Neuroanatomical distribution of disease-associated prion protein in cases of bovine spongiform encephalopathy detected by fallen stock surveillance in Japan.

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle characterized by accumulation of the disease-associated prion protein (PrP(Sc)) in the central nervous system (CNS). The immunohistochemical patterns and distribution of PrP(Sc) were investigated in the CNS, brains, and spinal cords of 7 naturally occurring BSE cases confirmed by the fallen stock surveillance program in Japan. No animals showed characteristic clinical signs of the disease. Coronal slices of 14 different brain areas in each case were immunohistochemically analyzed using an anti-prion protein antibody. Immunolabeled PrP(Sc) deposition was widely observed throughout each brain and spinal cord. Intense PrP(Sc) deposition was greater in the thalamus, brainstem, and spinal cord of the gray matter than in the neocortices. The topographical distribution pattern and severity of PrP(Sc) accumulation were mapped and plotted as immunohistochemical profiles of the different brain areas along the caudal-rostral axis of the brain. The distribution pattern and severity of the immunolabeled PrP(Sc) in the CNS were almost the same among the 7 cases analyzed, suggesting that the naturally occurring cases in this study were at the preclinical stage of the disease. Immunohistochemical mapping of the PrP(Sc) deposits will be used to clarify the different stages of BSE in cattle.

Conformational change in hamster scrapie prion protein (PrP27-30) associated with proteinase K resistance and prion infectivity.

The scrapie prion protein (PrP27-30) is a crucial component of the prion and is responsible for its transmissibility. Structural information on this protein is limited because it is insoluble and shows aggregated properties. In this study, PrP27-30 was effectively dispersed using sonication under the weak alkaline condition. Subsequently, the small PrP27-30 aggregates were subjected to different pH, heat, and denaturing conditions. The loss of proteinase K (PK) resistance of PrP27-30 and prion infectivity were monitored along with spectroscopic changes. Prion inactivation could not be achieved by the loss of PK resistance alone; a significant loss of the PrP27-30 amyloid structure, which was represented by a decrease in thioflavin T fluorescence, was required for the loss of transmissibility.

Tumor necrosis factor alpha is not a pathogenic determinant in acute lethal encephalitis induced by a highly neurovirulent strain of mouse hepatitis virus.

To investigate the role of tumor necrosis factor alpha (TNFalpha) in the pathogenesis of acute viral encephalitis, TNFalpha-deficient mice were infected with a highly neurovirulent strain of mouse hepatitis virus, JHM, and compared with JHM-infected C57BL/6 mice as controls. All the JHM-infected mice had succumbed to infection by 6 days postinfection. The virus replication kinetics, histopathological changes and mRNA expression levels of proinflammatory cytokines in the brain did not differ between TNFalpha-deficient and control C57BL/6 mice. These results suggest that TNFalpha is not a pathogenic determinant in JHM-induced acute lethal encephalitis.

A potential blood test for transmissible spongiform encephalopathies by detecting carbohydrate-dependent aggregates of PrPres-like proteins in scrapie-Infected hamster plasma.

PrPres has rarely been detected in blood (except in leukocytes) even in diseased animal models that are known to contain a large amount of PrPres in infected tissues. It seems likely that PrPres detection in blood is difficult because of the low titer of infectious material within the blood. Here, we demonstrate the detection of proteinase K-resistant 3F4-reactive protein in the plasma of scrapie-infected hamsters but not in the plasma of mock-infected hamsters by partial purification using a novel method termed "acidic SDS precipitation," in conjunction with a highly sensitive chemiluminescence detection system used to show the presence of PrP at a concentration equivalent to 1.4x10(-9) g of brain homogenate or 1.5x10(-12) g (6.5x10(-17) mol) of rPrP by conventional Western blotting. The 3F4-reactive proteins in scrapie-infected hamster plasma often resulted in multiple Mw protein bands occurring at higher Mw positions than the position of the di-glycosyl PrP molecule. Mixing scrapie-infected hamster brain homogenate with mock-infected hamster plasma resulted in the formation of similar Mw positions for multiple 3F4-reactive proteins. Predigestion of carbohydrate side chains from the proteins in the plasma or brain homogenate before mixing resulted in failure to obtain these multiple 3F4-reactive proteins. These observations indicate that PrPres aggregated with other proteins in the plasma through carbohydrate side chains and was successfully detected in the plasma of scrapie-infected hamsters. Counterparts in these aggregates with PrPres-like proteins in scHaPl are not known but any that exist should resist the PK digestion.

Assessment of prion inactivation by combined use of Bacillus-derived protease and SDS.

Prions, infectious agents causing transmissible spongiform encephalopathy, retain infectivity even after undergoing routine sterilization processes. We found that MSK103 protease, identified in our previous study, effectively reduces infectivity and the level of misfolded isoform of the prion protein in scrapie-infected brain homogenates in the presence of SDS. The treatment therefore can be applied to the decontamination of thermolabile instruments.

Urinary excretion and blood level of prions in scrapie-infected hamsters.

