New Law Could Open Up Lab Books
Awarded NIH grants already in public domain, expert says
NINDS solicitation for prion experiments on primates
Jan 1999 new book: "Prions: Molecular and Cellular Biology" D Harris, ed., Horizon Press
PrP expression in B lymphocytes
Screening of sheep for BSE: further details
Host range of CWD is altered on passage in ferrets
Jocelyn Kaiser 10 Dec 98 Science[The thrust of this article is that the new law is bad, that is, competition and careerism among researchers is vastly more important than scientific progress on disease or serving patients. This website disagrees. Over and over in prion research, we have seen labs holding up the release of data for 18-24 months for the sole purpose of hurting other researchers. Over 450 people die of CJD in the United States alone during this time frame. The public pays for this research and has a right to its prompt release. This web site will be among the first to take advantage of the new law requiring full and open disclosure of current research. -- whatever form this might take. webmaster]
Tucked into last month's giant spending bill is an unwelcome message to academic researchers: Their data may be fair game for anyone who asks.
A few words in the section funding the White House Office of Management and Budget (OMB) would extend the federal Freedom of Information Act (FOIA)--a 1966 law to make government more accountable to the public--to extramural grants. That opens the possibility that scientists at universities, hospitals, or nonprofit organizations might have to turn over the contents of their computer disks of data, or even their lab notebooks, in response to a request to the agency that funded their work. "We're all very troubled," says Wendy Baldwin, deputy director for extramural research at the National Institutes of Health.
The language, inserted by Senator Richard Shelby (R-AL), says OMB must revise its rules for administering federally funded research grants "to require Federal awarding agencies to ensure that all data produced under an award will be made available to the public through the procedures established under the Freedom of Information Act." Private parties requesting the data may be charged "a reasonable user fee." At present, only funding agencies themselves can ask grantees for data. The new language implies that federally funded researchers must turn over their data to anyone who files a FOIA request. "The taxpayers have a right to much of this information," says Shelby.
The roots of the provision go back to last year's controversy over new Environmental Protection Agency air pollution rules for fine soot. Industry groups and some legislators demanded that university researchers hand over their data on the health effects of the pollution, leading to an unsuccessful legislative proposal requiring public data release (Science, 8 August 1997, p. 758). This year, a separate funding bill containing a request for OMB to study the issue was vetoed by President Clinton for unrelated reasons, leading Shelby to insert more direct language in the massive spending bill passed before Congress adjourned (Science, 23 October, p. 598).
Some observers are outraged that this sweeping measure was passed with no hearings. "It is ironic that a provision described as a sunshine provision needed to be tucked into a 4000-page bill in the dead of night," says Representative George Brown (D-CA), ranking Democrat on the House Science Committee. And some health researchers are worried that the directive will give industry a new tool to stall health regulations. "If past history is any indication, vested interests will misuse [this provision] to discredit valid research results they don't like and to harass the researchers doing the work," says New York University environmental scientist George Thurston, whose studies helped form the basis for EPA's contested regulations.
Others worry that raw data will be requested before it has been analyzed and peer reviewed. "It's important that we have processes in place for data sharing, but this basically opens the door to anyone's data without any filters," Baldwin says. University researchers say that privacy and proprietary data might also be compromised.
The question facing OMB now is how to implement the new requirement. Agency officials say they hope to be consulted in a process likely to take many months.
William H. Goldwater Director of the Extramural Programs Management Office of the NIH Office of Extramural Research 1984 to 1993. Bethesda, MD"Jocelyn Kaiser's article "New law could open up lab books" (News of the Week, 6 Nov., p. 1023) presents a good picture of the perceived threat to the integrity of research laboratory and clinical data that could be wrought by the new law that would "require Federal awarding agencies to ensure that all data produced under an award [grant] will be made available to the public through the procedures established under the Freedom of Information Act [FOIA]." It is important to recognize, however, that the FOIA and subsequent court decisions establish protections that were used for many years when the National Institutes of Health (NIH) dealt with FOIA issues and policies, and that can still be helpful in guarding against disclosures.
First, the act provides several exemptions from disclosure, of which three covered most circumstances where NIH deemed it better not to apply mandatory disclosure (1):
1) "trade secrets and commercial or financial information obtained from a person and privileged or confidential";
2) "interagency or intra-agency memorandums or letters which would not be available by law to a party other than an agency in litigation with the agency"; and
3) "personnel and medical files and similar files the disclosure of which would constitute a clearly unwarranted invasion of personal privacy."
Second, besides these and related exemptions from mandatory disclosure, various court cases have protected against disclosure of peer review evaluations to third parties. Supreme Court decisions have protected data from release when such release would harm the competitive position of the person supplying those data, and have denied third parties access, leaving it to the awarding NIH institute to determine whether to release certain data in a grant-supported clinical trias, even though the institute had earlier released those data to an appropriate scientific organization to confirm the validity of the findings and conclusions.
In all cases, NIH established an advantageous system under which persons requesting information had to specify the information or data requested to avoid "fishing expeditions," for instance, and sought advice from the persons supplying those data or information as to whether they wished to withhold parts of the requested data from disclosure under one or more of the allowable exemptions. Subsequent negotiations usually yielded some degrees of flexibility in releasing the information, at minimal risk or discomfort to the persons supplying it.
Adaptations of the above and other restraints in the FOIA and in niceties of responses should serve to protect government agency grantees, contractors, and intramural researchers against undue hardships and agony, as otherwise perceived by some who now face this new and sudden intrusion into their research lives. Time may have brought some subsequent changes into the basic act and corresponding agency regulations, and careful attention may still forestall the catastrophe that some researchers and their institutions now envision."
