The effects of host age on follicular dendritic cell status dramatically impair scrapie agent neuroinvasion in aged mice.

Following peripheral exposure, many transmissible spongiform encephalopathy (TSE) agents accumulate first in lymphoid tissues before spreading to the CNS (termed neuroinvasion) where they cause neurodegeneration. Early TSE agent accumulation upon follicular dendritic cells (FDCs) in lymphoid follicles appears critical for efficient neuroinvasion. Most clinical cases of variant Creutzfeldt-Jakob disease have occurred in young adults, although the reasons behind this apparent age-related susceptibility are uncertain. Host age has a significant influence on immune function. As FDC status and immune complex trapping is reduced in aged mice (600 days old), we hypothesized that this aging-related decline in FDC function might impair TSE pathogenesis. We show that coincident with the effects of host age on FDC status, the early TSE agent accumulation in the spleens of aged mice was significantly impaired. Furthermore, following peripheral exposure, none of the aged mice developed clinical TSE disease during their lifespans, although most mice displayed histopathological signs of TSE disease in their brains. Our data imply that the reduced status of FDCs in aged mice significantly impairs the early TSE agent accumulation in lymphoid tissues and subsequent neuroinvasion. Furthermore, the inefficient neuroinvasion in aged individuals may lead to significant levels of subclinical TSE disease in the population.

Transmissions of variant Creutzfeldt-Jakob disease from brain and lymphoreticular tissue show uniform and conserved bovine spongiform encephalopathy-related phenotypic properties on primary and secondary passage in wild-type mice.

Prion strains are defined by their biological properties after transmission to wild-type mice, specifically by their incubation periods and patterns of vacuolar pathology ('lesion profiles'). Preliminary results from transmissions of variant Creutzfeldt-Jakob disease (vCJD) to wild-type mice provided the first compelling evidence for the close similarity of the vCJD agent to the agent causing bovine spongiform encephalopathy (BSE). Complete results from this investigation, including the transmission characteristics of vCJD from brain and peripheral tissues of 10 cases (after primary transmission and subsequent mouse-to-mouse passage), have now been analysed. All 10 vCJD sources resulted in consistent incubation periods and lesion profiles, suggesting that all 10 patients were infected with the same strain of agent. Incubation periods suggested that infectious titres may be subject to regional variation within the brain. Comparison of incubation periods and lesion profiles from transmission of brain and peripheral tissues showed no evidence of tissue-specific modification in the biological properties of the agent. Analysis of the protease-resistant prion protein (PrP(res)) by Western blotting from primary and subsequent passages in mice showed a glycosylation pattern closely resembling that of vCJD in humans, the so-called BSE 'glycoform signature'. Minor variations in PrP(res) fragment size were evident between mouse strains carrying different alleles of the gene encoding PrP both in primary transmissions and on further passages of vCJD brain. Overall, the results closely resembled those of previously reported transmissions of BSE in the same mouse strains, consistent with BSE being the origin of all of these vCJD cases.

The bank vole (Myodes glareolus) as a sensitive bioassay for sheep scrapie.

Despite intensive studies on sheep scrapie, a number of questions remain unanswered, such as the natural mode of transmission and the amount of infectivity which accumulates in edible tissues at different stages of scrapie infection. Studies using the mouse model proved to be useful for recognizing scrapie strain diversity, but the low sensitivity of mice to some natural scrapie isolates hampered further investigations. To investigate the sensitivity of bank voles (Myodes glareolus) to scrapie, we performed end-point titrations from two unrelated scrapie sources. Similar titres [10(5.5) ID50 U g(-1) and 10(5.8) ID50 U g(-1), both intracerebrally (i.c.)] were obtained, showing that voles can detect infectivity up to 3-4 orders of magnitude lower when compared with laboratory mice. We further investigated the relationships between PrPSc molecular characteristics, strain and prion titre in the brain and tonsil of the same scrapie-affected sheep. We found that protease-resistant PrPSc fragments (PrPres) from brain and tonsil had different molecular features, but induced identical disease phenotypes in voles. The infectivity titre of the tonsil estimated by incubation time assay was 10(4.8) i.c. ID50 U g(-1), i.e. fivefold less than the brain. This compared well with the relative PrPres content, which was 8.8-fold less in tonsil than in brain. Our results suggest that brain and tonsil harboured the same prion strain showing different glycoprofiles in relation to the different cellular/tissue types in which it replicated, and that a PrPSc-based estimate of scrapie infectivity in sheep tissues could be achieved by combining sensitive PrPres detection methods and bioassay in voles.

