Porphyrin inhibition of rogue prion formation
Doxycycline control of prion protein transgene expression
Collinge method to screen all sheep for BSE
Significant prion titres found outside the CNS in clinically normal hamsters
Italian man and pet cat: article review
Dot blot: rapid sheep polymorphisms?
Promoter polymorphism in Alzheimer
Richard Bessen at Creighton funded by $738,000 federal grants
13 Oct 98 Caughey PNAS article and webmaster commentaryThis is a nice bit of molecular pharmacology. It is good to have another class of compounds to look at, especially one with a long history as non-toxic and permeable to the blood-brain barrier. It is far too early to say how useful these compounds will prove in practise but the methods here could be used to screen other drug families. Hopefully, the public will not start dousing themselves just yet with Internet tetrapyrroles as self-therapy.
It might seem that a compound that blocked conversion of normal to rogue would have no use in nvCJD, where presumbably ample time has gone by for seed crystals to already form. But recent papers show this to be wrong: the toxic fibrils are not metabolically stable. Thus, in one optimistic scenario, conversion blockers (such as porphyrins) do not even have to be taken in perpetuity, only long enough for seed fibrils to completely turn over.
The authors of this PNAS paper do not supply any insight as why normal prion protein, but not rogue, should have a half micromolar binding constant for porphyrins and seem to have gotten on to it because of earlier sulphonylated or sulfated compounds and their own experience with this particular family of compounds. Of course, a crystallographic structure of congo red bound to protein was determined long ago. Birefringent CR binding has nothing whatsoever to do with electrostatic polyanion binding but instead requires the precise spacing afforded by cross-beta helix. Non-specific CR binding can occur but lacks relevence to disease.
Congo red (and its analogues) bind independently of conformational disease type whereas hemes would be probably be specific to prion disease. Thus CR might be of value in Huntington whereas heme would not. Note however that CR and heme could be synergistic in prion disease since they do not compete for a binding site.
Why do porphyrins bind normal prion but not rogue? The explanation may be quite simple. Over a year ago, the Zurich ETH group published a computer-generated observation that the closest neighbor in 3D to the structure ot mouse prion was hemoglobin itself, or rather certain non-contiguous domains of the globin superfamily. Globins had an uncanny root mean square error fit of 2.4 angstroms. A RasMol coordinate file is available for beta hemoglobin.
I noted at the time that the three relevent coils of hemoglobin were just those of critical to the heme binding pocket, that the prion protein thus contained, whether by happenstance or homology, a natural mechanism for dimerizing about the symmetric tetrapyrrole. I suggested too that it was high time for someone to release native prion protein from the cell surfact with phospholipase, immunoprecipitate, and determine the ratio of radioactive ferric iron and copper/zinc to polypeptide chain (1:2:2 expected). (There are no Scatchard plots in the Caughey's paper.)
There the matter sat. This will be hugely embarrassing to the prion research community if a prominent chromophore has gone undetected for 13 years and thousands of papers.
The Caughey paper might thus be taken as the much-needed experimental evidence for a heme-binding site. The main result of this paper is then no accident: prion protein, as usually purified, is an apo-enzyme. Adding back its heme prosthetic group thermodynamically stabilizes the holo-enzyme, as reported for thousands of proteins. [The same effect is reported with copper in the posthumous J Biol Chem. 1998 Oct 2;273(40):25545-7 paper of R. Marsh.] Rogue conformer has a substantially different structure and no longer binds porphyrin.
Now we all know what sort of proteins contain both copper and heme: neurotransmitter oxygenases. A complete set of these was posted at this site in August 1996. On 8 Oct 98, the 3D structure of the conserved neurotoxic fragment 106-126 was deduced from its tandem palindromes; it will be posted shortly along with the full quaternary structure suggested by a heme pocket. Its structure provides an immediate explanation for the conformational change to cross-beta that the apo-protein can undergo once it has gotten off the normal turn-over track.
Thus in the heme scenario, the normal structure/function of prion enzyme may finally be at hand, only the particular substrate is still lacking. Normal function, diagnostics, and therapy have always been a three-legged stool as this paper well illustrates.
PNAS Vol. 95, Issue 21, 12117-12122, October 13, 1998 Winslow S. Caughey, Lynne D. Raymond, Motohiro Horiuchi, and Byron Caughey [This is the fourth paper co-authored by the Caughey's: see below]A central aspect of pathogenesis in the transmissible spongiform encephalopathies or prion diseases is the conversion of normal protease-sensitive prion protein (PrP-sen) to the abnormal protease-resistant form, PrP-res. Here we identify porphyrins and phthalocyanines as inhibitors of PrP-res accumulation. The most potent of these tetrapyrroles had IC50 values of 0.5-1 µM in scrapie-infected mouse neuroblastoma (ScNB) cell cultures. Inhibition was observed without effects on protein biosynthesis in general or PrP-sen biosynthesis in particular.