Bill Goldwater, former director of the Extramural Programs Management Office at NIH wrote the webmaster on 11 Dec 98 saying:"Awarded NIH grant applications have always been available to requesters who identify those applications by number, or principal investigator/ institution, etc. Certain private/ proprietary information is often omitted, e.g. personnel salaries or other budget items, and scientific-technical information unique and private to the investigator/ institution, disclosure of which to third parties could place those third parties at an unfair advantage in competing with the original grantee or others for subsequent grants or contracts.
NIH also used to publish annual compendia of grant and contract recipients, catalogued by state and city. They also used to publish much more complete catalogues of grant and contract information by subjects, e.g., mad cow disease or related neurological or viral topics. These used to be available in book form but now might well be on-line in some handy form.
Here is how to approach NIH. Don't write to 'NIH grant administrator' or to ' GrantsInfo', which has only general information about grant processes, and no grant files. You should address the chief extramural officials of the institutions, National Institute of General Medical Sciences, National Institute of Neurological Diseases and Stroke, and National Institute on Aging..
Go the NIH opening web pages and locate each of those three institutions. If any have an organizational chart or list, try to identify the associate or deputy director of their extramural programs, or similar title. Write directly to him/her at that institutions. Try also to get exact addresses, and even email addresses, which are available from the NIH listings of those details in a mail and phone index featured among the items on the NIH home page. If you cannot find such name(s), at least try to identify the director of each institutions and write to him/her. If you cannot identify that person, write to the title, e.g., Director, National Institute of....., with the closest mail address you can find, and leave out the greeting in the letter, e.g., Dear Dr Jones.
When you do write, be sure to identify the pertinent grant numbers for that institutions, the grantee institutions, and principal investigators, and ask for a copy of that/ those awarded applications, at least as much as is properly available under provisions of the Freedom of Information Act. All these details and phraseologies should help them understand that you know whereof you speak and thus better lead to prompt action. You may wish to mention briefly your general motivation for wishing to see those applications, although that is not essential to your request. "
NIH offers nothing whatsoever about awarded grants on its huge web site. NINDS fails to provide any contact information whatsoever, not even an email for the site creator. On 10 Dec 98, I received rude and uninformative responses from both NINDS and NIH to a courteous letter for basic information about grants awarded in past years for prion research. Surely they track grants that they have awarded over the years in a database -- why not just put the database on the web?
In the past, grantees were required to acknowledge their sources of support at the end of published articles. This enabled tracking of what got done during the course of the grant as some versions of Medline carried this data. Today, especially with PNAS, government and university researchers are not listing specific grants or even the funding agency. This places an additional burden on individuals requesting information as there is no grant number.
Here are a few preliminary results on grant numbers, foundation EINs (id number for grant programs overseen by IRS), and biotech firms [no disclosure requirements] relevent to prion research:
National Institutes of Health: AG12925 AG16462 AG 10770 AG08967 AG02132 AG10770 GM39247 NS14069 NS22786 "GM" in a grant number refers to the National Institute of General Medical Sciences. "AG" is the National Institute on Aging. "NS" is National Institute of Neurological Diseases and Stroke. Sherman Fairchild Foundation [no web] EIN:131951698 Bernard Osher Foundation EIN: 942506257 G. Harold and Leila Y. Mathers Foundation [no web presence] EIN: unknownAmerican Health Assistance Foundation
"AHAF is one of America's leading supporters of scientific and medical investigations into Alzheimer's disease, glaucoma, heart disease, and stroke. Founded in 1973, AHAF has since awarded more than $38 million in research grants to the most promising scientific minds around the world. AHAF is currently supporting a total of 37 research efforts at some of the most respected universities, hospitals and medical centers across the country: 15 projects funded through Alzheimer's Disease Research; 16 through National Glaucoma Research; and 6 through the National Heart Foundation."
David A. Westaway, Ph.D. Centre for Research in Neurodegenerative Diseases Toronto, Ontario, Canada Transgenic Mice Expressing the Ch.1 FAD Gene Presenilin II Grant Period: April 1, 1997 - March 31, 1999 Stanley B. Prusiner, M.D. University of California San Francisco Inducible Prion Diseases Caused by Artificial Mutations AHAF Alzheimer's Disease Research Grants Scientific Review Committee Advisors:› Stanley B. Prusiner, M.D.› Pierluigi Gambetti, M.D.
King of Prussia PA Phone: 610-878-4693"Centeon is the global leader in the plasma protein industry, providing an unprecedented range of innovative, high quality therapies and unique support services to patients worldwide. Centeon is dedicated to the research and development of plasma proteins and emerging technologies. Centeon was formed through a joint venture of Hoechst AG and RhŔne-Poulenc Rorer Inc."
Attachment IA, Statement of Work„LCNSS NINDSSECTION C - DESCRIPTION/SPECIFICATION/WORK STATEMENT
ARTICLE C.1. BACKGROUND
This solicitation is a recompetition of an ongoing contract for animal care and housing support services to the National Institute of Neurological Disorders and Stroke (NINDS) sponsored long-term study of "slow, latent and temperate infections of the nervous system caused by conventional viruses and the abnormal isoform of the precursor protein of amyloid referred to as the prion protein (PrPres) conducted in chimpanzees and Old World and New World nonhuman primates. The University of Southwestern Louisiana, New Iberia Research Center, has provided the animal housing, veterinary care and brain biopsy/autopsy support and facilities which have made possible these discoveries. This support service at their New Iberia Research Center has been conducted for more than 30 years under contract and for the past three years under contract NO1-NS-5-2323. Included in this proposal is the establishment; veterinary care and housing and technical support of a rhesus monkey breeding colony (specific pathogen free) to serve the research requirements of NINDS Intramural scientists.
