseases

With the appearance of bovine spongiform encephalopathy (BSE) and a new variant of Creutzfeldt-Jakob disease (nvCJD) that seems to be caused by BSE, there is an increased need for improvement of diagnostic techniques and recognition of all variants of prion diseases in humans and animals. Publications on the immunohistochemical identification of PrP(Sc) in the tonsils and appendix in the incubation period of nvCJD indicate that new and more sensitive techniques for the detection of PrP(Sc) in various tissues may be a valuable tool for early diagnosis in prion diseases. We developed a new and sensitive technique to detect PrP(Sc) in formalin-fixed and paraffin-embedded tissue, the paraffin-embedded tissue blot (PET blot), and reinvestigated archival brain material from CJD as well as BSE and scrapie. In addition, C57/Bl6 mice experimentally infected with the ME7 strain were investigated sequentially during the incubation time to compare this new technique with conventional methodologies. The PET blot detects PrP(Sc) in idiopathic (sporadic) and acquired prion diseases, even in cases with equivocal or negative immunohistochemistry, and is more sensitive than the conventional Western blot and histoblot techniques. The PET blot makes possible the detection of PrP(Sc) during the incubation period long before the onset of clinical disease and in prion disease variants with very low levels of PrP(Sc). In mice experimentally infected with the ME7 strain, the PET blot detects PrP(Sc) in the brain 30 days after intracerebral inoculation-145 days before the onset of clinical signs. Its anatomical resolution is superior to that of the histoblot technique. It may therefore be of particular interest in biopsy diagnosis. Thus it complements other tissue-based techniques for the diagnosis of prion diseases in humans and animals.

The diagnosis of prion diseases.

Prion diseases of humans and other animals have clinical, economic and political significance. Pre-mortem diagnosis is therefore of great importance. Clinical diagnostic criteria and the current status of the available diagnostic tests are reviewed and possible future developments discussed. Presently, most diagnostic tests are indirect, relying on findings that may not be confined to prion diseases rather than on their particular intrinsic nature. They are therefore not absolutely specific nor are they 100% sensitive. The electroencephalogram (EEG), cerebral imaging and cerebrospinal fluid (CSF) analysis are the main techniques employed. However, there is hope that useful blood tests could be developed which would be simpler and less invasive. Also, there is hope that more specific tests will become available.

Prion: the infectious protein in neurodegenerative diseases

Prion particle is an infectious agent causing neurodegenerative diseases in humans and animals, such as Creut4feldt-Jakob disease, kuru, Gerstmann-Striiussler-Scheinker syndrome, and fatal familial insomnia in humans; mad cow disease, scrapie, and feline spongiform encephalopathy in animals. This particle is devoid of n The protein is converted into its abnormal isoform (PrP-) p PrP conformational change whereby the a-helical content decreases il of P anunknownproteinXwhichmightfunctionasamolecularchaperone. Manyprioncharacteristicshavebeenrevealedintheselasttvvo decades, such as physical, chemical, sftain, molecular biology, and immunological characteristics. Some investigators revealed the topologicalforms ofprionprotein in the endoplasmic reticulum membrane and theirrole inthe pathophysiologicalprocess of the disease. But still prion diseases continue 1o raise many unanswerable questions to be investigated. One of the unanswerable questions is prion muhiplication. Many prion multiplication models are sug constantftntlitry is that prion replication requires the interaction of PrPt-PrP. The studies of pathogenes ch more attention to develop an effective therapy for these neurodegenerative dise.ases. Diagnosis of prion diseases is based on the clinical and histopathological findings, and immunochemical tests of some proteins in cerebrospinal fluid. Immunochemical tests of proteins in cerebrospinal Jluid are important in developing premortem diagnosis of prion di.seases.

Early induction of protein nexin-I in scrapie.

We used RNA fingerprinting by arbitrary primed PCR to identify genes whose expression is up-regulated in the brain of hamsters affected by prion disease. One gene implicated by RNA fingerprinting encoded the hamster homologue of protein nexin-I (PN-I), a serine proteinase inhibitor, and was further investigated by Northern blot analysis. PN-I mRNA levels were increased at pre-clinical stages (19 days after inoculation) and remained elevated when the spongiform encephalopathy was anatomopathologically and clinically evident (at 50 and 80 days). Future RNA screening conducted as illustrated may help to reveal a spectrum of genes relevant for the etiopathogenesis and/or diagnosis of prion disease.