No major change in vCJD agent strain after secondary transmission via blood transfusion.

BACKGROUND: The identification of transmission of variant Creutzfeldt-Jakob disease (vCJD) by blood transfusion has prompted investigation to establish whether there has been any alteration in the vCJD agent following this route of secondary transmission. Any increase in virulence or host adaptation would require a reassessment of the risk analyses relating to the possibility of a significant secondary outbreak of vCJD. Since there are likely to be carriers of the vCJD agent in the general population, there is a potential for further infection by routes such as blood transfusion or contaminated surgical instruments. METHODOLOGY: We inoculated both wild-type and transgenic mice with material from the first case of transfusion associated vCJD infection. PRINCIPAL FINDINGS: The strain transmission properties of blood transfusion associated vCJD infection show remarkable similarities to the strain of vCJD associated with transmission from bovine spongiform encephalopathy (BSE). CONCLUSIONS: Although it has been hypothesized that adaptation of the BSE agent through secondary passage in humans may result in a greater risk of onward transmission due to an increased virulence of the agent for humans, our data presented here in two murine models suggest no significant alterations to transmission efficiency of the agent following human-to-human transmission of vCJD.

Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins.

Clinical diagnosis and research into transmissible spongiform encephalopathies are hampered by the lack of sufficiently sensitive and specific reagents able to adequately detect the normal cellular form of the prion protein, PrP(C), and the pathological isoform, PrP(Sc). In order to provide such reagents, we applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against a recombinant murine prion protein, to select single-stranded DNA ligands (aptamers) of high affinity. The SELEX protocol and subsequent aptamer characterisation employed protein immobilisation/partitioning using nickel-complexed magnetic particles and a novel SYBR Green-mediated quantitative real-time PCR technique. Following eight rounds of selection, the enriched aptamer pool was cloned and 24 clones sequenced. Seven of these were 'orphan' clones and the remainder were grouped into three separate T-rich families. All but four of the aptamer clones exhibited specific binding to the murine prion protein and the majority also bound to human and ovine prion proteins. Dissociation constants (K(d)) ranged from 18 to 79 nM. Flow cytometry with fluorescein-labelled aptamers confirmed that binding to cells was dependent on the expression of PrP(C). Preliminary studies also indicate that a trivalent aptamer pool is capable of binding the pathological isoform PrP(Sc) following guanidinium denaturation.

Efficient transmission and characterization of Creutzfeldt-Jakob disease strains in bank voles.

Transmission of prions between species is limited by the "species barrier," which hampers a full characterization of human prion strains in the mouse model. We report that the efficiency of primary transmission of prions from Creutzfeldt-Jakob disease patients to a wild rodent species, the bank vole (Clethrionomys glareolus), is comparable to that reported in transgenic mice carrying human prion protein, in spite of a low prion protein-sequence homology between man and vole. Voles infected with sporadic and genetic Creutzfeldt-Jakob disease isolates show strain-specific patterns of spongiform degeneration and pathological prion protein-deposition, and accumulate protease-resistant prion protein with biochemical properties similar to the human counterpart. Adaptation of genetic Creutzfeldt-Jakob disease isolates to voles shows little or no evidence of a transmission barrier, in contrast to the striking barriers observed during transmission of mouse, hamster, and sheep prions to voles. Our results imply that in voles there is no clear relationship between the degree of homology of the prion protein of the donor and recipient species and susceptibility, consistent with the view that the prion strain gives a major contribution to the species barrier. The vole is therefore a valuable model to study human prion diversity and, being susceptible to a range of animal prions, represents a unique tool for comparing isolates from different species.

Detection of type 1 prion protein in variant Creutzfeldt-Jakob disease.

Molecular typing of the abnormal form of the prion protein (PrP(Sc)) has come to be regarded as a powerful tool in the investigation of the prion diseases. All evidence thus far presented indicates a single PrP(Sc) molecular type in variant Creutzfeldt-Jakob disease (termed type 2B), presumably resulting from infection with a single strain of the agent (bovine spongiform encephalopathy). Here we show for the first time that the PrP(Sc) that accumulates in the brain in variant Creutzfeldt-Jakob disease also contains a minority type 1 component. This minority type 1 PrP(Sc) was found in all 21 cases of variant Creutzfeldt-Jakob disease tested, irrespective of brain region examined, and was also present in the variant Creutzfeldt-Jakob disease tonsil. The quantitative balance between PrP(Sc) types was maintained when variant Creutzfeldt-Jakob disease was transmitted to wild-type mice and was also found in bovine spongiform encephalopathy cattle brain, indicating that the agent rather than the host specifies their relative representation. These results indicate that PrP(Sc) molecular typing is based on quantitative rather than qualitative phenomena and point to a complex relationship between prion protein biochemistry, disease phenotype and agent strain.

