Advancing Prion Science: Guidance for the National Prion Research Program: Interim Report (2003)

New antemortem laboratory tests for the detection of PrP^Sc, the protease-resistant protein associated with prion disease, are imperative. Research considerations in improving those tests should proceed with full recognition that major breakthroughs are needed to achieve the levels of sensitivity and specificity required to test live animal and human tissues.

The committee believes that an ideal test would detect less than 1 infectious unit (IU) of prions in the relevant organism or sample. Prusiner and colleagues demonstrated that 1 IU equals approximately 10⁵ PrP^Sc molecules in a purified prion preparation (Prusiner et al., 1982). However, it is possible that the size of an IU differs depending on the host, the strain, and the mode of transmission. Laboratory tests designed to detect prions directly are unable to identify less than 1 IU.

Infectivity studies with animal bioassay models are among the most sensitive methods for demonstrating the presence of the PrP^Sc infectious agent, albeit indirectly. Yet, these animal tests, such as the murine bioassay, are hampered by the species barrier. For example, conventional mice used to bioassay the infectivity of the agent of bovine spongiform encephalopathy (BSE) could detect only 1,000 IU per inoculum (Wells et al., 1998). The sensitivity of the bioassay is also limited by the small inoculum size that can be given to these mice intracerebrally (Wadsworth et al., 2001).

Current technology does not allow detection of small enough numbers of prion proteins, ready detection of the conformation of the infectious form of the prion protein, or detection and distinction among different allelic and strain variants of the prion protein. Such distinctions could be made, in principle, by use of antibodies or other molecular affinity reagents, such as peptide or nucleic acid aptamers with high specificities for target recognition. When coupled with sensitive methods for detection of a reagent bound to a target, such as those that rely upon upconversion of phosphors with negligible natural background fluorescence or those mentioned below, a number of approaches offer significant potential. In general, researchers need to leverage novel and fast-breaking developments in biotechnology-for example, rapid advances in proteomics and mass spectrometry that enable high-throughput, precise characterization of proteins-if significant break-throughs in prion detection are to be achieved.

Practical detection schemes for the near term are likely to involve the use of molecules that recognize specific epitopes on prions, such as epitopes that are specific for the disease conformation or those that are specific for different alleles. In principle, these molecules could be antibodies, for example, 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 Köhler and Milstein (1975). At present, prion detection methods are dependent on a few antibodies selected in vivo that offer a

limited, if any, ability to distinguish between infectious and noninfectious prion protein conformations.

Antibodies can also be selected in vitro, for example, after display on the surface of a filamentous phage. The advent of recombinant DNA techniques has made it possible to construct useful antibody derivatives, including single-chain antibodies that contain the binding regions for the heavy and light chains on a single polypeptide (single-chain antibody variable region fragments [scFvs]), and derivatives that contain well-behaved constant regions (e.g., from mouse immunoglobulin G fetal calf serum [Fcs]) that can be recognized by secondary reagents such as staphylococcal proteins A and G.

Recognition molecules could also be nucleic acid (RNA or DNA) aptamers that bind to the target epitope. Aptamers are selected from large pools of nucleotides with different sequences. The aptamers' affinities are typically increased further after rounds of mutagenesis and selection for those that bind to epitopes more tightly (Ellington and Szostak, 1990; Tuerk and Gold, 1990). Protein aptamers are molecules that display conformationally constrained regions with variable sequences from a protein scaffold (Colas et al., 1996). Pools of nucleotides with random sequences encode the regions with variable sequences. Selection for binding targets is performed in vitro (for example, by selection of phages that display aptamers that bind to the desired target) or in vivo by yeast two-hybrid methods. RNA aptamers can readily be synthesized from DNA templates by transcription in vitro. In addition, aptamers can readily be synthesized by expression in bacteria, yeast, or other cell-based systems.

Developing new antibodies to PrP^Sc using the methods described above could significantly improve the sensitivity of current assay methods. For example, the radioimmunoassay (RIA) was developed in the 1950s (Yalow and Berson, 1959). Alternative assays, such as enzyme-linked immunosorbent assays (ELISAs), use the activity of a dissociated enzyme (such as alkaline phosphatase) on a fluorogenic or chromogenic substrate in lieu of radioactivity and have a lower limit of detection of millions to billions of epitopes (Engvall and Perlman, 1971).