The immunodetection of the abnormal isoform of prion protein.

Transmissible spongiform encephalopathies such as scrapie in sheep and goats, Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle, are neurodegenerative disorders. A proposed causative agent for these diseases is an infectious protein, the so called 'prion'. An abnormal isoform of prion protein (PrPSc) can be detected according to the prion propagation method used. As PrPSc appears to constitute the main, if not the only, infectious entity its detection for the diagnosis of prion diseases is important. Immunodetection methods for PrPSc analysis are popular tools for diagnosis and research studies. In this paper, a review of the present knowledge concerning immunodetection is presented and the enhancement of the immunoreactivity of antisera to mouse and hamster prion protein peptides using the techniques of Western blotting and immunohistochemistry is summarized.

Prion diseases in man.

Prion diseases are uncommon fatal neurodegenerative disorders which have gained scientific and public importance as a result of major advances in the understanding of the nature of the causative agent, and the emergence of new forms of these diseases in both animals and man. The transmissible agent in prion diseases is unique and is closely associated with an abnormal isoform of a widely distributed cell-surface glycoprotein, prion protein. The precise mechanisms of conversion to the abnormal isoform are unknown; changes in protein folding are of major importance. The abnormal isoform of the protein accumulates in the central nervous system in all prion diseases, but the processes involved in protein accumulation and the pathogenesis of neuronal dysfunction and cell death are poorly understood. Human prion diseases occur as sporadic, familial, and acquired disorders, the most recently identified of which is new variant Creutzfeldt-Jakob disease, which has been aetiologically linked to exposure to the bovine spongiform encephalopathy agent through the food chain. Surveillance of human prion diseases will be crucial in the assessment of the impact of this new disease in the United Kingdom and elsewhere. Effective surveillance depends on accurate diagnosis, which in turn places a high priority on autopsy in suspected cases; neuropathology is essential for the diagnosis of human prion diseases. Phenotypic variation is prominent in all forms of human prion disease; future classifications of these disorders are likely to incorporate genetic and biochemical data in addition to clinical and pathological parameters.

Prion protein expression in different species: Analysis with a panel of new mAbs

By immunizing prion knockout mice (Prnp-/-) with recombinant murine prion protein (PrPc), we obtained a panel of mAbs specific for murine PrPc. These mAbs can be applied to immunoblotting, cell surface immunofluorescent staining, and immunohistochemistry at light and electron microscopy. These mAbs recognize both the normal (PrPc) and protease-resistant (PrPres) isoforms of PrP. Some mAbs are species restricted, while others react with PrP from a broad range of mammals including mice, humans, monkeys, cows, sheep, squirrels, and hamsters. Moreover, some of the mAbs selectively recognize different PrP glycoforms as well as the metabolic fragments of PrPc. These newly generated PrPc antibodies will help to explore the biology of PrPc and to establish the diagnosis of prion diseases in both humans and animals.

Establishing a Pathological Diagnosis in Degenerative Dementias

While clinicopathological studies have confirmed that Alzheimer's disease (AD) is the most common neurodegenerative cause of dementia, these same studies have also revealed that other degenerative pathologies account for a significant proportion of patients with cognitive decline. Because pathological assessment of non-Alzheimer neurodegenerative diseases now demands routine use of a costly panel of immunohistochemical techniques a scheme for staged examination of brain tissue has been developed. This scheme is weighted to initially screen out cases of Alzheimer's disease, dementia with Lewy bodies and vascular dementia using conventional staining methods and established diagnostic protocols, bringing in immunochemical techniques to discriminate between non-Alzheimer degenerative dementias. Diagnosis of pathologies causing the clinical syndrome of frontotemporal dementia can be ascertained using conventional staining supplemented by immunochemical detection of ubiquitin, tau protein and alpha beta crystallin. The diagnosis of prion disease is reliably confirmed by immunohistochemical detection of prion protein. This morphological assessment complements emerging genetic insights into many of these neurodegenerative diseases.

