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From: TSS ()
Subject: Transmissible Spongiform Encephalopathies The Definitive American TSE meeting February 12-13 2007
Date: November 15, 2006 at 8:10 am PST

Transmissible Spongiform Encephalopathies The Definitive American TSE meeting February 12-13 2007

Transmissible Spongiform Encephalopathies continue to cause serious concern among researchers whose work utilizes materials from animals and/or humans which may be contaminated with the causative agent. The appearance of BSE in herds of cattle born after the introduction of the ruminant feed ban and the potential impact of BSE and CWD on human health in the U.S has raised new concerns and questions, as has the continued occurrence of Chronic Wasting Disease (CWD) in North American deer herds. The news that 3 of 17 recipients of blood from a vCJD donor contracted the disease has shown that transmission through blood transfusion is a reality. This conference will present the newest data on TSE’s in the context of its application to the pharmaceutical, biological, environmental and device industries.

SESSIONS INCLUDE:

EMERGING CONCERNS

NEW RESEARCH DIRECTION

INFECTIVITY AND TRANSMISSION

DETECTION AND REMOVAL

CONFORMATIONAL TRANSITION AND IFECTIVITY

SCIENTIFIC ADVISORY BOARD:
Larisa Cervenakova, M.D., Ph.D., Scientist II, Transmissible Diseases Department, J.H. Holland Laboratory for the Biomedical Sciences, American Red Cross

Kiki B. Hellman, Ph.D., President & Founder, The Hellman Group, LLC

Suzette A. Priola, Ph.D., Senior Investigator, Chief, TSE/Prion Molecular Biology Section, National Institutes of Health, NIAID, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories

PRESENTATIONS BY:
Judd M. Aiken, DVM, Professor, Animal Health & Biomedical Sciences, University of Wisconsin-Madison, School of Veterinary Medicine

Seong An, Ph.D., Research Fellow, Research and Development, People Bio Inc.

David M. Asher, M.D., Laboratory Chief, Laboratory of Methods Development, FDA

Helen Baxter, Ph.D., Senior University Fellow, School of Chemistry, University of Edinburgh

Paul W. Brown, M.D. Lisa Ferguson, DVM, Senior Staff Veterinarian, USDA-APHIS

Luisa Gregori, Ph.D., Assistant Director and Assistant Professor, VA Research Services and University of Maryland, BREF

Qingzhong Kong, Ph.D., Assistant Professor, Pathology, Case Western Reserve University

Anthony L. Lau, Ph.D., Post-Doctoral Fellow, Novartis Vaccines and Diagnostics, Inc.

Pascal LeBlanc, Ph.D., LaboRetro unité de virologie humaine, INSERM
Sylvain Lehmann, Professor, Institut de Génétique Humaine du CNRS

David M. Lynn, Ph.D., Assistant Professor, Chemical and Biological Engineering, Emory University

Maurizio Pocchiari, Ph.D., Director of Research-Virology, Istituto Superiore Di Sanita (Italy)

Burt Pritchett, DVM, Veterinary Medical Officer, FDA Center for Veterinary
Medicine

Alex Raeber, Ph.D., Director of Research, Prionics AG

Michael Stack, Senior Researcher, TSE Molecular Biology, Veterinary
Laboratories Agency

James Walker, Ph.D., Senior Project Manager, TSE Research Group, Health
Protection Agency

Charles Weissmann, Ph.D., Professor, Neurogenetics, Scripps Research
Institute, Florida

Robert G. Will, M.D., Consultant Neurologist, CJD Surveillance Unit, Western General Hospital

Wen-Quan Zou, M.D., Ph.D., Assistant Professor, Pathology, Case Western Reserve University

--------------------------------------------------------------------------------

For questions or suggestions about the meeting, please contact:
Elizabeth Lamb
Senior Conference Director
Cambridge Healthtech Institute
E-mail: elamb@healthtech.com

For exhibit and sponsorship information, please contact:
Suzanne Carroll
Manager, Business Development
Phone: 781-972-5452 • E-mail: scarroll@healthtech.com

http://www.healthtech.com/2007/tse/index.ASP

SUNDAY, FEBRUARY 11

5:00 - 6:00 pm Early Registration

MONDAY, FEBRUARY 12

7:30 am Registration, Morning Coffee


Emerging Concerns


8:30 Welcome by Session Chairperson
Paul W. Brown, M.D.
8:45 Variant CJD: The End of the Beginning or the Beginning of the End?
Robert G. Will, M.D., National CJD Surveillance Unit, University of Edinburgh
Variant CJD incidence has been in decline in the UK since 1999 and the feared epidemic of this zoonotic disease has not yet materialized. A number of other countries have identified cases of vCJD, notably in France, but the overall non-UK incidence of vCJD is not increasing markedly. There is good evidence indicating that vCJD can be iatrogenically transmitted through blood transfusion but it is unlikely that this route of transmission will lead to a self-sustaining outbreak. Public health concerns are therefore in decline, but because of the potentially extended incubation periods, it will be many years before further outbreaks of vCJD can be excluded, for example through alternative mechanisms of transmission.

