超声处理对羊瘙痒病脑组织中PrPSc聚集状态的影响

目的 研究超声处理对感染羊瘙痒症仓鼠脑组织中PrP^Sc聚集状态的影响，寻找产生PrP^Sc低聚体的条件。方法 裂解液制备脑组织提取物，用各种超声条件处理不同阶段的脑组织提取物；以蛋白酶消化后的Western blot方法和图象分析系统观测PrP^Sc蛋白的分布和聚集状态。结果 适当的超声处理(15s共30次)可增加脑组织匀浆上清中PrP^Sc含量1．29～1．58倍；同样条件下超声处理可明显增加羊瘙痒因子263K感染仓鼠脑组织匀浆上清中PrP蛋白总量，而对正常对照仓鼠脑组织匀浆上清中PrP蛋白总量影响不大；对经常规高速离心获得的PrP^Sc的超声处理显示约90％的PrP^Sc存在于离心上清液中。结论 对感染动物脑组织进行超声处理可增加PrP^Sc的提取量，利于实验室检测。适当的超声处理可破碎大的相对分子质量的PrP^Sc聚集物，产生小的相对分子质量的PrP^Sc产物。     

Panencephalopathic Creutzfeldt‐Jakob Disease with Distinct Pattern of Prion Protein Deposition in a Patient with D178N Mutation and Homozygosity for Valine at Codon 129 of the Prion Protein Gene

Prion diseases include sporadic, acquired and genetic forms linked to mutations of the prion protein (PrP) gene (PRNP). In subjects carrying the D178N PRNP mutation, distinct phenotypes can be observed, depending on the methionine/valine codon 129 polymorphism. We present here a 53‐year‐old woman with D178N mutation in the PRNP gene and homozygosity for valine at codon 129. The disease started at age 47 with memory deficits, progressive cognitive impairment and ataxia. The clinical picture slowly worsened to a state of akinetic mutism in about 2 years and the disease course was 6 years. The neuropathologic examination demonstrated severe diffuse cerebral atrophy with neuronal loss, spongiosis and marked myelin loss and tissue rarefaction in the hemispheric white matter, configuring panencephalopathic Creutzfeldt‐Jakob disease. PrP deposition was present in the cerebral cortex, basal ganglia and cerebellum with diffuse synaptic‐type pattern of immunoreactivity and clusters of countless, small PrP deposits, particularly evident in the lower cortical layers, in the striatum and in the molecular layer of the cerebellum. Western blot analysis showed the presence of type 1 PrP^Sc (Parchi classification). These findings underline the clear‐cut distinction between the neuropathological features of Creutzfeldt‐Jakob disease associated with D178N PRNP mutation and those of fatal familial insomnia.     

Complete Freund's adjuvant immunization prolongs survival in experimental prion disease in mice

We recently reported that immunization of mice with certain self-prion protein peptides induced specific T-cell and B-cell immune responses; importantly, this immunization was associated with a decrease in the number of protease-resistant PrP(Sc) particles recoverable in a transplanted, scrapie-infected syngeneic tumor. The present study was carried out to determine whether immunization with the immunogenic PrP peptides might influence the natural history of experimental scrapie in mice. We immunized C57BL/6 mice with self-prion peptides in complete Freund's adjuvant (CFA) or with CFA alone as a control and then infected the mice with mouse-adapted scrapie by injection either intraperitoneally or intracerebrally. We report here that immunization with CFA, irrespective of whether prion peptides were present in the inoculum, resulted in marked prolongation of survival of the mice, whether the challenge was intracerebral or intraperitoneal. Mice in the immunized and control groups that died contained equivalent amounts of PrP(Sc). Thus, CFA immunization has a therapeutic effect in experimental scrapie in mice, possibly by reducing the rate of PrP(Sc) accumulation in the brain.     

C‐reactive protein and IL‐6: new marker proteins for the diagnosis of CJD in plasma?

BACKGROUND: CJD is usually diagnosed by clinical and neuropathological findings. A number of proteins regarded as markers for neuronal damage in plasma or serum have recently been described. Markers typical for tissue damage, although not usually associated with CJD, are another possibility. An evaluation of the relative usefulness of markers of neuronal and tissue damage in identifying CJD could be beneficial. STUDY DESIGN AND METHODS: Plasma samples were collected from 46 patients with sporadic CJD and from a control group of 42 healthy subjects. The samples were analyzed with tests that were specific for C-reactive protein (CRP), IL-6, neuron-specific enolase (NSE), S-100 proteins, and cellular prion protein (PrP(c)). The results were compared, and a cutoff level for each test used was defined as the 90th percentile from the control group. RESULTS: The assay specific for NSE identified only 13 percent of the sporadic CJD patients as positive. The identification rate of the other markers was significantly higher: S-100, 76.1 percent; PrP(c), 76.1 percent; CRP, 78.3 percent; and IL-6, 73.3 percent. Only three of the samples were positive in all five tests. CONCLUSION: The markers for tissue damage, CRP and IL-6, are as useful as the previously described markers for neuronal damage in the diagnosis of CJD in plasma. All the markers tested are, however, of only limited value in the diagnosis of CJD in plasma. A combination of all surrogate markers improves the specificity but still provides no definitive diagnosis of the disease.     