The solicitation requests proposals for the continuation of required animal colony management support services to long term Division of Intramural Research (DIR), NINDS, NIH, studies that have been actively pursued for more than three decades at the New Iberia Research Center. Initially, the objective and goal of the research was to establish the etiology and the pathogenesis of kuru. However, it was early recognized that establishing the etiology of kuru and determining the pathogenesis of this rare disease would serve as a model for additional subacute progressive degenerative diseases of the nervous system of humans and animals which may have a similar etiology and such has been the case.
The results of these studies to date have established a new group of infectious diseases which have been identified as the transmissible spongiform encephalopathies (TSEs). In humans they are: kuru, Creutzfeldt-Jakob disease (CJD), new variant Creutzfeldt-Jakob disease (nvCJD), Gerstmann-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). In animals the diseases are: scrapie, transmissible mink encephalopathy (TME), chronic wasting disease of deer and elk (CWD), feline spongiform encephalopathy (FSE) and bovine spongiform encephalopathy (BSE).
The only macromolecule thus far associated with infectivity is a sialoglycoprotein, designated a prion protein (PrP) which is partially resistant to treatment with the enzyme proteinase-K and is designated PrPres. It is derived from a normal cellular protein common to all mammals which undergo a posttranslational modification resulting in a configurational change (unfolding) and shifts from an alpha-pleated to a beta-pleated structure. The underlying cause of the posttranslational modification is unknown as is the role of the normal cellular protein.
In addition to the discovery of this new group of transmissible diseases has been the demonstration that mutations on the prion protein gene which occur in an autosomal dominant genetic form are responsible for about 10% of CJD cases, 100% of GSS cases, and 100% of FFI cases. This is the first and only evidence that a disease can both be infectious and genetic in origin. Considerable controversy surrounds the theory that PrPres is the sole cause of the TSEs with other scientists suggesting either a conventional virus, a virino, a viroid or a protein-x component.
These theories have resulted in a redefinition of goals and objectives which replace the largely primary transmission studies which previously formed the basis of this contract. Moreover, the epidemic of bovine spongiform encephalopathy in the United Kingdom resulting in the death of more than 170,000 cattle and the description of so-called new variant Creuzfeldt-Jakob disease in humans probably associated with the disease in cattle has caused a redefinition of goals and objectives. Finally, significant concern both nationally and internationally has been raised concerning the safety of blood and blood derivatives particularly concerning possible contamination with new variant CJD.
Specific Goals and Objectives:
The following types of studies on the spongiform encephalopathies and other neurological diseases:
1. To better determine the pathogenesis of the spongiform encephalopathies in order to isolate and identify the mechanism underlying the posttranslational modification and configurational change of the host cell associated amyloid precursor protein (normal PrP).
2. To determine the safety of human blood supply in the United States through the in vivo testing of human blood and blood derivatives derived from patients with spongiform encephalopathies. This study is of national and international urgency.
3. To determine when in the course of illness infectivity can be detected in human blood and in which blood derivatives does it occur.
4. If infectivity is found in blood what cells serve as carriers of the infectious agent and can this be removed by leucodepletion. This must be done in chimpanzees using human leukocytes.
5. A monoclonal antibody has been reported to distinguish between PrP and PrPres. Can this antibody be used in a diagnostic test such as antibody-antigen capture in blood?
6. Analysis of species specificity and studies of interactions between heterologus PrP molecules which block infectivity.
7. Analysis of strain specificity at the molecular level.
8. Testing of drug candidates.
9. Susceptibility of chimpanzees fed head meat and brain/spinal cord from cattle with BSE.
10. Development and improvement of diagnostic test.
This competitive renewal of the existing contract is proposed for a five year period during which time there will be a phase out of Old World monkeys currently on hand and a reduction of New World monkeys that have been on long term studies and which now must be reused/replaced. The rhesus monkey breeding colony will continue as will the closed chimpanzee colony.
* Copies of reprints of the results thus far published may be requested from:
Dr. Clarence J. Gibbs, Jr., Chief, Laboratory of Central Nervous System Studies, Division of Intramural Research, National Institute of Neurological Disorders and Stroke, Building 36, Room 4A-05, 36 Convent Drive MSC4122, Bethesda, Maryland 20892-4122.
ARTICLE C.2. STATEMENT OF WORK
... Proposed Number of Animals to be Housed Under New Contract for TSE research
Chimpanzees 57 African Green 3 › Capuchin 38 › Cynomolgus 10 › Pigtail 2 › Squirrel 112Gang caging of apes will be permitted provided that there is access to combined indoor/outdoor runs. Outdoor gang caging of Old World and New World monkeys according to species in corn-crib type structures equipped with resting platforms and perches will be permitted provided that the geographic location is: (1) isolated from the public; (2) enclosed with restricted admittance; and, (3) climatically acceptable year round to the welfare of the animals. Nonhuman primates shall be provided high protein, vitamin-enriches dry biscuits, fresh fruits, and green vegetables daily; vitamin supplements shall be administered as required. All animals shall have access to fresh water on a continuous twenty-four (24) hour/day basis. All caging and indoor/outdoor runs shall be thoroughly washed and disinfected daily. Unconsumed food shall be removed from all areas before the end of the normal working day. Horses shall be housed in individual pen-type stalls indoors in a facility that is free of flying insects. They shall be fed available commercial food of high quality as recommended by an on-site veterinarian.
