散发性克-雅病PrP基因129密码子基因型与临床表型14例研究

目的：探讨散发性克－雅病（Creutzfeldt-Jakob disease,CJD)PrP基因129位点密码子基因型与临床表型的关系。方法：对14例散发性CJD患者进行PrP基因129例密码子的检测，并与临床表现进行了分析。结果：（1）根据诊断标准，14例散发性CJD中8例诊断为肯定CJD，6例诊断为很可能CJD。（2）8例诊断肯定CJD组中，PrP基因129例位点密码子为甲硫氨酸纯合型6例，甲充氨酸／缬氨酸2例，6例诊断很可能CJD组的PrP基因129密码子均为甲硫氨酸纯合型。（3）12例PrP基因129位点为甲硫氨酸纯合型的患者以认知障碍起病8例，共济失调1例；视觉障碍2例；肌阵挛1例，病程最长20个月，最短2．5个月，病程中有癫痫5例，肌阵挛6周，视觉障碍6例。7例有典型周期性同步放电（PSD）脑电改变。（4）2例甲硫氨酸／缬氨型患者均以共济失调起病。2个月后才出现痴呆，病理程分析为16个月和20个月，均无典型的PSD。（2）本组散发性CJDPrP基因129位点密码子甲硫氨酸／甲硫氨酸，甲硫氨酸／缬氨酸，分布比例与日本相同，但与西方不同，而且没有缬氨酸纯合型。     

Creutzfeldt‐Jakob disease with an M232R substitution: report of a patient showing slowly progressive disease with abundant plaque‐like PrP deposits in the cerebellum

Patients with genetic Creutzfeldt‐Jakob disease in which arginine is substituted for methionine at codon 232 (M232R) of the prion protein gene (CJD232) have been described in Japan, and a recent study has revealed the presence of two clinical phenotypes: a rapidly progressive type (rapid‐type) and a slowly progressive type (slow‐type). Although the former is known to show pathologic features similar to those of classical CJD, the neuropathology of the latter still remains unclear. We report the autopsy findings of slow‐type CJD232 of 37 months' duration in a 73‐year‐old man who had methionine homozygosity at codon 129 of the prion protein gene (129MM). His initial symptoms included agraphia and memory disturbance, followed by relatively slowly progressive dementia. Myoclonus and akinetic mutism became evident 5 and 23 months after disease onset, respectively. The electroencephalogram revealed periodic sharp wave complexes at 7 months before death. The neuropathologic features were partly reminiscent of those of MM2‐cortical‐type sporadic CJD, showing spongiform change of the large confluent vacuole type, neuronal loss with gliosis, and coarse, perivacuolar prion protein deposits, which were later shown to consist of protease‐resistant type 2 prion protein, in the cerebral cortex and striatum. It was of considerable interest that not only was the medial thalamus severely involved, but also that the cerebellar cortex showed loss of Purkinje cells and abundant plaque‐like prion protein deposits. These findings are not a feature of MM2‐cortical‐type sporadic CJD. Whether or not the M232R substitution, in combination with the genetic polymorphism and the molecular type of pathological prion protein, really participates in the development of CJD232 and its different phenotypes awaits further studies.     

羊瘙痒因子263K感染的金黄仓鼠脑中PrP~(Sc)的动态沉积特点

目的　观察羊瘙痒因子 2 63K感染的金黄仓鼠脑中PrPSc的沉积特点。方法　颅内接种羊瘙痒因子 2 63K感染金黄仓鼠 ,分别于接种后第 2 0天、40天、50天、60天和 70天取脑组织 ,用免疫组化法检测PrPSc的沉积 ,HE染色观察病理变化 ,Westernblot检测蛋白酶抗性PrPSc。结果　颅内接种 2 63K后第 2 0天 ,脑组织中即可检测到耐热和耐蛋白酶K的PrPSc的沉积 ,呈散在分布 ;第 40天可见细胞内PrPSc的沉积 ,第 50天脑组织中出现灶性、颗粒状、细胞内PrPSc的沉积 ,累及部位扩大 ,第60 - 70天可见弥漫性呈桑葚状、斑块状沉积物。PrPSc主要沉积于大脑皮质Ⅲ、Ⅳ、Ⅴ层 ,海马锥体细胞层 ,小脑皮质颗粒层。HE染色结果显示大脑皮质海绵样病变在接种后 40天出现 ,随着接种时间的延长而加剧。Westernblot检测显示PrPSc在接种后 40天即可检出 ,PrP总量和PrPSc含量随着接种时间的延长而增加。结论　随着接种时间延长 ,羊瘙痒因子 2 63K感染的金黄仓鼠脑中PrPSc沉积累及的范围、程度扩大 ,IHC检测脑组织中PrPSc的沉积早于临床症状的出现 ,甚至其它检测方法的检出。     