Tetrapyrroles also inhibited PrP-res formation in a cell-free reaction composed predominantly of hamster PrP-res and PrP-sen. Inhibitors were found among phthalocyanines, deuteroporphyrins IX, and meso-substituted porphines; examples included compounds containing anionic, neutral protic, and cationic peripheral substituents and various metals. We conclude that certain tetrapyrroles specifically inhibit the conversion of PrP-sen to PrP-res without apparent cytotoxic effects. The inhibition observed in the cell-free conversion reaction suggests that the mechanism involved direct interactions of the tetrapyrrole with PrP-res and/or PrP-sen. These findings introduce a new class of inhibitors of PrP-res formation that represents a potential source of therapeutic agents for transmissible spongiform encephalopathies.
The bovine spongiform encephalopathy epidemic and the appearance of the new variant of Creutzfeldt-Jakob disease in humans has heightened the urgency to develop therapies for the transmissible spongiform encephalopathies (TSE) or prion diseases. TSE pathogenesis appears to result from the accumulation in the central nervous system of the abnormal protease-resistant form of prion protein (PrP-res), which is derived from its normal protease-sensitive isoform, PrP-sen. The PrP-sen-to-PrP-res conversion involves changes in conformation and/or monomer aggregation without apparent modifications of amino acid residues.
One approach to TSE therapy is to inhibit PrP-res formation in the infected host. Sulfated glycans and the sulfonated amyloid stain Congo red are known inhibitors of PrP-res formation and scrapie agent replication in scrapie-infected neuroblastoma (ScNB) cells (2-4). These polyanions are also protective against scrapie in rodents if administered near the time of infection but, unfortunately, have no therapeutic benefit after the infection has reached the central nervous system (5-8). Their therapeutic ineffectiveness postinfection may be a result of an inability to cross the blood-brain barrier to the brain where most of the PrP-res accumulates and TSE pathogenesis occurs. This problem and/or inherent toxicity also limit the utility of other classes of potential drugs, the polyene antibiotics (9) and anthracycline (10).
Porphyrins and phthalocyanines (Pcs) are tetrapyrrole compounds that possess characteristics that make them of interest as potential inhibitors. These tetrapyrroles bear some structural resemblance to Congo red in that they all contain hydrophobic aromatic rings and can be synthesized with sulfonate groups. Tetrapyrroles can bind strongly and selectively to proteins and affect changes in protein conformation (11-18), potentially critical properties of an effective inhibitor. Tetrapyrroles are available with wide variations in structure, low toxicities in medical applications (19-22), and the apparent ability to cross the blood-brain barrier (23-26).
In the present study, we identified tetrapyrroles that inhibit the formation of PrP-res in ScNB cells and in a cell-free system. Included were deuteroporphyrins IX (DPs) that are analogs of the natural hemes A, B, C, and S (13), meso-substituted porphines, and Pcs. Surprisingly, the structures of some effective inhibitors were inconsistent with the structural features thought to be important in Congo red and other known inhibitors of PrP-res formation. ...
The present results show that tetrapyrroles inhibit PrP-res formation in both mouse ScNB cells and the hamster PrP cell-free conversion system. The ScNB cell experiments indicated that this inhibition occurred without apparent cytotoxicity or effects on the rate of PrP-sen biosynthesis. Compared with the prototypic inhibitor Congo red (34), the PcTS-Fe3+ is about 10-fold more potent as an inhibitor in the cell-free conversion reaction (Fig. 6). On the other hand, PcTS-Fe3+ is about 100-fold less potent than Congo red as an inhibitor in the ScNB cell system (Figs. 1 and 2; ref. 2).
The basis for the discrepancy in the relative potencies of these inhibitors in these two experimental systems is not known, but may be a result of differences in the PrP molecules involved (mouse vs. hamster) or differences in the extent to which these compounds engage in nonproductive binding to unrelated plasma or cellular proteins in the ScNB system. Both plasma proteins, such as albumin, and cytosolic proteins are known to bind some tetrapyrroles avidly (20, 35-37), which would reduce the tetrapyrrole molecules available for binding to PrP-sen and PrP-res. Furthermore, the self-association of some tetrapyrroles may also reduce the effective tetrapyrrole concentration significantly (38-42).