Prior to the initiation of any experiment sponsored by the NINDS, an approved animal use protocol shall be on file with the Office of the NINDS Veterinarian, in accordance with MI 3040-2. The use of chimpanzees will be subject to the written approval of the PHS AIDS Animal Model Committee, currently known as the Animal Model Committee (AMC) or other designated NIH committee. Animals shall be identified by species, sex, tattoo number and ISIS number, and shall be bled for base line hematological, clinical chemistry, immunological studies, serum storage, and separated lymphocytes in DMSO storage. All nonhuman primates shall be skin-tested for tuberculosis at intervals recommended by current veterinary care standards prior to and at intervals following inoculation.
In addition to the general housing instructions prescribed earlier in this document, initiation of new NINDS-sponsored experiments in animals already in the colony may require modified housing, including separation of animals used from routine animal housing. The types of studies covered by this contract shall require housing of animals according to specific studies, e.g. human retroviruses separate housing, spongiform encephalopathies and other neurological disease separate housing. Other special conditions will be discussed with the Contractor prior to the initiation of such studies by the NINDS.
The physical condition of all animals shall be evaluated daily for the detection of early clinical signs of disease in response to the inoculum or to an intercurrent infection, trauma, or other cause. While well-trained animal care technicians are permitted to make these daily observations, animals showing any of the above conditions shall be immediately evaluated, treated as necessary, and followed daily by a veterinarian on the Contractorľs staff. The veterinarian shall be responsible for notifying the NINDS Project Officer at the first detection of such overt signs and shall keep the Project Officer fully informed as to the course of the animalľs illness/disease and response to treatment. Such animals shall be placed on the following "Special Observation Protocol":
... b. Criteria for Placement of Animals on Special Observation:
1. Spongiform encephalopathies and other neurological diseases.
(a) Changing in personality or attitude
(1) May be more withdrawn
(2) May be more aggressive
(3) May exhibit tremors and/or myoclonus
(4) May exhibit limb neglect
(b) Subtle progressive neurological signs, e.g. disorientation, paresis, ataxia
(1) Muscle spasms, tremors and/or incoordination
(2) Lower than normal activity
(3) Negative clinical and laboratory findings with presence of one or nore of the above criteria
(4) Any one or combination of the above criteria may be sufficient for placement of animal on special observation.
.... e. Cleanup
1. Disposal of carcass and decontamination.
(a) Apes and monkeys„incinerate remains.
(b) All instruments contaminated during the procedure shall be first autoclaved in distilled H2O a minimum of 60 minutes before final cleaning process is accomplished.
(c) All disposable materials used shall be carefully placed in plastic bags and taken to incinerator immediately.
(d) All surfaces in contact with the animal or tissues thereof must be cleansed with 1N NaOH or undiluted sodium hypochlorite (bleach).
(e) Necropsy room floor and lab bench tops must be cleansed with bleach solution.
(f) All table surfaces shall be decontaminated with bleach and then cleansed with Micro Bact solution (8 ml per quart water).
webmaster 11 Dec 1998. More extensive comments will appear upon receiving a review copy promised by Horizon Press.We don't need another book of cannibal anecdotes or BSE policy fumbles. Fortunately, this one is scientific: "Prions: Molecular and Cellular Biology". A distinguished cast of veteran researchers providing 11 substantial sounding chapters. The subject cannot be exhaustively covered; instead there is an in-depth discussion of a particular topic within general areas such as CJD point mutations.
The issue with books like this is when did the authors turn in their chapters, did they integrate results from other labs with their own, and will the articles retain value as new results pour in to the journals. There is value however in trying to pull things together at a certain point in time. As this book is 'hot off the press' and the authors are all very active in the field, I would guess that this book is about as fresh as as a book can be (the publisher does not yet have a page count yet).
Hardcover (January 1,1999 but available now) Horizon Scientific Press ISBN: 1898486077 price 74.99 UK pounds or 130 US dollars Tel: 01953-601106 Fax: 01953-603068 Amazon.com also lists it for $125, early January deliveryHorizon very decently provides abstracts of each chapter on their web site, which is important for someone thinking about ordering a somewhat expensive book. Some quick initial observations: A general role for met 138 hamster/mouse species barrier (chap 3) may be a new development. There is a lot more going on with cell-free conversion (chap 2) than we have seen in journals. Is chap 6 just to be a memoire -- why 6 authors? Chap 7 sounds very familiar; chap 10 terribly dated. Surely there will be further news in chap 8; it does not take 14 authors to review residues 106-126. Chap 11 says there is a 3rd yeast prion.
