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A pedigree of familial CJD with five octapeptide repeat insert (50RI) is reported. In this family seven out of nine members from one generation had dementia. Three showed slowly progressive course, two with typical clinical features of CJD, and two without details. The probaband is a 62 year old man, who initially presented with abnormal behavior at age 52. The symptoms gradually deteriorated into a bed-ridden state with no verbal expression at age 59. There was no 14-3-3- protein in CSF or PSD in EEG. MRI showed cerebral atrophy with T2 high intensity of the white matter. At age 62, he still responds to a call for his name. The proband’s oldest brother showed similar clinical features, starting difficulty in word recall around age 64, followed by progressive mental deterioration but was ambulatory even just prior to death. He died unexpectedly and no autopsy was done. The other two brothers showed typical clinical features of CJD, with onset at age 41 and 51, respectively. Their clinical course was about one year, accompanying myoclonus, PSD and akinetic mutism. The brain weight was 1,320g and 1,260g respectively. In the cerebral cortex, severe neuronal loss and gliosis with spongiformic changes were present. Hippocampus was relatively spared. The cerebellum showed severe depletion of granular cells. Immunocytochemically the deposition of prion protein was of synaptic pattern in the cerebral cortex and confluent granular one in the cerebellar cortex. In addition, degeneration of the cerebral white matter was diffuse in one case but focal in another. In the latter case, 5ORI was detected. So far, the results of the sequence analysis differ in two other reported families with 5ORI. A current family may provide a direct evidence that two distinct phenotypes of rapidly or slowly progressive clinical course may arise from the same 5ORI sequence abnormality.

The central event in molecular prion pathogenesis is the conformational change of the cellular prion protein (PrP^C) to an abnormal prion protein (PrP^Sc), and the subsequent accumulation of PrP^Sc in infected human and animals. Recently, reports have shown that the exposure of scrapie-infected cells (ScN2a) to an anti-prion protein (PrP) antibody inhibited this conformational change and accumulation of PrP^Sc. This immunological approach has some problems for in vivo applications because of the difficulty of the blood brain barrier and the resulting lack of accessibility of the antibody to brain tissue. We developed an alternative intervention system using gene expression. In this study, we demonstrated that PrP^Sc accumulation in ScN2a cells could be prevented by this expression system in vitro. We cloned a cDNA of the single chain antibody variable region fragment (ScFv) of an anti-PrP monoclonal antibody T2, and transfected it into a mouse neuroblastoma cell, N2a58 (N2a58-T2ScFv). The T2-ScFv was expressed stably in the N2a58-T2ScFv cells and gave no effects for the PrP^C expression. The cells were co-cultivated with ScN2a cells for 4days or 7days, and the quantity of PrP^Sc was assayed by western blotting and enzyme-linked immunosorbent assay (ELISA). PrP^Sc in ScN2a cells reduced significantly with co-cultivation. This suggests that insertion of a PrP-specific anti-body gene into a neuronal cell will be a potential therapeutic tool for prion diseases.

The new variant of human Creutzfeldt-Jakob disease is transmitted orally via food contaminated with prions of bovine spongiform encephalopathy from cattle, a fact that demands the development of vaccines, in particular those enhancing mucosal immunity, to prevent the transmission of prions between species. Prions are thought to consist mainly of the proteinase K-resistant isoform of prion protein (PrP) encoded on the host genome. There have been reports showing that the mucosal immunogenicity of a peptide could be enhanced by fusion with the B-subunit of E. coli heat-labile enterotoxin (LTB) or cholera toxin. In the present study, to determine whether PrP can be also immunogenically enhanced by fusion with LTB, we compared the mucosal immunogenicity of LTB-fused mouse (mo) and bovine (bo) PrP (LTB-mo or boPrP) with that of non-fused mo and boPrP by intranasally immunizing C57BL/6 and Balb/c mice three times every two weeks in the presence of recombinant LT as an adjuvant. In the fusion protein, the C-terminal half of mo and boPrP was linked to the C-terminus of LTB by hinge sequence of Gly-Pro-Gly-Pro. The biochemical properties of these fusion proteins were similar to those of LTB, including pentameric protein structure and GMl ganglioside-binding competence. Immune sera were collected one week after the final immunization and subjected to ELISA to detect the PrP-specific antibodies. MoPrP induced only a weak immune response in both mouse lines. No immunogenic enhancement could be detected in the mice immunized with LTB-moPrP. This poor immune response against moPrP and LTB-moPrP is attributable to the self-tolerance of mice to self-antigen. In contrast, boPrP was highly immunogenic in Balb/c mice but not in C57BL/6 mice. This indicates that the antibody response against PrP can differ widely in individuals. However, the fusion with LTB markedly enhanced immonogenicity of boPrP in both mouse lines, producing more than 100-fold IgG and IgA capable of cross-reacting with human and sheep PrPs but not with mo or hamster PrPs. This striking enhancement of the mucosal immunogenicity of boPrP in mice by fusion with LTB points to LTB-fused PrPs as a possible mucosal vaccine to elicit antibodies prophylactic for the oral transmission of prions from one species to another.

Prion is a host-encoded protein and folds into at least two conformations, physiological PrP^C and pathogenic PrP^Sc. PrP^Sc mediates the conversion of PrP^C into PrP^Sc. Since prion-binding agents are expected to show inhibitory or stimulating effects on the conversion of prion protein, the binding compounds are promising candidates for the anti-prion disease agents. We established an ELISA assay system using the prion peptide PrP(106–126) and an anti-prion monoclonal antibody 3F4 to detect the prion-binding activity, and tested the activity of the extracts of medical plants and microbial culture broth samples. Some of the samples that showed prion-binding activity in this ELISA assay were advanced to the next assay to detect the inhibition on conversion of PrP^C into PrP^Sc using ScN2a cells. Finally we found that some compounds derived from chlorophylls showed clear inhibition for the prion protein conversion. The mechanism of inhibition by these chemicals may stand on the binding to the 3F4 epitope area of prion protein and interrupting the PrP^C-PrP^Sc interaction. The safety of these chemicals are so sufficient, since they have already used as food additives (coloring agents). Furthermore, one of, them, sodium copper chlorophyllin, has managed as a drug to cure bad breath. They are expected to be good lead compounds to develop the new anti-prion drugs.

Prion diseases are a group of fatal neurodegenerative disorders, and thought to be caused by the accumulation of an infectious protease-resistant isoform of prion protein (PrP^Sc). Mysteriously, the cattle exposed to infectious feed do not necessarily catch bovine spongiform encephalopathy (BSE). For one reason, we assumed that some constituents in forage grasses inhibit the infection of BSE. We therefore investigated whether some forage grasses show inhibitory effects on the formation of PrP^Sc.

Amyloid deposition in the brain is assumed to correlate with pathogenesis of human neurodegenerative disorders, including transmissible spongiform encephalopathies (TSE). To date, many researchers have devoted them-selves to early detection of pathological amyloids, and PET neuroimagings of beta-amyloid aggregates in Alzheimer’s disease have been developed to be under clinical trials.