Colorado prion meeting in July
Prion Biosafety at UCSF
Tonsil test for nvCJD questioned
Progress on species barrier
AMYLOID AND OTHER ABNORMAL PROTEIN ASSEMBLY PROCESSESİ
July 13-18İ FASEB Copper Mtn, Colorado Anthony L. Fink, Chairİ Peter Lansbury, Co-chairİ 13 July. Keynote Addresses: Amyloid Diseases. D. Selkoe, B. Chesebro.İ 14 July. Methods for Analysis of Amyloid and Aggregregated Protein. A.L. Fink, D. Wemmer, C. Lieber, G. Millhauser, P. Fraser, H. Saibil.İ 15 July. b Sheet and Amyloid Fibril Structure. J. Nowick, S. Roderick. Abnormal Protein Folding. J. King, T. Baldwin, J. Kelly, R. Rudolf, W. Englander.İ 16 July. Accessory Proteins. S. Lindquist, A. Horwich, A. Gragerov. Mechanism of Amyloid Formation by A b. P. Lansbury, D. Teplow, R. Wetzel, M. Zagorski. 17 July. Mechanism for Prion Formation. B. Caughey, R. Glockshuber, M. Baldwin. Animal Models of Amyloid Diseases. A. Aguzzi, W. Soello, K. Hsiao.İ 18 July Amyloid as a Therapeutic Target. C. Post, M. Findeis, B. Solomon. İ On-site accommodation (includes registration, room, meals) Single Occupancy (1 paid registrant per unit)İİİİİİİİİİİİİİİ$680 Off-site accommodations (registration and meals only)İİİİİİ $450 Off-site participants are responsible for paying their own housing costs.
2 June 1997 Glenn A. Funk, Ph.D. Biosafety Officer UC San francisco 415-476-2097
Tom, we do indeed have some specific safety procedures in place for our prion research labs and animal facilities. I've only been at UCSF for six months but one of my first chores was to rewrite the campus biosafety manual, which hadn't been reissued since 1982!! I've just completed that task and will be happy to share the prion sections (or the entire manual, for that matter) with the Listees but first I'm going to ask the Prusiner lab folks to review those sections to make sure I've included the latest and greatest and got it all right in the process..
For your general info, we have at UCSF a suite of laboratories for studying prions wherein most operations are carried out under BSL2 physical containment with elevated safety practices more typical of a BSL3 lab. We also have a remote animal facility used primarily for breeding but which also contains several ABSL3 rooms in a barrier suite used for handling animals inoculated with materials from human prion disease. I believe most but not all of our work here is with CJD and scrapie
The following text is from the new version of the UCSF Biosafety Manual; it has been reviewed by Stan Prusiner and Darlene Groth, his senior technologist, and represents current UCSF policy and practice.
Transmissible spongiform encephalopathies are caused by a unique infectious agent called a prion, composed of a proteinaceous material devoid of detectable amounts of nucleic acid. Prions are unusually resistant to standard means of inactivation, including formaldehyde, ethanol and UV radiation. However, they can be inactivated by fresh household bleach, 1.0M sodium hydroxide (NaOH), 4.0M guanidine reagents, phenol and autoclaving. Procedures involving brain tissue from patients with neurological degenerative disorders (such as CJD and Alzheimer's disease) pose special challenges in reducing potential exposure to prions; such material should be handled with at least the same precautions as HIV+ or HBV+ [hepatitus B virus] human tissue.
The CDC classifies prions as Risk Group 2 agents requiring Biosafety Level 2 (BSL 2) containment. All researchers working with these agents are required by the University of California, San Francisco (UCSF) to have a valid Biological Use Authorization; Biosafety Committee approval must be granted before any research can be initiated.
In 1988, Cal-OSHA issued a "Special Order" to the University of California, San Diego outlining numerous safety and recordkeeping procedures to be used at that institution. The following current UCSF prion procedures have evolved from those Cal-OSHA edicts.
