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~ Diagnostics for Transmissible ~ soone`torm Encephalopathies Diagnostic tests lie at the forward edge of all efforts to control hu- man and animal diseases, for it is the diagnostic test that clarifies the specific clinical problem. A reliable and speedy diagnosis leads to appropriate clinical management and intervention. Yet very few diagnos- tic tests are available to detect the infectious agents associated with trans- missible spongiform encephalopathies (TSEs). A number of tests have been developed for the rapid detection of PrP specific to bovine spongiform encephalopathy (BSE) in the central nervous system (CNS) tissue of cattle. At present, the European Community has approved five such tests to screen slaughtered cattle for BSE. Similarly, two rapid postmortem tests are available in the United States to diagnose chronic wasting disease (CWD) in deer and elk. However, there is no test approved by the U.S. Department of Agriculture (USDA) or the Food and Drug Ad- ministration (FDA) to detect TSEs in live animals or people. This chapter first addresses the unique challenges involved in develop- ing antemortem clinical diagnostics for TSEs. This is followed by a discus- sion of the diagnostic tools used at present and some of the newer tech- niques being explored. The final section presents the committee's recommendations for research that is likely to yield sensitive, reliable, and cost-effective antemortem TSE diagnostics in the future. In the case of animals, the ability to diagnose or detect an infection drives food safety interventions, which can prevent the introduction of con- taminated food into the food chain and offset economic damage to the food production industry. In the case of people, the detection of infection can avert the introduction of potentially infectious blood into the blood supply 72

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DIA GNOSTICS FOR TSEs 73 system and be used to direct appropriate treatment. In addition, the use of diagnostic tests for mass screening has epidemiological utility since it can reveal the extent and distribution of a prior-related disease within a herd or population, and can be used to monitor the effects of animal and public health intervention strategies. Most infectious diseases, such as malaria, tuberculosis, hepatitis, and AIDS, can be diagnosed by established methods. This is not the case with TSE. A prion cannot be identified by direct visualization under a micro- scope, cultivation in a laboratory, detection of specific antibodies or anti- gens by standard immunology methods, or detection of its nucleic acid by molecular methods such as polymerase chain reaction (PCR). It consists of host protein with an altered conformation such that the body does not recognize it as foreign and does not produce antibodies against it. It also lacks identifiable DNA or RNA, so it cannot be identified by PCR or other nucleic acid-based tests. These factors make detecting the agent very difficult. TSE agents have other peculiarities that confound detection. They are largely insoluble, distributed unevenly in body tissues, and found in a lim- ited set of tissues by currently available tests. PrPSc (the pro/ease-resistant protein associated with prion disease; see Chapter 2) is neurotropic, so it ultimately affects cells of the nervous system tissues. Where and how PrPSc progresses through the body before its final assault on the nervous system is largely unclear, however, complicating the ability to locate and detect it. The similarities between host prpC (the pro/ease-sensitive cellular pro- tein) and PrPSc pose a fundamental problem. Since it is normal to find prpC in healthy individuals, detection tests must differentiate between the two proteins. The strategy thus far has been to mix the test material with the proteinase K (PK) enzyme, which digests normal prion protein but only a portion of the abnormal protein. Then various techniques, described below, detect the residual PrPSc after digestion. Since this process incidentally re- duces the small amount of original PrPSc captured, it is inherently less sensi- tive than methods that do not rely on PK digestion.) Thus two major factors challenge the designer of any useful antemor- tem 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), necessitating an exquisitely sensitive test. Sec- ond, since hosts incubating a TSE lack overt evidence of illness during most of the period between infection and death, both infected and uninfected animals and people would need to undergo antemortem diagnostic testing. iThe degree to which PrPSc resists PK digestion depends on its strain. The limit of resistance to PK digestion may relate to the conformation of each strain (Safer et al., 1998). Some loss of PrPSc is also due to the test process itself.