Within the past two decades, numerous detection methods based on physical phenomena have also been devised. They include evanes-cent-wave methods (such as those based on surface plasmon resonance), methods that detect resonances in the microwave range, meth-

ods 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 last method, none of these are as sensitive as RIAs and ELISAs, but they offer advantages, as they can be used with underivatized recognition and target molecules and can be directly coupled to optical or electrical readouts. Evanescent-wave devices, which are used widely, 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 molecules with a given mass/charge ratio and with unambiguous identification.

More recently, wet methods have been developed, and these possibly have even greater sensitivities for the detection of prions. One of these is the protein complementation technique (Remy and Michnick, 1999). Another method couples recognition proteins with polymerase chain reaction (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.Carlson, and R.Brent, unpublished results, 2002).

Use of any of these methods for the reliable detection of prions in clinical and environmental samples requires that the prions be purified and concentrated. This requirement can be addressed by a variety of approaches.

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. Efforts to select antibodies specific for the conformation of prions have been hampered by the lack of immunogenicity of the revealed epitopes, the tolerance of the mammalian immune system to these epitopes, and the lack of an industrial-scale effort, among perhaps other reasons.

Whatever the reason for the failure of past efforts, the reasonable response to the problem is to select more modern kinds of recognition molecules in vitro, bypassing the vertebrate immune system completely. Some of these issues are common to many areas of application in the biological sciences and have received high-level scientific and national attention (Desai et al., 2002).

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 may make their commercial application difficult until patent-sharing schemes can be devised. Nevertheless, neither technical nor legal barriers block government or philanthropic groups from funding the production of these reagents for use to detect prion particles.

In summary, a wealth of natural and engineered molecules along with a variety of detection technologies have been developed for recognition of biological targets and have been used for other applications. Now prion investigators must apply them to the development of selective, sensitive tools that target PrP^Sc. 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.

Diagnostic approaches based on detection of indirect disease markers have a long and checkered history. In general, they have been stigmatized by their lack of specificity (e.g., tests for the erythrocyte sedimentation rate and C-reactive protein) or their dependence on the generation of a specific antibody-which is delayed in all disease processes and which is absent in some disease processes, including transmissible spongiform encephalopathies (TSEs). Today, powerful methods for detection of robust surrogate markers of disease create new opportunities for diagnosis and force reconsideration of these approaches. These methods are based on genomic or proteomic tech-

niques, focus on complex biological patterns, and depend on pattern recognition algorithms.

All forms of mammalian pathophysiology and pathology are accompanied by sterotyped and highly choreographed intra- and extracellular changes in the diversity, abundance, and spatial distributions 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, 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 different changes in parallel. For example, changes in the abundance of RNA transcripts for nearly all genes expressed in humans can be measured simultaneously 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 or prognostic signatures can be defined by surveying the complex patterns of the abundance of biomolecules in different pathologies. Pattern recognition algorithms fall into two groups: those that discover classes of disease (or genes), such as clustering and self-organizing maps, and those that predict different classes of elements from predefined signatures, such as support vector machines and f-test algorithms (for example, diagonal linear discriminant analysis). Some of the most compelling examples of this approach concern efforts to classify cancer subtypes and predict survival and the response to therapy. Specific patterns of RNA transcript abundance predict the outcomes for patients with various malignancies, such as breast and lung cancer, lymphoma, and leukemia (Alizadeh et al, 2000). Stereotyped, discriminant patterns of transcript abundance may also be characteristic of the mammalian response to infection (Boldrick et al., 2002).

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 absent 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 to whose diagnosis the investigators were blinded and correctly identify 177 of 228 patients without prostate cancer (Petricoin et al., 2002b).

In diagnosing prion infections, it seems 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 characteristic of infection. One path is a blind search for such protein patterns by 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.

Research gains leading toward better diagnostics would be accelerated if better cell culture systems were in place. These systems have significant advantages over animal bioassay systems, with the most important advantage 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 prions. The efficiency of infection is low, the rate of PrP^Sc accumulation is slow, and the yield of PrP^Sc is limited. In addition, the factors that determine susceptibility to infection are poorly understood. Therefore, investigators must find new cell cultures or model systems susceptible to prions in vitro and new ways to enhance the efficiency of the initiation and propagation of infection (e.g., molecules that enhance the conversion of PrP^C to PrP^Sc). This work will not only improve the possibility for the use of cultured cells to assay prions, but also will shed light on the cellular mechanisms underlying prion replication.