Prions, infectious agents causing transmissible spongiform encephalopathy (TSE), are composed primarily of the pathogenic form (PrP(Sc)) of the host-encoded prion protein. Although very low levels of infectivity have been detected in urine from scrapie-infected rodents, no reports of urinary PrP(Sc) have been substantiated. Studies on the dynamics of urinary PrP(Sc) during infection are needed to ensure the safety of urine-derived biopharmaceuticals and to assess the possible horizontal transmission of prion diseases. Using the protein misfolding cyclic amplification technique, a time-course study of urinary excretion and blood levels of PrP(Sc) was performed in Sc237-infected hamsters and a high rate of PrP(Sc) excretion was found during the terminal stage of the disease. Following oral administration, PrP(Sc) was present in all buffy coat samples examined; it was also present in most of the plasma samples obtained from hamsters in the symptomatic stage. PrP(Sc) was excreted in urine for a few days after oral administration; subsequently, urinary PrP(Sc) was not detected until the terminal disease stage. These results represent the first biochemical detection of PrP(Sc) in urine from TSE-infected animals.

Assessment of prion inactivation by fenton reaction using protein misfolding cyclic amplification and bioassay.

An abnormal isoform of the prion protein, associated with transmissible spongiform encephalopathies, retains infectivity even after undergoing routine sterilization processes. We found that a formulation of iron ions combined with hydrogen peroxide effectively reduced infectivity and the level of abnormal isoforms of the prion protein in scrapie-infected brain homogenates. Therefore, the Fenton reaction has potential for prion decontamination.

Prion inactivation by the Maillard reaction.

Since variant Creutzfeldt-Jakob disease (vCJD) has been suspected to be attributable to the infectious agents associated with bovine spongiform encephalopathy (BSE), it is important to prevent the transmission of pathogenic forms of prion protein (PrP(Sc)) through contaminated feeding materials such as meat and bone meal (MBM). Here, we demonstrate that the Maillard reaction employing a formulation of glucose in combination with sodium hydrogen carbonates effectively reduced the infectivity (approximately 5.9-log reduction) of a scrapie-infected hamster brain homogenate. In addition to a bioassay, a protein misfolding cyclic amplification (PMCA) technique, in which PrP(Sc) can be amplified in vitro, was used as a rapid test for assessing PrP(Sc) inactivation. The PMCA analysis also indicated that the PrP(Sc) level in the infected material significantly decreased following the Maillard reaction. Therefore, the Maillard reaction can be employed for the decontamination of large amounts of byproducts such as MBM.

Microglial cell line established from prion protein-overexpressing mice is susceptible to various murine prion strains.

Several lines of evidence suggest that microglia have important roles in the pathogenesis of prion diseases. Here, we establish a novel microglial cell line (MG20) from neonatal tga20 mice that overexpress murine prion protein. After exposure to Chandler scrapie, we observed the replication and accumulation of disease-associated forms of the prion protein in MG20 cells up to the 15th passage. Furthermore, MG20 cells were susceptible to ME7, Obihiro scrapie, and bovine spongiform encephalopathy agents. Thus, MG20 cell lines persistently infected with various murine prion strains provide a useful model for the study of the pathogenesis of prion diseases.

Protein misfolding cyclic amplification as a rapid test for assessment of prion inactivation.

Abnormal isoform of prion proteins (PrP(Sc)), which are infectious agents associated with prion diseases, retain infectivity after undergoing routine sterilization processes. A sensitive method to detect the infectivity is a bioassay, and it has been used for assessing prion inactivation. However, the result is obtained after several hundred days. Here, protein misfolding cyclic amplification (PMCA) in which PrP(Sc) can be amplified in vitro was applied for assessing prion inactivation by dry heating and autoclaving. Scrapie-infected hamster brains were inactivated under various conditions, and residual infectivity and PrP(Sc) were detected by the bioassay and PMCA, respectively. The PMCA results were in good agreement with those of the bioassay. In samples autoclaved at temperatures below 150 degrees C, while infected mice died in the bioassay, protease-resistant PrP (PrP(res)) signals were detected in the second or third round of PMCA. Three rounds of PMCA require only 6 days, which means that the PMCA method is much faster than the bioassay.

Rapid PrP(Sc) detection in lymphoid tissue and application to scrapie surveillance of fallen stock in Japan: variable PrP(Sc) accumulation in palatal tonsil in natural scrapie.

Rapid western blot (WB) procedure for an abnormal isoform of prion protein (PrP(Sc) ) detection in lymphoid tissues was established and has been applied to the surveillance of fallen stock. In this program, brain and palatal tonsil were examined by WB and three cases of sheep scrapie were detected. While one clinically scrapie-infected sheep harbored PrP(Sc) in the brain and palatal tonsil, the two sheep in the pre-clinical stage harbored PrP(Sc) in the brain, but not in the palatal tonsil. This study shows that PrP(Sc) accumulation in palatal tonsil is variable in natural scrapie, even among genetically susceptible sheep.