Resistance of neonatal mice to scrapie is associated with inefficient infection of the immature spleen.

Previous studies demonstrated that neonatal mice up to about a week old are less susceptible than adult mice to infection by intraperitoneal inoculation with mouse-passaged scrapie. In peripherally inoculated adult mice, scrapie replicates in lymphoid tissues such as the spleen before invading the central nervous system. Here, we investigated scrapie susceptibility in neonatal mice in more detail, concentrating on spleen involvement. First, we demonstrated that neonatal mice are about 10 times less susceptible than adults to intraperitoneal scrapie inoculation. Then we injected mice intraperitoneally with a scrapie dose that produced disease in all mice inoculated at 10 days or older but in only about a third of neonatally inoculated mice. In this experiment, spleens collected 70 days after scrapie injection of mice 10 days old or older almost all contained pathological prion protein, PrPSc, and those that were bioassayed all contained high infectivity levels. In contrast, at this early stage, only two of six spleens from neonatally inoculated mice had detectable, low infectivity levels; no PrPSc was detected, even in the two spleens. Therefore, neonatal mice have an impaired ability to replicate scrapie in their spleens, suggesting that replication sites are absent or sparse at birth but mature within 10 days. The increase in susceptibility with age correlated with the first immunocytochemical detection of the normal cellular form of prion protein, PrPc, on maturing follicular dendritic cell networks. As lymphoid tissues are more mature at birth in sheep, cattle, and humans than in mice, our results suggest that in utero infection with scrapie-like agents is theoretically possible in these species.

Follicular dendritic cell dedifferentiation reduces scrapie susceptibility following inoculation via the skin.

Transmissible spongiform encephalopathies (TSEs) are a group of subacute infectious neurodegenerative diseases that are characterized by the accumulation in affected tissues of PrP(Sc), an abnormal isoform of the host prion protein (PrPc). Following peripheral exposure, TSE infectivity and PrP(Sc) usually accumulate in lymphoid tissues prior to neuroinvasion. Studies in mice have shown that exposure through scarified skin is an effective means of TSE transmission. Following inoculation via the skin, a functional immune system is critical for the transmission of TSEs to the brain, but until now, it has not been known which components of the immune system are required for efficient neuroinvasion. Temporary dedifferentiation of follicular dendritic cells (FDCs) by treatment with an inhibitor of the lymphotoxin-beta receptor signalling pathway (LTbetaR-Ig) 3 days before or 14 days after inoculation via the skin, blocked the early accumulation of PrP(Sc) and TSE infectivity within the draining lymph node. Furthermore, in the temporary absence of FDCs before inoculation, disease susceptibility was reduced and survival time significantly extended. Treatment with LTbetaR-Ig 14 days after TSE inoculation also significantly extended the disease incubation period. However, treatment 42 days after inoculation did not affect disease susceptibility or survival time, suggesting that the infection may have already have spread to the nervous system. Together these data show that FDCs are essential for the accumulation of PrP(Sc) and infectivity within lymphoid tissues and subsequent neuroinvasion following TSE exposure via the skin.

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.

Complement component C5 is not involved in scrapie pathogenesis.

During transmissible spongiform encephalopathy (TSE) infections the accumulation of abnormal prion protein within the brain is often accompanied by severe neurodegeneration. Studies have implicated complement, including the membrane attack complex (MAC, C5b-C9), in inducing pathology in some neurodegenerative diseases. Recent studies show the MAC is localized on neurons in the brains of TSE patients implicating complement-mediated cell lysis in TSE neuropathology. To determine the role of the MAC in TSEs, we compared scrapie pathogenesis in C5-deficient and C5-sufficient mice. C5-deficient mice developed clinical scrapie with incubation periods similar to C5-sufficient mice. Furthermore, the severity of the neuropathology was not significantly different between C5-deficient and C5-sufficient mice. These data show that C5, and the MAC, are not involved in TSE pathogenesis.

Scrapie transmission following exposure through the skin is dependent on follicular dendritic cells in lymphoid tissues.