Potential Mechanism of Inhibition. Because of the complexity of the ScNB cell culture system, many possible mechanisms of inhibition by tetrapyrroles in this system can be envisioned, ranging from direct effects on PrP-sen PrP-res interactions to indirect effects on the biology of the cells. However, since several of these compounds also inhibit the cell-free conversion reaction, which is composed predominantly of PrP species, it is likely that the inhibition by the tetrapyrroles is due to their direct interactions with PrP molecules. The binding of tetrapyrroles to either form of PrP might sterically hinder PrP-resPrP-sen interactions or affect the conformations of the molecules in ways that interfere with the conversion reaction. Nonetheless, since the PrP-res preparations presumably are not completely pure, it remains possible that tetrapyrrole interactions with other molecules might play a role in inhibition.
Since Congo red and most of the other known polyanionic inhibitors of PrP-res formation are sulfonated or sulfated, we anticipated that the sulfonated porphyrins and phthalocyanines might be the best inhibitors. Surprisingly, however, the sulfonates or other anionic groups were not required for inhibition by the porphyrins (Figs. 3 and 4). Indeed, porphyrins with neutral glycol, or even cationic, substituents were effective inhibitors. This aspect of the porphyrins stands in contrast to the polysulfated glycans, which are ineffective when the sulfates are removed or substituted with cationic groups (3).
Previous studies of a variety of tetrapyrrole systems provide a firm basis for predicting the important types of bonding interactions that contribute to both tetrapyrroleprotein binding and tetrapyrrole self-associations. These are electrostatic interactions of groups on the periphery of the core ring, protic interactions at the central nitrogens if metal-free, axial ligand binding at metal in metal complexes, and -bonding of the core aromatic ring and any peripheral aromatic rings (38, 39, 41, 43). The large planar core aromatic ring system is likely to be an important feature because it is common to all the tetrapyrrole inhibitors, whereas the peripheral substituents and metals ions (or lack thereof) can vary widely. The identification of the most effective inhibitor and therapeutic agent among tetrapyrrole structures will require the optimization of the combination of core structures and substituents.
Important therapeutic parameters likely will include not only the specificity and affinity of PrP binding of these compounds, but also the pharmacokinetics, side effects, toxicity, and delivery to the brain. Many of the tetrapyrrole inhibitors found here are known to be well tolerated in animals, e.g., PcTS-Al3+, PcTrS-Al3+, T(N-Me-4-Py)P-Fe3+, T(Ph-4-SO3)P, and T(Ph-4-SO3)P-Fe3+ (19, 21, 44-48). An ability to penetrate the blood-brain barrier is expected to be helpful although an inhibitor could be useful prophylactically by preventing PrP-res formation outside the central nervous system. Data on the penetration of the blood-brain barrier by tetrapyrroles are limited. One inhibitor studied here, PcTS-Al3+, and several DP analogs appear to enter the brain (23-26, 48). The intrinsic lipophilicity of tetrapyrroles favors the development of effective modalities for their delivery to the brain.
Tetrapyrroles, TSEs, and Other Amyloidoses. The mechanism of conversion of PrP-sen to PrP-res appears to resemble the pathogenic processes of amyloid formation associated with a variety of other diseases including Alzheimer's disease and Type 2 diabetes (1). Thus, it is possible that these tetrapyrroles might serve as inhibitors not only of PrP-res formation, but also of other types of amyloid formation. A recent report showed that another porphyrin, hemin, can inhibit Alzheimer's peptide polymerization and cytotoxicity (49). This observation and the present study showing that a broad spectrum of porphyrins and phthalocyanines inhibit PrP-res formation make tetrapyrroles attractive candidates for more extensive study. Fortunately, in the case of TSE diseases, excellent animal models are available for testing their potential therapeutic effects.
