Yuko Ushiki

Expression of cellular prion-related protein by murine Langerhans cells and keratinocytes.

Transmissible spongiform encephalopathies are characterized by the accumulation of a proteinase-resistant isoform of the cellular prion-related protein (PrP(c)) within the central nervous system (CNS). The accumulation of scrapie-associated PrP (PrP(Sc)) within cells of the lymphoreticular system prior to its accumulation in the CNS is regarded as important for the development of neurological diseases after peripheral inoculation. Little, however, is known as to which cells are the targets for peripheral inoculation. Here, the presence of PrP(c) on murine Langerhans cells (LC), dendritic cells in the skin and mucosa, and keratinocytes (KC) is demonstrated by immunohistochemical staining, Western-blotting and FACS analysis. The expression of PrP(c) mRNA in freshly purified LC and KC was also detected by reverse transcriptase-polymerase chain reaction. The expression of PrP(c) on LC was slightly increased during culture. These data suggest that LC and KC may be the targets for peripheral infection with prions.

An advantageous method utilizing new homogenizing device BioMasher and a sensitive ELISA to detect bovine spongiform encephalopathy accurately in brain tissue.

A new screening method was developed to detect bovine spongiform encephalopathy (BSE). This method is advantageous because it has a simpler and safer protocol than commercial kits. A new device was developed for this method; it was named the BioMasher, to homogenize brain tissue by passing it through a porous rigid polypropylene filter. In this system, a purification step was eliminated in the sample preparation. Thus, the time needed for sample pretreatment is substantially shortened, and the risk of infection during sample processing is effectively reduced. Monoclonal antibodies to prion protein were created and used to construct a sensitive sandwich enzyme-linked immunosorbent assay system. The sensitivity of this assay kit using frozen BSE-positive brain is comparable or more sensitive than commercial kits. Moreover, the detection sensitivity for deteriorated samples, which were kept at 37 degrees C for 1 day, is 10- to 30-fold more sensitive than a commercial kit.

Western blot assessment of prion inactivation by alkali treatment in the process of horticultural fertilizer production from meat meal

Abstract Western blot detection of the abnormal isoform of the prion protein (PrPSc) was used to assess prion inactivation by heating under alkaline conditions during the manufacturing process used to produce horticultural fertilizer from meat meal. PrPSc was detected in the sample prepared from the horticultural fertilizer spiked with a 0.25 µg equivalent of scrapie-infected mouse brain. In contrast, PrPSc was not detected in a sample containing 0.25 g brain equivalent prepared from a small-scale processed mixture of scrapie-infected mouse brain and meat meal using the same method as that used to produce horticultural fertilizer. Our results indicated that the amount of PrPSc decreased to at least 1/106 by processing with heating under alkaline conditions. Although the bioassay suggests that prion infectivity was reduced under these conditions, this procedure did not completely remove high-titer prion infectivity.

A Novel BSE screening kit with simplified preparation method for EIA

Introduction In Japan, all slaughtered and deceased bovine materials are tested for BSE infection. The primary screening test is undertaken by the meat inspection office or live stock hygiene service center in each prefecture. In these circumstances, BSE kits adapted for relatively small number of samples are required. Here we describe the development of a neŵ BSE screening system, which has simpler and safer protocol for sample preparation steps for EIA. This assay system has been named the NippIBL BSE Assay system.

Partial characterization of monoclonal antibodies which bind to disease-associated prion protein in immunoprecipitaion assay

Conformational conversion of cellular prion protein (PrPC) to scrapie-form prion protein (PrPSc) 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 PrPC and PrPSc or mechanism of prion replication. Therefore the purpose of this study is to generate such mAbs that would react with PrPSc but not with PrPC.

Surveillance of chronic wasting disease (CWD) in Japan

Chronic wasting disease (CWD) in cervids including elk, mule deer, and white-tailed deer, is a member of the transmissible spongiform encephalopathies (TSEs). CWD is a serious problem in North America. The detection of abnormal isoforms of prion protein (PrPSc) is a key factor for the diagnosis of CWD, similar to other TSEs. The surveillance program for TSEs in animals is conducted by the Ministry of Agriculture, Forestry, and Fishery (MAFF) and is targeted to sheep, goats, and deer. In Japan, several different anti-prion protein (PrP) monoclonal antibodies (mAbs) are utilized for bovine spongiform encephalopathy (BSE) confirmation. Since CWD does not occur naturally in Japan, the immunoreactivity of the antibodies against PrPSc found in deer was not known. In this study, we examined the immunoreactivities of these antibodies against PrPSc found in CWD. The protocols that are used in Japan for confirmation of BSE cases are Western blot (WB) and immunohistochemistry (IHC). We used these same protocols to examine CWD positive brain samples which were provided by Dr. A. Davis of the National Veterinary Service Laboratory, USA. Mabs. T1, T2, 44B1, and 72-5, were used successfully to detect PrPSc in CWD affected mule deer brains by WB. In IHC, PrPSc was detected with mAbs T2, 44B1, and polyclonal antibody B103. These results determined that the antibodies used for BSE confirmation are also applicable to CWD, as for scrapie. These same antibodies could detect PrPC from Japanese deer by WB without proteinase digestion. The amino acid sequence of PrP of Japanese deer was found to be the same as sequence as the one reported for mule deer. These antibodies were then used for CWD surveillance in Japan. When 127 of hunter-killed deer from Hokkaido were examined, PrPSc was not detected in any of the animals.