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Emerging Concerns:
De novo Formation of Prions

8:00 Welcome by Session Chairperson
Paul W. Brown, M.D.

8:15 Ultra-Sensitive Prion Assays Based on Seeded Conversions of Recombinant Prion Protein
Byron W. Caughey, Ph.D., Senior Investigator, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIH
PrPSc can seed the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). Soto and colleagues have shown that this seeding activity allows ultrasensitive detection of prions using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Building on the PMCA approach, we have developed faster, simpler prion detection methods using recombinant PrP-sen (rPrP-sen) which can discriminate normal hamster brain homogenates from scrapie brain homogenates containing <1 intracerebral lethal dose within 2-3 days. In periodically sonicated or shaken cell-free reactions, sub-femptogram equivalents of PrPSc seeded the conversion of rPrP-sen into easily detected quantities of specific protease-resistant PrP fibrils. Diseased and normal hamsters were also distinguished using 2-μl of cerebral spinal fluid as seeds. The relative speed, simplicity, replicability and sensitivity of these reactions should facilitate both the development of practical prion assays and structural analyses of prion-seeded PrP polymers.

8:40 De Novo Formation of Purified Native Prions
Nathan R. Deleault, Ph.D., Department of Biochemistry, Dartmouth Medical School
To study the mechanism of prion formation biochemically, we conducted a series of serial Protein Misfolding Cyclic Amplification (sPMCA) reactions using purified native PrP^C and synthetic polyanionic molecules as substrates.  For the first time, we demonstrate that infectious, wild type prions can be: (1) propagated in vitro using purified substrates, and (2) generated de novo from non-infectious components.  Furthermore, we have observed that polyanionic molecules are selectively incorporated into physical complexes with PrP during the formation of purified prions in vitro.

9:05 De novo Generation of Prion Infectivity in a Cell-Free System
Joaquin Castilla, Ph.D., Assistant Professor, Department of Infectology, Scripps Research Institute-Florida
Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative disorders affecting both humans and animals. There is no available treatment or therapy for these fatal diseases. The infectious agent associated with TSEs (termed prion) appears to be composed uniquely of a protein, which is a conformationally-modified version (PrPSc) of the cellular prion protein (PrPC). The disease is propagated by the conversion of host PrPC into PrPSc induced by small quantities of PrPSc. Interestingly, prions occur in the form of different strains that show distinct biological and physicochemical properties. TSEs can have diverse origins, including genetic, sporadic (putatively spontaneous) and infectious. The occurrence of sporadic cases of prion diseases in humans and maybe in other species, i.e. atypical bovine spongiform encephalopathy (BSE) in European and USA cattle and atypical scrapie cases in sheep suggest that spontaneous prion diseases may happen infrequently but ubiquitously. However, there are no reported cases of spontaneously-occurring prion disease in experimental wild-type rodent models. We have used a novel technique, Protein Misfolding Cyclic Amplification (PMCA) to rapidly propagate prions in the test tube, using normal brain homogenate as substrate. Prions propagated in vitro are infectious in vivo and maintain their prion strain specificity. PMCA has been used to efficiently amplify a variety of prion strains from mouse, hamster, bank vole, deer, cattle, sheep and human. Therefore, to mimic spontaneous generation of infectivity in vitro becomes one of the most important challenges in the prion field. We show here, for the first time, the de novo generation of infectious prions from bank voles (Clethrionomys glareolus) starting with non-infectious brain homogenates. Several biochemically different prion strains were generated using two different wild-type vole genotypes. The de novo in vitro generated PrPSc was highly infectious after its inoculation in bank voles. We show an extensive characterization of this "spontaneous" phenomenon.

9:30 PMCA Amplification of Prion Amyloid without Amplification of Infectivity
Robert G. Rohwer, Ph.D., Director, Molecular Neurovirology Laboratory, Veterans Affairs Medical Center; and Associate Professor of Neurology, School of Medicine, University of Maryland at Baltimore
Employing the original protocol for PMCA developed by Soto and colleagues, we obtained a 16 to 32 fold amplification of PK resistant PrP as determined by two fold serial dilution to the starting concentration on Western blot. In comparison, there was no difference in titer, as measured by limiting dilution titration, between the frozen control, a sample that was incubated at 37°C without sonication and the sonicated sample that produced the amyloid amplification. The limiting dilution titration method is sufficiently sensitive to have detected even a 20% difference in titer between the samples. A two fold increase in titer would have caused the infection of nearly every animal at the limiting dilution and could not have been missed. If there is amplification of infectivity during PMCA, it must follow very different kinetics from the amyloid.

9:55 Silent Prions in Normal Brains
Wen-Quan Zou, M.D., Ph.D., Assistant Professor, Neuropathology, Case Western Reserve University
The co-existence of cellular prion protein (PrPC) and its pathological isoform (PrPSc) is a prerequisite for the pathogenesis of prion diseases. However, molecular mechanism of PrPSc formation in the spontaneous prion diseases including sporadic and familial forms remains poorly understood. Our recent studies indicate that in the uninfected brain there are small amounts of abnormal PrP species that may be involved in the pathogenesis of spontaneous prion diseases.