2. All tissues, infectious waste and instruments (e.g., specimen containers, knives, blades, cutting boards, and centrifuge tubes) used in the processing of such samples must be decontaminated by 1N NaOH or undiluted fresh household bleach (5.25% sodium hypochlorite) and autoclaving at 132o C for 4.5 hours (see Section C below for UCSF prion decontamination procedures).
3. Personnel must wear gloves and gowns while handling tissues which are potentially contaminated. All protective clothing must be removed before leaving the laboratory.
4. Sonication or homogenization of tissues must be performed in a properly certified Class II biosafety cabinet.
5. Microtome blades and knives used for cutting tissue must be cleaned with an instrument that does not put the hand or finger of the operator in or near contact with the blade.
6. Any skin contact with possibly infectious materials should be followed by washing with 1.0 N sodium hydroxide for two to three minutes, followed by extensive washing with water.
7. The Principal Investigator (PI) must contact the Office of Environmental Health and Safety (EH&S) in writing regarding spills and accidents which result in overt exposure to tissues. The report must include the following:
a. Specification of amount released, time involved, and explanation of procedures used to determine the amount involved.
b. Description of the area involved and the extent of employee exposure.
c. Report of medical treatment provided.
d. Corrective action taken to prevent the reoccurrence of the incident.
2. Upon death, retirement or other termination of the employee's employment or in the event the employer ceases to do business with a successor; records or notarized true copies must be forwarded to the Director of National Institute of Occupational Safety and Health (NIOSH).
3. Records must be provided upon request by representatives of the Chief and/or Director of NIOSH.
4. Any physician who conducts a medical examination must furnish the employer a statement of the employee's suitability for employment.
5. Access to the laboratory must be restricted to trained personnel when work is being conducted on tissue.
6. Personnel handling tissue must be trained in the following:
a. Nature of CJD
b. Route of transmission of CJD
c. Specific hazards associated with handling of the tissue.
2. Contaminated surfaces that can withstand the treatment are cleaned with 1.0N NaOH, allowing 5 minutes of contact time, followed by wipedown with 1.0N HCl, then thorough washing with clear water.
3. Contaminated surfaces that cannot withstand NaOH/HCl treatment are cleaned with 10% houshold bleach, allowing 10-15 minutes contact time, then washed with clear water.
4. Contaminated skin surfaces are washed with 1.0N NaOH or 10% bleach for 2-3 minutes, followed by rinsing with copious amounts of water. Splashes to the eye are rinsed with copious amounts of water or saline.
5. Contaminated dry waste is autoclaved at 1320 C for 4.5 hours, then discarded as solid waste (trash).
6. Sharps waste is autoclaved at 132ş C for 4.5 hours before being picked up as medical waste for incineration.
Note: This is a departure from the UCSF policy of not autoclaving sharps but is acceptable in this case because of the elevated risks associated with prion exposure.
Subhash C Arya
Centre for Logistical Research and Innovation, M-122 (of Part 2), Greater Kailash-II, New Delhi 110048, India
Sir--Hill and colleagues' (Jan 11, p 99)1 immunohistochemistry and western blot analysis on tonsillar tissue obtained at necropsy in a 35-year-old woman who had a diagnosis of new variant Creutzfeldt-Jakob disease (nvCJD) revealed abnormal replication of protease-resistant prion protein (PrP) in the tonsillar germinal centre. Apart from a tonsillar biopsy for an early clinical or preclinical diagnosis of CJD or nvCJD by immunohistochemistry and western blot analysis, it would be better to use aspirated core tissue from tonsils for these investigations.
During bacteriological analysis of recurrent tonsillitis in 34 patients, Timon and colleagues2 found that the qualitative and quantitative data from aspirated core tissue and excised tissue were similar. Furthermore, even in individuals who had early tonsillectomy lymphoid tissue could be localised by ultrasonography or computed tomography. Both techniques would guide the needle for aspiration of tonsillar tissue through the peroral route.