Dong A, Huang P, Caughey B, Caughey WS Infrared analysis of ligand- and oxidation-induced conformational changes in hemoglobins and myoglobins. Arch Biochem Biophys. 1995 Feb 1;316(2):893-8. Dong A, Caughey B, Caughey WS, Bhat KS, Coe JE Secondary structure of the pentraxin female protein in water determined by infrared spectroscopy: effects of calcium and phosphorylcholine. Biochemistry. 1992 Oct 6;31(39):9364-70. Caughey BW, Dong A, Bhat KS, Ernst D, Hayes SF, Caughey WS Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy. Biochemistry. 1991 Aug 6;30(31):7672-80.Abstract: A protease-resistant form of the protein PrP (PrP-res) accumulates in tissues of mammals infected with scrapie, Creutzfeldt-Jakob disease, and related transmissible neurodegenerative diseases. This abnormal form of PrP can aggregate into insoluble amyloid-like fibrils and plaques and has been identified as the major component of brain fractions enriched for scrapie infectivity. Using a recently developed technique in Fourier transform infrared spectroscopy which allows protein conformational analysis in aqueous media, we have studied the secondary structure of the proteinase K resistant core of PrP-res (PrP-res 27-30) as it exists in highly infectious fibril preparations. Second-derivative analysis of the infrared spectra has enabled us to quantitate the relative amounts of different secondary structures in the PrP-res aggregates. The analysis indicated that PrP-res 27-30 is predominantly composed of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn (31%) and alpha-helix (17%) were identified, indicating that amyloid-like fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within PrP-res 27-30. Published erratum appears in Biochemistry 1991 Oct 29;30(43):10600
PNAS Vol. 95, Issue 21, 12580-12585 Patrick Tremblay, Zeev Meiner,..., Stephen J. DeArmond, and Stanley B. PrusinerHighlights of article:
To control the expression of PrPC in transgenic (Tg) mice, we used a tetracycline controlled transactivator (tTA) driven by the PrP gene control elements and a tTA-responsive promoter linked to a PrP gene [Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. Adult Tg mice showed no deleterious effects upon repression of PrPC expression (>90%) by oral doxycycline, but the mice developed progressive ataxia at 50 days after inoculation with prions unless maintained on doxycycline. Although Tg mice on doxycycline accumulated low levels of PrPSc, they showed no neurologic dysfunction, indicating that low levels of PrPSc can be tolerated. Use of the tTA system to control PrP expression allowed production of Tg mice with high levels of PrP that otherwise cause many embryonic and neonatal deaths. Measurement of PrPSc clearance in Tg mice should be possible, facilitating the development of pharmacotherapeutics. ....
The function of PrPC is unknown. In two lines of PrP-deficient (Prnp0/0) mice, no clinical signs of illness have been reported, and the animals seem to develop normally (2, 3). Brain slices from these two Prnp0/0 lines have been reported to show defective neurotransmission at GABAergic synapses and diminished long-term potentiation (4, 5). In two other studies, no detectable electrophysiological defects could be identified in one of the Prnp0/0 lines (6, 7). However, defective sleep-wake cycles and altered circadian rhythms have been reported for both of these Prnp0/0 lines (8). Subsequently, an additional line of Prnp0/0 mice was reported to develop ataxia due to Purkinje cell degeneration at about 70 weeks of age (9). Whether PrP has a role in Cu2+ metabolism is unclear, but studies of Cu2+ in Prnp0/0 mice support such a hypothesis (10). Several studies have suggested that PrP may bind Cu2+ in the octarepeat region (11). Two Cu2+ ions appear to be bound per PrP molecule through a square planar geometry (12).
To challenge the hypothesis that PrPSc is required for transmission and pathogenesis of prion disease (13), Prnp0/0 mice were inoculated with prions. These PrP-deficient mice neither developed disease (14) nor replicated prions (15, 16). Moreover, mice hemizygous for PrP gene ablation showed prolonged incubation times (15, 17, 18).
On this background, we undertook development of a system where the level of PrP expression could be regulated to modulate the rate of prion formation. We chose the tetracycline-responsive gene system that was developed by using the Escherichia coli tetracycline resistance Tn10 operon (19). It makes use of a transactivator (tTA) obtained by fusing the tetracycline repressor with the transactivation domain of the herpes simplex virus VP16 transcription factor. The tTA binds specifically with high affinity to the tetracycline operator (tetO) and activates transcription from a minimal promoter linked to the target gene. Binding of doxycycline, a tetracycline analog, to tTA prevents the protein from binding to the tetO region, thereby preventing target gene expression.
To test the hypothesis that developmental compensation in PrP-deficient mice prevented any recognizable dysfunction in adult mice, we used the tTA system in Tg mice to regulate PrP expression. Doxycycline administered to adult Tg(tTA:PrP) mice acutely repressed the expression of PrPC but did not produce any recognizable adverse effects in the mice over a 30-day period. Neither the viability nor the neurological status of the mice was compromised, and histological examination of the brains did not reveal any abnormalities. These results indicate that high levels of PrPC are not essential for short-term neuronal survival, as its expression can be repressed over 20-fold without adverse effects. It is noteworthy that adult Tg(tTA:PrP)3 mice were placed on oral doxycycline to repress their PrPC expression and have remained well for >380 days with continual administration of doxycycline (Table 5).
The accumulation of PrPSc in the brains of animals and humans is a specific hallmark of prion disease. Often, but not always, proteolytic fragments of PrPSc coalesce in the extracellular space to form amyloid plaques. Such extracellular accumulations of PrPSc were thought to be of little consequence in the pathogenesis of prion disease because they are a nonobligatory feature of the disease (28). Moreover, PrPSc either within caveolae or within an intracellular compartment has been implicated in the pathogenesis of prion disease (29, 30), a conclusion supported by neuronal cell grafts producing PrPSc in the CNS of Prnp0/0 mice (31).