Neuro-ophthalmological aspects of prion diseases

Prion brain diseases (Kuru, Creutzfeldt-Jakob disease, familial fatal insomnia (FFI), Gerstman-Straussler-Scheinker syndrome) are conditioned by the host’s genome. Susceptibility (hereditary) is related to the PrPc gene polymorphism (insertion and/or point mutations at different codons). The normal harmless PrPc (neuron) is transformed into a rogue structurally altered PrPsc (scrapie) invading neurocells. Human consumption of protein from bovine spongiform encephalopathy (BSE) cattle, fed scrapie-sheep offal, may be responsible for prion disease. The clinical features are mirrored by a wide spectrum of brain grey matter involvement in middle-aged patients. Progressive dementia and supranuclear (ocular) motor symptoms are characteristic; at onset visual symptoms may occur in 20–25% of cases. In dementing patients, diagnosis of prion disease may only be confirmed by brain biopsy, demonstrating the typical spongiosis and pleated prion plaques (methacarn fixation). Care should be taken to never use tissue from dementing patients for substitutive therapy or transplants.

Generation of Monoclonal Antibodies against Human Prion Proteins in PrP0/0 Mice

Prion diseases belong to a group of neurodegenerative disorders affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). The pathogenic mechanisms of the prion diseases are not yet understood. Monoclonal antibodies provide valuable tools in the diagnosis, as well as in the basic research, of several diseases; however, monospecific antisera or monoclonal antibodies (mAbs) against human prion proteins were, until now, not available. We have developed an immunization protocol based on nucleic acid injection into nontolerant PrP0/0 mice. DNA or RNA coding for different human prion proteins including the mutated sequences associated with CJD, GSS, and FFI were injected into muscle tissue. Mice were primarily inoculated with DNA plasmids encoding the prion protein (PRNP) gene and boosted either with DNA, RNA, or recombinant Semliki Forest Virus particles expressing PRNP. Hybridomas were then prepared. Different mAbs against human prion proteins were obtained, and their binding behavior was analyzed by peptide enzyme-linked immunosorbent assay, Western blot, immunofluorescence, and immunoprecipitation. Their cross-reactivity with prion protein from other species was also determined. Our mAbs are directed against four different linear epitopes and may also recognize discontinuous regions of the native prion protein. These antibodies should allow us to address questions concerning the nature of the prion protein as well as the initiation and progression of prion diseases. Moreover, these mAbs can now be used for the diagnosis of prion diseases of humans and animals.

Diagnosis and Incidence of Prion (Creutzfeldt-Jakob) Disease: A Retrospective Archival Survey with Implications for Future Research

Reliable identification of Creutzfeldt-Jakob disease (CJD) in the UX has become essential following the suggestion that prion disease in cattle (BSE) might transmit, accidentally, to humans who eat contaminated beef. Recent data suggest that some cases of CJD may be clinically unrecognized; in order to examine this proposal we reviewed all cases of dementia ( n=1000+) collected in the Runwell Hospital Brain Archive between 1964 and 1990. We identified 19 cases of spongiform encephalopathy of which only 11 were diagnosed before death. These 11 individuals had a characteristic clinical history of CJD (relentless mental deterioration, prominent motor signs and death within a year). Their brains showed little or no external abnormality. In contrast, only two of the eight clinically unrecognized cases had characteristic symptoms. The remaining six presented atypically; their illness lasted 3 years or more, motor signs were much less evident, and simple dementia was the most prominent feature. The brains showed moderate or severe cerebral atrophy. Our data indicate that only about 60% of prion disease cases with pathologically typical spongiform encephalopathy were identified clinically during life. This suggests that human prion disease may be more common than previously supposed and that a further review of the epidemiology of the disease is required