实验动物传递证实密码子129甲硫氨酸纯合子Heidenhain型Creutzfeldt-Jakob病

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Neurodevelopmental expression and localization of the cellular prion protein in the central nervous system of the mouse

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders caused by PrP^Sc, or prion, an abnormally folded form of the cellular prion protein (PrP^C). The abundant expression of PrP^C in the central nervous system (CNS) is a requirement for prion replication, yet despite years of intensive research the physiological function of PrP^C still remains unclear. Several routes of investigation point out a potential role for PrP^C in axon growth and neuronal development. Thus, we undertook a detailed analysis of the spatial and temporal expression of PrP^C during mouse CNS development. Our findings show regional differences of the expression of PrP, with some specific white matter structures showing the earliest and highest expression of PrP^C. Indeed, all these regions are part of the thalamolimbic neurocircuitry, suggesting a potential role of PrP^C in the development and functioning of this specific brain system. J. Comp. Neurol. 518:1879–1891, 2010. © 2010 Wiley‐Liss, Inc.     

Early and late pathogenesis of natural scrapie infection in sheep

The pathogenesis of scrapie infection was studied in sheep carrying the PrP(VRQ)/PrP(VRQ) genotype, which is associated with a high susceptibility for natural scrapie. The sheep were killed at sequential time points during a scrapie infection covering both the early and late stages of scrapie pathogenesis. Various lymphoid and neural tissues were collected and immunohistochemically examined for the presence of the scrapie-associated prion protein PrP(Sc), a marker for scrapie infectivity The first stage of scrapie infection consisted of invasion of the palatine tonsil and Peyer's patches of the caudal jejunum and ileum, the so-called gut-associated lymphoid tissues (GALT). At the same time, PrP(Sc) was detected in the medial retropharyngeal lymph nodes draining the palatine tonsil and the mesenteric lymph nodes draining the jejunal and ileal Peyer's patches. From these initial sites of scrapie replication, the scrapie agent disseminated to other non-GALT-related lymphoid tissues. Neuroinvasion started in the enteric nervous system followed by retrograde spread of the scrapie agent via efferent parasympathetic and sympathetic nerve fibres innervating the gut, to the dorsal motor nucleus of the vagus in the medulla oblongata and the intermediolateral column of the thoracic spinal cord segments T8-T10, respectively.     

A direct relationship between the partitioning of the pathogenic prion protein and transmissible spongiform encephalopathy infectivity during the purification of plasma proteins

BACKGROUND: Experimental evidence from rodent models indicates that blood can contain transmissible spongiform encephalopathy (TSE) infectivity, which suggests a potential risk for TSE transmission via proteins isolated from human plasma. Because methods that can reduce TSE infectivity typically are detrimental to protein function, infectivity must be removed to ensure the safety of these therapeutic proteins. Animal bioassays are conventionally used to detect infectivity, but the pathogenic form of the prion protein (PrP^Sc) can serve as a marker for TSE infectivity. STUDY DESIGN AND METHODS: Seven plasma protein‐purification steps were performed after the plasma intermediates were spiked with TSE‐infected material. Resulting fractions were analyzed for PrP^Sc by using a Western blot assay and for TSE infectivity by using an animal bioassay. Western blots were quantitated by an endpoint dilution analysis, and infectivity titers were calculated by the Spearman‐Kärber method. RESULTS: PrP^Sc partitioning paralleled TSE infectivity partitioning, regardless of the nature of the protein‐purification step. The detection ranges for PrP^Sc and infectivity were 0 to 5.3 log and 1.1 to 8.9 log median infectious dose per unit, respectively. Clearance of PrP^Sc and infectivity ranged from 1.0 to 6.0 log. CONCLUSION: Purification steps for isolating therapeutic proteins from human plasma showed the removal of both PrP^Sc and TSE infectivity. PrP^Sc partitioning coincided with infectivity partitioning, which showed a close relationship between PrP^Sc and TSE infectivity. By exploiting this association, the in vitro Western blot assay for PrP^Sc was valuable for estimating the partitioning of TSE infectivity during plasma protein purification.     

Prion proteins: a biological role beyond prion diseases

The biological role of the scrapie isoform of prion protein (PrP(Sc)) as an infectious agent in numerous human and non-human disorders of the central nervous system is well established. In contrast, and despite decades of intensive research, the physiological function of the endogenous cellular form of the prion protein (PrP(C)) remains elusive. In mammals, the ubiquitous expression of PrP(C) suggests biological functions other than its pathological role in propagating the accumulation of its misfolded isotype. Other functions that have been attributed to PrP(C) include signal transduction, synaptic transmission and protection against cell death through the apoptotic pathway. More recently, immunoregulatory properties of PrP(C) have been reported. We review accumulating in vitro and in vivo evidence regarding physiological functions of PrP(C).