Recent improvements in clinical neuroimaging have shown increasing utility in clinical diagnostics for TSEs. Magnetic resonance imaging (MRI) is able to visualize the brain pathology of patients with Creutzfeldt-Jakob disease (CJD) and can even help in differentiating variant Creutzfeldt-Jakob disease (vCJD) from sporadic Creutzfeldt-

Jakob disease (sCJD), as mentioned in Chapter 3. Newer scanning devices and tissue uptake reagents will further increase the utility of this clinical tool.

MRI of vCJD patients has been helpful from a diagnostic point of view because of the frequent and specific pulvinar sign, an abnormality that has been described (Zeidler et al., 2000). Symmetric hyperintense signals have been reported in the basal ganglia of patients with sCJD; however, this finding is frequently absent and lacks specificity, making it less useful. For this reason, investigators have examined new imaging methods that enhance the capabilities of present methods or provide very new technical approaches, such as multiphoton microscopy.

Multiphoton microscopy uses near-infrared light, which penetrates more deeply than visible or UV light and which permits imaging of microscopic structures within the cortex of the living animal at an extraordinarily high resolution with no apparent deleterious effects. To visualize β-amyloid deposits in living transgenic mice with Alzheimer's disease, researchers used multiphoton microscopy with locally applied fluorescently labeled antibody against β-amyloid or systemically administered fluorescent derivatives of chemicals that bind to β-amyloid, such as thioflavine A and Congo red (Bacskai et al., 2001; Christie et al., 2001; Klunk et al., 2002). This in vivo imaging approach has allowed characterization 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 PrP^Sc. The technique would enable characterization of the progression of PrP^Sc accumulation and localization 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 β-amyloid-binding probes into the central nervous system may allow similar kinds of β-amyloid-imaging approaches by MRI and positron emission tomography for studies with humans (Bacskai et al., 2002; Mathis et al., 2002; Shoghi-Jadid et al., 2002). 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 antiprion therapies.

The committee believes that the fastest way to improve diagnostic tools is not through applied research to improve existing detection methods but through basic research that fills in the gaps in the fundamental knowledge of prions and their disease-causing abilities. The European experience provides evidence that applied research alone is insufficient. The European Union has spent approximately 30 million euros (30.7 million dollars) over the past 10 years to attempt to develop satisfactory postmortem diagnostic tests for BSE, yet the Western blot test, which has been around for three decades, is still the most commonly used method (Personal communication to the committee, A. Aguzzi, University Hospital of Zurich, July 15, 2002).

The history of medicine also provides examples of basic discoveries that were essential precursors to the development of diagnostics. For instance, before inexpensive and rapid diagnostics were available for non-A, non-B hepatitis, the hepatitis C virus had to be identified and cloned. Likewise, before an effective blood test became available for screening for human immunodeficiency virus, the virus first had to be identified and isolated.

Many brilliant and dedicated scientists have been working for more than 20 years to solve the mysteries related to disease-causing prions. They have collectively made great progress, yet many fundamental questions remain. Basic research on prions also will serve as a foundation for new and better evidence-based surveillance and public health policies.

The most critical areas of basic prion research include solving the structure of PrPSc and relating the structure to prion strain differences (Box 4-1); determining endogenous and exogenous mechanisms of prion replication (Box 4-2); elucidating prion epidemiology and natural history (Box 4-3); clarifying the pathways and pathogenic mechanisms used by prions (Box 4-4); and elucidating the physiological function of PrP^C (Box 4-5).

Current models of prion conformation and tertiary structure are neither complete nor conclusive. Defining the critical structural differences between infectious and noninfectious forms of the prion protein could provide the basis for TSE diagnostics and elucidate the correlations between PrP structures and strains. The committee recommends that the National Prion Research Program (NPRP) of the U.S. Department of Defense support research to these ends.

Defining the structural differences between PrP isoforms might enable scientists to synthesize a PrP^Sc-specific antibody probe or aptamer, opening the door to a TSE diagnostic tool. Antibody probes are increasingly used to detect infectious agents in tissue, but their application to prion detection is limited because no independently validated antibody binds exclusively to PrP^Sc without prior digestion of PrP^C. One group of investigators reported an antibody specific to PrP^Sc (Korth et al., 2001).

Many of the tests for detection of PrP^Sc use antibody probes, as described in Chapter 3. Most tests, however, must use proteinase K to distinguish PrP^C from PrP^Sc. Proteinase K digestion reduces the already miniscule amount of material usually available for prion detection. The committee looks forward to the development of new test methods that exclude proteinase K.