The authorship is not evenly distributed. That is, there are many Italian, Swiss, and Rocky Mtn names but no Japanese. Prusiner's group is not represented (though several dozen reviews and after-dinner speeches probably suffice) nor is NIH. There is only so much that can be included before this becomes a $1000 book. Overall, I don't expect to be thrilled by every section but that there will be some useful information here and there:
Rudi Glockshuber, Simone Hornemann, Roland Riek, Martin Billeter, Gerhard Wider, Susanne Liemann, Ralph Zahn and Kurt WłthrichThe NMR structure of the recombinant mouse prion protein in the cellular form (mPrPC) contains a flexible disordered N-terminal segment of residues 23-125 and a novel globular fold with three a-helices and a small Ģ-sheet formed by the C-terminal residues 126-231. The presumed infectious agent of transmissible spongiform encephalopathies (TSEs), PrPSc, differs from PrPC at least with respect to the conformation of the segment 90-125, which is resistant to proteolysis in PrPSc but disordered and accessible in PrPC. At acidic pH the fragment mPrP(121-231) can adopt an alternative conformation with increased Ģ-sheet content, which may relate to the presumed conversion of PrPC to PrPSc in acidic endosomes. Biochemical experiments and analysis of the locations in the structure of mPrPC of the point mutation sites linked with familial human TSEs indicate that destabilization of PrPC cannot be a general mechanism favoring formation of PrPSc in inherited TSEs. Amino acid substitutions among the highly conserved mammalian prion proteins are clustered in four distinct regions in the three-dimensional structure of mPrP(121-231). Three of these regions represent potential surface recognition sites that may be involved in intermolecular interactions related to the species barrier for infectious transmission of TSEs.
Byron CaugheyIn transmissible spongiform encephalopathies (TSE) or prion diseases, the endogenous protease-sensitive prion protein (PrP-sen) of the host is converted to an abnormal pathogenic form that has a characteristic partial protease resistance (PrP-res). Recent studies with cell-free reactions indicate that the PrP-res itself can directly induce this conversion of PrP-sen. This PrP-res induced conversion reaction is highly specific in ways that might account at the molecular level for TSE species barriers, polymorphism barriers, and strains. Not only has this reaction been observed using mostly purified PrP-sen and PrP-res reactants, but also in TSE-infected brain slices. The conversion mechanism appears to involve both the binding of PrP-sen to polymeric PrP-res and a conformational change that results in incorporation into the PrP-res polymer.
Suzette A. Priola and Bruce ChesebroIn the transmissible spongiform encephalopathies (TSE), the conversion of the normal protease-sensitive host protein PrP-sen to an abnormal protease-resistant form, PrP-res, is a critical step in disease pathogenesis. Amino acid mismatches between PrP-sen and PrP-res can dramatically decrease the amount of PrP-res made and modulate the resistance to crossspecies transmission of TSE infectivity. In mouse scrapie-infected neuroblastoma cells, a hamster-specific methionine at postion 138 in the mouse PrP gene was found to inhibit the species-specific formation of mouse PrP-res. In vivo, PrP-sen and PrP-res molecules which mismatch at this position may have a reduced potential to form PrP-res. This might account for the difficulty in transmitting scrapie and bovine spongiform encephalopathy (BSE) to hamsters, as well as sheep scrapie to certain goats which have a methionine at the homologous residue (position 142 in goat PrP). Conversely, matching of amino acid residues at this position may facilitate cross-species transmission of TSE diseases such as BSE to new species.
David A. HarrisStudying PrPC and PrPSc in cell culture systems is advantageous because such systems contain all the organelles, membranes, and molecular cofactors that are likely to play an important role in the biology of the proteins. Using cultured cells expressing PrPC, we have discovered that this isoform constitutively cycles between the cell surface and an endocytic compartment, a process that is mediated by clathrin-coated pits and a putative PrPC receptor. We have also constructed stably transfected lines of CHO cells that express PrP molecules carrying mutations that are associated with familial prion diseases. The mutant PrP molecules in these cells are spontaneously converted to the PrPSc state, a phenomenon which has allowed us to analyze several key features of prion formation.
Piero Parchi, Sabina Capellari, Gianluigi Zanusso, Neena Singh, Pierluigi Gambetti and Robert B. PetersenThe prion protein is a normal cellular glycoprotein which, after conversion to a protease resistant pathogenic form appears to be the major, if not the only, component of the infectious agent known as the prion. Numerous strains of prions, differing in their incubation period and neuropathology, have been isolated in the same host genotype, indicating that prions carry information which is independent from the host. Molecular analysis of prion strains has recently focused on human prion diseases and support the view that the prion protein can specify disease phenotypes by differences in its conformation and glycosylation. The observation that approximately 10-15% of all human prion diseases are familiar and result from mutations in the prion protein gene provided a basis for pursuing the factors that favor, or result in, the de novo conversion of the prion protein to a pathogenic form. A cell culture model was established to study the effects of the pathogenic mutations on the metabolism of the mutant prion protein. One of the consistent changes found as a result of the pathogenic mutations clustered in the region of post-translational modification was an alteration of the glycoisoform ratio; an observation that also applies to prion protein derived from prion diseased brain samples.
Charles Weissmann, Alex J. Raeber, Doron Shmerling, Antonio Cozzio, Eckhard Flechsig and Adriano AguzziLinkage between the infectious scrapie agent and PrP was first established at the biochemical level and subsequently reinforced by genetic evidence. This led to the prediction that animals devoid of PrP should be resistant to experimental scrapie and fail to propagate infectivity which was then experimentally borne out, adding support to the "protein only" hypothesis. In addition, the availability of PrP knockout mice provided an approach to new lines of investigation.