The N-terminal cleavage site of PrPSc from BSE differs from that of PrPSc from scrapie.

Heterogeneity in transmissible spongiform encephalopathy is thought to have derived from conformational variation in an abnormal isoform of the prion protein (PrPSc). To characterize PrPSc in bovine spongiform encephalopathy (BSE) and scrapie, we analyzed the newly generated N-terminus of PrPSc isoforms by digestion with proteinase K (PK). With a lower concentration of PK, the terminal amino acid of BSE PrPSc converged at N96. Under the same conditions, however, the terminal amino acid of scrapie PrPSc was G81 or G85. Furthermore, with an increase of PK concentration, the N-terminal amino acid was shifted and converged at G89. The results suggest that the PK cleavage site of BSE PrPSc is uniform and is different from the cleavage site of scrapie PrPSc.

Effect of tissue deterioration on postmortem BSE diagnosis by immunobiochemical detection of an abnormal isoform of prion protein.

Surveillance for bovine spongiform encephalopathy (BSE) in fallen stock in Japan is conducted with a commercial enzyme-linked immunosorbent assay (ELISA) for mass screening, with Western blotting (WB) and immunohistochemistry performed for confirmation of the ELISA. All tests are based on immunological detection of an abnormal isoform of the prion protein (PrP(Sc)) in brain tissues, which have sometimes deteriorated by the time samples from fallen stock reach a diagnostic laboratory. To evaluate BSE surveillance procedures for fallen stock, we examined PrP(Sc) detection from artificially deteriorated BSE-affected bovine brain tissues with a commercial ELISA kit and compared the results with those of WB. The optical density (OD) values of the ELISA decreased with advancing deterioration of the tissues, whereas no reduction in the signal for PrP(Sc) was observed in WB, even when performed after 4 days of incubation at 37 degrees C. The progressive decrease in the OD values in the ELISA appear to be caused by a partial loss of the N-terminal moiety of PrP(Sc) due to digestion by endogeneous and/or contaminated microbial enzymes, and by the presence of ELISA inhibitors that are generated in deteriorated tissues. These results suggest that WB is the most reliable test for fallen stock, especially for cattle brains within decaying carcasses.

Susceptibility of heat shock protein 70.1-deficient C57BL/6 J, wild-type C57BL/6 J and A/J mice to Trypanosoma congolense infection.

The heat shock protein (HSP) 70.1 gene lies on mouse chromosome 17 among the candidates for Tir1, the major quantitative trait locus associated with response to Trypanosoma congolense infection. To evaluate whether the HSP70.1 gene is involved in the response, we compared the susceptibility of HSP70.1-deficient C57BL/6 J, resistant wild-type C57BL/6 J and susceptible A/J mice. No differences were observed between HSP70.1-deficient and wild-type C57BL/6 J mice in survival time, levels of parasitemia and anemia, suggesting that there is no involvement of the HSP70.1 gene in control of T. congolense infection. The course of infection was markedly different between A/J and C57BL/6 J mice. A/J mice showed a bi-phasic survival pattern, which seemed to be associated with two waves of high parasitemia, but developed only moderate anemia. C57BL/6 J mice controlled parasitemia well but developed severe anemia in the late stage of infection.

Susceptibility of transgenic mice expressing chimeric sheep, bovine and human PrP genes to sheep scrapie.

The use of Transgenic (Tg) mice expressing chimeric sheep/mouse (Sh/Mo) prion protein (PrP) and chimeric bovine/mouse (Bo/Mo) PrP genes was evaluated as a sheep scrapie model. We also investigated the potential for the transmission of sheep scrapie to a human/mouse (Hu/Mo) PrP Tg mouse line. The Sh/Mo PrP and Bo/Mo PrP Tg Prnp(+/+) or Prnp(0/0) mouse lines were inoculated intracerebrally with brain homogenates from three sheep with natural scrapie (KU, Y5 or S2). Incubation periods were slightly shorter in Sh/Mo PrP Tg Prnp(+/+), than in non-Tg mice inoculated with KU brain homogenate. In contrast, the incubation period was significantly prolonged (p<0.05) in Bo/Mo PrP Tg Prnp(+/+) mice inoculated with KU brain homogenate. The incubation period was significantly longer in all Tg Prnp(+/+) and Prnp(0/0), than in non-Tg mice (p<0.01) inoculated withY5 brain homogenate. None of the Tg Prnp(0/0) mice inoculated with S2 brain homogenate developed clinical signs and PrP(Sc) was undetectable in their brains. These results suggested that expression of the Sh/Mo PrP or Bo/Mo PrP transgenes does not confer susceptibility to sheep prions upon mice, and thus none of the Tg mouse lines could be a suitable model of sheep scrapie. Hu/Mo PrP Tg Prnp(0/0) mice inoculated with natural and experimental scrapie or mouse prions did not develop clinical signs of scrapie and PrP(Sc) was undetectable. These results suggested that neither sheep nor mouse strains of scrapie are highly transmissible to humans.