Immunohistochemistry for PrPSc in natural scrapie reveals patterns which are associated with the PrP genotype

Immunohistochemistry for PrPSc is used widely in scrapie diagnosis. In natural scrapie cases the use of immunohistochemistry (IHC) has revealed the existence of up to 12 different morphological types of immunostained deposits. The significance of this pattern variability in relation to genotype has not been studied extensively in natural disease. In this study we recorded in detail PrPSc patterns at the obex level of the medulla oblongata from 163 animals derived from 55 flocks which presented through passive surveillance in the UK and Italy. A strong association was seen between PrPSc patterns and PrP genotype, particularly in relation to codon 136. In a blind assessment of this association we were able to predict, with over 80% accuracy, the genotype of 151 scrapie cases which were presented through passive surveillance from 13 farms. The genotype of these cases was ARQ/ARQ or VRQ/VRQ. The association of PrPsc patterns with genotype was generally stronger in those farms where all the affected animals belonged to a single genotype compared with farms where both genotypes were identified, with the exception of one farm in which the genotype of all affected sheep was ARQ/ARQ and the PrPSc patterns were of the VRQ/VRQ type. Our observations support the hypothesis that the observed association between specific IHC patterns and genotypes may in fact be strain driven but in natural disease individual scrapie strains may demonstrate a genotypic tropism.     

Disease-associated prion protein is not detectable in human systemic amyloid deposits

Cerebral and cardiac amyloid deposits have been reported after scrapie infection in transgenic mice expressing variant prion protein (PrP(C)) lacking the glycophosphatidylinositol anchor. The amyloid fibril protein in the systemic amyloid deposits was not characterized, and there is no clinical or pathological association between prion diseases and systemic amyloidosis in humans. Nevertheless, in view of the potential clinical significance of these murine observations, we tested both human amyloidotic tissues and isolated amyloid fibrils for the presence of PrP(Sc), the prion protein conformation associated with transmissible spongiform encephalopathy (TSE). We also sequenced the complete prion protein gene, PRNP, in amyloidosis patients. No specific immunohistochemical staining for PrP(Sc) was obtained in the amyloidotic cardiac and other visceral tissues of patients with different types of systemic amyloidosis. No protease-resistant prion protein, PrP(res), was detectable by Western blotting of amyloid fibrils isolated from cardiac and other systemic amyloid deposits. Only the complete normal wild-type PRNP gene sequence was identified, including the usual distribution of codon 129 polymorphisms. These reassuringly negative results do not support the idea that there is any relationship of prions or TSE with human systemic amyloidosis, including cardiac amyloid deposition.     

Deletion of Kif5c Does Not Alter Prion Disease Tempo or Spread in Mouse Brain

In prion diseases, the spread of infectious prions (PrPSc) is thought to occur within nerves and across synapses of the central nervous system (CNS). However, the mechanisms by which PrPSc moves within axons and across nerve synapses remain undetermined. Molecular motors, including kinesins and dyneins, transport many types of intracellular cargo. Kinesin-1C (KIF5C) has been shown to transport vesicles carrying the normal prion protein (PrPC) within axons, but whether KIF5C is involved in PrPSc axonal transport is unknown. The current study tested whether stereotactic inoculation in the striatum of KIF5C knock-out mice (Kif5c^−/−) with 0.5 µL volumes of mouse-adapted scrapie strains 22 L or ME7 would result in an altered rate of prion spreading and/or disease timing. Groups of mice injected with each strain were euthanized at either pre-clinical time points or following the development of prion disease. Immunohistochemistry for PrP was performed on brain sections and PrPSc distribution and tempo of spread were compared between mouse strains. In these experiments, no differences in PrPSc spread, distribution or survival times were observed between C57BL/6 and Kif5c^−/− mice.     

Evaluation of depth filtration to remove prion challenge from an immune globulin preparation