Defining the prion structure could also reveal structure-based phenotypic differences among prion strains. This is important because it is thought that PrP^C binds to PrP^Sc before it is converted into PrP^Sc (Caughey, 2002). If the structure of the prion strain differs too greatly from the host's PrP^C, binding may occur but conversion will not (Bessen et al., 1995).

Defining the structures of PrP^C and PrP^Sc at the sites where they interact during binding and conformational change would support the development of molecules to block those interactions.

Since prions are insoluble, it is particularly difficult to study their structures with standard proteomic tools. A newer technique called solid-state nuclear magnetic resonance (NMR) overcomes the solubility problem and is in the early stages of application to research on the three-dimensional structure of prions (Laws et al., 2001; Wemmer, 2002). In addition, electron crystallography is being used to probe the two-dimensional structures of PrP^Sc crystals (Wille, 2002). Nobel laureate Kurt Wuthrich produced three-dimensional models of PrP^C using liquid-phase NMR experiments (Zahn et al., 2000). Nevertheless, the research community must do much more to obtain a comprehensive understanding of the structural differences between infectious PrP and noninfectious PrP.

It is believed that both the conversion of cellular PrP to PrP^Sc and the accumulation of prions depend on the help of one or more molecules (Caughey, 2001). These ancillary or chaperoning factors could serve as surrogate markers for prion detection and as drug targets for TSE therapeutics and prophylaxes. Therefore, it is critical that NPRP fund research designed to identify the molecules that facilitate PrP^Sc formation and accumulation in vivo.

Experiments have demonstrated that the chaperone proteins GroEL and hsp104 can stimulate the cell-free conversion reaction, as can sulfated glycans (Wong et al., 2001) and partial denaturants (DebBurman et al., 1997). Other chaperone proteins thought to modulate PrP conversion include hsp73 (Tatzelt et al., 1995), members of the hsp60 class (DebBurman et al., 1997; Edenhofer et al., 1996; Stockel and Hartl, 2001), and BiP (Jin et al., 2000). There are at least a half dozen apparently natural PrP ligands or conversion modulators: copper (II) (Hornshaw et al., 1995), the laminin receptor (Weiss and Randour, 2002), laminin (Graner et al., 2000), stress-induced protein 1 (Zanata et al., 2002), and nucleic acids (Cordeiro et al., 2001; Gabus et al., 2001; Nandi and Leclerc, 1999).

The molecule or molecules associated with prion conversion may be easier to detect than prions themselves. For instance, there may be a known antibody that binds specifically to a chaperone protein involved with PrP formation or accumulation. Ancillary or chaperoning factors

could potentially amplify PrP^Sc, helping to overcome current prion detection limits.

A related goal of NPRP should be to fund research aimed at isolating the multiprotein complexes that contain prions. Such studies might identify new cofactors that are important in the formation and stabilization of PrP^Sc and infectivity. For instance, molecules such as sulfated glycosaminoglycans appear to be associated with PrP^Sc deposits in vivo (Snow et al., 1990) and may play a role in their formation in vivo, as can be the case with a variety of other amyloid protein deposits. Further understanding of the identities and roles of PrP^Sc-associated molecules might suggest new therapeutic and diagnostic approaches.

To develop better diagnostics, much more understanding about the nature and dynamics of prion infection must be gained. Studying the pathogenesis of TSEs holds the keys to such understanding. Researchers, clinicians, and public health officials must know which tissues are infectious and when, the mechanisms by which the infectious agent enters and disseminates in the body and then invades the brain, what causes cellular toxicity (prions, prions plus another molecular species, or a totally different molecular entity), the mechanisms by which the toxic events in TSEs lead to cellular dysfunction and clinical symptomatology, how the infectious agent spreads from host to host, and host determinants of susceptibility to infection. Understanding these issues will lead to improved characterization of diagnostic targets, better diagnostic strategies, greater target discrimination, and improved diagnostic sensitivity and specificity.

The answers to these questions may vary among strains and hosts. Therefore, all pathogenetic studies must be interpreted in their specific contexts, which will make the development of diagnostics more difficult. At the same time, what investigators learn about the pathogenesis of one prion disease will yield information relevant to the understanding of other prion diseases.

The study of TSEs should not be limited to mammalian species. Much can be learned from the study of prions found in other, nonmammalian organisms.

