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We established a rapid bioassay method for transmissibility of human prions to knock-in mouse expressing human/mouse chimeric prion protein (Ki-ChM mouse). In Ki-ChM mice, we detected accumulation of abnormal isoform (PrP^Sc) of prion protein in follicular dendritic cells (FDCs) in lymphoid organ within 14 days following intraperitoneal administration of human prions. Intraperitoneal administration of the inoculum provides an advantage over intracerebral injection, considering only a limited volume of 20µl can be inoculated with the intracerebral route. To overcome this limitation and to confirm dose dependent effect on PrP^Sc accumulation with intraperitoneal injection, we made an attempt to inoculate brain homogenate of human sporadic CJD (sCJD) with large dose and repetitive injection of a volume up to ∼5000µl. Fifty micro liter of 10^−2, 10^−3, and 10^−4 diluted sCJD were inoculated respective groups intraperitoneally. Five hundred µl of 10^−3 and 10^−4 dilution of sCJD inoculated with one shot were shown as equivalent infectivity to 50µl of 10^−2 and 10^−3 dilution of them respectively. Five hundred micro liter of 10^−4 dilution of sCJD inoculated with once a day during 10 days were shown as an equivalent infectivity to 50µl of 10^−2 dilution of it. These results indicate that the detection of PrP^Sc in FDCs has increased in proportion to the amount of prion inoculated. It is conceivable that the prion of a low density piles up the intake frequency and the pathogenicity rises as well as a high density prion.

Because of the emergence of variant CJD and accumulation of iatrogenic CJD cases, the establishment of therapeutics for prion disease is urgently needed. One possible strategy for therapeutics is an inhibition of an abnormal isoform of prion protein (PrP^Sc) formation in the infected host. The C-terminal portion of prion protein (PrP), corresponding to a protease-resistant core fragment of PrP^Sc, is essential for prion propagation. Antibodies to the C-terminal portion of PrP are known to inhibit PrP^Sc accumulation in prion-persistently-infected cells. Here we show that, in addition to monoclonal antibodies (mAbs) to the C-terminal portion of PrP, a mAb recognizing the octapeptide repeat region in the N-terminal portion of PrP that is dispensable for PrP^Sc formation, reduced PrP^Sc accumulation in cells persistently infected with prion; mAb 110 and 31C6, recognizing linear epitope aa 59–90 and aa 143–149, respectively, and mAbs 44B1 and 72, recognizing discontinuous epitope located within aa 155–231 and aa 89–231, respectively, inhibited the PrP^Sc formation when the cells were cultured with medium containing these mAbs. The 50% effective doses were as low as ∼1 nM, and, regardless of their epitope specificity, the inhibitory mAbs shared the ability to bind cellular prion protein (PrP^C) expressed on the cell surface. Flow cytometric analysis revealed that mAbs which bound to the cell surface during cell culture were not internalized even after their withdrawal from the growth medium. Retention of the mAb-PrP^C complex on the cell surface was also confirmed by the fact that internalization was enhanced by treatment of cells with dextran sulfate. These results suggest that anti-PrP mAb antagonized PrP^Sc formation by interfering with the regular PrP^C degradation pathway.

The pathogenesis of prion disease involves a structural change of prion protein (PrP). A series of antibodies recognizing a distinct conformational change of prion is useful for not only understanding the mechanism of molecular conversion but also for diagnostic and therapeutic reagents. Prions with various conformations can be prepared in vitro under varying physicochemical condition. Antibody-displaying phage library enables us to isolate such antibodies by simple biopanning procedure. This feature is superior to conventional animal-based approach. Recently, Prusiner et al. reported that the in vitro-refolded recombinant prion could be infectious in mouse model (Science 305: 673–676, 2004).

Conformational conversion of cellular prion protein (PrP^C) to scrapie-form prion protein (PrP^Sc) is thought to be the central event in prion pathogenesis. Several studies have shown that their distinct conformation should cause different immunogenic properties, however, almost all mAbs generated against PrP are unable to recognize each ones separately. Such characters of mAbs make it diffcult to study conformational difference between PrP^C and PrP^Sc or mechanism of prion replication. Therefore the purpose of this study is to generate such mAbs that would react with PrP^Sc but not with PrP^C.

Creutzfeldt-Jacob disease (CJD) is a neurodegenerative disorder caused by piron, which mainly consists of abnormal prion protein (PrP^Sc). As PrP^Sc was detected recently in two patients who received transfusion from preclinical variant CJD (vCJD), concerns about the theoretical risk of transmission of vCJD through blood products increased. In order to contribute the blood products-safety against prion, we aimed at obtaining monoclonal antibodies (mAbs) eligible for screening procedures.

Since the in vitro selection method, which chooses nucleic acid molecules of demands from huge scale clot of randomized nucleic acid molecules, has been developed (Tuerk, et al., Science.; 249 (4968):505-10, 1990 etc.), various species of functional nucleic acid molecules have been developed. Notably, one of those molecules called “Aptamer” can specifically recognizes and binds to its target from small molecule such as amino acid to large molecules such as proteins or virus particles. As its property is almost same as antibody, aptamer has been applied in basic study of nucleic acid/protein interaction and clinical or diagnostic drugs. (Allen, et al., Virology, 209, 327–336, 1995 etc.)