Cross-contamination or iatrogenic transmission of abnormal PrP from tonsillar tissue should not be ignored. Apart from a disposable tonsil-biopsy kit,1 surgical instruments meant for intervention in tonsils, epiglottis, soft palate, tongue, pharynx, and nasopharynx should be sterilised by autoclaving at 132C for 5 h or by use of 2N NaOH for about 12 h. Sterilisation would interrupt inadvertent spread of abnormal PrP-laden germinal centres in lymphoid tissue in the region.
1 Hill AF, Zeidler M, Ironside J, Collinge J. Diagnosis of new variant Creutzfeldt-Jakob disease by tonsil biopsy. Lancet 1997; 349: 99100.
2 Timon CI, Cafferkey MI, Welsh M. Fine-needle aspiration in recurrent tonsillitis. Arch Otolaryngol Head Neck Surg 1991; 117: 65356.
David J Evans:
Imperial College School of Medicine at St Mary's, London W2 1PG, UK
Sir--The title used by Hill and colleagues1 somewhat oversells their findings because they report protease-resistant prion protein (PrP) in tonsillar tissue obtained in a single necropsy case. The frequency of tonsillar positivity and its temporal relation to brain disease is not yet established, though it is true that Schreuder's work2 shows that in sheep with scrapie tonsillar involvement occurs during the early stages of the disease.
The presence of PrP antigen in lymphoid follicles is of interest; it would have been useful to confirm by a monoclonal antibody that the dendritic reticulum cells were involved. As the investigators must be aware, dendritic reticulum cells are present in various sites, but are conspicuously absent from the central nervous system. Thus, direct migration of these cells is unlikely. They are however noted scavengers of circulating antigen, which raises the question of whether antigen is present in the blood. Now that a monoclonal antibody technology for PrP is available, examination of the immunohistochemistry of other sites that contain dendritic reticulum cells is needed.
1 Hill AF, Zeidler M, Ironside J, Collinge J. Diagnosis of new variant Creutzfeldt-Jakob disease by tonsil biopsy. Lancet 1997; 349: 99100. 2 Schreuder BEC, van Keulen LJM, Vromans MEW, Langeveld JPM, Smits MA. Preclnical test for prion diseases. Nature 1996; 381: 563.
John Collinge, Andrew F Hill, James Ironside, Martin Zeidler Neurogenetics Unit, Imperial College School of Medicine at St Mary's, London W2 National CJD Surveillance Unit, Western General Hospital, EdinburghSir--We are grateful for Arya's comments, but are concerned that tonsillar-tissue aspiration may not yield sufficient tissue for Western blot analysis, which can be a technically difficult procedure that requires a series of blots to achieve a satisfactory result. It seems unlikely that aspiration would be helpful for immonocytochemistry because the germinal-centre architecture would not be well-preserved in all cases. However, tonsillar-tissue aspiration could be a useful adjunct in patients who had undergone earlier tonsillectomy and in whom little remaining tissue was available for biopsy.
With respect to the issue of inadvertent iatrogenic transmission of new variant Creutzfeldt-Jakob disease (nvCJD), it must be remembered that, at present, the incidence of nvCJD is low. It is impossible to estimate how many individuals might be incubating nvCJD and at what stage infectivity would be present in tissues. Guidelines already exist for the handling and decontamination of surgical instruments used on patients with suspected CJD,1 and are currently under revision in light of recent information on nvCJD.
We accept Evans' point that our finding was preliminary on a single necropsy case and that further research is needed to determine the clinical usefulness of this investigation. However, since such tissues are not routinely taken at necropsy, we felt it important to report our findings at this early stage. Analysis of several further cases and an extensive control series is now in progress. Further studies to characterise the immunophenotype of cells positive for protease-resistant prion proteins in the tonsil in nvCJD are also underway. We also aim to investigate other tissues in which dendritic reticulum cells are present. As Evans suggests, interaction between the immune system and peripheral nervous system may be important in prion spread to the central nervous system.2
1 Precautions for work with human and animal transmissible spongiform encephalopathies, Advisory Committee on Dangerous Pathogens. London: HM Stationery Office, 1994.
