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.

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.

Production and characterization of a panel of monoclonal antibodies against native human cellular prion protein.

The human prion diseases, such as variant Creutzfeldt-Jakob disease (vCJD), are characterized by the conversion of the normal cellular prion protein (PrP(C)) into an abnormal disease associated form (PrP(Sc)). Monoclonal antibodies (MAbs) that recognize these different PrP isoforms are valuable reagents both in the diagnosis of these diseases and in prion disease research in general but we know of no attempts to raise MAbs against native human PrP(C). We immunized prion protein gene ablated (PrP(-/-)) mice with native human PrP(C) purified from platelets (pHuPrP) generating a predominantly IgG isotype anti-pHuPrP polyclonal antibody response in all mice. Following fusion of splenocytes from the immunized mice with SP2/0 myeloma cells, we were able to identify single cell clone and cryopreserve 14 stable hybridoma cell lines producing MAbs that reacted with pHuPrP. The properties of these MAbs (such as isotype, binding to native/denatured pHuPrP, and HuPrP epitopes recognized) are described. Furthermore, several of these MAbs showed a selectivity in their ability to immunoprecipitate disease associated PrP(Sc) and its corresponding protease resistant core (PrP(res)).

An antibody to the aggregated synthetic prion protein peptide (PrP106-126) selectively recognizes disease-associated prion protein (PrP) from human brain specimens.

Human prion diseases are characterized by the conversion of the normal host cellular prion protein (PrP(C)) into an abnormal misfolded form [disease-associated prion protein (PrP(Sc))]. Antibodies that are capable of distinguishing between PrP(C) and PrP(Sc) may prove to be useful, not only for the diagnosis of these diseases, but also for a better understanding of the molecular mechanisms involved in disease pathogenesis. In an attempt to produce such antibodies, we immunized mice with an aggregated peptide spanning amino acid residues 106 to 126 of human PrP (PrP106-126). We were able to isolate and single cell clone a hybridoma cell line (P1:1) which secreted an IgM isotype antibody [monoclonal antibody (mAb P1:1)] that recognized the aggregated, but not the monomeric form of the immunogen. When used in immunoprecipitation assays, the antibody did not recognize normal PrP(C) from non-prion disease brain specimens, but did selectively immunoprecipitate full-length PrP(Sc) from cases of variant and sporadic Creutzfeldt-Jakob disease and Gerstmann-Straussler-Scheinker disease. These results suggest that P1:1 recognizes an epitope formed during the structural rearrangement or aggregation of the PrP that is common to the major PrP(Sc) types found in the most common forms of human prion disease.

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.

Purification of normal cellular prion protein from human platelets and the formation of a high molecular weight prion protein complex following platelet activation.

A method for the extraction and purification of PrP(C), in its native monomeric form, from outdated human platelet concentrates is described. Both calcium ionophore platelet activation and lysis in Triton X-100 were evaluated as methods for the extraction of soluble platelet PrP(C) in its monomeric form. Following platelet activation, the majority of released PrP(C) was detected as a disulphide linked high molecular weight complex, which under reducing conditions could be separated into what appear to be stable non-disulphide linked PrP dimers or PrP covalently linked to another as yet unidentified protein. This phenomenon appears to be unique to activation since only monomeric PrP(C) was detected following lysis of resting platelets. Subsequently, PrP(C) was purified from the Triton X-100 lysate by sequential cation ion exchange and Cu2+ affinity chromatography. From 10 L of outdated platelet concentrate, we were able to recover 1.29 mg PrP(C) at a purity of 92%.

The transmissible spongiform encephalopathies: pathogenic mechanisms and strategies for therapeutic intervention.

Primary neurodegenerative diseases tend to be intractable and largely affect the elderly. There is rarely the opportunity to identify individuals at risk and the appearance of clinical symptoms usually signifies the occurrence of irreversible neurological damage. This situation describes sporadic Creutzfeldt-Jakob disease which occurs world-wide, affecting one person per million per annum. The epidemic of bovine spongiform encephalopathy in the UK in the 1980s and the subsequent causal appearance of variant Creutzfeldt-Jakob disease in young UK residents in the 1990s has refocused attention on this whole group of diseases, known as the transmissible spongiform encephalopathies or prion diseases. The potentially lengthy incubation period of variant Creutzfeldt-Jakob disease, including perhaps an obligate peripheral phase, prior to neuroinvasion, marks variant Creutzfeldt-Jakob disease out as different from sporadic Creutzfeldt-Jakob disease. The formal possibility of detecting individuals infected with the bovine spongiform encephalopathy agent during this asymptomatic peripheral phase provides a strong incentive for the development of therapies for transmissible spongiform encephalopathies. This review focuses on recent advances in the understanding of the pathogenesis of these diseases, with particular reference to in vitro and animal model systems. Such systems have proved invaluable in the identification of potential therapeutic strategies that either specifically target the prion protein or more generally target peripheral pathogenesis. Furthermore, recent experiments in animal models suggest that even after neuroinvasion there may be pharmacological avenues to explore that might retard or even halt the degenerative process.

Comparative analysis of normal prion protein expression on human, rodent, and ruminant blood cells by using a panel of prion antibodies.

BACKGROUND: It is not known whether variant CJD can be transmitted within the human population by blood transfusion. The expression of normal cellular prion protein (PrPC) by different blood cell types may permit selective uptake and dissemination of infectivity. STUDY DESIGN AND METHODS: The normal distribution of PrPC on the major blood cell types of species known to be susceptible to natural or experimental transmissible spongiform encephalopathies was studied. Blood from healthy humans, mice, hamsters, cattle, and sheep was examined by flow cytometry by using a large panel of antibodies with different prion protein (PrP) epitope specificities to maximize the detection of PrP variants across species and cell type. RESULTS: PrP was detected on all major human blood cells types except eosinophils, but was not detected as ubiquitously or uniformly on major blood cell types of different animal species. CONCLUSION: Different animal species have unique patterns of expression of PrPC on blood cell types, with none equivalent to the human pattern. This needs to be considered when extrapolating from animal models of blood-borne transmissible spongiform encephalopathy infectivity, particularly in regard to the risk assessment of potential variant CJD spread within the human population. The relationship between PrP distribution and infectivity distribution in blood needs further investigation.

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.