Selective propagation of mouse-passaged scrapie prions with long incubation period from a mixed prion population using GT1-7 cells.

In our previous study, we demonstrated the propagation of mouse-passaged scrapie isolates with long incubation periods (L-type) derived from natural Japanese sheep scrapie cases in murine hypothalamic GT1-7 cells, along with disease-associated prion protein (PrPSc) accumulation. We here analyzed the susceptibility of GT1-7 cells to scrapie prions by exposure to infected mouse brains at different passages, following interspecies transmission. Wild-type mice challenged with a natural sheep scrapie case (Kanagawa) exhibited heterogeneity of transmitted scrapie prions in early passages, and this mixed population converged upon one with a short incubation period (S-type) following subsequent passages. However, when GT1-7 cells were challenged with these heterologous samples, L-type prions became dominant. This study demonstrated that the susceptibility of GT1-7 cells to L-type prions was at least 105 times higher than that to S-type prions and that L-type prion-specific biological characteristics remained unchanged after serial passages in GT1-7 cells. This suggests that a GT1-7 cell culture model would be more useful for the economical and stable amplification of L-type prions at the laboratory level. Furthermore, this cell culture model might be used to selectively propagate L-type scrapie prions from a mixed prion population.

Neuroanatomical distribution of disease-associated prion protein in experimental bovine spongiform encephalopathy in cattle after intracerebral inoculation.

The pathologic disease-associated prion protein (PrP(Sc)) has been shown to be expressed in the central nervous system of Holstein cattle inoculated intracerebrally with 3 sources of classical bovine spongiform encephalopathy (BSE) isolates. Several regions of the brain and spinal cord were analyzed for PrP(Sc) expression by immunohistochemical and Western blotting analyses. Animals euthanized at 10 months post-inoculation (mpi) showed PrP(Sc) deposits in the brainstem and thalamus, but no vacuolation; this suggested that the BSE agent might exhibit area-dependent tropism in the brain. At 16 and 18 mpi, a small amount of vacuolation was detected in the brainstem and thalamus, but not in the cerebral cortices. At 20 to 24 mpi, when clinical symptoms were apparent, heavy PrP(Sc) deposits were evident throughout the brain and spinal cord. The mean time to the appearance of clinical symptoms was 19.7 mpi, and the mean survival time was 22.7 mpi. These findings show that PrP(Sc) accumulation was detected approximately 10 months before the clinical symptoms of BSE became apparent. In addition, the 3 sources of BSE prion induced no detectable differences in the clinical signs, incubation periods, neuroanatomical location of vacuoles, or distribution and pattern of PrP(Sc) depositions in the brain.

Immunohistochemical detection of disease-associated prion protein in the intestine of cattle naturally affected with bovine spongiform encephalopathy by using an alkaline-based chemical antigen retrieval method.

An alkaline-based chemical antigen retrieval pretreatment step was used to enhance immunolabeling of disease-associated prion protein (PrP(Sc)) in formalin-fixed and paraffin-embedded tissue sections from cattle naturally affected with bovine spongiform encephalopathy (BSE). The modified chemical method used in this study amplified the PrP(Sc) signal by unmasking PrP(Sc) compared with the normal cellular prion protein. In addition, this method reduced nonspecific background immunolabeling that resulted from the destruction of the residual normal cellular form of prion protein, and reduced the treatment time compared with the usual autoclave pretreatment step. Immunolabeled PrP(Sc) was thereby clearly detected in the myenteric plexus of the ileum in naturally occurring BSE cattle.

[Clinical analysis of time-course changes in minerals and parathyroid hormone levels during treatment with phosphate binders in hemodialysis patients with secondary hyperparathyroidism].

146 hemodialysis (HD) patients with secondary hyperparathyroidism (2 degrees HPT) were studied about the therapeutic effects of phosphate binders. We divided these patients into four groups; Group I: 59 patients treated with CaCO(3) (1.5-6.0 g/day), Group II: 42 cases with sevelamer hydrochloride (0.75-9.0 g/day), Group III: 30 with both CaCO(3) and sevelamer, Group IV: 15 with both CaCO(3) and cholestimide (1.5-6.0 g/day). These patients were prescribed several phosphate binders for at least 18 months. The serum levels of P, albumin-corrected Ca (Ca), Ca x P product and intact parathyroid hormone (iPTH) were serially determined. In Group I and IV, these four parameters showed no significant difference between at before administration and at after 1, 3, 6 and 12 months. In Group II, the values of iPTH, Ca and Ca x P product between at before sevelamer administration and at after 9 months were 199.43+/-94.40 vs 159.86+/-96.03 pg/mL (p<0.05), 9.48+/-1.12 vs 9.01+/-1.00 mg/dL (p<0.05) and 62.72+/-19.62 vs 50.44+/-25.97 mg(2)/dL(2) (p<0.05), respectively. In Group III, P showed significant decrease from 7.16+/-1.33 to 6.72+/-1.69 mg/dL (p<0.05) between at the time of adding sevelamer to CaCO(3) and at after nine months. These results indicate that sevelamer plays an excellent role in the treatment of 2 degrees HPT mainly by controlling Ca level and the combination therapy with CaCO(3) is useful for improvement of P level in patients undergoing HD.