There is no safe dose of prions

Understanding the circumstances under which exposure to transmissible spongiform encephalopathies (TSEs) leads to infection is important for managing risks to public health. Based upon ideas in toxicology and radiology, it is plausible that exposure to harmful agents, including TSEs, is completely safe if the dose is low enough. However, the existence of a threshold, below which infection probability is zero has never been demonstrated experimentally. Here we explore this question by combining data and mathematical models that describe scrapie infections in mice following experimental challenge over a broad range of doses. We analyse data from 4338 mice inoculated at doses ranging over ten orders of magnitude. These data are compared to results from a within-host model in which prions accumulate according to a stochastic birth-death process. Crucially, this model assumes no threshold on the dose required for infection. Our data reveal that infection is possible at the very low dose of a 1000 fold dilution of the dose that infects half the challenged animals (ID50). Furthermore, the dose response curve closely matches that predicted by the model. These findings imply that there is no safe dose of prions and that assessments of the risk from low dose exposure are right to assume a linear relationship between dose and probability of infection. We also refine two common perceptions about TSE incubation periods: that their mean values decrease linearly with logarithmic decreases in dose and that they are highly reproducible between hosts. The model and data both show that the linear decrease in incubation period holds only for doses above the ID50. Furthermore, variability in incubation periods is greater than predicted by the model, not smaller. This result poses new questions about the sources of variability in prion incubation periods. It also provides insight into the limitations of the incubation period assay.     

Genetic control of scrapie and Creutzfeldt-Jakob disease in mice

Genetic control of experimental scrapie and Creutzfeldt-Jakob disease (CJD) was studied in inbred strains of mice by measuring the times from intracerebral inoculation with the agents to the onset of neurological dysfunction. Every strain of mice examined was susceptible to infection; however, a wide range of incubation times was found for both scrapie and CJD. New Zealand (NZ) mice, which eventually develop an autoimmune disorder, were inoculated intracerebrally with 10(6) ID50 units of the scrapie agent in a Chandler isolate. NZW mice showed incubation periods of less than 95 days; this is the shortest period recorded for any murine host with scrapie. In NZB and NZB X W F1 mice, the incubation periods were approximately 130 days and were similar to those in BALB/c and C57BL mice. Male and female NZ mice exhibited scrapie incubation periods of the same length. Similar results were obtained when B10.Q and C57BL/6J mice were inoculated intracerebrally with 10(4) ID50 units of the CJD agent in a K.Fu. isolate. These observations define a genetic locus or loci controlling the length of scrapie and CJD incubation periods; alleles coding for longer incubation times appear to be autosomal dominant. When congenic mice with a C57BL/10J background differing only in their H-2 haplotypes were studied, the results showed that the D subregion of the H-2 complex played a central role in controlling the length of the CJD incubation period. The q allele at the D subregion resulted in shorter incubation times, whereas the d allele resulted in long incubation times. The p, s, b, and k alleles gave intermediate incubation times. We propose the symbol PID-1 for designating this genetic locus which is located within the D subregion of the major histocompatibility (H-2) complex on murine chromosome 17. In addition, observations on congenic mice provide evidence for the influence of sex on CJD incubation periods. In some strains of inbred mice, males showed significantly shorter incubation periods compared with those for females with experimental CJD. These studies with inbred mice have defined previously unrecognized genes that control the length of scrapie and CJD incubation periods.     

Linkage of prion protein and scrapie incubation time genes

A single gene (Prn-i) that affects scrapie incubation period in mice has been identified. I/LnJ mice have a very long incubation period after inoculation of scrapie prions (200-385 days) and NZW/LacJ mice have a short one (113 +/- 2.8 days). (NZW X I/Ln)F1 hybrid mice had incubation times of 223 +/- 2.8 days indicating longer incubation times were dominant. Incubation periods in the backcross progeny of (NZW/LacJ X I/LnJ)F1 X NZW/LacJ segregated into two groups (64 mice, 130 +/- 1.1 d; 66 mice, 195 +/- 1.9 d) indicating single gene control. NZW/LacJ and 20 other inbred strains have the Prn-pa allele which is identified as a 3.8 kb Xbal fragment using a hamster PrP (prion protein) cDNA probe. I/LnJ and three other Prn-pb mouse strains have a 5.5 kb Xbal restriction fragment. Analysis of DNA from 66 backcross mice indicated Prn-i is tightly linked to Prn-p, the structural gene for PrP.     

Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques

Three transgenic mouse lines designated Tg 69, 71, and 81 were produced harboring a Syrian hamster (Ha) prion protein (PrP) gene; all expressed the cellular HaPrP isoform in their brains. Inoculation of Tg 81 mice or hamsters with Ha prions caused scrapie in integral of 75 days; nontransgenic control mice failed to develop scrapie after greater than 500 days. Tg 71 mice inoculated with Ha prions developed scrapie in integral of 170 days. Both Tg 71 and Tg 81 mice exhibited spongiform degeneration and reactive astrocytic gliosis, and they produced the scrapie HaPrP isoform in their brains. Tg 81 brains also showed HaPrP amyloid plaques characteristic of Ha scrapie and contained integral of 10(9) ID50 units of Ha prions based on Ha bioassays. Our findings argue that the PrP gene modulates scrapie susceptibility, incubation times, and neuropathology; furthermore, they demonstrate synthesis of infectious scrapie prions programmed by a recombinant DNA molecule.     

Competition between Different Scrapie Agents in Mice

STRAINS of scrapie agent differ in a number of their biological properties, one of which is average incubation period^1,2. Incubation period is inversely proportional to dose for all known strains of scrapie and some differ so markedly that there is no overlap in their dose-incubation period curves. A large dose of one agent takes longer to produce the disease than the LD₅₀ dose of another agent³. Two such scrapie agents are 22A and 22C when compared by intracerebral injection of VM mice. Agent 22A has a much shorter incubation period than 22C in VM mice because they carry the p7 allele of the sine gene that controls scrapie incubation. It should therefore be possible to produce mixed infections in VM mice, but to ensure that the clinical disease is produced by the 22A agent. It can be checked that 22A causes the disease, rather than 22C, by examining the distribution of brain lesions². Interaction during pathogenesis between the two agents—competition or synergism—would be seen as a difference in the incubation period of 22A. We achieved mixed infection by injecting 22C first and 22A on a later occasion.     

Scrapie infection activates the replication of ecotropic, xenotropic, and polytropic murine leukemia virus (MuLV) in brains and spinal cords of senescence-accelerated mice: implication of MuLV in progression of scrapie pathogenesis

Senescence-accelerated mice (SAMP8) have a short life span, whereas SAMR1 mice are resistant to accelerated senescence. Previously it has been reported that the Akv strain of ecotropic murine leukemia virus (E-MuLV) was detected in brains of SAMP8 mice but not in brains of SAMR1 mice. In order to determine the change of MuLV levels following scrapie infection, we analyzed the E-MuLV titer and the RNA expression levels of E-MuLV, xenotropic MuLV, and polytropic MuLV in brains and spinal cords of scrapie-infected SAM mice. The expression levels of the 3 types of MuLV were increased in scrapie-infected mice compared to control mice; E-MuLV expression was detected in infected SAMR1 mice, but only in the terminal stage of scrapie disease. We also examined incubation periods and the levels of PrPSc in scrapie-infected SAMR1 (sR1) and SAMP8 (sP8) mice. We confirmed that the incubation period was shorter in sP8 (210+/-5 days) compared to sR1 (235+/-10 days) after intraperitoneal injection. The levels of PrPSc in sP8 were significantly greater than sR1 at 210+/-5 days, but levels of PrPSc at the terminal stage of scrapie in both SAM strains were virtually identical. These results show the activation of MuLV expression by scrapie infection and suggest acceleration of the progression of scrapie pathogenesis by MuLV.     