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74 ADVANCING PRION SCIENCE Therefore, this test must be of sufficient specificity to differentiate accu- rately between normal and misfolded PrP. For some purposes, the test would also need to discriminate among one or more prion strains a challenge heightened by basic deficiencies in our understanding of prion strain diver- . , . . . silty ant t" be nature ot strain variation. The ultimate objective of a TSE diagnostic test is to detect a single infectious unit (IU) while avoiding a false-positive result. In this report, we define an IU as the smallest amount of infectious agent leading to an infec- tion in a single person or animal exposed. Achieving the ideal TSE diagnos- tic test will be complex because a single IU may not be equivalent to a single infectious particle. In other words, a single IU of a TSE agent likely consists of many aggregates of PrPSc, not just one aggregate. For further explana- tion, please refer to the detailed definition of the term infectious unit in the glossary at the beginning of this volume. The quest for antemortem diagnostics will play a fundamental role in controlling the spread of TSEs, yet current tests are largely unvalidated and not readily available. Although the ultimate objective is far from being achieved, scientists continue to invest much worthwhile effort into improv- ing TSE detection methods. CLINICAL DIAGNOSTICS In general, diagnoses of prion diseases by clinical description or ancil- lary 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. Differentiation of prion disease from other neurodegenerative diseases and differentiation among different prion strains on clinical grounds are problematic because affected individuals exhibit similar symptoms. Clinical diagnostic criteria have nevertheless been established for sporadic Creutzfel~t-Takob disease (sCTD) and variant Creutzfel~t-Takob disease (vCJD) (Will et al., 20001. Some general clinical differences distinguish sCJD from vCJD (see Table 4-11. For example, vCJD, unlike Creutzfel~t-Jakob disease (CJD), occurs in patients generally younger than 40 years old; often presents with early psychiatric and sensory neurological symptoms; and has a longer duration of illness prior to death, usually more than a year (WHO, 2001a). Spencer and colleagues (2002) reviewed the early psychiatric mani- festations of vCTD. They describe the clinical characteristics of the first 100 vCJD patients identified and conclude that "the combination of a psychiat- ric disorder with affective or psychotic features and persistent pain, dysar- thria, gait ataxia, or sensory symptoms should at least raise the suspicion of vCTD, particularly if this is combined with any suggestion of cognitive im- pairment" (Spencer et al., 2002:14821. Despite these differences between vCTD and COD, they are not sufficient to establish a definitive diagnosis.

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DIA GNOSTICS FOR TSEs TABLE 4-1 Clinical Differentiation of sCTD and vCTD 75 Clinical Feature or Supporting Clinical Procedures Classical sCJD (M/M or M/V 1) vCJD Average age at clinical onset Length of survival from date of clinical onset Early psychiatric symptoms El ectro enc ep hat ography (EEG) Magnetic resonance imaging (MRI) Cerebrospinal fluid (CSF) Histopathology of brain tissue PrP immunohistochemical . . .. . . 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 . . stalmng pattern PrP present in tissue. especially toward late stage of disease Type 2B NOTE: M/M = methionine homozygous at codon 129; MIV 1 = heterozygous at codon 129, subtype 1 (see Table 4-2). Ancillary clinical testing typically supplements the medical work-up for vCTD or CTD. The most helpful noninvasive tests have been electro- encephalography (EEG); neuroimaging; examination of CSF; and, more re- cently, tonsillar biopsy and prion strain identification by immunoblotting. Evaluation of tissue obtained by brain biopsy establishes or excludes the diagnosis of TSE in almost all cases, but brain biopsy is highly invasive and is limited to cases in which a treatable condition must be excluded. Electroencephalography In typical cases of sCTD (see Table 4-2), the EEGs of more than 80 percent of patients show distinctive changes (Parch) et al., 1999~. The trac-