2 Aguzzi A. Neuro-immune connection in spread of prions in the body? Lancet 1997; 349: 74243.
Typing prion isoforms Nature 386 (6622): 232-234 (Mar 1997)[LETTER] Parchi P, Capellari S, Chen SG, ..., Kretzschmar H Biochemical typing of scrapie strains Nature 386 (6625): 564 (Apr 1997) Somerville RA, Chong A, Mulqueen OU, Birkett CR, Wood SC, Hope J
Proc. Natl. Acad. Sci. USA Vol. 94, pp. 4931-4936, May 13 1997 free fulltext online Alex Bossers, Peter Belt, Gregory Raymond, Byron Caughey, Ruth de Vries, and Mari A. SmitsWebmaster commentary: The authors seem to have a working in vitro test of the species barrier, here taken as an intra-species polymorphism barrier, that allows various prion recruiter/recruitee permutations to be tested fairly rapidly. The data seems with changes in the prion/prion dimer interface driving some of the subtleties of transmission at the molecular level. It seems quite possible to test cow-to-sheep and possibly cow-to-human using these same methods.
Prion diseases are natural transmissible neurodegenerative disorders in humans and animals. They are characterized by the accumulation of a protease-resistant scrapie-associated prion protein (PrPSc) of the host-encoded cellular prion protein (PrPC) mainly in the central nervous system. Polymorphisms in the PrP gene are linked to differences in susceptibility for prion diseases. The mechanisms underlying these effects are still unknown. Here we describe studies of the influence of sheep PrP polymorphisms on the conversion of PrPC into protease-resistant forms. In a cell-free system, sheep PrPSc induced the conversion of sheep PrPC into protease-resistant PrP (PrP-res) similar or identical to PrPSc. Polymorphisms present in either PrPC or PrPSc had dramatic effects on the cell-free conversion efficiencies. The PrP variant associated with a high susceptibility to scrapie and short survival times of scrapie-affected sheep was efficiently converted into PrP-res. The wild-type PrP variant associated with a neutral effect on susceptibility and intermediate survival times was converted with intermediate efficiency. The PrP variant associated with scrapie resistance and long survival times was poorly converted. Thus the in vitro conversion characteristics of the sheep PrP variants reflect their linkage with scrapie susceptibility and survival times of scrapie-affected sheep. The modulating effect of the polymorphisms in PrPC and PrPSc on the cell-free conversion characteristics suggests that, besides the species barrier, polymorphism barriers play a significant role in the transmissibility of prion diseases.
Several PrP polymorphisms of humans have been associated with incidence, susceptibility, and pathology of the disease (1, 8). For sheep, eight mutually exclusive PrP polymorphisms have been described (9-15), resulting in nine different allelic variants. The allelic variants with polymorphisms at codons 112, 137, 141, 154, or 211 are rare and have not been significantly associated with any disease phenotype yet. In contrast, the PrPVQ allele (polymorphic amino acids at positions 136 and 171 are indicated by superscript single-letter code) is associated with high susceptibility to scrapie and short survival times of scrapie-affected sheep (9-12, 15-18), whereas the PrPAR allele is associated with resistance or incubation times that span beyond the lifetime of sheep (9-12, 16, 17). In breeds where PrPVQ is rare, e.g. the Suffolk breed, the wild-type PrPAQ allele is associated with susceptibility to scrapie, although with a low or incomplete penetrance (18, 19). The mechanisms by which the different PrP allelic variants contribute to differences in scrapie susceptibility and survival time are not yet understood. However, it is possible that the various PrPC variants differ in their conversion kinetics into PrPSc. Such differences may be due to differences in expression levels, in cotranslational or posttranslational modifications, and/or differences in conformational structures of the various PrP variants. .. In this paper we report, for the first time to our knowledge, the cell-free conversion of sheep PrPC into protease-resistant forms similar or identical to ShPrPSc. In addition we report that polymorphisms that are associated with differences in scrapie susceptibility and differences in survival times of scrapie affected sheep also account for comparable differences in cell-free conversion efficiencies. This suggests that the PrP conversion kinetics are directly related to scrapie susceptibility and the length of survival times of sheep affected by natural scrapie. Because there is a good correlation between in vitro cell-free conversion data and in vivo scrapie susceptibility data thus far (9-12, 16, 17), this assay may be useful for determining the relative susceptibility of individual allelic forms of PrP to different prion sources and/or the relative transmissibility of these prion sources.
