传染性海绵状脑病的诊断进展和存在的问题

传染性海绵状脑病是由朊病毒引起的人和多种哺乳动物以神经退行性变化为主要特征的一种慢性致死性传染病。引起这类疾病的病原因子是一种编码宿主蛋白的PrPC转变为异常的PrPSC沉积在大脑,导致传染性海绵状脑病的发生。本文从临床症状识别、组织病理学诊断、致病性朊蛋白检测、生物学测定以及毒株鉴定等几个方面作一回顾和总结,为揭示朊病毒疾病致病机理和诊断研究提供借鉴。     

A role for His155 in binding of human prion peptide144-167 to immobilised prion protein

The interactions and conformational changes that lead to the conversion of the normal prion protein (PrP(c)) to its pathogenic form, PrP(sc), are still being elucidated. Using Surface Plasma Resonance (SPR), we provide evidence that a synthetic peptide (PrP(144-167)) corresponding to residues comprising the alpha helix 1-beta strand 2 domain of PrP(c) is able to interact and bind to immobilised recombinant human PrP (rHuPrP) in a dose-dependent manner. The interaction is pH dependent with an increase in binding observed as the pH is lowered, particularly between pH 6.5 and pH 5.5 suggesting a specific role for His(155) in the interaction, confirmed by covalent modification of this residue in the peptide with diethylpyrocarbonate (DEPC). Circular dichroism analysis of PrP(144-167) revealed no secondary structure motifs across the pH range investigated. Possible pH related structural changes of immobilised rHuPrP are also discussed with regard to the increased affinity for PrP(144-167).     

Transmissible spongiform encephalopathy strain-associated diversity of N-terminal proteinase K cleavage sites of PrPSc from scrapie-infected and bovine spongiform encephalopathy-infected mice

Assessment of the different conformational states of the abnormal prion protein (PrP^Sc) in the CNS provides an established basis for distinguishing transmissible spongiform encephalopathy (TSE) strains. PrP^Sc conformers are variably resistant to N-terminal proteinase K (PK) digestion, and analysis of the consensus products (PrP^res) by immunoassay enables effective, but relatively low-resolution differentiation. Determination of the precise N-terminal amino acid profile (N-TAAP) of PrP^res presents a potential high-resolution means of TSE-strain typing, and thus of differential disease diagnosis. This approach was evaluated using individual mice affected by model scrapie (22A, ME7, 87V and 79A) and bovine spongiform encephalopathy (BSE) (301V) strains. Nano liquid chromatography–mass spectrometry (LC-MS) was used to determine PrP^res N-terminal tryptic digestion products. Four major N-terminal tryptic peptides were generated from all mouse TSE strains investigated, corresponding with predominant N-termination of PrP^res at G⁸¹, G⁸⁵, G⁸⁹ and G⁹¹. Both the mass spectrometric abundance of the individual peptides and the ratios of pairs of these peptides were evaluated as markers of conformation in relation to their potential for strain discrimination. The yield of peptides was significantly greater for BSE than scrapie strains and the relative quantities of particular peptide pairs differed between strains. Thus, whereas peptide G⁹¹–K¹⁰⁵ was a dominant peptide from 301V, this was not the case for other strains and, significantly, the ratio of peptides G⁹¹–K¹⁰⁵:G⁸⁹–K¹⁰⁵ was substantially higher for BSE-infected compared with scrapie-infected mice. These data support the potential of the N-TAAP approach for high-resolution TSE strain typing and differential diagnosis.     

The “brave new world” of transmissible spongiform encephalopathy (infectious cerebral amyloidosis)

The story of transmissible human spongiform encephalopathy, from its origins to the present time, enjoys the commentary of a cast of characters from Shakespeare's imaginary island inThe Tempest, with a brief visit to the real island of Tasmania for a bird's eye view of the prion, and some concluding thoughts about the current state of research in the netherworlds of molecular biology and physical chemistry.     

Alkaline serine protease produced by Streptomyces sp. degrades PrP(Sc)

A PrP(Sc)-degrading enzyme was isolated from the culture medium of Streptomyces sp. using perchloric acid-soluble protein (PSP) as a substrate. The media of 500 microbial species were screened to obtain the PSP-degrading enzyme. The medium containing the protease secreted from strain 99-GP-2D-5 showed the highest PSP-degrading activity. Strain 99-GP-2D-5 was assigned as the genus Streptomyces by its morphological and chemotaxonomic characteristics. When scrapie prion was used as the substrate, it was completely digested by the enzyme. The amino acid sequence of the enzyme was identical to that of the C-terminal region of alkaline serine protease (ASP) I. ASP I may be the precursor of the enzyme, and the enzyme seems to be the mature type of ASP I. The maximal activity of the enzyme was observed at 60 degrees C and pH 11, and the scrapie prion was degraded within 3 min under the optimum conditions.     