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DIA GNOSTICS FOR TSEs 77 ing shows biphasic and triphasic periodic complexes in the clinical course (Parch) et al., 1999), which are evident more than 90 percent of the time with repeated tracings (Chiofalo et al., 19801. These periodic complexes are observed less frequently in patients with the other subtypes of sC]D and in familial C]D (Gambetti et al., 1999; Parchi et al., 1999) and have never been found in patients with vC]D, although nonspecific siow-wave abnor- malities can be seen. Neuroimaging Neuroimaging by computed tomography (CT) and magnetic resonance imaging (MRI) can be useful, especially to rule out non-prior-related neu- rological diseases. The CT result is usually normal, although the scan may show atrophy in patients with a protracted clinical course (WHO, 19981. This finding may be absent and is nonspecific. The MRI scan may also show atrophic changes in patients with late-course disease. When patients are evaluated by T2 MRI, proton- density-weighted MRI, or fluid- attenu- ated-inversion-recovery MRI, there is an increased signal in the basal gan- glia about 80 percent of the time (WHO, 1998), as well as gray matter hyperintensities noted in diffusion-weighted imaging sequences in patients with C]D (Mendez et al., 20031. MRI also can be used to help differentiate vC]D from sC]D because the posterior puivinar region of the thalamus shows a hyperintense signal in patients with vC]D. This puivinar sign is present in 90 percent of patients with vC]D and is more than 95 percent specific in selected cases, making it the best available in viva test for the diagnosis of vC]D (~0, 2001b). CSF Protein A neuronal protein called 14-3-3 can be detected in CSF, particularly in individuals who have diseases, such as sC]D, involving rapid neuronal de- struction (Zerr et al., 20001. This protein may help differentiate TSE from Alzheimer's disease and other dementias, where it is usually not detectable. In necrotizing diseases such as stroke, viral encephalitis, and transverse myelitis, the test for this protein may also be positive (Johnson and Gibbs, 1998; WHO, 19981. In more slowly progressing forms of C]D such as fa- milial cases of human TSEs and vC]D, this CSF protein test is often nega- tive. A WHO study (2001a) found the 14-3-3 test to be only 53 percent sensitive when used to diagnose vC]D. Tonsi! Biopsy More recently, tonsil biopsy has been used for the presumptive identi- fication of vC]D. Immunohistochemical testing for the prion protein in

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78 ADVANCING PRION SCIENCE these tissues has demonstrated that the protein is present in patients with vC]D but not in those with sC]D (Hill et al., 1999; WHO, 2001a). The postulated reasons for this difference include a strain effect, a species-bar- rier effect, or the oral route of exposure in vC]D (Hill et al., 19991. There have been too few case series to determine the sensitivity or specificity of this ancillary test. Although controversial, tests of tonsil and appendix lymphoid tissues are being used to screen large, asymptomatic populations for TSEs. The largest study to date was conducted in the United Kingdom (Hilton et al., 20021. Between 1995 and 1999, Hilton and colleagues tested 8,318 tonsil and appendix tissue samples from 10- to 50-year-old individuals and found one appendix tissue sample that tested positive for PrPSc. From that result, they estimated the prevalence of vC]D to be 120 per million in the United Kingdom, with a 95 percent confidence limit of 0.5 to 900 cases (Hilton et al., 20021. It remains unknown how long before the onset of symptoms PrPSc ac- cumulates in human tonsils or the human appendix and whether all positive individuals will inevitably progress to the fatal CNS disease. Nevertheless, studies of sheep naturally infected with the scrapie agent and of mice ex- perimentally infected with that same agent have demonstrated that PrPSc is detectable in lymphoid tissues long before clinical signs of neurological dis- ease appear. Brain Biopsy In atypical cases of C]D, brain biopsy with histological examination for spongiform changes, immunocytochemical staining, and Western blotting for PrPSc, as well as analysis of the PrP gene, is diagnostic in virtually all cases. However, this approach is seldom needed to diagnose patients with a typical clinical course and consistent findings of classical sC]D by LEG, MRI, and CSF analysis. Histological examination of brain tissue should be performed for all patients with possible and probable cases of TSE, as well as for all individuals with questionable neurodegenerative diseases at au- topsy, to ensure that a new phenotype of prion disease is not missed. In both sC]D and vC]D, histology typically reveals the spongiform ap- pearance of the CNS tissues. However, amyloid plaque formations with the characteristic morphology known as florid plaques are seen in all patients with vCTD, whereas kuru plaques (without the characteristics of the florid plaques) are observed only in patients with the sCJD subtype methionine/ valine 2 (M/V 2), which accounts for about 10 percent of all cases of sCJD (Tohnson and Gibbs, 1998; Parchi et al., 19991.