The efficiency of the cell-free conversion reaction was strongly dependent on both the type of PrPC variant and on the source of PrPSc used to induce the conversion. The PrPCVQ variant, which is associated with high susceptibility and short survival times of scrapie-affected sheep, was very efficiently converted into protease-resistant forms. The wild-type PrPCAQ variant, which is associated with a neutral effect on susceptibility and intermediate survival times, was converted into protease-resistant forms with intermediate efficiency. The PrPCAR variant, which is associated with resistance and long survival times, was poorly converted into protease-resistant forms. Although in some breeds, i.e. Suffolk and Romanov, PrPAQ is associated with an incomplete penetrance to scrapie susceptibility, probably due to the low incidence of PrPVQ (16, 19, 32), PrPVQ carriers of these breeds still have the shortest scrapie survival time (16, 32). Another point of interest is the finding that PrPCAR can be converted, although with a very low efficiency, into protease-resistant forms suggesting the possibility of scrapie agent replication in PrPAR-carrying sheep as has been described by Ikeda et al. (32).
Not only the primary PrPC sequence was found to determine the conversion characteristics but also the primary amino acid sequence of PrPSc. PrPC(VQ/VQ) converted PrPCVQ, PrPCAQ, and PrPCAR with decreasing efficiencies. In contrast, PrPSc(AQ/AQ) converted PrPCVQ almost as efficiently as the PrPCAQ variant. The PrPCAR variant was poorly converted by both PrPSc isolates. This suggests that scrapie susceptibility is not only determined by the PrP genotype of the acceptor animal but also by the PrP genotype of the animal that produced the infectious PrPSc. This is consistent with the finding that the SSBP/1 scrapie isolate obtained from PrPVQ NPU-Cheviot sheep is best transmitted to PrPVQ sheep (12, 17). It is also consistent with the striking behavior of the CH1641 scrapie isolate, which was primarily isolated from a positive line (mainly PrPVQ-carrying) NPU-Cheviot sheep, when passaged in positive-line or negative-line (non-PrPVQ) Cheviot sheep. The first (primary) intracerebral passage of this positive-line material to positive-line Cheviot sheep resulted in short incubation times. Passage of the primary CH1641 isolate into negative-line Cheviot sheep resulted in longer incubation times (33) probably due to polymorphism barriers. If the negative-line passaged isolates were subsequently passaged in negative-line Cheviot sheep the incubation times in this line of sheep decreased (17, 33). A subsequent passage from these negative-line to positive-line Cheviot sheep increased the incubation times dramatically (17, 33) again probably due to the polymorphism barrier.
Modification of scrapie isolate properties were also found in mice scrapie transmission experiments in which the properties of PrPSc could be modified by passage of scrapie isolates through mice with different PrPC amino acid sequences (34). Further support is derived by the transmission of human Creutzfeldt-Jakob disease or GSS to mice expressing chimeric mouse/human PrP transgenes carrying specific mutations. Mice carrying the Glu-to-Lys mutation at position 200 (E200K) were resistant to human prions from a patient with GSS carrying a Pro-to-Leu mutation at position 102 (P102L) but were susceptible to prions from familial Creutzfeldt-Jakob disease patients harboring the E200K mutation. However, mice carrying the mouse/human transgene with the P102L mutation were susceptible to GSS prions (24).
Interestingly, a homogenate of bovine spongiform encephalopathy, of which the primary amino acid sequence (at the polymorphic amino acid positions of sheep PrP) is best comparable with the sheep PrPAQ genotype, gives the shortest incubation times in PrPAQ sheep if inoculated by the intracerebral route. If inoculated via the longer oral route however, PrPVQ sheep have the shortest incubation time (17). Probably inoculation via the oral route, compared with inoculation by the intracerebral route, extends the incubation time long enough to overcome the polymorphism barrier and subsequently allows the agent to spread more quickly using PrPCVQ instead of PrPCAQ.
