Advancing Prion Science Guidance for the National Prion Research Program (2004) / Chapter Skim
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4 Diagnostics for Transmissible Spongiform Encephalopathies
4 Diagnostics for Transmissible Spongiform Encephalopathies
Pages 72-107
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From page 72... ...
Yet very few diagnostic tests are available to detect the infectious agents associated with transmissible spongiform encephalopathies (TSEs)
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From page 73... ...
Thus two major factors challenge the designer of any useful antemortem diagnostic test for a TSE agent. First, only small amounts of prions may be available for detection in accessible living tissues, such as blood, urine, and cerebrospinal fluid (CSF)
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From page 74... ...
CLINICAL DIAGNOSTICS In general, diagnoses of prion diseases by clinical description or ancillary clinical tests are not specific enough to confirm a specific prion disease. In important circumstances, however, they give the clinician some clues that may help support or question the diagnosis of a prion disease.
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From page 75... ...
. stalmng pattern or oram tissue 63 yr 4 mo Unusual Bi- or triphasic periodic complexes Increased signal in basal ganglia, caudate nucleus, and putamen 14-3-3 protein levels usually elevated No amyloid plaques Punctate pattern Immunohistochemical staining Negative of tonsil or appendix tissue PrPSc isotype by Western blot Type 1A 29 yr 14 mo Common Nonspecific, slow Hyperintense signal in pulvinar region of the thalamus 14-3-3 protein levels not usually elevated 100% florid plaques Widespread plaque .
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From page 77... ...
D such as familial cases of human TSEs and vC] D, this CSF protein test is often negative.
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From page 78... ...
Nevertheless, studies of sheep naturally infected with the scrapie agent and of mice experimentally infected with that same agent have demonstrated that PrPSc is detectable in lymphoid tissues long before clinical signs of neurological disease appear. Brain Biopsy In atypical cases of C]
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From page 79... ...
In addition to the PrPSc type, the phenotype of human prion diseases is influenced by the genotype at codon 129 of the PRNP, the site of a common M/V polymorphism. A classification of sporadic prion diseases has been generated on the basis of the combination of the genotype at codon 129 and the PrPSc type (Parch)
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From page 80... ...
. All three tests are designed to detect the infectious agent of CWD in peripheral lymphoid tissues of select cervids.2 2The Bio-Rad test is approved for use on tissue samples from mule deer, white-tailed deer, and elk (personal communication, Rick Hill, USDA APHIS Center for Veterinary Biologics, November 25, 2003; USDA APHIS, 2002)
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From page 81... ...
; VMRD Inc.
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From page 82... ...
Test kits are available for the diagnosis of scrapie in sheep and BSE in cattle. The test uses gel electrophoresis with a specific antibody against PrP after the prpC in homogenized brain tissue has been digested by PK.
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From page 83... ...
(The tests all use postmortem brain tissue samples.) Consequently, these approved tests are sufficiently sensitive for the detection of BSE only in clinically sick cattle or in apparently healthy cattle that are near the clinical onset of prion disease.
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From page 84... ...
(The tests all use postmortem brain tissue samples.) Consequently, these approved tests are sufficiently sensitive for the detection of BSE only in clinically sick cattle or in apparently healthy cattle that are near the clinical onset of prion disease.
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From page 85... ...
. A breakthrough in the pace of TSE research occurred when investigators successfully infected mice with the scrapie agent by intracranial inoculation (Chandler, 19611.
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From page 86... ...
Both sheep and human prions have been propagated in this cell system after the agent was first passaged through mice (Kingsbury et al., 1984; Race et al., 19871. Other cells reported to have been used in cell culture systems include the GT-1 cell line, which is derived from mouse hypothalamic neurons and has been used successfully to study the scrapie agent (Schatz!
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From page 87... ...
, known for its reliability in remaining chronically infected with the same Chandler scrapie agent over multiple generations. In one study, investigators used pentosan sulfate to cure a scrapie infection in SMB cells, then reinfected the cell culture with two different strains of scrapie agents.
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From page 88... ...
Saborio and colleagues reported more than 30fold increases in the amount of PrPSc over that provided in the infected brain extract. This new conversion system should improve the chances of detecting any new infectivity associated with conversion and may also be exploited to enhance the detection of PrPSc in TSE diagnostic tests.
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From page 89... ...