Molecular genetic studies of Creutzfeldt-Jakob disease

Genetic study of over 200 cases of Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), fatal familial insomnia (FFI), and kuru have brought a reliable body of evidence that the familial forms of CJD and all known cases of GSS and FFI are linked to germline mutations in the coding region of the PRNP gene on chromosome 20, either point substitutions or expansion of the number of repeat units. No pathogenic mutations have so far been found in sporadic or infectious forms of CJD, although there are features of genetic predisposition in iatrogenic CJD and kuru. In FFI and familial CJD, clinically and pathologically distinct syndromes that are both linked to the 178^Asp→Asn substitution, phenotypic expression is dependent on a polymorphism at codon 129. Synthetic peptides homologous to several regions of PrP spontaneously form insoluble amyloid fibrils with unique morphological characteristics and polymerization tendencies. Peptides homologous to mutated regions of PrP exhibit enhanced fibrilogenic properties and, if mixed with the wild-type peptide, produce even more abundant and larger fibrous aggregates. A similar process in vivo may lead to amyloid accumulation and disease, and transmission of “baby fibrils” may induce disease in other hosts.     

Comparison of DNA variants in the PRNP and NF1 regions between bovine spongiform encephalopathy and control cattle

DNA from 252 bovine spongiform encephalopathy (BSE) cattle and 376 non-diseased control cattle were genotyped for nine loci in the prion protein (PRNP) gene region, three loci in the neurofibromin 1 (NF1) region and four control loci on different chromosomes. The allele and genotype frequencies of the control loci were similar in BSE and control cattle. In the analysed 7.4 Mb PRNP region, the largest differences between BSE and control cattle were found for the loci REG2, R16 and R18, which are located between +300 and +5600 bp, spanning PRNP introns 1 to 2. Carriers of the REG2 genotype 128/128 were younger at BSE diagnosis than those with the other genotypes (128/140 or 140/140). The predominant haplotype REG2 128 bp-R18 173 bp occurred more frequently (P < 0.001), and the second-most frequent haplotype (REG2 140 bp-R18 175 bp) occurred less frequently (P < 0.05) in BSE than in control cattle. The largest frequency differences between BSE and control groups were observed in the Brown Swiss breed. Across all breeds, most of the same alleles and haplotypes of the PRNP region were associated with BSE. In the 23-cM NF1 region, associations with BSE incidence were found for the RM222 allele and for the DIK4009 genotype frequencies. Cattle carrying RM222 genotypes with the 127- or 129-bp alleles were about half a year older at BSE incidence than those with other genotypes. Across the breeds, different alleles and genotypes of the NF1 region were associated with BSE. The informative DNA markers were used to localize the genetic disposition to BSE and may be useful for the identification of the causative DNA variants.     

Polymorphisms of Two Indels at the PRNP Gene in Three Beef Cattle Herds

Bovine spongiform encephalopathy (BSE) is a transmissible fatal neurodegenerative disorder, presenting a characteristic spongiform degeneration of cattle brain due to the accumulation of a pathogenic and protease-resistant infectious protein (prion). Two deletion/insertion polymorphisms of the prion protein gene (23 bp at the promoter region and 12 bp at intron 1) were analyzed in three beef cattle herds (Aberdeen Angus, Charolais, and Franqueiro) to verify allele frequencies for possible use in selection of resistant animals. High frequencies of susceptibility alleles (23 and 12 bp deletion) and haplotype (23 del/12 del) were observed in the Aberdeen Angus and Charolais herds, but Franqueiro presented one of the highest frequencies of resistant alleles so far described. These data indicate the need for selection in Aberdeen Angus and Charolais breeds to increase the frequency of resistant animals in order to reduce the probabilities of BSE outbreaks in these populations.     