With the production of Tg(tTA:PrP) mice, it is possible to examine the effects of low or intermediate levels of PrPSc in the CNS. We found that low levels of PrPSc did not produce any deleterious clinical or histological effects up to 380 days after inoculation of RML prions in Tg(tTA:PrP)3 mice (Fig. 3C). Studies of Prnp+/0 mice with one functional PrP allele show greatly prolonged incubation times (15) but at a higher accumulation of PrPSc than was anticipated (17). Studies with Tg(tTA:PrP) mice where the levels of PrPC expression are held at different levels throughout the incubation time should help to clarify this issue.
The findings reported here clearly show that repression of PrPC expression in young adult Tg(tTA:PrP) mice is not deleterious, whereas accumulation of PrPSc in the same line of animals is lethal (Table 5). Even though Purkinje cell degeneration in 70-week-old Prnp0/0 mice has been found (9), our data continue to argue that the accumulation of PrPSc and not a loss of PrPC function is responsible for the pathogenesis of prion disease.
The use of the tTA-regulated PrP transgene expression revealed that high levels of even wild-type PrPC are often incompatible with neonatal development, as most Tg(tTA:PrP) mice died within the first 3 weeks of life (Table 4). Repression of PrPC expression by beginning doxycycline treatment during embryonic development was sufficient to prevent this mortality. The ability to control PrPC expression should allow the establishment of Tg mice expressing higher levels of wild-type or mutant PrP than was previously possible. Such mice may demonstrate unique sensitivities to prion infection and provide the basis for a truly rapid bioassay.
Reversing the course of prion diseases by blocking the production of PrPSc through repression of PrPC expression will allow us to measure the removal of PrPSc. Such clearance studies, which were not previously possible, are a prelude to the development of effective therapies where drugs that block PrPSc formation are administered at the earliest onset of symptoms to patients with sporadic Creutzfeldt-Jakob disease. At present, we have no understanding of how much PrPSc can be tolerated by the CNS and how rapidly it will disappear once synthesis of its precursor, PrPC, is repressed.
Microbiol Immunol 1998;42(8):579-82 Ishiguro N, Shinagawa M, Onoe S, Yamanouchi K, Saito TA rapid method to determine the allelic variants of the sheep PrP gene was developed. DNA samples from 128 Suffolk sheep (39 rams and 89 ewes) were screened by using polymerase chain reactions and dot-blot hybridization with 32P-labeled nine allele-specific oligonucleotide probes corresponding to the polymorphic PrP codons 112, 136, 154 and 171. Three allelic variants of the PrP gene, PrP(MARQ), PrP(TARQ) and PrP(MARR), were found in the flocks. Among those variants, nearly half of the ewes had alleles of the 171-Arg variant that is closely associated with resistance to natural scrapie. Assessments of allelic mutations of the PrP gene may help to select the scrapie-resistant progenitors in the flocks.
Alex Bossers comments: "Macro- or micro-arrays (DNAchips) will be the future of these kind of techniques. Or a fast technique allready available like TaqMan (PE) which we routinely perform nowadays from blood sample to PrP genotype (incl. PCR/DNA isolation etc.) in less than 4 hours."
Molecular Psychiatry September 1998, Volume 3, Issue 5 Table of Contents Medline is only up to July 98 for this journal
405 Two commonly expanded CAG/CTG repeat loci: involvement in affective disorders? K Lindblad et al. 438 The short variant of the polymorphism within the promoter region of the serotonin transporter gene is a risk factor for late onset Alzheimer's disease JRM Oliveira et al. 449 An association study of a functional polymorphism of the serotonin transporter gene with personality and psychiatric symptoms AF Jorm et al.