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DIA GNOSTICS FOR TSEs 79 Isotype by Western Blotting Additional diagnostic precision has been made possible by the intro- duction of PrPSc isotypes on the basis of the mobility of the PrPSc fragment, which is PK-resistant, after gel electrophoresis and Western blotting (Collinge et al., 1996; Monari et al., 1994; Parchi et al., 19971. According to a widely used typing method, there are two major types (or strains) of PrPSC in all forms of COD and fatal insomnia, including sCTD, iatrogenic COD, vCTD, fatal familial insomnia (FFI), and sporadic fatal insomnia (sFI) (Parch) et al., 19971. PrPSc type 1 migrates to 21 kilodaltons (kDa) on gels after treatment with PK and deglycosylation. PrPSc type 2 migrates to 19 kDa under the same conditions. The different gel mobilities of the two PrPSc types are due to the different sites of PrPSc cleavage by PK, resulting in PK-resistant frag- ments of differing sizes. These two types codistribute with distinct disease phenotypes and are conserved upon transmission to receptive animals. Therefore, they fulfill the definition of prion strains, and this strongly indi- cates that they have distinct conformations. Additional subtypes of PrPSc have been distinguished on the basis of both the ratios of the three PrPSc glycoforms (Parch) et al., 1997) and the profile generated by two-dimen- sional gel electrophoresis (Pan et al., 20011. 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 com- mon 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) et al., 1996, 19991. This classification in- cludes five subtypes of sCTD and sFI (see Table 4-21. Each of these subtypes has distinct clinical and pathological features. Despite these advances, clinical methods remain supportive rather than diagnostic. As in virtually all other disease conditions, the diagnosis is most reliable when obtained by combining information from the clinical exami- nation, ancillary clinical tests, and laboratory tests. Yet even when all this information is combined, present diagnostic tools lack sufficient sensitivity and specificity. The development of tests to improve the early diagnosis of human prion disease and to detect presymptomatic infections in humans and animals more reliably is a major priority. CURRENT LABORATORY DIAGNOSTICS Histopathology and Immunohistochemistry The first method used to confirm the diagnosis of a TSE is postmortem neuropathogical examination of brain tissue from an animal or a human,

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80 ADVANCING PRION SCIENCE and this method remains the gold standard. WHO's position is that "a definitive diagnosis of C}D including nvC}D New variant C}D] is estab- lished only by neuropathological examination" (WHO, 1998:13~. Tissue is collected; preserved in formalin; sectioned; stained; and then examined with a light microscope, which is used to look for the characteristic pathological abnormalities on histological examination. This procedure is generally aug- mented with immunohistochemical staining of the tissue, which uses a PrP antibody-tagged stain that affixes onto PrP. The stain will be abnormally dark or dense in areas where an abnormal amount of PrP is present (see Plate 4-1~. Electron microscopy can also be used to observe fibrils, called scrapie-associated fibrils, in fresh postmortem tissue (Merz et al., 1983) as well as in autolytic tissue (see Figure 4-1~. Immunochemical Detection Methods Five standardized commercial screening tests (two by Prionics AG and one each by Enfer Scientific Ltd., Bio-Rad Laboratories Inc., and InPro Biotechnology Inc.) have been approved by the European Commission (EC) for use in the direct and rapid detection of PrPSc (Moynagh and Schimmel, 1999; EC, 2003~. The test by San Francisco-based InPro was developed in the United States, while the other four tests were developed in Europe. Those tests now on the market are used primarily in Europe. In the United States, no rapid postmortem or antemortem diagnostic test for human TSEs had been submitted to FDA for approval as of August 2003 (personal communication, D. Asher, FDA, 2003~. Furthermore, there are no USDA-approved rapid postmortem or antemortem tests to diagnose TSEs in sheep or cattle. USDA has approved three rapid postmortem diag- nostic tests for CWD: the Bio-Rad enzyme-linked immunosorbent assay (ELISA) by Bio-Rad of Hercules, California; the Dot Blot ELISA by Veteri- nary Medical Research and Development (VMRD) Inc. of Puliman, Wash- ington; and the IDEXX Her3Chek~ CWD Antigen Test Kit from IDEXX Laboratories Inc. of Westbrook, Maine (IDEXX, 2003; personal communi- cation, Rick Hill, USDA APHIS Center for Veterinary Biologics, November 25, 2003; USDA APHIS, 2002; VMRD Inc., 2003a). All three tests are designed to detect the infectious agent of CWD in peripheral lymphoid tis- sues 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). The VMRD test is approved for use on tissue samples from white-tailed deer and mule deer (VMRD Inc., 2003a). The IDEXX test is ap- proved for use on white-tailed deer tissue. All tests are approved for use exclusively in APHIS-

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DIA GNOSTICS FOR TSEs 81 FIGURE 4-1 Electron micrograph of negatively stained fibrils composed of PrP 27-30 from scrapie-infected Syrian hamster brains. SOURCE: H. Wille, Institute for Neurodegenerative Diseases, University of California San Francisco, September 9, 2003. approved veterinary laboratories and for surveillance purposes only. The sensitivities and speci- ficities are as follows: Sensitivity ( % ) Specificity ( % ) No. Specimens Bio-Rad 99.6 99.9 2,892 VMRD 91.5 100.0 298 IDEXX N/A N/A N/A NOTE: N/A = not available. SOURCE: Bio-Rad Laboratories Inc. (2003); VMRD Inc. (2003b).