Adriano Aguzzi, Thomas BlĒttler, Michael A. Klein, Alex J. RĒber, Ivan Hegyi, Rico Frigg, Charles Weissmann, and Sebastian BrandnerAlthough the prime role of PrP in prion diseases is undisputed, the mechanisms of brain damage, and those that control the affinity of the agent for the central nervous system, are unclear. These questions can be addressed by selectively expressing prnp in specific tissues of mice. Towards this goal, we have developed neurografting and bone marrow grafting strategies. Availability of mice overexpressing the prnp gene (which encodes the normal prion protein), along with prnp knockout mice, allows for selective reconstitution experiments aimed at expressing PrP in specific portions of the brain. Here, we summarize how such studies can offer insights into how prions administered to extracerebral sites can gain access to central nervous tissue, and into the molecular requirements for spongiform brain damage.
F. Tagliavini, M. Salmona, G. Forloni, P. Malesani, G. Giaccone, B. Canciani, R. McArthur, J. Lansen, P. Piccardo, S.R. Dlouhy, B. Ghetti, B. Frangione, F. Prelli, O. BugianiThe central event in prion diseases is the conformational conversion of the cellular prion protein (PrPC) into abnormal forms (PrPres) that have high content of (-sheet secondary structure and high tendency to form insoluble aggregates and amyloid fibrils. Amyloid formation occurs to the highest degree in Gerstmann-StrĒussler-Scheinker disease (GSS). Deposition of PrPres and PrP amyloid in the brain is accompanied by degeneration of neurons and activation of glial cells. Biochemical studies have shown that the GSS amyloid protein is a fragment of PrP, whose N-terminus is located within the octapeptide repeat region and C-terminus corresponds to residue (150.
Studies with synthetic peptides homologous to consecutive segments of this fragment indicate that the region spanning residues 106-126 is able to adopt different conformations in distinct environments, although has high propensity to form stable (-sheet structures and assemble into amyloid fibrils which are partially resistant to protease digestion. The peptide PrP106-126 causes neuronal death by apoptosis, hypertrophy and proliferation of astrocytes, and activation of microglial cells in vitro. These data suggest that the region corresponding to residues 106-126 may be critical for the structural transition PrPC>PrPres, and that cerebral accumulation of peptides including this sequence may be responsible for the tissue changes that occur in prion diseases. The role of PrP in the pathogenesis of these disorders is further supported by the observation that compounds able to interact with disease-specific PrP isoforms delay the accumulation of PrPres and PrP amyloid and the appearance of nerve cell degeneration and glial cell reaction in experimental scrapie.
Katherine Young, Pedro Piccardo, Stephen Dlouhy, Orso Bugiani, Fabrizio Tagliavini, and Bernardino GhettiThe genetic prion diseases are autosomal dominant neurodegenerative diseases associated with mutations in the prion protein gene (PRNP). As with the sporadic and transmitted prion diseases, the genetic prion diseases are thought to result from a conformational change in the prion protein (PrP). These diseases are marked at the biochemical level by the accumulation of PrP that is in an abnormal conformation.
There are 5 classical genetic prion diseases: Creutzfeldt-Jakob disease (CJD), Gerstmann- StrĒussler-Scheinker disease (GSS), fatal familial insomnia (FFI), prion protein cerebral amyloid angiopathy (PrP-CAA), and atypical dementia. Approximately 15% of CJD cases are familial and 6 different point mutations have been found in CJD. All GSS cases are genetic and 6 GSS mutations have been found. FFI is caused by a mutation at PrP residue 178 and PrP-CAA is caused by a stop codon mutation at residue 145. Heterogeneous phenotypes have been found associated with mutations at PrP residues 171 and 183 and with 5, 6, 7, 8 and 9 insertions in the PrP octapeptide repeat region.
The classic CJD phenotype is one of rapidly progressing dementia often accompanied by myoclonus and periodic synchronized discharges (PSDs) in the electroencephalogram (EEG). In contrast, GSS is a more slowly progressing disease in which ataxia is the predominant sign and dementia usually occurs at a later stage. The pathologic phenotype of CJD consists of spongiform changes, neuronal loss, and gliosis. The hallmark of GSS is the presence of unicentric and multicentric amyloid deposits immunoreactive with antibodies to PrP. Amyloid deposition is accompanied by gliosis, neuronal loss, and occasionally, spongiform degeneration. To these clinical and pathologic criteria we can now add biochemical (immunoblot) and molecular genetic (PRNP mutation screen analysis) assays. As new techniques are applied and more familial cases are identified, more exceptions to the classic CJD and GSS definitions are found, as can be seen in some of the descriptions below.
It is not unusual for a patient with a genetic prion disease to be diagnosed at the onset of symptoms as having olivopontocerebellar atrophy, Huntington disease, or amyotrophic lateral sclerosis (ALS). Several studies have been done to search for pathologic PrP in patients with dementia or other neurodegenerative diseases. Although several PRNP mutations have been found in these patients, in general there is not a significant number of patients with prion diseases who are hidden in other diagnostic groups.
In PRNP there are both pathogenic mutations and benign polymorphisms. Formally speaking, a mutation is defined as a change in a gene. The DNA mutation can be a "silent" mutation which does not change the protein, it can lead to a non-pathogenic amino acid change, or it can lead to a pathogenic amino acid change. A site is defined as polymorphic when there are 2 or more alleles, each at a frequency of at least 1%, in a given population. Since the genetic prion diseases are rare, and therefore a pathogenic mutation does not exist at polymorphic frequencies, we will use the term mutation when we refer to a disease-causing change and polymorphism when we speak about a benign change. However, it should be kept in mind that some of the PRNP mutations found in prion disease patients have not been formally linked to the disease and so may turn out to be benign mutations, or "rare polymorphisms".