Tumor necrosis factor alpha-deficient, but not interleukin-6-deficient, mice resist peripheral infection with scrapie

In most peripheral infections of rodents and sheep with scrapie, infectivity is found first in lymphoid tissues and later in the central nervous system (CNS). Cells within the germinal centers (GCs) of the spleen and lymph nodes are important sites of extraneural replication, from which infection is likely to spread to the CNS along peripheral nerves. Here, using immunodeficient mice, we investigate the identity of the cells in the spleen that are important for disease propagation. Despite possessing functional T and B lymphocytes, tumor necrosis factor alpha-deficient (TNF-alpha(-/-)) mice lack GCs and follicular dendritic cell (FDC) networks in lymphoid tissues. In contrast, lymphoid tissues of interleukin-6-deficient (IL-6(-/-)) mice possess FDC networks but have impaired GCs. When the CNSs of TNF-alpha(-/-), IL-6(-/-), and wild-type mice were directly challenged with the ME7 scrapie strain, 100% of the mice were susceptible, developing disease after closely similar incubation periods. However, when challenged peripherally (intraperitoneally), most TNF-alpha(-/-) mice failed to develop scrapie up to 503 days postinjection. All wild-type and IL-6(-/-) mice succumbed to disease approximately 300 days after the peripheral challenge. High levels of scrapie infection and the disease-specific isomer of the prion protein, PrP(Sc), were detectable in spleens from challenged wild-type and IL-6(-/-) mice but not from TNF-alpha(-/-) mice. Histopathological analysis of spleen tissue demonstrated heavy PrP accumulations in direct association with FDCs in challenged wild-type and IL-6(-/-) mice. No PrP(Sc) accumulation was detected in spleens from TNF-alpha(-/-) mice. We conclude that, for the ME7 scrapie strain, mature FDCs are critical for replication in lymphoid tissues and that in their absence, neuroinvasion following peripheral challenge is impaired.     

Disease phenotype in sheep after infection with cloned murine scrapie strains

Prion diseases exhibit different disease phenotypes in their natural hosts and when transmitted to rodents, and this variability is regarded as indicative of prion strain diversity. Phenotypic characterization of scrapie strains in sheep can be attempted by histological, immunohistochemical and biochemical approaches, but it is widely considered that strain confirmation and characterization requires rodent bioassay. Examples of scrapie strains obtained from original sheep isolates by serial passage in mice include ME7, 79A, 22A and 87V. In order to address aspects of prion strain stability across the species barrier, we transmitted the above murine strains to sheep of different breeds and susceptible Prnp genotypes. The experiment included 40 sheep dosed by the oral route alone and 36 sheep challenged by combined subcutaneous and intracerebral routes. Overall, the combined route produced higher attack rates (~100%) than the oral route (~50%) and 2–4 times shorter incubation periods. Uniquely, 87V given orally was unable to infect any sheep. Overall, scrapie strains adapted and cloned in mice produce distinct but variable disease phenotypes in sheep depending on breed or Prnp genotype. Further re-isolation experiments in mice are in progress in order to determine whether the original cloned murine disease phenotype will reemerge.     

Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication

Transgenic (Tg) mice expressing both Syrian hamster (Ha) and mouse (Mo) prion protein (PrP) genes were used to probe the mechanism of scrapie prion replication. Four Tg lines expressing HaPrP exhibited distinct incubation times ranging from 48 to 277 days, which correlated inversely with HaPrP mRNA and HaPrPC. Bioassays of Tg brain extracts showed that the prion inoculum dictates which prions are synthesized de novo. Tg mice inoculated with Ha prions had approximately 10(9) ID50 units of Ha prions per gram of brain and less than 10 units of Mo prions. Conversely, Tg mice inoculated with Mo prions synthesized Mo prions but not Ha prions. Similarly, Tg mice inoculated with Ha prions exhibited neuropathologic changes characteristic of hamsters with scrapie, while Mo prions produced changes similar to those in non-Tg mice. Our results argue that species specificity of scrapie prions resides in the PrP sequence and prion synthesis is initiated by a species-specific interaction between PrPSc in the inoculum and homologous PrPC.     