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82 ADVANCING PRION SCIENCE TABLE 4-3 Estimated Detection Limits of the First Three EC-Approved Postmortem Tests for BSE Dilution of Homogenatea Number of BSE-Infected Brain-Homogenate Samples Scoring Positive Prionics Check Western o lo-l .s lo-2.o lows 0-3. 0-3.5 Test by Enfer Scientific 6/6 15/20 (+2?)b 0/20 6/6 20/20 20/20 0/20 Test by Bio-Rad 6/6 20/20 20/20 20/20 18/20 1/20 0/20 NOTE: The data represent the number of samples testing positive/total number of samples tested. a ". . . positive brain homogenate of known infectivity titer was tested at dilutions in negative brain" (Moynagh and Schimmel, 1999:105). b Two samples rated inconclusive at this dilution. SOURCE: Adapted from Moynagh and Schimmel (1999). Tests Produced by Prionics, Enfer, and Bio-Rad The rapid test most widely used to screen for BSE in Europe is the Western blot test called Prionics Check Western, produced by Schlieren, Switzeriand-based Prionics. 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. The Enfer and Bio-Rad tests use slightly different mechanisms for de- tection. The test by Cashel, Ireland-based Enfer is an ELISA. After digestion by PK, residual PrPSc binds to a capture antibody at the bottom of the ELISA plate wells. After the wells have been washed, a detection antibody complexed with an activating enzyme is added. This second antibody binds to PrPSc and colors the substrate if PrPSc is present. The Bio-Rad test, cre- ated in France, is also an ELISA, but in the initial step after digestion, it uses two different antibodies to bind to different epitopes of PrPSc. This is the most sensitive of these first three approved tests (see Table 4-3~. Prionics-Check Luminescence Immunoassay (LIAJ and InPro Automated Conformationally Dependent Immunoassay (aCDIJ In April 2003, the EC approved two more rapid tests for the postmor- tem detection of BSE Prionics-Check LIA and InPro aCDI (EC, 2003~.

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DIA GNOSTICS FOR TSEs 97 oafs (such as those based on surface plasmon resonance), methods that de- tect resonances in the microwave range, methods that detect changes in the frequency of surface acoustic waves, methods that detect changes in the frequency of piezoelectric cantilevers, microcalorimetric methods, methods based on the field effect in transistors and capacitors, and methods that use evanescent wave-dependent changes in Raman scattering on metallic nanoparticles. With the exception of the latter, however, none of these meth- ods is as sensitive as radioimmunoassays and ELISAs; nonetheless they of- fer advantages, as they can be used with underivatized recognition and tar- get molecules and can be coupled directly to optical or electrical readouts. Evanescent wave devices, which are widely used, solve the issue of coupling wet and dry elements by making the part of the apparatus that comes into contact with the biological sample disposable. Modern Fourier transform ion cyclotron resonance methods are capable of detecting about 1,000 mol- ecules with a given mass:charge ratio and with unambiguous identification. Wet Methods More recently, wet methods have been developed that may have even greater sensitivity for the detection of priors. One of these is the protein- fragment complementation assay (Remy and Michnick, 19991. Another couples recognition proteins with PCR-amplifiable DNA tails (protein PCR). The resulting chimeric molecules can be used with existing real-time PCR techniques and may allow extension of PCR to protein detection at a level of 1 to 10 arbitrarily designated epitopes (I. Burbulis, R. Carison, and R. Brent, unpublished results, 20021. Use of any of these methods for the reliable detection of prions in clini- cal and environmental samples requires that the prions be purified and con- centrated. 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 mol- ecules specific for prion species, strain, and allelic variants and for the infec- tious conformation. Efforts to select antibodies specific for the conforma- tion of prions have been hampered by the lack of immunogenicity of the revealed epitopes, the tolerance of the mammalian immune system to these epitopes, the lack of an industrial-scale effort, and perhaps other factors as well. Whatever the reason for the failure of past efforts, the reasonable re- sponse to the problem is to select more modern kinds of recognition mol- ecules in vitro, bypassing the vertebrate immune system completely. Some