Science Daily Magazine 10-8-98 [Contact info: Bessen, Richard Dept. of Medical Microbiology & Immunology, Creighton University Omaha, NE Tel (402) 280-3072; Fax (402) 280-1875OMAHA, Neb. -- Proteins that may cause "mad cow" disease, chronic wasting disease in mule deer and elk, and Creutzfeldt Jakob disease in humans, all of which are fatal neurological diseases, are the subject of two studies at Creighton funded by federal grants. Ý
Richard Bessen, assistant professor of medical microbiology and immunology, has received a five-year $488,000 National Institutes of Health grant and a three-year $250,000 grant from the United States Department of Agriculture to study prions. Ý Prions are proteins that all humans possess in their normal form. The disease forms are chemically identical but are configured in different shapes. Ý
"What makes prions unique is that these infectious agents do not appear to contain a nucleic acid molecule which is the genetic basis of all life forms," Bessen said. "Prion diseases are caused by the misfolding of normal prion proteins. There is debate about how this misfolding occurs and how that pattern is replicated." Ý
It appears that "mad cow" disease, or bovine spongiform encephalopathy (BSE), adapted from sheep scrapie. Ý
"If you eat processed foods, such as sausage or products with animal-derived food additives, you may have been exposed to sheep scrapie, with no effect," Bessen said. "However, cattle apparently are susceptible upon oral exposure to scrapie-contaminated feed additives. Once they infected cattle, the prions may have adapted further to create an agent that posed danger to humans. Ý
"The BSE epidemic in the United Kingdom indicates that these agents, transmissible spongiform encephalopathies or TSEs, can adapt unpredictably to new hosts and potentially can cause widespread neurodegenerative disease," Bessen said. "There have been more than 175,000 cases of BSE in Europe, mainly in the United Kingdom." ÝÝ
Initially, the potential danger of BSE to humans was downplayed, but BSE now has been linked to 27 cases of variant Creutzfeldt-Jakob disease in humans. The cattle industry has changed feeding and herd management approaches to avoid possible transmission of prion disease to humans and animals. No cases of BSE have been confirmed in the United States. ÝÝ
Bessen is examining the molecular basis of prion strain diversity and how prions replicate. He also will investigate the ability of drugs to inhibit the conversion of normal prions into the disease forms. ÝÝ
Creighton is an independent Catholic university operated by the Jesuits. It recently was ranked No. 1 for the third consecutive year among Midwestern universities in the U.S. News and World Report magazine's 1999 "America's Best Colleges" edition.
Raeber AJ, et al. Astrocyte-specific expression of hamster prion protein (PrP) renders PrP knockout mice susceptible to hamster scrapie. EMBO J. 1997 Oct 15;16(20):6057-65. Bessen RA, et al. In situ formation of protease-resistant prion protein in transmissible spongiform encephalopathy-infected brain slices. J Biol Chem. 1997 Jun 13;272(24):15227-31. Race RE, et al. Neuron-specific expression of a hamster prion protein minigene in transgenic mice induces susceptibility to hamster scrapie agent. Neuron. 1995 Nov;15(5):1183-91. Bessen RA, et al. Inhibition of murine retrovirus-induced neurodegeneration in the spinal cord by explant culture. J Virol. 1995 Nov;69(11):7300-3. Bessen RA, et al. Non-genetic propagation of strain-specific properties of scrapie prion protein. Nature. 1995 Jun 22;375(6533):698-700. Bessen RA, et al. Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy. J Virol. 1994 Dec;68(12):7859-68. Bartz JC, et al. Transmissible mink encephalopathy species barrier effect between ferret and mink: PrP gene and protein analysis. J Gen Virol. 1994 Nov;75 ( Pt 11):2947-53. Marsh RF, et al. Physicochemical and biological characterizations of distinct strains of the transmissible mink encephalopathy agent. Philos Trans R Soc Lond B Biol Sci. 1994 Mar 29;343(1306):413-4. Marsh RF, et al. Epidemiologic and experimental studies on transmissible mink encephalopathy. Dev Biol Stand. 1993;80:111-8. Bessen RA, et al. Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent. J Virol. 1992 Apr;66(4):2096-101. Bessen RA, et al. Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters. J Gen Virol. 1992 Feb;73 ( Pt 2):329-34. Marsh RF, et al. Epidemiological and experimental studies on a new incident of transmissible mink encephalopathy. J Gen Virol. 1991 Mar;72 ( Pt 3):589-94.
October 3, 1998 The Lancet p. 1116 webmaster reviewThis is a four paragraph, five cite research letter about a 60 year old man in Italy who died of CJD in early January, 1994 and the 7 yar old female short-hair cat with FSE that was killed in mid-January of the same year. Both man and cat began showing serious symptoms in Nov 93.
This simultaneity was falsely reported in the press as the cat succumbing 3 months later, hinting at disease caught from the man, ie, to disfavor tainted cat food. Pet food companies advertise heavily in the newspaper food coupon section. It must be a fascinating job to be the editor in charge of falsifying the news wire.
The cat was usually fed canned pet food and slept on the man's bed. There was no record of bites. The brand and type of cat food are not noted (fish, chicken, pork, beef?), no unopened cans were saved or purchased post-diagnosis by researchers. Cats of course hate to change brands. There is no mention of whether the man lived alone or with family who would know more or whether he was poor.
Observe that the paper is being published 56 months after the fact without explanation.
Their only cite to FSE is also odd, a 1993 review said to describe 3 cats with the BSE strain of FSE, Pearson GR Vet Annual 1993 33:1-10 (off-Medline). Only 1 cat was strain-typed by the M Bruce method and that was in 1997 as far as I can recollect; the Collinge method was not available in 1993, so they apparently just mean"diffuse spongiosis and vacuolation of brainstem neurons" and different behavioral onset, which in my opinion are completely unsatisfactory for a differential strain diagnosis.