, wheat germ agglutinin, heparin, and various antibodies, have been used to bind selectively to PrPSc and thus concentrate the abnormal protein for further characterization (Harris, 20021. Protein Misfolding Cyclic Amplification (PMCA)
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From page 90... ...
At least one group of investigators has reported the use of capillary electrophoresis to detect PrPSC in sheep and elk blood (Schmerr and Tenny, 1997, 1998; Schmerr et al., 19991. This method has been used to study other proteins in the past (Tsuji, 1994)
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From page 91... ...
Recently, a team of investigators experimented with a similar immunocompetitive capillary electrophoresis assay test in humans and chim
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From page 92... ...
for the direct detection of PrPSc, as well as for the differentiation of disparate prion strains (Rubenstein et al., 19981. Fourier Transform Infrared (FTIR)
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From page 93... ...
They reported a sensitivity of 97 percent and a specificity of 100 percent; the predictive value was 100 percent for a positive test result and 98 percent for a negative test result. The investigators suggest that the test needs to be assessed with species other than hamsters, and caution that the differences observed between the scrapie-infected animals and the controls may not be specific for detection of the scrapie agent.
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From page 94... ...
However, the ability to leverage novel technologies to develop better diagnostics and the chance of success in doing so will improve significantly by first advancing fundamental knowledge of prion biology through basic research. RESEARCH RECOMMENDATIONS FOR TSE DIAGNOSTICS Exploitation of New Technology New antemortem laboratory tests for the detection of TSE agents are imperative.
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From page 95... ...
In principle, these molecules could be monoclonal antibodies, such as monoclonal mouse antiprion antibodies, which are made by immunizing mice with a preparation of a protein containing the desired epitopes and isolating hybridomas after cell fusion, as described by Kohier and Milstein (19751. Due to the difficulties of producing monoclonal antibodies to PrP, however, prion detection methods presently are dependent on relatively few antibodies produced in viva.
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From page 96... ...
Since PrPSc can be found in follicular dendritic cells within peripheral lymph nodes, Paramithiotis and colleagues postulated that tyrosine-tyrosine-arginine monoclonal antibodies may have a benefit in targeting this PrPSc and blocking neural invasion, if administered during the incubation period of a prion infection. They also believe these antibodies could lead to a better understanding of the structural peculiarities of PrPSc, which in turn, could result in the development of new diagnostics and therapeutic approaches.
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From page 97... ...
This requirement can be addressed by a variety of approaches. Conclusions Regarding Reagents and Detection Methods In broad terms, the present limitations to prion detection lie not in the lack of methods but in the paucity of antibody and other recognition molecules specific for prion species, strain, and allelic variants and for the infectious conformation.
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From page 98... ...
[Priority 11 Surrogate Markers and Signatures of Prion Disease Diagnostic approaches based on detection of indirect disease markers have a long and inconsistent history. In general, these approaches have been hindered by their lack of specificity (e.g., tests for the erythrocyte sedimentation rate and C-reactive protein)
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Recommendation 4.4: Fund research to identify surrogate markers or signatures for the detection of prions or prion diseases. [Priority 31
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From page 100... ...
Recommendation 4.5: Fund research to improve techniques for propagating prions in cultured cells and develop new in vitro cell systems as a means to assay and study priors. [Priority 21 Clinical Neuroimaging Recent improvements in clinical neuroimaging have shown increasing utility in clinical diagnostics for TSEs.
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From page 101... ...
These methods may be valuable in the diagnosis of humans with prion disease, especially individuals who are at risk for inherited or iatrogenic prion disease, and the evaluation of TSE therapies. Recommendation 4.6: Fund research to develop functional imaging for the presence of PrPSc in brain tissue, leading to an early diagnostic test similar to the imaging diagnostics being developed for Alzbeimer's disease.
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From page 102... ...
2003. Failure of immunocompetitive capillary electrophoresis assay to detect diseasespecific prion protein in buffy coat from humans and chimpanzees with CreutzieldtJakob disease.
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1999. Investigation of variant Creutzieldt-Jakob disease and other human prion diseases with tonsil biopsy samples.
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From page 104... ...
2001. Novel differences between two human prion strains revealed by two-dimensional gel electrophoresis.
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2002. Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice.
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From page 106... ...
Laude H.2001. Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein.
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From page 107... ...
2001b. Working Group on International Reference Materials for Diagnosis and Study of Transmissible Spongiform Encephalopathies (TSEs)
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