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98 ADVANCING PRION SCIENCE of these problems are common to many areas of application in the biologi- cal sciences and have received high-level scientific and national attention (Desai et al., 20021. A daunting number of people and organizations own the rights to the intellectual property needed to generate modern molecular reagents with affinities for prion proteins and to use those molecules in detection schemes. This situation may make the commercial application of such reagents diffi- cult until patent-sharing schemes can be devised. Nevertheless, neither tech- nical nor legal barriers block government or philanthropic groups from funding the production of these reagents for use in detecting prion particles. In summary, a wealth of natural and engineered molecules, along with a variety of detection methods, have been developed for recognition of bio- logical targets. Prion investigators must now apply these molecules and methods to the development of selective, sensitive tools that can target PrPSc. Once bound by specific reagents, prions become detectable and susceptible to attack. That attack might employ catalytically active binding reagents, such as ribozymes, that offer the potential for target inactivation. Recommendation 4.3: Fund research to develop novel methods and reagents that detect or bind to priors, including new antibodies, peptides, nucleic acids, synthetic derivatives, and chimeric mol- ecules. This research could lead not only to better diagnostics, but also to better therapeutic and prophylactic strategies. [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 sedimen- tation rate and C-reactive protein) or by their dependence on the generation of a specific antibody that is delayed in all disease processes and absent, altogether in some, including TSEs. Today, powerful methods for detection of robust surrogate markers of disease create new opportunities for diagno- sis and force reconsideration of these approaches. These methods are based on genomic or proteomic techniques, focus on complex biological patterns, and depend on pattern recognition algorithms. All forms of mammalian pathophysiology and pathology are accompa- nied by stereotyped and highly choreographed intra- and extracellular changes in the diversity, abundance, and spatial distribution of biomolecules. Mammalian biological systems are particularly sophisticated and sensitive in their recognition of and response to perturbations. These responses can be defined by complex changes in many classes of molecules,

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DIA GNOSTICS FOR TSEs 99 including changes in DNA structure, RNA transcript abundance, protein abundance and modification, and protein localization. Modern genomic techniques have greatly facilitated comprehensive measurement of these various changes in parallel. For example, changes in the abundance of RNA transcripts for nearly all genes expressed in humans can be measured simul- taneously and repeatedly over short periods of time by using DNA microarrays. Similarly, changes in the abundance of oligosaccharides or proteins among a massive number of species can be measured either by a method that uses a solid-state format or by mass spectrometry. Diagnostic and prognostic signatures can be identified by examining complex patterns of biomolecules that occur in various disease conditions. Pattern recognition methods fall into two categories: those that discover possible signatures (class discovery methods) based on the association of specific patterns with an outcome of interest, and those that test and vali- date these signatures (class prediction methods). Both of these methods have been used successfully to classify cancer subtypes, to predict survival, and to evaluate response to therapy. Specific patterns of RNA transcript abun- dance predict the outcomes for patients with various malignancies, such as breast and lung cancer, lymphoma, and leukemia (Alizadeh et al., 20001. Stereotyped, discriminant patterns of transcript abundance may also be characteristic of the mammalian response to infection (Boldrick et al., 20021. In a recent study using mass spectrometry, investigators were able to identify a group of surrogate proteins in 50 of 50 patients with ovarian cancer, including 18 patients with early-stage disease. This pattern was ab- sent from 60 of 63 patients with a noncancer diagnosis (Petricoin et al., 2002a). The same technique was used to diagnose prostate cancer in 36 of 38 patients whose diagnosis was blind to the investigators to identify cor- rectly 177 of 228 patients without prostate cancer (Petricoin et al., 2002b). In diagnosing prion infections, it appears reasonable to postulate that there are patterns of altered transcript abundance or protein expression in, for example, blood, lymph nodes, or cerebrospinal fluid that are character- istic of infection. One approach is to conduct a blind search for such pro- tein patterns by means of protein mass spectrometry (Petricoin et al., 2002b) followed by isolation of recognition molecules directed against the proteins that have been identified. The components that make up the diagnostic pattern need not necessarily be directly involved in pathogenesis, nor must they have a known function. This kind of approach, however, must rely on rigorous evaluation with well-chosen control samples and on predictions obtained from the results of tests with sets of test samples. 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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100 ADVANCING PRION SCIENCE Cell Culture Systems Research gains leading to better diagnostics would be accelerated if better cell culture systems were in place. These systems have significant advantages over animal bioassay systems, the most important being that they can greatly shorten the length of time required to complete the test. At present, only a few lines of cultured cells can be infected with priors. The efficiency of infection is low, the rate of PrPSc accumulation is slow, and the yield of PrPSc is limited. In addition, the factors that determine susceptibility to infection are poorly understood. Therefore, investigators must find new cell cultures or mode! systems with bona fide CNS properties that are susceptible to prions in vitro, as well as new ways to enhance the efficiency of the initiation and propagation of infection (e.g., molecules that enhance the conversion of prpC to PrPSc). This work would not only en- hance the potential for the use of cultured cells to assay priors, it would also shed light on the cellular mechanisms underlying prion replication and nerve-cell death. 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. MRI is able to visualize the brain lesions of patients with C]D and can even help in differentiating vC]D from sC]D (as noted in Chapter 31. Newer scanning devices and tissue uptake reagents will further increase the utility of this clinical tool. MRI of vC]D patients has been helpful from a diagnostic point of view because of the frequent and specific puivinar sign, an abnormality described by Zeidler and colleagues (20001. Symmetric hyperintense signals have been reported in the basal ganglia of patients with sC]D; however, this finding is frequently absent and lacks specificity, making it less useful. For this rea- son, investigators have examined new imaging methods that can enhance the capabilities of present methods or provide very new technical ap- proaches, such as multiphoton microscopy. Multiphoton microscopy uses near-infrared light, which penetrates more deeply than visible or ultraviolet light and permits imaging of micro- scopic structures within the cortex of the living animal at an extraordinarily high resolution with no apparent deleterious effects. To visualize p-amyloid deposits in living transgenic mice with lesions like Alzheimer's disease, re- searchers have used multiphoton microscopy with locally applied fluorescently labeled antibody against p-amyloid or systemically adminis-