The role of glycophosphatidylinositol anchor in the amplification of the scrapie isoform of prion protein in vitro

Transmissible spongiform encephalopathies are associated with an autocatalytic conversion of normal prion protein, PrP^C, to a protease-resistant form, PrPres. This autocatalytic reaction can be reproduced in vitro using a procedure called protein misfolding cyclic amplification (PMCA). Here we show that, unlike brain-derived PrP^C, bacterially-expressed recombinant prion protein (rPrP) is a poor substrate for PrPres amplification in a standard PMCA reaction. The differences between PrP^C and rPrP appear to be due to the lack of the glycophosphatidylinositol anchor in the recombinant protein. These findings shed a new light on prion protein conversion process and have important implications for the efforts to generate synthetic prions for structural and biophysical studies.     

Inhibition of PrPSc formation by synthetic O-sulfated glycopyranosides and their polymers

Sulfated glycosaminoglycans (GAGs) and sulfated glycans inhibit formation of the abnormal isoform of prion protein (PrPSc) in prion-infected cells and prolong the incubation time of scrapie-infected animals. Sulfation of GAGs is not tightly regulated and possible sites of sulfation are randomly modified, which complicates elucidation of the fundamental structures of GAGs that mediate the inhibition of PrPSc formation. To address the structure-activity relationship of GAGs in the inhibition of PrPSc formation, we screened the ability of various regioselectively O-sulfated glycopyranosides to inhibit PrPSc formation in prion-infected cells. Among the glycopyranosides and their polymers examined, monomeric 4-sulfo-N-acetyl-glucosamine (4SGN), and two glycopolymers, poly-4SGN and poly-6-sulfo-N-acetyl-glucosamine (poly-6SGN), inhibited PrPSc formation with 50% effective doses below 20 microg/ml, and their inhibitory effect became more evident with consecutive treatments. Structural comparisons suggested that a combination of an N-acetyl group at C-2 and an O-sulfate group at either O-4 or O-6 on glucopyranoside might be involved in the inhibition of PrPSc formation. Furthermore, polymeric but not monomeric 6SGN inhibited PrPSc formation, suggesting the importance of a polyvalent configuration in its effect. These results indicate that the synthetic sulfated glycosides are useful not only for the analysis of structure-activity relationship of GAGs but also for the development of therapeutics for prion diseases.     

In vitro amplification of H-type atypical bovine spongiform encephalopathy by protein misfolding cyclic amplification

The in vitro amplification of prions by serial protein misfolding cyclic amplification has been shown to detect PrP^Sc to levels at least as sensitive as rodent bioassay but in a fraction of the time. Bovine spongiform encephalopathy is a zoonotic prion disease in cattle and has been shown to occur in 3 distinct forms, classical BSE (C-BSE) and 2 atypical BSE forms (L-BSE and H-BSE). Atypical forms are usually detected in asymptomatic, older cattle and are suggested to be spontaneous forms of the disease. Here, we show the development of a serial protein misfolding cyclic amplification method for the detection of H-BSE. The assay could detect PrP^Sc from 3 distinct experimental isolates of H-BSE, could detect PrP^Sc in as little as 1×10^?12 g of brain material and was highly specific. Additionally, the product of serial protein misfolding cyclic amplification at all dilutions of seed analyzed could be readily distinguished from L-BSE, which did not amplify, and C-BSE, which had PrP^Sc with distinct protease K-resistance and protease K-resistant PrP^Sc molecular weights.     

Molecular aspects of disease pathogenesis in the transmissible spongiform encephalopathies

The transmissible spongiform encephalopathies (TSE), or prion diseases, are a group of rare, fatal, and transmissible neurodegenerative diseases of mammals for which there are no known viral or bacterial etiological agents. The bovine form of these diseases, bovine spongiform encephalopathy (BSE), has crossed over into humans to cause variant Creutzfeldt-Jakob disease. As a result, BSE and the TSE diseases are now considered a significant threat to human health. Understanding the basic mechanisms of TSE pathogenesis is essential for the development of effective TSE diagnostic tests and anti-TSE therapeutic regimens. This review provides an overview of the molecular mechanisms that underlie this enigmatic group of diseases.     

克雅氏病———医患双方的挑战

克雅氏病是由朊蛋白病毒感染人类引起的罕见的、可传播性的、致命性的神经系统疾病,目前已成为令全球恐慌的不可治愈性疾病,其患者在全球范围的增加,给医患双方带来新的挑战.本文就克雅氏病的临床分型、流行病学、病理、诊断及护理作一系统介绍,使大家对该病的认识更加深刻.     