Pathogenesis

11:10 Accumulation of Prion Protein in the Brain That is Not Associated with Transmissible Disease
Pedro Piccardo, M.D., Senior Investigator, OBRR / DETTD / LBPUA, FDA

11:35 High Levels of TSE Infectivity Can Be Associated with Little or No Detectable PrPSc in Vivo
Rona Barron, Ph.D., Neuropathogenesis Unit, Roslin Institute and Royal (Dick) School of Veterinary Studies
This work examines the relationship between TSE infectivity and the abnormal prion protein, PrPSc. In a mouse model of disease we have shown high titres of TSE infectivity in brain tissue which contains little or no PrP-res. We also found no evidence of other abnormal PrP isofoms such as PK-sen PrPSc. These data question the true relationship between PrPSc and TSE infectivity, and the current reliance on PrPSc as the sole diagnostic marker for TSE disease.

12:00 Conversion of the BASE Prion into the BSE Prion: The Origin of BSE?
Fabrizio Tagliavini, Ph.D., Director, Division of Neurology 5 & Neuropathology, Neurological Institute "Carlo Besta"
Twenty years after the identification of bovine spongiform encephalopathy (BSE), the origin of the causal agent is still unknown. This issue is of fundamental importance, since knowledge of the origin of the BSE agent is essential for prevention of future outbreak of the disease or variants thereof in cattle and other mammals. We carried out transmission studies with transgenic mice expressing bovine PrP and four lines of non-transgenic mice and found that an atypical form of spongiform encephalopathy of cattle, termed BASE or BSE-L, is caused by a prion strain distinct from that of classical BSE. Noteworthy, this newly characterized prion strain has the ability to convert into the classical BSE strain upon serial transmission to inbred mouse lines. According to these results, BASE--which is regarded as a sporadic form of prion disease in cattle--may be the origin of BSE, following conversion of the causal agent in an intermediate host.

12:45 Luncheon Technology Workshop
(Sponsorship Available) or Lunch on Your Own

2:00 Sporadic CJD and Atypical BSE: Two Children of One Protein
Maurizio Pocchiari, M.D., Director of Research, Virology, Istituto Superiore Di Sanita
The identification of forms of TSE diseases in cattle caused by prion strains different from BSE has raised new concerns on the possibility that these novel agents might induce disease in humans with a phenotype resembling sporadic CJD. The analysis of the distribution of the different molecular subtypes of sporadic CJD might give some answers.

2:25 Variant CJD: Residual Uncertainties
Robert Will, M.D., National CJD Surveillance Unit, Edinburgh, UK

Mortality from variant CJD continues to decline, but concerns for public health persist. These are based on uncertainty on the population prevalence of infection, the incubation period of vCJD and the potential for further cases of secondary transmission. Information from epidemiology, molecular biology and transmission studies may　provide new insights into these issues.　
2:50 Panel Discussion

Detection

3:05 Comments by Session Chairperson
Byron W. Caughey, Ph.D., Senior Investigator, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIH

3:10 Biochemical Detection of Prions in Blood and Urine
Claudio Soto, Ph.D., Professor, Neurology, University of Texas-Galveston

3:35 The Elusive Precursor of PrPSc
Teresa Pinheiro, Ph.D., Professor, Biological Sciences, University of Warwick
A key molecular event in prion diseases is the conversion of the cellular prion protein, PrPC, to the aberrant disease-associated state, PrPSc. The details of this molecular transformation are not fully understood, but it has been suggested that an intermediate on the normal folding pathway of PrPC may be recruited to form PrPSc. Using conventional analyses of folding transition data determined by fluorescence and circular dichroism, and novel phase diagram analyses, we present compelling evidence for the presence of an intermediate species on the folding pathway of PrP. This state is structurally close to the native state and upon incubation produces off-pathway aggregates. The results explain the difficulties in prion diagnosis and suggest possible new directions for test development.

4:30 Detection of Blood Prions with Epitope
Protection Technology
Neil Cashman, M.D., Professor, Medicine, Amorfix Life Sciences
In order to detect prions, an assay must distinguish between the normally folded prion protein PrPC and its aggregated disease-causing conformation PrPSc. We have developed a chemical means to differentiate between PrPC and PrPSc, called Epitope Protection technology. PrPC is selectively modified with short-lived and highly-reactive chemicals which modify selected amino acids in the protein. Such chemical modification efficiently blocks immunological epitopes on PrPC and leaves them unrecognizable to many PrP antibodies. PrP molecules within prion particles are "protected" from chemical modification, and can then be detected by conventional immunoassay after disaggregation of the sample. We now report detection of the very small concentration of PrPSc, which has been estimated in the low femtogram range per mL of infected blood. We have tested this methodology by screening blinded panels of vCJD brain and spleen material spiked into plasma, and we can reliably detect vCJD brain homogenate after diluting a 10% homogenate more than 105-fold into plasma. A high-throughput version of the assay has been developed and allows the processing of thousands of samples per day.

4:55 PrionScreen - All TSEs on a Plate
Martin Mehl, Ph.D., Product Manager BSE, Marketing, R-Biopharm AG

PrionScreen, the Roche Diagnostics last generation ELISA kit, provides the actual demands to identify all relevant typical and atypical TSEs from various species within the same assay. The overall changes in test kit requirements and the data of the recent EU approvals are summarized from the industries view.

6:40 Close of Day One