Prion Protein Transgenes and the Neuropathology in Prion Diseases

The concept that prions are novel pathogens which are different from both viroids and viruses has received increasing support from many avenues of investigation over the past decade. Enriching fractions from Syrian hamster (SHa) brain for scrapie prion infectivity led to the discovery of the prion protein (PrP). Prion diseases of animals include scrapie and "mad cow" disease; those of humans present as inherited, sporadic and infectious neurodegenerative disorders, two of which are called Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker disease (GSS). The inherited human prion diseases are genetically linked to mutations in the PrP gene that result in non-conservative amino acid substitutions. Transgenic (Tg) mice expressing PrP carrying a GSS mutation developed neurodegeneration spontaneously and produced prions de novo. In other studies, Tg mice expressing both SHa and mouse (Mo) PrP genes were used to demonstrate that the "species barrier" for scrapie prions resides in the primary structure of PrP. This concept was strengthened by the results of studies in which mice expressing chimeric Mo/human (Hu) PrP transgenes were constructed which differ from MoPrP by nine amino acids between residues 96 and 167. All of the Tg(MHu2M) mice developed neurologic disease approximately 200 days after inoculation with brain homogenate from three patients who died of CJD. About 10% of Tg(HuPrP) mice expressing HuPrP and non-Tg mice developed neurologic disease > 500 days after inoculation with CJD prions. The different susceptibilities of Tg(HuPrP) and Tg(MHu2M) mice to human prions indicate that additional species specific factors such as chaperone proteins are involved in prion replication. Diagnosis, prevention and treatment of human prion diseases should be facilitated by study of Tg(MHu2M) mice. Our findings and those from other studies suggest that mutant and wtPrP interact, perhaps through a chaperone-like protein, during the pathogenesis of the prion diseases.

Transmission of Creutzfeldt-Jakob disease from humans to transgenic mice expressing chimeric human-mouse prion protein.

Transgenic (Tg) mice were constructed that express a chimeric prion protein (PrP) in which a segment of mouse (Mo) PrP was replaced with the corresponding human (Hu) PrP sequence. The chimeric PrP, designated MHu2MPrP, differs from MoPrP by 9 amino acids between residues 96 and 167. All of the Tg(MHu2M) mice developed neurologic disease approximately 200 days after inoculation with brain homogenates from three patients dying of Creutzfeldt-Jakob disease (CJD). Inoculation of Tg(MHu2M) mice with CJD prions produced MHu2MPrPSc (where PrPSc is the scrapie isoform of PrP); inoculation with Mo prions produced Mo-PrPSc. The patterns of MHu2MPrPSc and MoPrPSc accumulation in the brains of Tg(MHu2M) mice were different. About 10% of Tg(HuPrP) mice expressing HuPrP and non-Tg mice developed neurologic disease > 500 days after inoculation with CJD prions. The different susceptibilities of Tg(HuPrP) and Tg(MHu2M) mice to Hu prions indicate that additional species-specific factors are involved in prion replication. Diagnosis, prevention, and treatment of Hu prion diseases should be facilitated by Tg(MHu2M) mice.

Diagnosis and incidence of prion (creutzfeldt-jakob) disease: a retrospective archival survey : G. W. Roberts ∗ C. J. Bruton ∗∗, R. K. Bruton ∗∗ and S.M. Gentleman ∗∗∗. ∗Dept. of Molecular Neuropathology, SmithKline Beecham Pharmaceuticals, Harlow, Essex, CM19 5AD,UK. ∗∗MRC Dept. of Neuropathology, Runwell Hospital, Wickford Essex, UK. ∗∗∗Depts. of Psychiatry and Anatomy, Charing Cross & Westminster Medical School, Dunstan's

Diagnosis and incidence of prion (creutzfeldt-jakob) disease: a retrospective archival survey : G. W. Roberts ∗ C. J. Bruton ∗∗, R. K. Bruton ∗∗ and S.M. Gentleman ∗∗∗. ∗Dept. of Molecular Neuropathology, SmithKline Beecham Pharmaceuticals, Harlow, Essex, CM19 5AD,UK. ∗∗MRC Dept. of Neuropathology, Runwell Hospital, Wickford Essex, UK. ∗∗∗Depts. of Psychiatry and Anatomy, Charing Cross & Westminster Medical School, Dunstan's