Dominique DormontBSE epidemic has started in the UK in the middle eighties. Today, more than 170,000 cattle have been affected by this disease in Great Britain, and only few cases have been reported outside of British islands. BSE agent has particular biological and physico-chemical properties that are distinct from those of known scrapie agents.; in particular, it can resist to certain procedures that inactivates scrapie agent. Moreover, the oral route is almost as efficient as the parental route for infection, suggesting specific biological properties. The origin of the spread of BSE in the UK is due to the meat and bone meal (MBM) which manufacturing process has been changed in the late seventies and early eighties, the new processes being unable to inactivate the BSE agent. Ban of MBM from the feeding of ruminants has demonstrated its efficacy in reducing the incidence of BSE. A new form of Creutzfeldt-Jakob disease has appeared in the UK and France recently; this new variant has specific characteristics at both clinical and molecular levels. Several experimental data indicate that BSE agent could be the origin of this new form of Creutzfledt-Jakob disease.
Daniel C. Masison, Herman K. Edskes, Marie-Lise Maddelein, Kimberly L. Taylor and Reed B. Wickner[URE3] and [PSI] are two non-Mendelian genetic elements discovered over 25 years ago and never assigned to a nucleic acid replicon. Their genetic properties led us to propose that they are prions, altered self-propagating forms of Ure2p and Sup35p, respectively, that cannot properly carry out the normal functions of these proteins. Ure2p is partially protease-resistant in [URE3] strains and Sup35p is aggregated specifically in [PSI] strains supporting this idea. Overexpression of Hsp104 cures [PSI], as does the absence of this protein, suggesting that the prion change of Sup35p in [PSI] strains is aggregation. Strains of [PSI], analogous to those described for scrapie, have now been described as well as an in vitro system for [PSI] propagation. Recently, two new potential prions have been described, one in yeast and the other in the filamentous fungus, Podospora .
Recent Medline abstracts of interest
Nat Med 1998 Dec;4(12):1429-33 Klein MA, Frigg R, Raeber AJ, Flechsig E, Hegyi I, Zinkernagel RM, Weissmann C, Aguzzi APrion diseases are typically initiated by infection of peripheral sites, as in the case of bovine spongiform encephalopathy, new variant Creutzfeldt-Jakob disease, kuru and most cases of iatrogenic Creutzfeldt-Jakob disease. In mouse scrapie, prion infectivity accumulates in lymphoid organs, and the absence of mature B lymphocytes prevents peripherally administered prions from inducing central nervous system disease. We have now assessed whether expression of the cellular prion protein, PrPc, is required for B lymphocytes to mediate neuroinvasion. We found that repopulation of SCID and Rag-1(-/-) mice with fetal liver cells from either PrP-expressing or PrP-deficient mice and from T-cell deficient mice, but not from B-cell deficient mice, is equally efficient in restoring neuroinvasion after intraperitoneal inoculation of scrapie prions. These results indicate that cells whose maturation depends on B cells or their products, such as follicular dendritic cells, may enhance neuroinvasion. Alternatively, B cells may transport prions to the nervous system by a PrP-independent mechanism.
Nat Med 1998 Dec;4(12):1369-70 Collinge J, Hawke S commentary on abstract above
Am J Pathol 1998 Dec;153(6):1695-700 Ikeda K, Kawada N, Wang YQ, Kadoya H, Nakatani K, Sato M, Kaneda K firstname.lastname@example.orgSuppression subtractive hybridization was used to clone genes associated with the activation of hepatic stellate cells and 13 genes were found to be dominantly expressed in activated stellate cells. Among them, one was identical to the 421-837th base pairs of cDNA sequence reported for rat prion-related protein (PrP). In cultured stellate cells, PrP mRNA expression increased in a time-dependent manner in parallel with smooth muscle (SM) alpha-actin mRNA expression. In situ hybridization demonstrated that PrP mRNA was localized in and around the fibrous septa of carbon tetrachloride (CCl4)-treated liver. Cellular PrP (PrPc) was produced by culture-activated stellate cells, and immunohistochemically detected in the fibrous septa of CCl4-damaged liver and sinusoidal linings of common bile duct-ligated liver, consistent with the localization of SM alpha-actin. Immunoelectron microscopy revealed that PrPc resided on the plasma membrane of stellate cells. These results indicate that PrP expression is closely related to stellate cell activation associated with fibrogenic stimuli.
J Biol Chem 1998 Dec 11;273(50):33107-33110 Pauly PC, Harris DAPrion diseases result from conformational alteration of PrPC, a cell surface glycoprotein expressed in brain, spinal cord, and several peripheral tissues, into PrPSc, a protease-resistant isoform that is the principal component of infectious prion particles. Although a great deal is known about the pathogenic role of PrPSc, the physiological function of PrPC has remained a mystery. Several lines of evidence have recently suggested the possibility that PrPC may play a role in the metabolism of copper. To further investigate the interaction of PrPC and copper, we have analyzed the effect of this metal ion on the endocytic trafficking of PrPC in cultured neuroblastoma cells. We report here that copper rapidly and reversibly stimulates endocytosis of PrPC from the cell surface. This effect may be physiologically relevant and suggests the hypothesis that PrPC could serve as a recycling receptor for uptake of copper ions from the extracellular milieu.
