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Well-known member
Advances in Understanding the Nature and
Behavior of the Agent
9:00 Welcome by Session Chairperson
Dr. Paul W. Brown
9:15 De novo Generation of Mammalian Prions
Dr. Giuseppe Legname, Institute for Neurodegenerative Diseases, University of California at San Francisco
A long-standing issue in the study of prion diseases, or transmissible spongiform encephalopathies, has been the formal proof that such disorders are indeed caused solely by conformation changes in the prion protein (PrP). In order to test the protein-only hypothesis we polymerized N-terminally truncated MoPrP(89-230) into amyloid and bioassayed the samples in transgenic (Tg) mice expressing MoPrP(89-230) (Tg(MoPrP,?23-88)9949/Prnp0/0). All of these Tg mice developed neurologic dysfunction between 380 and 660 days after inoculation, while control Tg mice inoculated with phosphate-buffered saline solution remained healthy for 670 days before sacrifice. Western blotting of brain extracts showed protease-resistant PrPSc; serial transmission of the brain extracts to wt FVB and Tg(MoPrP)4053 mice resulted in incubation times of 160 and 90 days, respectively. Prions generated de novo were compared to RML, which represents a well-characterized mouse scrapie strain. At least two new strains with different biochemical, biophysical and neuropathological characteristics were created. Our results support the propositions that prions are infectious proteins and that sporadic prion disease requires only the spontaneous conversion of PrPC into PrPSc.
9:45 In Vitro Generation of Infectivity by Protein Misfolding Cyclic Amplification (PMCA) and Use for Prion Detection in Blood
Dr. Claudio Soto, Professor, Neurology, University Of Texas Galveston
10:15 Early Events during TSE Infection
Dr. Suzette Priola, National Institutes of Health, NIAID, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories
Despite the fact that normal protease-sensitive prion protein (PrP-sen) is required for infection with transmissible spongiform encephalopathy (TSE) agents, not all PrP-sen expressing cells are susceptible to TSE infection. In order to determine why TSE infection appears to be restricted to certain cell types, we have developed a tissue culture system that allows us to monitor both acute and persistent formation of protease-resistant prion protein (PrP-res), the form of PrP associated with TSE diseases. Our results demonstrate that, while any cell type can make PrP-res acutely, only certain cell types can make PrP-res persistently and become chronically infected with TSE agents. The data suggest that infection with TSE agents is a two-stage process involving a cell type-independent phase of acute PrP-res formation followed by a cell type-dependent phase of persistent PrP-res formation and infection.
10:45 Coffee Break, Poster and Exhibit Viewing
11:15 Transmission of Human TSE Diseases to Bank Voles
Dr. Maurizio Pocchiari, Director of Research Virology, Istituto Superiore di Sanita, Italy
Rodent models of human transmissible spongiform encephalopathy (TSE) represent a valuable tool for characterising human isolates, but primary inoculations have always resulted in a low rate of transmission and long incubation periods. We found that the wild rodent bank vole (C. glareolus) is highly susceptible to some forms of sporadic and genetic Creutzfeldt-Jakob disease with incubation periods shorter than 180 days. These rodents may be valuable for strain typing human TSE diseases. Co-Authors: Vaccari G., Di Bari M.A., Simson S., Frassanito P., Nonno R., Cartoni C., Borroni R., Cardone F, Agrimi U.
11:45 Studies on Pathogenesis, with Special Attention to Muscle Involvement
Dr. Michael Beekes, Robert Koch Institute, Germany
PrPTSE, the biochemical marker for infectious agents causing transmissible spongiform encephalopathies (TSEs) or prion diseases, can be detected in muscle tissue, not only at late stages of disease, but already before clinical symptoms become visible. Using different detection methods, such as Western blotting, immunohistochemistry or PET blotting the location of disease-specific prion protein in muscles, nerves and lymphoid tissue can be visualized in order to track down the routing pathways and reservoirs of TSE agents in the body. Experimental rodent studies are expected to provide further conceptual insights into the pathophysiology of TSE infections and may contribute to a better assessment of the risks for public health possibly emanating from "prions in skeletal muscle".
12:15 Pathologic PrP Deposition at the Cerebral Subcellular Level
Dr. Herbert Budka, Medical University of Vienna, Austria
We examined co-localization patterns of distinct PrPsc immunodeposits with cellular markers by confocal laser microscopy in CJD brains. PrPsc co-localizes most prominently with chemical synapses, but also electric synapses, neuronal somata, dendrites, axons, astrocytes, and microglia harbor PrPsc. Axons, shown for the first time in human disease to contain PrPsc, may be a relevant route of prion spread, and activated microglia and astrocytes may contribute to PrPsc processing or degradation.