The infectivity of transmissible spongiform encephalopathy agent at low doses: the importance of phospholipid

The issue of whether the mechanism of infection is independent or co-operative for low doses of transmissible spongiform encephalopathy (TSE) agent is critical for risk assessment. The susceptibility (and hence ID(50)) of individuals with the same prion protein (PrP) genotype may vary considerably with a small proportion being very susceptible. Assuming independent action, the incubation period (IP) would continue to increase until the dose is below the ID(50) of the most susceptible individuals in the experiment, at which point it would become constant. This may explain the observed increase in IP with decreasing dose below the apparent ID(50) in experiments with untreated TSE agent. In contrast, IPs for autoclaved or NaOH-treated TSE agent increase greatly at doses 相似文献   

Paradoxical shortening of scrapie incubation times by expression of prion protein transgenes derived from long incubation period mice.

Prolonged incubation times for experimental scrapie in I/LnJ mice are dictated by a dominant gene linked to the prion protein gene (Prn-p). Transgenic mice were analyzed to discriminate between an effect of the I/LnJ Prn-pb allele and a distinct incubation time locus designated Prn-i. Paradoxically, 4 independent Prn-pb transgenic mouse lines had scrapie incubation times shorter than nontransgenic controls, instead of the anticipated prolonged incubation periods. Aberrant or overexpression of the Prn-pb transgenes may dictate abbreviated incubation times, masking genuine Prn-p/Prn-i congruence; alternatively, a discrete Prn-i gene lies adjacent to Prn-p.     

Identification of two prion protein regions that modify scrapie incubation time

A series of prion transmission experiments was performed in transgenic (Tg) mice expressing either wild-type, chimeric, or truncated prion protein (PrP) molecules. Following inoculation with Rocky Mountain Laboratory (RML) murine prions, scrapie incubation times for Tg(MoPrP)4053, Tg(MHM2)294/Prnp(0/0), and Tg(MoPrP, Delta23-88)9949/Prnp(0/0) mice were approximately 50, 120, and 160 days, respectively. Similar scrapie incubation times were obtained after inoculation of these lines of Tg mice with either MHM2(MHM2(RML)) or MoPrP(Delta23-88)(RML) prions, excluding the possibility that sequence-dependent transmission barriers could account for the observed differences. Tg(MHM2)294/Prnp(0/0) mice displayed prolonged scrapie incubation times with four different strains of murine prions. These data provide evidence that the N terminus of MoPrP and the chimeric region of MHM2 PrP (residues 108 through 111) both influence the inherent efficiency of prion propagation.     

Extended scrapie incubation time in goats singly heterozygous for PRNP S146 or K222

Scrapie is the transmissible spongiform encephalopathy (TSE) of sheep and goats, and scrapie eradication in sheep is based in part on strong genetic resistance to classical scrapie. Goats may serve as a scrapie reservoir, and to date there has been no experimental inoculation confirming strong genetic resistance in goats. Two prion protein variants (amino acid substitutions S146 and K222) in goats have been significantly underrepresented in scrapie cases though present in scrapie-exposed flocks, and have demonstrated low cell-free protein conversion efficiency to the disease form (PrP(D)). To test degree of genetic resistance conferred in live animals with consistent exposure, we performed the first oral scrapie challenge of goats singly heterozygous for either PRNP S146 or K222. All N146-Q222 homozygotes became clinically scrapie positive by an average of 24months, but all S146 and K222 heterozygotes remain scrapie negative by both rectal biopsy and clinical signs at significantly longer incubation times (P<0.0001 for both comparisons). Recent reports indicate small numbers of S146 and K222 heterozygous goats have become naturally infected with scrapie, suggesting these alleles do not confer complete resistance in the heterozygous state but rather extend incubation. The oral challenge results presented here confirm extended incubation observed in a recent intracerebral challenge of K222 heterozygotes, and to our knowledge provide the first demonstration of extended incubation in S146 heterozygotes. These results suggest longer relevant trace-back histories in scrapie-eradication programs for animals bearing these alleles and strengthen the case for additional challenge experiments in both homozygotes to assess potential scrapie resistance.     