Autoantibody to glial fibrillary acidic protein in the sera of cattle with bovine spongiform encephalopathy

It is desirable to make the diagnosis in live cattle with bovine spongiform encephalopathy (BSE), and thus surrogate markers for the disease have been eagerly sought. Serum proteins from BSE cattle were analyzed by 2‐D Western blotting and TOF‐MS. Autoantibodies against proteins in cytoskeletal fractions prepared from normal bovine brains were found in the sera of BSE cattle. The protein recognized was identified to be glial fibrillary acidic protein (GFAP), which is expressed mainly in astrocytes in the brain. The antigen protein, GFAP, was also found in the sera of BSE cattle. The percentages of both positive sera in the autoantibody and GFAP were 44.0% for the BSE cattle, 0% for the healthy cattle, and 5.0% for the clinically suspected BSE‐negative cattle. A significant relationship between the presence of GFAP and the expression of its autoantibody in the serum was recognized in the BSE cattle. These findings suggest a leakage of GFAP into the peripheral blood during neurodegeneration associated with BSE, accompanied by the autoantibody production, and might be useful in understanding the pathogenesis and in developing a serological diagnosis of BSE in live cattle.     

Concentration of Disease-Associated Prion Protein with Silicon Dioxide

Reagents that can precipitate the disease-associated prion protein (PrP^Sc) are vital for the development of high sensitivity tests to detect low levels of this disease marker in biological material. Here, a range of minerals are shown to precipitate both ovine cellular prion protein (PrP^C) and ovine scrapie PrP^Sc. The precipitation of prion protein with silicon dioxide is unaffected by PrP^Sc strain or host species and the method can be used to precipitate bovine BSE. This method can reliably concentrate protease-resistant ovine PrP^Sc (PrP^res) derived from 1.69 μg of brain protein from a clinically infected animal diluted into either 50 ml of buffer or 15 ml of plasma. The introduction of a SiO₂ precipitation step into the immunological detection of PrP^res increased detection sensitivity by over 1,500-fold. Minerals such as SiO₂ are readily available, low cost reagents with generic application to the concentration of diseases-associated prion proteins.     

Effect of amphotericin B on wild-type and mutated prion proteins in cultured cells: putative mechanism of action in transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic, or genetic in origin. Although some doubts remain on the nature of the responsible agent of these diseases, it is clear that a protein called PrP(Sc) [the scrapie isoform of prion protein (PrP)] plays a central role. PrP(Sc) represents a conformational variant of PrP(C) (the cellular isoform of PrP), the normal host protein. Polyene antibiotics, such as amphotericin B, have been shown to delay the accumulation of PrP(Sc) and to increase the incubation time of the disease after experimental transmission in laboratory animals. Unlike agents such as Congo red, the inhibitory effect of amphotericin B on PrP(Sc) generation has not been observed in infected cultures. Using transfected cells expressing wild-type or mutated mouse PrPs, we show here that amphotericin B is able to interfere with the generation of abnormal PrP isoforms in culture. Its action seems related to a modification of PrP trafficking through the association of this glycosylphosphatidylinositol-anchored protein with detergent-resistant microdomains. These results represent a first step toward the comprehension of the mechanism of action of amphotericin B in transmissible spongiform encephalopathies.     

PrPC interacts with tetraspanin-7 through bovine PrP154-182 containing alpha-helix 1

The cellular prion protein (PrP^C) is highly conserved in the evolution of mammals, and therefore, thought to have important cellular functions. Despite decades of intensive research, the physiological function of PrP^C remains enigmatic. We carried out a yeast two-hybrid screen on a bovine brain cDNA expression library and identified the transmembrane protein tetraspanin-7 (CD231), as a PrP^C interacting protein. We confirmed the interaction between PrP^C and tetraspanin-7 by yeast two-hybrid assay, immunofluorescent co-localization, and immunocoprecipitation. Our mutational studies further demonstrated that PrP^C specifically binds tetraspanin-7 through the region corresponding to bovine PrP_154-182 containing alpha-helix 1.     

Intraspecies transmission of L‐type‐like bovine spongiform encephalopathy detected in Japan

It has been assumed that the agent causing BSE in cattle is a uniform strain (classical BSE); however, different neuropathological and molecular phenotypes of BSE (atypical BSE) have been recently reported. We demonstrated the successful transmission of L‐type‐like atypical BSE detected in Japan (BSE/JP24 isolate) to cattle. Based on the incubation period, neuropathological hallmarks, and molecular properties of the abnormal host prion protein, the characteristics of BSE/JP24 prion were apparently distinguishable from the classical BSE prion and closely resemble those of bovine amyloidotic spongiform encephalopathy prion detected in Italy.