12:45 Luncheon Technology Workshop Sponsored by
Enhancing Robust BSE Testing With Automation
Mr. Chris Neary, General Manager, Beckman Coulter, Inc
Behavior of the Agent
9:00 Welcome by Session Chairperson
Dr. Paul W. Brown
9:15 De novo Generation of Mammalian Prions
Dr. Giuseppe Legname, Institute for Neurodegenerative Diseases, University of California at San Francisco
A long-standing issue in the study of prion diseases, or transmissible spongiform encephalopathies, has been the formal proof that such disorders are indeed caused solely by conformation changes in the prion protein (PrP). In order to test the protein-only hypothesis we polymerized N-terminally truncated MoPrP(89-230) into amyloid and bioassayed the samples in transgenic (Tg) mice expressing MoPrP(89-230) (Tg(MoPrP,?23-88)9949/Prnp0/0). All of these Tg mice developed neurologic dysfunction between 380 and 660 days after inoculation, while control Tg mice inoculated with phosphate-buffered saline solution remained healthy for 670 days before sacrifice. Western blotting of brain extracts showed protease-resistant PrPSc; serial transmission of the brain extracts to wt FVB and Tg(MoPrP)4053 mice resulted in incubation times of 160 and 90 days, respectively. Prions generated de novo were compared to RML, which represents a well-characterized mouse scrapie strain. At least two new strains with different biochemical, biophysical and neuropathological characteristics were created. Our results support the propositions that prions are infectious proteins and that sporadic prion disease requires only the spontaneous conversion of PrPC into PrPSc.
9:45 In Vitro Generation of Infectivity by Protein Misfolding Cyclic Amplification (PMCA) and Use for Prion Detection in Blood
Dr. Claudio Soto, Professor, Neurology, University Of Texas Galveston
10:15 Early Events during TSE Infection
Dr. Suzette Priola, National Institutes of Health, NIAID, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories
Despite the fact that normal protease-sensitive prion protein (PrP-sen) is required for infection with transmissible spongiform encephalopathy (TSE) agents, not all PrP-sen expressing cells are susceptible to TSE infection. In order to determine why TSE infection appears to be restricted to certain cell types, we have developed a tissue culture system that allows us to monitor both acute and persistent formation of protease-resistant prion protein (PrP-res), the form of PrP associated with TSE diseases. Our results demonstrate that, while any cell type can make PrP-res acutely, only certain cell types can make PrP-res persistently and become chronically infected with TSE agents. The data suggest that infection with TSE agents is a two-stage process involving a cell type-independent phase of acute PrP-res formation followed by a cell type-dependent phase of persistent PrP-res formation and infection.
10:45 Coffee Break, Poster and Exhibit Viewing
11:15 Transmission of Human TSE Diseases to Bank Voles
Dr. Maurizio Pocchiari, Director of Research Virology, Istituto Superiore di Sanita, Italy
Rodent models of human transmissible spongiform encephalopathy (TSE) represent a valuable tool for characterising human isolates, but primary inoculations have always resulted in a low rate of transmission and long incubation periods. We found that the wild rodent bank vole (C. glareolus) is highly susceptible to some forms of sporadic and genetic Creutzfeldt-Jakob disease with incubation periods shorter than 180 days. These rodents may be valuable for strain typing human TSE diseases. Co-Authors: Vaccari G., Di Bari M.A., Simson S., Frassanito P., Nonno R., Cartoni C., Borroni R., Cardone F, Agrimi U.
11:45 Studies on Pathogenesis, with Special Attention to Muscle Involvement
Dr. Michael Beekes, Robert Koch Institute, Germany
PrPTSE, the biochemical marker for infectious agents causing transmissible spongiform encephalopathies (TSEs) or prion diseases, can be detected in muscle tissue, not only at late stages of disease, but already before clinical symptoms become visible. Using different detection methods, such as Western blotting, immunohistochemistry or PET blotting the location of disease-specific prion protein in muscles, nerves and lymphoid tissue can be visualized in order to track down the routing pathways and reservoirs of TSE agents in the body. Experimental rodent studies are expected to provide further conceptual insights into the pathophysiology of TSE infections and may contribute to a better assessment of the risks for public health possibly emanating from "prions in skeletal muscle".
12:15 Pathologic PrP Deposition at the Cerebral Subcellular Level
Dr. Herbert Budka, Medical University of Vienna, Austria
We examined co-localization patterns of distinct PrPsc immunodeposits with cellular markers by confocal laser microscopy in CJD brains. PrPsc co-localizes most prominently with chemical synapses, but also electric synapses, neuronal somata, dendrites, axons, astrocytes, and microglia harbor PrPsc. Axons, shown for the first time in human disease to contain PrPsc, may be a relevant route of prion spread, and activated microglia and astrocytes may contribute to PrPsc processing or degradation.
12:45 Luncheon Technology Workshop Sponsored by
Enhancing Robust BSE Testing With Automation
Mr. Chris Neary, General Manager, Beckman Coulter, Inc