Preliminary data from cell-free conversion experiments with the three PrPC variants using PrPSc isolated from a PrPVQ/AQ sheep suggest that this PrPSc isolate mainly consists of PrPCVQ because this PrPSc(VQ/AQ) isolate converted PrPCVQ at least three times as efficiently as PrPCAQ into protease-resistant forms (Fig. 4). This again is consistent with the finding that PrPCVQ is more readily converted into PrP-res than PrPCAQ. Thus in sheep containing the mutant PrPVQ allele, it is likely that the PrPCVQ variant will be the preferred converted variant, similar to what has been found for the mutant human PrP allele in GSS (35). Consequently, after infection of flocks of sheep having the PrPVQ allele, the agent pool would be predicted to become enriched for PrPVQ.
This study shows that the cell-free system is an excellent system to measure the relative transmissibility of a prion source to animals or humans with known PrP genotypes. Although the mechanism by which PrPC is converted into PrPSc and the mechanism by which polymorphisms in PrP modulate the conversion efficiency is not yet clear, studies with the cell-free conversion reaction (36) and small synthetic PrP peptides (37) are consistent with a nucleated polymerization mechanism (38, 39). The conversion of PrPC to PrPSc involves a transition from a state that is predominantly -helical to one that is largely -sheet (4, 5, 40). PrPC may rapidly interchange between these two conformations in its normal monomeric state but only be stabilized and accumulated in the -sheet conformation by binding to a preformed PrPSc polymer (37, 38, 41). Alternatively, the transition to the PrPSc conformation may only be induced (catalyzed) upon direct binding of PrPC to the PrPSc polymer. PrP polymorphisms may influence the equilibrium between the -helical and -sheet conformations in PrPC and/or the ease with which PrPSc induces PrPC to switch to the -sheet conformation. Polymorphisms that destabilize the -helical conformation of PrPC would be expected to have these effects.
In this study we have tested the cell-free conversion of three (PrPVQ, PrPAQ, and PrPAR) of the nine PrP variants found in sheep, including the two allelic variants that are associated with the extremes in susceptibility to scrapie (highly susceptible or resistant). From the other six allelic variants: PrPT112AQ, PrPAT137Q, PrPAF141Q, PrPAH154Q, PrPAH, and PrPAQQ211, it is not known whether they are significantly associated with susceptibility to natural or experimental scrapie in sheep. Using the recently published high-resolution NMR structure of the mouse PrPC domain containing residues 121-232 together with Novotny secondary structure predictions, it might be possible to rationalize the effects of certain of the sheep PrP polymorphisms on PrPC conformation. At least two other polymorphisms in the sheep PrP gene could be associated, by these predictions, with scrapie susceptibility. The PrPAT137Q variant could be grouped with the PrPVQ variant, because both give a prediction of more -sheeted structure and a change in hydrophobicity in the loop between -sheet-1 and -helix-1, which may indicate helix breaking or hydrophobic core destabilizing properties as found in theoretical studies of the Ala to Val mutation at position 117 in the human PrP sequence (42). The PrPAH154Q variant is protective against scrapie, and no scrapie-affected sheep with this genotype have been found (10, 12, 15, 32). This variant could be grouped with the PrPAR variant, because both involve a charge inversion compared with the wild-type PrPAQ variant. The latter two polymorphisms are located in the loops between -helix-1 and -sheet-2, and between -sheet-1 and -helix-3, respectively, and may influence the stabilization of the hydrophobic core or the dipolar character of PrPC. The other four alleles did not show differences in Novotny secondary structure predictions other than the PrPAQ variant and therefore probably may be grouped with this variant. Additional cell-free conversion data with all known sheep PrPC variants may enable us in the near future to determine more exactly the relative scrapie susceptibility between sheep having different PrP alleles.