9:15 Sporadic CJD: Does Transmission through Blood Occur?
Maurizio Pocchiari, Ph.D., Director of Research-Virology, Istituto Superiore Di Sanita (Italy)

9:45 The 'Spontaneous' BSE Issue: Pros, Cons, and Strategies to Test the Hypothesis
Paul W. Brown, M.D.

10:15 Coffee Break, Poster and Exhibit Viewing in the Exhibit Hall

10:45 Discrimination between CWD, BSE and Scrapie Strains: An Evaluation of Tests
Michael Stack, Senior Researcher, TSE Molecular Biology, Veterinary Laboratories Agency
Bovine spongiform encephalopathy (BSE) has been implicated as the cause of the appearance in humans of new variant In Great Britain, during the 1980s, it is possible that BSE-contaminated meat and bone meal may have been fed to sheep, raising the possibility that BSE could have been transmitted to the national sheep flock, and could therefore be a potential secondary threat to public health. Molecular and immunohistochemical techniques which can discriminate between experimental BSE in sheep, and natural scrapie cases have been developed and evaluated in blinded ring trials. Increased surveillance for Transmissible Spongiform Encephalopathies (TSEs) in cervid populations across Europe is planned to start in 2007, and if a disease such as Chronic Wasting Disease (CWD) is discovered, one of the first questions would be whether the molecular profile is similar to that found for North American CWD cases, or could there have been contact with the agents of BSE or scrapie resulting in a change of disease profile? The tests evaluated in the blinded ring trials in Europe also offer potential discrimination between CWD and other animal TSEs, and could therefore be used to rule out BSE in cervids as another possible secondary threat to public health. This presentation will describe the principles of discrimination and the evaluation results obtained so far.

New Research Directions


Session Chairperson:
Suzette A. Priola, Ph.D., Senior Investigator, Chief, TSE/Prion Molecular Biology Section, National Institutes of Health, NIAID, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories
11:15 Developing Cell Cultures for Prion Studies
Sylvain Lehmann, Professor, Institut de Génétique Humaine du CNRS
Cell cultures susceptible to TSE agents represent relevant and useful experimental models for Prion studies. They have been used in particular to study TSE molecular events and to develop and validate innovative therapeutic approaches. More recently, prion infected cell culture models have been considered as an alternative to animal bioassays to detect the presence of infectivity. In fact, it was possible to obtain in specific cell culture paradigms a reliable and highly sensitive detection of prions that was much faster and at a lesser cost than in animals. Nevertheless, many problems persist with the use of these cell culture models including their standardization or the range of prion species and strains that they can detect.

11:45 Retroviral Infection Strongly Enhances the Release of Scrapie Infectivity in Cell Culture
Pascal LeBlanc, Ph.D., LaboRetro unité de virologie humaine, INSERM
Although there is much evidence to suggest that PrPSc, a misfolded form of the cellular prion protein PrPC, is the infectious agent of prion diseases, the mechanism of PrPSc transmission and the factors that affect its spread remain unknown. Here we show that the release of PrPSc from scrapie-infected cells is markedly enhanced by retroviral infection, implicating retroviruses in the spread of prion diseases and providing mechanistic insights into prion transmission.

12:15 Lunch on Your Own (Luncheon Technology Workshops Available)

Infectivity and Transmission


1:20 Comments by Session Chairpersons
Kiki B. Hellman, Ph.D., President & Founder, The Hellman Group, LLC
Suzette A. Priola, Ph.D., Senior Investigator, Chief, TSE/Prion Molecular Biology Section, National Institutes of Health, NIAID, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories
1:30 Infectivity in Urine of Hamsters Infected with Scrapie
Luisa Gregori, Ph.D., Assistant Director and Assistant Professor, VA Research Services and University of Maryland, BREF
Horizontal transmission from animal to animal of Transmissible Spongiform Encephalopathies (TSE) has been documented in the wild and in laboratory animals. The cause(s) of this transmission is not fully understood, although bodily excretions such as urine and feces are often proposed as potential sources of secondary exposure leading to horizontal transmissions. We report here on our investigation on infectivity in urine of TSE infected animals. Urine from hamsters infected with the 263K strain of scrapie was collected using metabolic cages and titered by intracerebral inoculation in the same animal species with the limiting dilution method. Five-ml equivalents of urine (diluted 1:3) were inoculated. After 357 days post inoculation, 11 animals developed scrapie with a titer of 2.3 +/- 0.4 ID/ml. No infectious have been observed in the cohort of animals inoculated with urine from control animals. Ten percent bladder and kidney tissue homogenates from infected hamsters were also titered using the end point titration method. At 343 days, the titers are 10(5.5) and 10(4.8) ID50/g for bladder and kidney, respectively. These results propose a new pathway of infection of the natural environment. The implications are far reaching from environmental contamination of pastures in the wild to risk management of domestic livestock and the safety of urine-derived therapeutic products.