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DIA GNOSTICS FOR TSEs 101 tered fluorescent derivatives of chemicals that bind to p-amyloid, such as thioflavine A and Congo red (Bacskai et al., 2001; Christie et al., 2001; Klunk et al., 20021. This in viva imaging approach has allowed character- ization of the natural history of senile plaques and evaluation of antiplaque therapy in mouse models of the disease. One could envision the application of similar studies to transgenic mouse models of prion disease, especially since thioflavine A and Congo red bind to PrPSc. The technique would en- able characterization of the progression of PrPSc accumulation and localiza- tion in animals or patients with disease by repeatedly imaging the same diseased region of the brain over time. Although multiphoton microscopy requires a portion of the skull to be thinned or removed for the passage of light, modifications to this technique may obviate this need. In addition, advances in detection sensitivity and improved means of entry of p-amyloid-binding probes into the central ner- vous system may allow the use of similar kinds of p-amyloid-imaging by MRI and positron emission tomography for studies with humans (Bacskai et al., 2002; Mathis et al., 2002; Shoghi-Jadid et al., 20021. 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 imag- ing 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. [Priority 31 REFERENCES Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, Boldrick JC, Sabet H. Tran T. Yu X, Powell JI, Yang L, Marti GE, Moore T. Hudson J Jr, Lu L, Lewis DB, Tibshirani R. Sherlock G. Chan WC, Greiner TC, Weisenburger DD, Armitage JO, Warnke R. Staudt LM, Levy R. Wilson W. Grever MR, Byrd JC, Botstein D, Brown PO, Staudt LM. 2000. Distinct types of diffuse large B-cell lymphoma identified by gene expression profil- ing. Nature 403(6769):503-511. Asante EA, Collinge J. 2001. Transgenic studies of the influence of the PrP structure on TSE diseases. Advances in Protein CI7emistry 57:273-311. Bacskai BJ, Kajdasz ST, Christie RH, Carter C, Games D, Seubert P. Schenk D, Hyman BT. 2001. Imaging of amyloid-beta deposits in brains of living mice permits direct observa- tion of clearance of plaques with immunotherapy. Nature Medicine 7(3):369-372. Bacskai BJ, Klunk WE, Mathis CA, Hyman BT. 2002. Imaging amyloid-beta deposits in vivo. Journal of Cerebral Blood Flow and Metabolism 22(9):1035-1041. Baron GS, Caughey B. 2003. Effect of glycosylphosphatidylinositol anchor-dependent and -independent prion protein association with model raft membranes on conversion to the pro/ease-resistant isoform. Journal of Biological CI7emistry 278(17):14883-14892.

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