Efficient transmission of two different sheep scrapie isolates in transgenic mice expressing the ovine PrP gene

We produced transgenic mice expressing the sheep prion protein to obtain a sensitive model for sheep spongiform encephalopathies (scrapie). The complete open reading frame, with alanine, arginine, and glutamine at susceptibility codons 136, 154, and 171, respectively, was inserted downstream from the neuron-specific enolase promoter. A mouse line, Tg(OvPrP4), devoid of the murine PrP gene, was obtained by crossing with PrP knockout mice. Tg(OvPrP4) mice were shown to selectively express sheep PrP in their brains, as demonstrated in mRNA and protein analysis. We showed that these mice were susceptible to infection by sheep scrapie following intracerebral inoculation with two natural sheep scrapie isolates, as demonstrated not only by the occurrence of neurological signs but also by the presence of the spongiform changes and abnormal prion protein accumulation in their brains. Mean times to death of 238 and 290 days were observed with these isolates, but the clinical course of the disease was strikingly different in the two cases. One isolate led to a very early onset of neurological signs which could last for prolonged periods before death. Independently of the incubation periods, some of the mice inoculated with this isolate showed low or undetectable levels of PrPsc, as detected by both Western blotting and immunohistochemistry. The development of experimental scrapie in these mice following inoculation of the scrapie infectious agent further confirms that neuronal expression of the PrP open reading frame alone is sufficient to mediate susceptibility to spongiform encephalopathies. More importantly, these mice provide a new and promising tool for studying the infectious agents in sheep spongiform encephalopathies.     

Accelerated prion disease pathogenesis in Toll-like receptor 4 signaling-mutant mice

Prion diseases such as scrapie involve the accumulation of disease-specific prion protein, PrP(Sc), in the brain. Toll-like receptors (TLRs) are a family of proteins that recognize microbial constituents and are central players in host innate immune responses. The TLR9 agonist unmethylated CpG DNA was shown to prolong the scrapie incubation period in mice, suggesting that innate immune activation interferes with prion disease progression. Thus, it was predicted that ablation of TLR signaling would result in accelerated pathogenesis. C3H/HeJ (Tlr4(Lps-d)) mice, which possess a mutation in the TLR4 intracellular domain preventing TLR4 signaling, and strain-matched wild-type control (C3H/HeOuJ) mice were infected intracerebrally or intraperitoneally with various doses of scrapie inoculum. Incubation periods were significantly shortened in C3H/HeJ compared with C3H/HeOuJ mice, regardless of the route of infection or dose administered. At the clinical phase of disease, brain PrP(Sc) levels in the two strains of mice showed no significant differences by Western blotting. In addition, compared with macrophages from C3H/HeOuJ mice, those from C3H/HeJ mice were unresponsive to fibrillogenic PrP peptides (PrP residues 106 to 126 [PrP(106-126)] and PrP(118-135)) and the TLR4 agonist lipopolysaccharide but not to the TLR2 agonist zymosan, as measured by cytokine production. These data confirm that innate immune activation via TLR signaling interferes with scrapie infection. Furthermore, the results also suggest that the scrapie pathogen, or a component(s) thereof, is capable of stimulating an innate immune response that is active in the central nervous system, since C3H/HeJ mice, which lack the response, exhibit shortened incubation periods following both intraperitoneal and intracerebral infections.     

Two different scrapie prions isolated in Japanese sheep flocks

Two different scrapie prion strains with different characteristics were obtained from two sheep naturally infected with scrapie in Japan. In mice transmission, one (Tsukuba-1) showed shorter incubation periods (133+/-2 days) than the other (Tsukuba-2) (288+/-5 days). Spongiform changes and accumulation of an abnormal isoform of prion protein (PrP(Sc)) were observed throughout the brain in Tsukuba-1 inoculated mice, while the lesions and the PrP(Sc) accumulation were localized in the brain stem of mice with Tsukuba-2. Western blot analysis showed that there was no strain-specific glycoform of PrP(Sc) within these two strains. A super-infection experiment revealed that neither strain interfered with the other's propagation.     