2:00 Soil Minerals Enhance Prion Infectivity
Judd M. Aiken, DVM, Professor, Animal Health & Biomedical Sciences, University of Wisconsin-Madison, School of Veterinary Medicine
We have recently demonstrated that prions bind clay and silica. The binding of PrPSc to a common soil clay (montmorillonite) is avid and this interaction enhances infectivity. The implications of this enhancement of transmission are far-reaching and include how scrapie and CWD are environmentally transmitted. The ramifications of these findings with regard to food safely will also be discussed.

2:30 Transmission of BSE-301v Following Infection from the Small Intestine: A New Model for Investigating Iatrogenic Transmission Risks for vCJD
James Walker, Ph.D., Senior Project Manager, TSE Research Group, Health Protection Agency
The study explores the use of a novel challenge model designed to provide information on the levels of infectivity of tissues following infection from the small intestine. The model uses direct challenge via the small intestine to prevent the contamination of the oral cavity by the primary inoculum. Groups of VM mice (n=10) were inoculated with 100ul of 2% w/v BSE-301v infected brain homogenate directly into the small intestine and sacrificed at 3, 6, 9, 12, 15, 18, 21 weeks post-inoculation. Tissues including spleen, saliva, salivary gland, trigeminal ganglia, gingival margin, alveolar bone, dental pulp, posterior tongue, anterior tongue, and brain were removed at the appropriate time points and re-inoculated (intra-cranially) into groups of mice (n=6) which were sacrificed at a clinical end point. Results to date indicate that brain and spleen from mice inoculated via this route became infectious very early in the course of disease progression and achieved maximum infectious titers well before clinical symptoms became apparent. Western blot analysis for the presence of PrPSc in the brain samples does not correlate well with the levels of infectivity and this is currently being investigated further. These results will be discussed in connection with our understanding of the iatrogenic transmission.

3:00 Afternoon Refreshment Break, Poster and Exhibit Viewing in the Exhibit Hall

3:30 Transmission of the Italian Atypical BSE (BASE) in Humanized Mouse Models
Qingzhong Kong, Ph.D., Assistant Professor, Pathology, Case Western Reserve University
Bovine Amyloid Spongiform Encephalopathy (BASE) is an atypical BSE strain discovered recently in Italy, and similar or different atypical BSE cases were also reported in other countries. The infectivity and phenotypes of these atypical BSE strains in humans are unknown. In collaboration with Pierluigi Gambetti, as well as Maria Caramelli and her co-workers, we have inoculated transgenic mice expressing human prion protein with brain homogenates from BASE or BSE infected cattle. Our data shows that about half of the BASE-inoculated mice became infected with an average incubation time of about 19 months; in contrast, none of the BSE-inoculated mice appear to be infected after more than 2 years. These results indicate that BASE is transmissible to humans and suggest that BASE is more virulent than classical BSE in humans.

4:00 Regulatory Panel Discussion:
Answers to the Regulatory Questions Regarding TSEs
David M. Asher, M.D., Laboratory Chief, Laboratory of Methods Development, FDA
Burt Pritchett, DVM, Veterinary Medical Officer, FDA Center for Veterinary Medicine
Lisa Ferguson, DVM, Senior Staff Veterinarian, USDA-APHIS TBA, EPA

5:30 Networking Reception in Exhibit Hall

6:30 Close of Day One

http://www.healthtech.com/2007/tse/day1.asp

TUESDAY, FEBRUARY 13


Detection and Removal


8:30 Morning Coffee
9:00 Comments by Session Chairperson
Larisa Cervenakova, M.D., Ph.D., Senior Scientist, Transmissible Diseases Department, J.H. Holland Laboratory for the Biomedical Sciences, American Red Cross