For example, the prions found in fungi have been studied extensively, including two prions of the yeast Saccharomyces cerevisiae. In 1994, those two prions, [URE3] and [PSI+], were discovered to be infectious forms of their normal proteins, Ure2P and Sup35p, respectively (Wickner, 1994). The first prion-inducing domain was defined and the protease resistance of the Ure2P in [URE3] prion strains gave the first hint of the mechanism (Masison & Wickner, 1995). Soon after, it was discovered that, as in mammals, multiple strains of yeast prions could exist (Derkatch et al., 1996). Ter-

Avanesyan's group was the first to show that the [PSI+] prion is a self-propagating aggregation of Sup35p in vivo and in vitro (Paushkin 1997).

Later that year, King and Wuthrich showed that the prion domain of Sup35p could form amyloid in vitro (King 1997). Then Glover and Lindquist showed that the full length of Sup35p could form filaments in vitro having characteristics of amyloid that were stimulated by extracts of [PSI+] cells but not the extract of [psi-] cells (Glover 1997). Also in 1997, a prion was found in the filamentous fungus Podospora anserine (Coustou et al., 1997). Another prion, [PIN+], of the Rnq1p protein of S. cerevisiae was reported in 2001 (Derkatch et al., 2001).

Work with these prions has been very rewarding. The experiments with S. cerevisiae described above provided the first evidence that chaperone proteins are involved in prion propagation (Chernoff et al., 1995). Additionally, the Mks1 protein was shown to be necessary for generation of the [URE3] protein (Edskes and Wickner, 2000). Also, the Ras-cyclic AMP pathway was found to negatively regulate generation of the [URE3] prion protein (Edskes and Wickner, 2000).

The presence of one prion in a cell can promote the generation of a second prion (Derkatch et al., 2001). Amyloid of the HET-s protein formed in vitro was shown to be infectious for fungal colonies, whereas nonspecific aggregates or the soluble form of the protein had no effect (Maddelein et al., 2002). Efforts to replicate this process in mammals have not yet been successful. In addition, investigators have shown that artificial prions can be constructed by using a prion domain of one protein and a reporter domain of another (Li and Lindquist, 2000). Another landmark discovery from a study of S. cerevisiae that used [Het-s] was that prions can be advantageous to the prion host (Coustou et al., 1997).

Drosophila and Caenorhabditis elegans have also proved to be superb models for the study of a variety of cellular and molecular processes and should be exploited to study prion diseases. These organisms have recently been used to model several human neurodegenerative conditions, including Parkinson's disease, tauopathies, and polyglutamine disorders. These model systems may clarify the basis for neurodegeneration in prion diseases, as they have in other neurodegenerative processes.

Despite the number of TSE transmission studies performed to date, many unanswered questions in addition to those discussed above

remain. Is chronic wasting disease (CWD) transmissible to humans? Is it transmissible to cattle or sheep? How is CWD transmitted among cervids? Further epidemiological and natural history studies would help answer these questions and would help shape appropriate measures to protect the public's health.

Several transmission studies are underway in Europe and the United States. In an ongoing experiment, Nora Hunter and colleagues at the United Kingdom's Institute for Animal Health recently demonstrated for the first time that healthy sheep can become infected with prions through transfusion of blood from BSE agent-infected sheep (Hunter et al., 2002). In a study funded by the European Union, scientists at the German Primate Center in Göttingen are performing transmission studies with rhesus monkeys to elucidate the pathogenesis of TSE in lymphoid tissue (Personal communication, A.Aguzzi, University Hospital of Zurich, October 12, 2002). Baxter International Inc., a Deerfield, Illinois-based pharmaceutical company, is conducting transmission studies with rhesus monkeys to understand the potential for prion infection from blood products (Personal communication, A. Aguzzi, University Hospital of Zurich, October 12, 2002).

Several additional transmission studies are planned. Scientists at the Rocky Mountain Laboratories of the National Institutes of Health expect to use squirrel monkeys to study prion infectivity in blood (Personal communication, R.T.Johnson, Johns Hopkins University, 2002). The Commissariat à l'Energie Atomique in Paris, France, plans

to build a large, new facility that would house 60 macaques for TSE-related studies, including the infectivity of different prion strains, such as those that cause vCJD (Deslys, 2002).

Because the primary structure of PrP^Sc is virtually identical to that of normal PrP, understanding as much as possible about PrP^C would be very helpful in the development of TSE diagnostic tests. A successful test must discriminate between these two closely related molecules. Moreover, understanding the normal role of PrP^C may reveal associated molecules and pathways that are appropriate detection targets. It remains unclear whether the basis for nerve cell dysfunction and death in prion disease is related to the toxicity of PrP^Sc, to the loss of function of PrP^C as a result of its conversion to PrP^Sc and its aggregation during a prion infection, or to other factors.

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