Detection of the abnormal prion protein in blood and cerebral spinal fluid of transmissible spongiform encephalophathy (TSE) infected individuals has not been possible by Western blot, immunohistochemistry or the present ELISA tests. We used an analytical approach in conjugation with a fluorescence immunoassay to develop methods to measure the abnormal prion in blood and cerebral spinal fluid. Monoclonal antibodies (mabs) and the corresponding fluorescein-labeled peptides and magnetic beads coupled to Protein A (PA) were used. The mabs, 12F10 and 3B5 reacted with the prion protein or the fluorescein-labeled peptides very rapidly in a liquid phase assay using a 0.15M TAPS buffer pH 8.8 containing 0.1% bovine serum albumin (BSA) and 0.1% N-Octyl-glucoside. The magnetic beads coupled to PA were mixed with the mab and recombinant prion protein (rPrP) or a sample and incubated for 30 min at 25 C. After washing the beads, the appropriate fluorescein labeled peptide was added and incubated for 15 min at 25 C. An aliquot of the supernatant fraction was removed and analyzed by capillary electrophoresis using laser-induced detection. Blood samples from sheep exposed to sheep scrapie were collected in 10 ml tubes containing 17.55 mg of EDTA. Buffy coats were prepared from the blood samples in the usual manner and were washed twice with 0.04 M Tris, pH 8.35 containing 0.135 M NH_4Cl. The prepared buffy coats were frozen and thawed twice, incubated with 50 µg/ml of DNase and then incubated with Proteinase K (50µg/ml). The samples were then incubated with an equal volume of hexafluoro-2-propanol (HFIP) at 56 C for 5 min. The HFIP layer was dried in a vacuum centrifuge. The sample was re-suspended and used in temperature and pH. The optimal pH and temperature for the reaction of the antibody to the fluorescein labeled peptide was 8.8 and 25 C respectively. When a standard curve was made with the rPrP, 10 pg of rPrP could be easily detected. The blood samples correlated with the scrapie status of the sheep as was determined by postmortem examination of the brain by western blot. Further development of this assay and validation will make possible a rapid test for early detection of the TSEs.

Immunohistochemistry for prion protein (PrP) is essential for pathological diagnosis of prion diseases, however, certain pretreatment procedures are required for antigen retrieval of formalin-fixed paraffin-embedded sections. Although hydrolytic autoclaving and formic-acid treatment are usually employed, these methods tend to result in nonspecific high background staining and poorly preserved sections. Thus, we tried autoclaving of the sections with nonionic detergent as follows: autoclave sections with 0.1% Triton X-100 in 50mM Tris-HCI buffer pH7.6, 121°C 20 minutes. Both monoclonal antibody (clone 3F4) and polyclonal anti-PrP-C-terminal antibody were used for human cases with Creutzfeldt-Jakob disease and mouse models of transmissible spongiform encephalopathies. This detergent autoclaving method resulted in significantly higher signal/noise ratio for accumulated PrP in the follicular dendritic cells compared to conventional pretreatment methods. Although synaptic staining in the brain was obtained rather weakly than with conventional methods, pretreatment with detergent autoclaving contributed to lower background and well preserved sections so that we could recognize PrP deposition well. Normal-form cellular PrP was not immunostained in control cases. These results were observed for both monoclonal and polyclonal antibodies, and both human and murine materials. The lower background staining, obtained with detergent autoclaving, enabled double immunofluorescence efficiently on the same sections. We, thus, propose the detergent autoclaving method as a useful choice of the pretreatment for PrP immunohistochemistry

Previous studies have reported a neuroprotective role for cellular prion protein (PrP^C) against apoptosis induced by serum deprivation in an immortalized prion protein gene deficient neuronal cell line. These cell lines were derived from Rikn-type prion protein gene-deficient (Prnp^-/-) (type-2) mice, which produce a prion protein (PrP)-like glycoprotein named as doppel protein (Dpl). To conclude that PrP^C inhibits apoptosis without Dpl, several immortalized cell lines were established from the brain of type-1 Prnp^-/- mice (Zrch I). The data showed that PrP^C had anti-apoptotic function in both neuronal and glial cells under serum free condition. On the other hand, the Prnp^-/- cells expressed Dpl showed apoptosis as with Prnp^-/- cells. The results suggest that the functions of PrP involve anti-apoptotic effect without reference to ectopic production of Dpl. Also to investigate whether PrP production effect neurite outgrowth of neuronal cell in vitro, the extension of the neurite was measured with time after passage. The PrP^C expressed cells showed longer neurite outgrowth than that of Prnp^-/- cells. The data suggest that PrP^C affects the subsequent growth of neurite.

To know the mechanisms in which cellular isoform of prion protein (PrP^C) is involved for the neuroprotection, we compared death signals in Prnp^-/- primary cultivated neurons to those in wild-type (WT) primary cultivated neurons. These results are also confirmed by immortarized neuronal cells. When copper was exposed to these neurons, the both of type-1 and type-2 Prnp^-/- neurons were more sensitive and underwent apoptotic cell death more readily than WT neurons. The cell death in Prnp^-/- neurons was effectively inhibited by anti-oxidants or some caspase-inhibitors as seen in wild-type neurons. In Prnp^-/- neurons, copper toxicity was enhanced more significantly by deprivation of anti-oxidants, and then induced more increasing apoptotic features. To know the level of the reactive oxygen species, the level of the intracellular hydrogen peroxide (H_2O_2) were measured. Intracellular H_2O_2 in Prnp^-/- cells decreased more rapidly than that in WT cells. These data demonstrate that in Prnp^-/- neurons, copper may be a strong mediator to convert H_2O_2 to more toxic free radicals in a fenton-like reaction, which induces more caspase-mediated apoptosis. These results suggest that PrP^C may moderate the intracellular H_2O_2 level as a copper binding protein or an anti-oxidant to protect neuronal cells from apoptotic death.