Other cats with confirmed FSE and affected zoo felids are not discussed. The MAFF site is showing cheetah 5+, puma 3, tiger 1, ocelot 2, house cats 85 (plus 1 in N Ireland, 1 in Norway, 1 in Lichtenstein) with cat cases holding steady at 6 _reported_ cases per year. If 5% of these have been examined for strain type, it would come as a big surprise to me.
The authors say that the man and cat were met/met at 129 and that "no pathogenic mutations in the patient's PrP gene were found." No experimental method is mentioned. They don't exactly say that they sequenced either gene. My guess is that they did not, that they only looked at a restriction fragment covering D178N and E200K. The cat gene was published by Liverpool on 02-Oct-1997 but this is not cited nor is their(partial?) cat sequence compared to it. They do not discuss dementia in the patient's lineage or surgical history or lifestyle. He is said to have "no unusual dietary habits."
They will later conclude that the man and cat had the same strain of sporadic CJD. I find this completely unacceptable because for starters they certainly did not determine if the cat had familial FSE. Numerous new pathogenic mutations have been found in human since 1994. The prion literature is rife with sequencing errors -- show me some data, as supplementary web material if there is no space in Lancet.
Next they say both man and cat had type I glycoform patterns (Collinge test) "comparable to those observed in sporadic CJD (details available from author)." The corresponding author's email is then wrongly given as Rizzuto@Gorgorna.univr.it -- it should apparently be email@example.com No fax or tel is provided. Are details really available -- in what form, to whom, and for how long? Was it included in peer-review?
There is no mention of control cats, either healthy or type IV. No one has ever shown cat prion to be glycosylated and if so, with what. (If you think these are the same then maybe you think cats have ABO blood groups too.) They do not mention any gels run after deglycosylation.
They do however provide sufficient immunohistochemistry to establish TSE in both patient and cat.
Where does this leave things? (Besides that Lancet did a disgraceful job of editing.) In my opinion, by far the likliest scenario is contaminated UK beef in the canned pet food. This could either be the well-known UK strain or some other epidemic strain-- only a miniscule percentage of animals has been tested so 'hyper-drowsy' is still quite tenable. M .Bruce is quoted as saying further strain-typing is essential in any event.
Or it could be another Stetsonville, or should I say Villa Stetsoni? (This might explain the 56 month delay and not saving a sample of the cat food.)
Italy reportedly imported massive amounts of contaminated MBM and used it as fish meal. The fish prion is so different that there is not the slightest reason to believe that BSE strains would passage faithfully in fish -- basically the 105-126 kernel is all that would survive. A large Italian research project on fish was funded some time back but nothing has surfaced from it.
CJD and FSE are each so rare that the cases cannot be independent. In the US, it is supposedly quite common for the elderly poor to eat pet food. There is the matter of the fork used to dish it out. Or the man could have fed the cat contaminated table scraps. Or he could have been inhaled dust from dry cat food. These scenarios have both man and cat orally infected from a common source.
There is also potential for horizontal transfer by eye or nasal route etc. in either direction. The one accepted case of conjugal CJD has not been strain-typed. Either man or cat could have sporadic, iatrogenic, or familial TSE and transmitted it to the other.
There is no real explanation for simultaneity of symptoms in any of the scenarios. The prion sequences involved are quite different and there is no reason to expect similar exposure titre or similar incubation times. (Think about linc and sinc mice, which differ at only two amino acids.) Possibly they both received an absolutely massive exposure at the same time that overwhelmed these subtleties.