9:10 Prion Strain Identification by the Scrapie Cell Assay using Cell Panels
Sukhi Mahal, Ph.D. and Charles Weissmann, Ph.D., Department of Infectology, Scripps Florida

9:40 Detection of PrPsc in Sheep Plasma using SRA
Seong An, Ph.D., Research Fellow, Research and Development, PeopleBio Inc.
We have developed a system called Multimer Detection System (MDS) for the detection of the PrPSc in blood, as previously presented on hamster plasma samples. We improved our MDS system to the Simultaneous Reaction Assay (SRA) using strepavidin conjugated magnetic beads to concentrate the sample. The SRA worked by adding the plasma sample, biotinylated entrapping antibody and HRP conjugated detection antibody simultaneously. SRA reduced the assay time by eliminating several incubation steps. We were able to differentiate the signal from scrapie from normal sheep blood samples. Hence, SRA is a very simple and robust method for detecting PrPSc in plasma.

10:10 Update on the 15B3 CJD Blood Test
Alex Raeber, Ph.D., Director of Research, Prionics AG
The detection of PrPSc in blood of TSE infected animals and humans have been hampered by the low levels and lack of protease resistance of PrPSc in blood. We have developed proprietary technologies based on the ELISA and FACS platform for the detection of disease associated prion proteins in plasma and serum using the conformation specific antibody 15B3 which recognizes both protease resistant and protease sensitive forms of abnormal, disease associated PrP. Analytical sensitivity studies using vCJD and scrapie brain homogenates suggest that the limit of detection is in the range of 10 - 100 infectious units per millilitre which is consistent with the levels of prion infectivity found in blood of infected animals and humans. Results of the analysis of serum and plasma panels from clinical and preclinical sheep infected with scrapie as well as screening of normal human plasma samples with the prototype blood test will be presented and the application of this test for use in the blood transfusion service and in the plasma fractionation process will be discussed.

10:40 Coffee Break, Poster and Exhibit Viewing in the Exhibit Hall

11:10 Detection and Removal of Residual Contamination on Surgical Instruments
Helen Baxter, Ph.D., Senior University Fellow, School of Chemistry, University of Edinburgh
Residual contamination on reprocessed surgical and dental instruments is a significant problem in contamination control in the health service. Of particular concern is the risk of iatrogenic transmission of CJD since it is well documented that TSE infectivity adhering to metal surfaces can survive conventional cleaning and sterilization procedures. As there is now clear evidence of cases of vCJD arising from blood transfusion, the potential risk from undiagnosed CJD carriers in the population make it imperative that effective new procedures for cleaning 'high risk' instruments be developed and put into practice. The results of an interdisciplinary study on new methods of contaminant detection and decontamination for surgical instruments will be described. Specifically: (a) the effectiveness of RF gas-plasma methods in removing TSE infectivity from stainless steel using an animal model; (b) application of RF gas-plasma decontamination for removal of residual contaminants from surgical instruments and comparison of these procedures to current methods; (c) development of low background covalent fluorescent probes for derivatization of proteins on surfaces; (d) development and application of rapid surface fluorescent scanning techniques for the detection and quantification of residual protein contamination on surgical instruments at the sub femptomole level.

Conformational Transition and Infectivity


11:40 Characterization of PrP-Derived Peptides Capable of Discriminating between and PrPSc in Plasma
Anthony L. Lau, Ph.D., Post-Doctoral Fellow, Novartis Vaccines and Diagnostics, Inc.
Development of reagents that specifically bind PrPSc has been a long sought after milestone in the study of prion biology. Prion sequences grafted into an antibody scaffold have previously been shown by Morocini et al. to interact with PrPSC. Upon discovering that peptides were capable of capturing PrPSC without being conformationally constrained within scaffold, we hypothesized that other segments of the PrP peptide sequence may also bind preferentially to PrPSC. After screening the biotinylated PrP-derived peptides, we found two that were capable of binding full-length PrPSC in plasma, which contains a vast excess of PrPC and other proteins. Using paramagnetic beads coated with active peptide to selectively capture PrPSc followed by a sandwich ELISA for detection, a prototype assay was developed and used to study the mechanism of interaction between the active peptides and PrPSc. Through extensive analyses, we show that positively charged amino acids play an important, but not exclusive, role in the interaction between the most active peptides and PrPSC. Binding occurs through a structural domain that is only present in the abnormal conformer, is species independent, and is not affected by proteinase K digestion. PrPC levels in plasma and the amount of PrPSC captured from brain homogenate of four different species were quantitated. The limit of detection was calculated to be ~10 attomoles of PrPSc (based on the mass of PrP27-30) from a ~106-fold dilution of human vCJD brain homogenate. We believe the sensitivity of this assay and the elimination of the need for protease digestion will provide prion researchers a powerful new tool to study new areas of prion biology. Co-Authors: Alice Yam, Melissa Michelitsch, Xuemei Wang, Carol M. Gao, Robert Goodson, Robert Shimizu, Gulliver Timoteo, John Hall, Angelica Medina-Selby, Doris Coit, Colin McCoin, Celine Hu, Bruce Phelps, Ping Wu, David Chien, and David Peretz
12:10 Abnormal Prion Protein Conformers in Normal Human Brain
Wen-Quan Zou, M.D., Ph.D., Assistant Professor, Pathology, Case Western Reserve University
Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPc) through an á-helix to b-sheet transition. Here we demonstrate, using sedimentation, gel filtration and enrichment of abnormal PrP by gene 5 protein, that small amounts of detergent-insoluble PrP aggregates and PK-resistant PrP species are present in normal human brains. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains.