Molecular screening of sheep for bovine spongiform encephalopathy. Neurosci Lett 1998 Oct 23;255(3):159-62 Hill AF, Sidle KC, Joiner S, Keyes P, Martin TC, Dawson M, Collinge J[The full text which consists mainly of introduction and discussion. They sought first to look at archival scrapie strains in the 5 common sheep prion genotypes. Unbelievably, hardly any material was saved over decades of research and this mostly from the cerebral cortex, not cerebellum. What they got from NPU (archival, pre-BSE) and CVL (current field isolates) was:
136 154 171 1964-69 (6) VV RR QQ AA RR QQ AV RR QQ 1989-93 (7) VV RR QQ AA RR QQ AV RR QQ AA HH QQ experimental (5) AV RR QQ AV RH QQ AA HH QQ AA RH QQ contemporary (16) VV RR QQ AA RR QQ AV RR QQ AA RH QQ AA RR QQ AA RH QQ AV RR RQThey note that the statutory BSE to ruminant ban came in 1988, that 0.5 gm orally suffices to transfer BSE to sheep [Foster JD et al. Vet Rec. 133 339-41 1993], and that perhaps 8 sheep strains can be serially propagated in inbred mice.
The only experiment was to compare fragment size and glycoform ratio of experimental BSE sheep to these archival and contemporary field cases. (Prionics monoclonal 6H4 was substituted in their usual procedure; this is not the notorious 15B3 but one with DYEDRY.. as epitope.) The result, subject to the limitations beyond the control of the authors, is experimental BSE in sheep could be readily distinguished from all 16 contemporary field cases, 7 recent field cases, and 6 archival samples. So there is no support for BSE originating in sheep nor for BSE having been transmitted to sheep; note sample sizes are very small.
The main feature of BSE in sheep is a low molecular weight [18k?] unglycosylated fragment. This is also seen in cattle BSE and nvCJD but has not been characterized further (like the 8k fragment of GSS). This fragment would be the basis of rapid and cheap screening sheep or cattle more extensively. They first intend to see if sheep BSE retains this distinct feature after serial passage in various sheep genotypes and breeds. Abstract:
Bovine spongiform encephalopathy (BSE) may have transmitted to sheep through feed and pose a risk to human health. Sheep BSE cannot be clinically distinguished from scrapie, and conventional strain typing would be impractical on a significant scale. As human prion strains can be distinguished by differences in prion protein (PrPsc) conformation and glycosylation we have applied PrP(Sc) typing to sheep. We found multiple Western blot patterns of PrP(Sc) in scrapie, consistent with the known scrapie strain diversity in sheep. Sheep passaged BSE showed a PrP(Sc) banding pattern similar to BSE passaged in other species [Collinge, J., Sidle, K.C.L., Meads, J., Ironside, J. and Hill, A.F., Nature, 383 (1996) 685-690], both in terms of fragment size following proteinase K cleavage and abundance of diglycosylated PrP. However, none of the historical or contemporary scrapie cases studied had a PrP(Sc) type identical to sheep BSE. While more extensive studies, including sheep of all PrP genotypes, will be required to fully evaluate these findings, these results suggest that large scale screening of sheep for BSE may be possible.
webmaster commentary 11 Dec 98Recall that K200K homozygotes could be important for testing gene dosage effects in humans and that this seems to be the only CJD locus where homozygotes have been recorded.
Looking at the full text of the K200K homozygous CJD paper, Gabizon et al. Am J Hum Genet 1993 Oct;53(4):828-35, the text states that there was "no substantial differences in the clinical course and age at disease onset were observed when patients were homozygous for K200K." [pg 832]. The original mutation seems to have occurred on an M129 allele as all K200 were found in this setting [thus no V129V E200K exists though M129V K200E is common].
However, only 1 homozygote was really studied, a 42 year old patient with M129M. Age of onset was, contrary to the text, anomalously early as the mean age of onset was 56 and the patient was the youngest of 18 M129M patients in the sample. [One case with age of onset 35 had M129V.] No clinical details or early history of the patient was provided.
Table 2 refers to two inferred ancestral homozygotes, one an individual with a negative spouse but 5/5 carrier children and a second with 3/3 carrier children. The authors write, "unfortunately, no tissue for genotyping from these probable homozygotes was available." [pg 830]. The odds of homozygosity in the parent are 1/32 and 1/8, by no means statistically signficant as dozens of families were considered.
It seems like the single K200K Libyan homozygote is the only known case of homozygous familial CJD. There may be others in Israel or Slovakia. These are worth a careful review for earlier age of onset etc. because many models envision mass-action driven equilibrium and gene dosage effects are clearly seen in mice.
Virology 1998 Nov 25;251(2):297-301 Bartz JC, Marsh RF, McKenzie DI, Aiken JMChronic wasting disease (CWD), a member of the transmissible spongiform encephalopathies (TSEs), was first identified in captive mule and black-tail deer in 1967. Due to the failure to transmit CWD to rodents, we investigated the use of ferrets (Mustela putorius furo) as a small animal model of CWD. The inoculation of CWD into ferrets resulted in an incubation period of 17-21 months on primary passage that shortened to 5 months by the third ferret passage.
The brain tissue of animals inoculated with ferret-passaged CWD exhibited spongiform degeneration and reactive astrocytosis. Western blot analysis of ferret-passaged CWD demonstrated the presence of PrP-res. Unlike mule deer CWD, ferret-passaged CWD was transmissible to Syrian golden hamsters (Mesocricetus auratus). Increasing the passage number of CWD in ferrets increased the pathogenicity of the agent for hamsters. This increase in host range of a field isolate on interspecies transmission emphasizes the need for caution when assessing the potential risk of transmission of TSEs, such as bovine spongiform encephalopathy, to new host species.