Cats at GenBank (use accession 3287889):
cat 3287889 MVKSHIGSWI LVLFVAMWSD VGLCKKRPKP GGGWNTGGSR YPGQGSPGGN cat 2406626 MVKSHIGSWI LVLFVAMWSD VGLCKKRPKP GGGWNTGGSR YPGQGSPGGN cat 2463040 .......... .......... .......... .......... .......... Consensus mvkshigswi lvlfvamwsd vglckkrpkp gggwntggsr ypgqgspggn 51 100 cat RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGGSHS cat RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGGSHS cat .......... .......... .......... .......... .......... Consensus ryppqggggw gqphgggwgq phgggwgqph gggwgqphgg ggwgqggshs 101 150 cat QWNKPSKPKT NMKHVAGAAA AGAVVGGLGG YMLGSAMSRP LIHFGNDYED cat QWNKPSKPKT NMKHVAGAAA AGAVVGGLGG YMLGSAMSRP LIHFGNDYED cat .......... .MKHVAGAAA AGAVVGGLGG YMLGSAMSRP LIHFGNDYED Consensus qwnkpskpkt nMKHVAGAAA AGAVVGGLGG YMLGSAMSRP LIHFGNDYED 151 200 cat RYYRENMYRY PDQVYYRPVD QYSNQNNFVR DCVNITVKQR TVTTTTKGEN cat RYYRENMYRY PNQVYYRPVD QYSNQNNFVH DCVNITVKQH TVTTTTKGEN cat RYYRENMYRY PDQVYYRPVD QYSNQNNFVR DCVNITVKQR TVTTTTKGEN Consensus RYYRENMYRY P#QVYYRPVD QYSNQNNFVr DCVNITVKQr TVTTTTKGEN 201 250 cat FTETDMKIME RVVEQMCVTQ YQKESEAYYQ RRASAILFSS PPVILLISFL cat FTETDIKIME RVVEQMCITQ YQRESEAYYQ RGASVILFSS PPVILLISFL cat FTETDMKIME RVVEQMCVTQ YQKESEAYYQ RRASA..... .......... Consensus FTETDmKIME RVVEQMC!TQ YQkESEAYYQ RrASailfss ppvillisfl 251 cat IFLIVG cat IFLIVG cat124 ...... cat iflivg
October 23, 1998 Electronic Telegraph Roger Highfield, Science EditorA technique has, according to this story, been developed that for the first time makes it practical to screen the national flock of 42 million sheep for mad cow disease. Prof Jeffrey Almond, a member of the Government's spongiform encephalopathy advisory committee, SEAC, was cited as saying that so far only nine sheep have been checked for BSE and he urged the Government to test the national flock "as a matter of urgency". British sheep may have been fed BSE-infected bonemeal made from cattle which had the disease until at least 1988.
Two years ago it was confirmed that BSE can be passed to sheep through their food. Until now there has been no simple way to distinguish the effects of BSE on sheep from those of scrapie, a related spongiform disease that has affected the national flock for centuries with no apparent consequences for human health.
Today, in the journal Neuroscience Letters, another SEAC member, Prof John Collinge, and colleagues at St Mary's Hospital, London, working with the Central Veterinary Laboratory, Weybridge, Surrey, published the results of a preliminary survey to establish a new screening method. Their work demonstrates that "molecular screening" for BSE in sheep should only cost a few pounds per test.
Prof Collinge was cited as explaining that the current method is a bioassay, which involves inserting suspected infectious tissue into the brains of laboratory mice and observing them. However, it takes two years for the mice to succumb to the disease, under careful observation and carefully controlled conditions. The overall cost, approaching 30,000 per sheep, and the time, makes the method impractical to use. The new method is based on a type of test called Western Blot analysis, which smashes up the abnormal prion protein thought to cause the disease into fragments and detects different strains by the different pattern of fragments. More work must be done to perfect the test but "it looks as though it is going to be an effective means of differentiating BSE in sheep from sheep scrapie", said Prof Collinge.
Recently, another method with the potential for mass screening was announced by the Nobel laureate Prof Stan Prusiner, Dr Jiri Safar and colleagues at the University of California, San Francisco.
Quick Comment (webmaster):
This sounds like welcome news -- "it looks as though it is going to be an effective means of differentiating BSE in sheep from sheep scrapie." Surely there is some sort of assumption here about the number and nature of strains of scrapie because the mapping of strains to fragment patterns need scarcely be 1:1. In fact it could easily be many:1.
How would a hypothetical second strain of BSE in sheep be distinguised from an obscure strain of scrapie in sheep? Is the method resilient to the vast number of sheep haplotypes? Can 5-repeat BSE be told from 6-repeat BSE? We shall need to take a close look at the resolving power of the signature.
No abstract yet on Medline nor at Neurorscience Letters web site, which they advertise as the 'fastest letters journal in the field' though they are only up to 21 Aug 98 in their own archive of title pages (unlike Medline which has September full abstracts of this journal already). It would be one of the weekly October 98 issues, volume 255.
J Gen Virol 1998 Oct;79 (Pt 10):2557-62 Bolton DCPrion titres were measured in the lungs and brains of Syrian hamsters after intraperitoneal inoculation with sucrose gradient-purified 263K prions (approximately 10(8) LD50). Prions were detected in the lung of one hamster on day 7, but were not detected in the lungs of any other hamster until day 71. Prions were detected in the lungs of all hamsters sampled thereafter but titres remained low through day 127. Prions were first detected in the brain on day 35 and brain titres increased exponentially until day 127 with a doubling time of about 4.5 days. On day 133, titres averaged 108 LD50/g in brain and 105 LD50/g in lung.
Two out of the five remaining hamsters were clinically normal but prion titres were not significantly different from those in the clinically affected hamsters. Thus, significant prion titres may be found outside the CNS in clinically normal hamsters.