12:40 Controlling Amyloid Growth in all Dimensions
David G. Lynn, Ph.D., Center for the Analysis of Supramolecular Self-assemblies, Center for Neurodegenerative Diseases, Departments of Chemistry and Biology, Emory University
The great progress made in defining the structure of protein and peptide amyloid assemblies, particularly the arrangement of peptides in b-sheets, is counterbalanced by the still poor understanding of the higher organization of b-sheets within the fibril and overall fibril/fibril associations. The assembly pathway and basis of amyloid toxicity may well depend on these higher-order structural features. For example, significant evidence points to association between sheets as the rate limiting step in fibril assembly, and a critical metal binding site has now been identified that involves residues from different individual sheets. Here we review experiments that are identifying some of the issues associated with sheet-sheet association by investigating simple model peptides derived from the central core of the Ab peptide implicated in Alzheimer’s Disease. These peptides transition between fibril/ribbon/nanotube morphologies in response to assembly conditions, laying the foundation for understanding the folding landscape for these higher order assemblies, revealing potential targets for therapeutic intervention, and opening strategies for the design of highly ordered peptide self-assembled microscale morphologies. Co-Authors: Jijun Dong, Kun Lu, Anil K. Mehta, Yan Liang, W. Seth Childers, and James A. Simmons

1:10 Close of TSE Conference


http://www.healthtech.com/2007/tse/day2.asp

SEE STEADY INCREASE IN SPORADIC CJD IN THE USA FROM
1997 TO 2006. SPORADIC CJD CASES TRIPLED, with phenotype
of 'UNKNOWN' strain growing. ...


http://www.cjdsurveillance.com/resources-casereport.html


There is a growing number of human CJD cases, and they were presented last week in San Francisco by Luigi Gambatti(?) from his CJD surveillance collection.

He estimates that it may be up to 14 or 15 persons which display selectively SPRPSC and practically no detected RPRPSC proteins.

http://www.fda.gov/ohrms/dockets/ac/06/transcripts/1006-4240t1.htm


http://www.fda.gov/ohrms/dockets/ac/06/transcripts/2006-4240t1.pdf

[Docket No. FSIS-2006-0011] FSIS Harvard Risk Assessment of Bovine
Spongiform Encephalopathy (BSE)


http://www.fsis.usda.gov/OPPDE/Comments/2006-0011/2006-0011-1.pdf

[Docket No. 03-025IFA] FSIS Prohibition of the Use of Specified Risk
Materials for Human Food and Requirement for the Disposition of
Non-Ambulatory Disabled Cattle

03-025IFA
03-025IFA-2


http://www.fsis.usda.gov/OPPDE/Comments/03-025IFA/03-025IFA-2.pdf

THE SEVEN SCIENTIST REPORT ***


http://www.fda.gov/ohrms/dockets/dockets/02n0273/02n-0273-EC244-Attach-1.pdf

Full Text

Diagnosis and Reporting of Creutzfeldt-Jakob Disease

Singeltary, Sr et al. JAMA.2001; 285: 733-734.


http://jama.ama-assn.org/cgi/content/full/285/6/733?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=dignosing+and+reporting+creutzfeldt+jakob+disease&searchid=1048865596978_1528&stored_search=&FIRSTINDEX=0&journalcode=jama

http://www.neurology.org/cgi/eletters/60/2/176#535


BRITISH MEDICAL JOURNAL


BMJ


http://www.bmj.com/cgi/eletters/319/7220/1312/b#EL2


BMJ


http://www.bmj.com/cgi/eletters/320/7226/8/b#EL1

Terry S. Singeltary Sr.

P.O. Box 42

Bacliff, Texas USA 77518




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