BACKGROUND: Transmissible spongiform encephalopathies (TSEs) are chronic infectious neurodegenerative diseases that are characterized by the accumulation in affected tissues of PrP(Sc), an abnormal isoform of the host prion protein (PrP(c)). Following peripheral exposure, PrP(Sc) usually accumulates on follicular dendritic cells (FDCS) in lymphoid tissues before neuroinvasion. Studies in mice have shown that TSE exposure through scarified skin is an effective means of transmission. Following inoculation via the skin, a functional immune system is critical for the transmission of scrapie to the brain as severe combined immunodeficiency (SCID) mice are refractory to infection. Until now, it was not known which components of the immune system are required for efficient scrapie neuroinvasion following skin scarification. OBJECTIVE: To determine which cells are critical for the transmission of scrapie to the brain following inoculation via the skin. METHODS: A chimeric mouse model was used, which had a mismatch in PrP(c) expression between FDCs and other bone marrow-derived cells within lymphoid tissues. These chimeric mice were challenged with scrapie by skin scarification to allow the separate roles of FDCs and lymphocytes in peripheral scrapie pathogenesis to be determined. RESULTS: We show that mature FDCs are essential for the accumulation of scrapie within lymphoid tissues and the subsequent transmission of infection to the brain following TSE exposure by this route. Furthermore, we show that the accumulation of PrP(Sc) and infectivity in the spleen is independent of PrP expression by lymphocytes or other bone marrow-derived cells. CONCLUSION: Following inoculation with scrapie by skin scarification, replication in the spleen and subsequent neuroinvasion is critically dependent upon mature FDCs.

TSE strain variation.

Studies in mice have revealed considerable strain variation in the agents causing transmissible spongiform encephalopathies (TSEs). TSE strains interact with genetic factors in the host (in particular PrP genotype) to influence characteristics of the disease such as incubation period and neuropathology. TSE strains can retain their identity after propagation in different host species or PrP genotypes, showing that these agents carry their own strain-specific information. It is not known whether this information resides in specific self-perpetuating modifications of PrP, or whether a separate informational molecule is required. Strain typing in mice can be used to explore links between TSEs occurring naturally in different species. Such studies have demonstrated that the strain causing BSE in cattle has also infected domestic cats and exotic ungulates. Most importantly, the BSE strain has also been isolated from patients with variant CJD. In contrast, different TSE strains are associated with sporadic CJD and sheep scrapie.

Follicular dendritic cell dedifferentiation by treatment with an inhibitor of the lymphotoxin pathway dramatically reduces scrapie susceptibility.

Transmissible spongiform encephalopathies (TSEs) may be acquired peripherally, in which case infectivity usually accumulates in lymphoid tissues before dissemination to the nervous system. Studies of mouse scrapie models have shown that mature follicular dendritic cells (FDCs), expressing the host prion protein (PrP(c)), are critical for replication of infection in lymphoid tissues and subsequent neuroinvasion. Since FDCs require lymphotoxin signals from B lymphocytes to maintain their differentiated state, blockade of this stimulation with a lymphotoxin beta receptor-immunoglobulin fusion protein (LT beta R-Ig) leads to their temporary dedifferentiation. Here, a single treatment with LT beta R-Ig before intraperitoneal scrapie inoculation blocked the early accumulation of infectivity and disease-specific PrP (PrP(Sc)) within the spleen and substantially reduced disease susceptibility. These effects coincided with an absence of FDCs in the spleen for ca. 28 days after treatment. Although the period of FDC dedifferentiation was extended to at least 49 days by consecutive LT beta R-Ig treatments, this had little added protective benefit after injection with a moderate dose of scrapie. We also demonstrate that mature FDCs are critical for the transmission of scrapie from the gastrointestinal tract. Treatment with LT beta R-Ig before oral scrapie inoculation blocked PrP(Sc) accumulation in Peyer's patches and mesenteric lymph nodes and prevented neuroinvasion. However, treatment 14 days after oral inoculation did not affect survival time or susceptibility, suggesting that infectivity may have already spread to the peripheral nervous system. Although manipulation of FDCs may offer a potential approach for early intervention in peripherally acquired TSEs, these data suggest that the duration of the treatment window may vary widely depending on the route of exposure.

Follicular dendritic cells as targets for intervention in transmissible spongiform encephalopathies.