Neuroinvasion by scrapie following inoculation via the skin is independent of migratory Langerhans cells

Many natural transmissible spongiform encephalopathy (TSE) infections are likely to be acquired peripherally, and studies in mice show that skin scarification is an effective means of scrapie transmission. After peripheral exposure, TSE agents usually accumulate in lymphoid tissues before spreading to the brain. The mechanisms of TSE transport to lymphoid tissues are not known. Langerhans cells (LCs) reside in the epidermis and migrate to the draining lymph node after encountering antigen. To investigate the potential role of LCs in scrapie transportation from the skin, we utilized mouse models in which their migration was blocked either due to CD40 ligand deficiency (CD40L-/- mice) or after caspase-1 inhibition. We show that the early accumulation of scrapie infectivity in the draining lymph node and subsequent neuroinvasion was not impaired in mice with blocked LC migration. Thus, LCs are not involved in TSE transport from the skin. After intracerebral inoculation with scrapie, wild-type mice and CD40L-/- mice develop clinical disease with similar incubation periods. However, after inoculation via skin scarification CD40L-/- mice develop disease significantly earlier than do wild-type mice. The shorter incubation period in CD40L-/- mice is unexpected and suggests that a CD40L-dependent mechanism is involved in impeding scrapie pathogenesis. In vitro studies demonstrated that LCs have the potential to acquire and degrade protease-resistant prion protein, which is thought to be a component of the infectious agent. Taken together, these data suggest that LCs are not involved in scrapie transport to draining lymphoid tissues but might have the potential to degrade scrapie in the skin.     

High incidence of scrapie induced by repeated injections of subinfectious prion doses

To clarify the mechanisms leading to the development of Creutzfeldt-Jakob disease in some recipients of pituitary-derived human growth hormone (hGH), we investigated the effects of repeated injections of low prion doses in mice. The injections were performed, as in hGH-treated children, by a peripheral route at short intervals and for an extended period. Twelve groups of 24 mice were intraperitoneally inoculated one, two, or five times per week for 200 days with 2 x 10(-5) to 2 x 10(-8) dilutions of brain homogenate containing the mouse-adapted C506M3 scrapie strain. Sixteen control mice were injected once a week for 200 days with a 2 x 10(-4) dilution of normal brain homogenate. Of mice injected in a single challenge with a scrapie inoculum of a 2 x 10(-4), 2 x 10(-5), or 2 x 10(-6) dilution, 2/10, 1/10, and 0/10 animals developed scrapie, respectively. Control mice remained healthy. One hundred thirty-five of 135 mice injected with repeated prion doses of a 2 x 10(-5) or 2 x 10(-6) dilution succumbed to scrapie. Of mice injected with repeated scrapie doses of a 2 x 10(-7) or 2 x 10(-8) dilution, 52/59 and 38/67 animals died of scrapie, respectively. A high incidence of scrapie was observed in mice receiving repeated doses at low infectivity, whereas there was no disease in mice that were injected once with the same doses. Repeated injections of low prion doses thus constitute a risk for development of prion disease even if the same total dose inoculated in a single challenge does not induce the disease.     

Propagation of prion strains through specific conformers of the prion protein.

Two prion strains with identical incubation periods in mice exhibited distinct incubation periods and different neuropathological profiles upon serial transmission to transgenic mice expressing chimeric Syrian hamster/mouse (MH2M) prion protein (PrP) genes [Tg(MH2M) mice] and subsequent transmission to Syrian hamsters. After transmission to Syrian hamsters, the Me7 strain was indistinguishable from the previously established Syrian hamster strain Sc237, despite having been derived from an independent ancestral source. This apparent convergence suggests that prion diversity may be limited. The Me7 mouse strain could also be transmitted directly to Syrian hamsters, but when derived in this way, its properties were distinct from those of Me7 passaged through Tg(MH2M) mice. The Me7 strain did not appear permanently altered in either case, since the original incubation period could be restored by effectively reversing the series of passages. Prion diversity enciphered in the conformation of the scrapie isoform of PrP (PrP(Sc)) (G. C. Telling et al., Science 274:2079-2082, 1996) seems to be limited by the sequence of the PrP substrates serially converted into PrP(Sc), while prions are propagated through interactions between the cellular and scrapie isoforms of PrP.