Transmissible spongiform encephalopathies (TSEs) are often acquired peripherally, for example by ingestion or iatrogenic exposure. After entry, TSE agents, as identified by disease-specific protein accumulation, usually accumulate on follicular dendritic cells (FDCs) in lymphoid tissues long before infection spreads to the brain. Neuroinvasion of TSE agents is significantly impaired in the absence of mature FDCs. Treatments that interfere with the integrity or function of FDCs extend survival time by blocking replication in lymphoid tissues and spread to the brain. The identification of FDCs as critical for TSE pathogenesis provides a cellular target to which therapies can be specifically directed.

Temporary blockade of the tumor necrosis factor receptor signaling pathway impedes the spread of scrapie to the brain.

Although the transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases, their agents usually replicate and accumulate in lymphoid tissues long before infection spreads to the central nervous system (CNS). Studies of a mouse scrapie model have shown that mature follicular dendritic cells (FDCs), which express the host prion protein (PrP(c)), are critical for replication of infection in lymphoid tissues. In the absence of mature FDCs, the spread of infection to the CNS is significantly impaired. Tumor necrosis factor alpha (TNF-alpha) secretion by lymphocytes is important for maintaining FDC networks, and signaling is mediated through TNF receptor 1 (TNFR-1) expressed on FDCs and/or their precursors. A treatment that blocks TNFR signaling leads to the temporary dedifferentiation of mature FDCs, raising the hypothesis that a similar treatment would significantly delay the peripheral pathogenesis of scrapie. Here, specific neutralization of the TNFR signaling pathway was achieved through treatment with a fusion protein consisting of two soluble human TNFR (huTNFR) (p80) domains linked to the Fc portion of human immunoglobulin G1 (huTNFR:Fc). A single treatment of mice with huTNFR:Fc before or shortly after intraperitoneal injection with the ME7 scrapie strain significantly delayed the onset of disease in the CNS and reduced the early accumulation of disease-specific PrP in the spleen. These effects coincided with a temporary dedifferentiation of mature FDCs within 5 days of huTNFR:Fc treatment. We conclude that treatments that specifically inhibit the TNFR signaling pathway may present an opportunity for early intervention in peripherally transmitted TSEs.

Strain characterization of natural sheep scrapie and comparison with BSE.

Scrapie was transmitted to mice from ten sheep, collected in the UK between 1985 and 1994. As in previous natural scrapie transmissions, the results varied between scrapie sources in terms of the incidence of disease, incubation periods and neuropathology in challenged mice. This contrasted with the uniformity seen in transmissions of BSE to mice. The scrapie and BSE isolates were characterized further by serial passage in mice. Different TSE strains were isolated from each source according to the Sinc or PrP genotype of the mouse used for passage. The same two mouse-passaged strains, 301C and 301V, were isolated from each of three BSE sources. Despite the variation seen in the primary transmissions of scrapie, relatively few mouse-passaged scrapie strains were isolated and these were distinct from the BSE-derived strains. The ME7 scrapie strain, which has often been isolated from independent sheep sources in the past, was identified in isolates from four of the sheep. However, a new distinct strain, 221C, was derived from a further four scrapie sheep. These results suggest that there is agent strain variation in natural scrapie in sheep and that the spectrum of strains present may have changed over the last 20 years. The tested sample is too small to come to any conclusions about whether the BSE strain is present in sheep, but the study provides a framework for further more extensive studies.

Migrating intestinal dendritic cells transport PrP(Sc) from the gut.

Bovine spongiform encephalopathy, variant Creutzfeldt-Jakob disease (vCJD) and possibly also sheep scrapie are orally acquired transmissible spongiform encephalopathies (TSEs). TSE agents usually replicate in lymphoid tissues before they spread into the central nervous system. In mouse TSE models PrP(c)-expressing follicular dendritic cells (FDCs) resident in lymphoid germinal centres are essential for replication, and in their absence neuroinvasion is impaired. Disease-associated forms of PrP (PrP(Sc)), a biochemical marker for TSE infection, also accumulate on FDCs in the lymphoid tissues of patients with vCJD and sheep with natural scrapie. TSE transport mechanisms between gut lumen and germinal centres are unknown. Migratory bone marrow-derived dendritic cells (DCs), entering the intestinal wall from blood, sample antigens from the gut lumen and carry them to mesenteric lymph nodes. Here we show that DCs acquire PrP(Sc) in vitro, and transport intestinally administered PrP(Sc) directly into lymphoid tissues in vivo. These studies suggest that DCs are a cellular bridge between the gut lumen and the lymphoid TSE replicative machinery.