BACKGROUND AND OBJECTIVES: Plasma-derived therapeutic proteins have the potential to contain transmissible spongiform encephalopathy (TSE) infectivity. This study evaluated the effectiveness and characterized the mechanism of abnormal prion protein removal during a depth-filtration step used in the manufacture of an immunoglobulin preparation. MATERIALS AND METHODS: Scrapie brain homogenate was treated with lysolecithin, sonicated and sequentially filtered through 0.45-, 0.22- and 0.1-microm membrane filters. The scrapie brain homogenate was then added (at a 1:51 dilution) to the Supernatant III fraction used in the manufacture of Rho(D) immune globulin (human). The spiked immunoglobulin preparation was then filtered through a depth filter under the same conditions used in full-scale production. After filtration, the depth filter was washed with hypertonic NaCl solutions to elute the abnormal prion protein (PrPSc) from the filter. A Western blot assay for PrPSc was used to quantify removal from the filtrate and recovery from the filter washes. A second run was performed whereby the PrPSc-spiked Supernatant III was filtered through a 0.22-microm membrane filter prior to depth filtration. A third run evaluated depth filtration of PrPSc in Tris-buffered saline (TBS). RESULTS: The depth filter removed greater than four logs of PrPSc from the Supernatant III filtrate. A significant portion of the PrPSc could be recovered from the depth filter by elution with high-molarity NaCl solutions. Prefiltration (through a 0.22-microm membrane filter) of the spiked Supernatant III prior to depth filtration removed all detectable PrPSc. Depth filtration removed less than one log of PrPSc from TBS. CONCLUSIONS: Depth filtration appears to remove PrPSc from the immunoglobulin preparation by mechanical straining rather than by adsorption to the filter matrix. The immunoglobulin preparation caused the PrPSc to aggregate from particles <0.1 microm in size to particles of >0.22 microm, probably as a result of the presence of methanol in the preparation. The depth filter failed to remove PrPSc from a purely aqueous environment.     

Sporadic fatal insomnia with spongiform degeneration in the thalamus and widespread PrPSc deposits in the brain

We report a case of human prion disease of 29 months duration in a 74‐year‐old Japanese man. The disease started with progressive sleeplessness and dementia. MRI showed gradually progressive cerebral atrophy. Neuronal loss, spongiform change and gliosis were evident in the thalamus and cerebral cortex, as well as in the striatum and amygdaloid nucleus. In the cerebellar cortex, mild‐to‐moderate depletion of Pukinje cells and spongiform change were observed. Mild neuronal loss in the inferior olivary nucleus was also seen. Immunohistochemistry revealed widespread perivacuolar deposits of abnormal prion protein (PrP^Sc) in the cerebral cortex, thalamus, basal ganglia, and brainstem, and minimal plaque‐like deposits of PrP^Sc in the cerebellar cortex. In the cerebellar plaque‐like deposits, the presence of amyloid fibrils was confirmed ultrastructurally. The entire pathology appeared to lie halfway between those of CJD and fatal insomnia, and further demonstrated the relationship between spongiform degeneration and PrP^Sc deposits, especially in the diseased thalamus. By immunoblotting, the thalamus was shown to contain the lowest amount of PrP^SC among the brain regions examined. The PrP^Sc of type 2, in which the ratio of the three glycoforms was compatible with that of sporadic fatal insomnia (MM2‐thalamic variant) reported previously, was also demonstrated. Analysis of the prion protein gene (PRNP) showed no mutation, and homozygosity for methionine at codon 129. In conclusion, we considered that this patient had been suffering from sporadic, pathologically atypical fatal insomnia.     

Risk of Prion Disease Transmission through Bovine‐Derived Bone Substitutes: A Systematic Review

Background: Despite the causal association between variant Creutzfeldt – Jakob disease and bovine spongiform encephalopathy (BSE), bovine origin graft materials are widely used during dental surgical procedures. The aim of this study was to assess the risk of BSE transmission through anorganic bovine bone substitutes. Methods: Electronic database of MEDLINE was searched to identify relevant studies regarding our focused questions, presence of BSE prion infectivity in raw bovine bone, BSE prion inactivation by bone substitute manufacturing process, protein contents in anorganic bovine bone substitutes, and validity of current BSE diagnostic methods. Search terms yielded 1,704 titles. After title/abstract screening and duplicates removal, 36 full‐text articles were screened for inclusion. Results: A total of 16 studies were included in the final analysis. No eligible studies were identified regarding the efficacy of BSE prion inactivation by the treatments used for anorganic bovine bone manufacturing. BSE infectivity and PrP^Sc, pathological prion, were detected in bovine bone marrow and serum samples. Proteins were detected in Tutoplast® (bovine), Bio‐Oss®, and tibia samples treated at the similar condition for Bio‐Oss deproteinization. Inconsistent results of different BSE diagnostic tests were not unusual findings (Iwata et al. 2006; Arnold et al. 2007; Murayama et al. 2010), and a study by Balkema‐Buschmann and colleagues showed an apparent discrepancy between BSE infectivity and detection of PrP(27‐30), the current surrogate marker for prion disease infectivity. Conclusion: This review indicates that bovine‐derived graft biomaterials may carry a risk of prion transmission to patients.     

Tetrapyrroles as anti-amyloidogenic drugs in the treatment of transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative conditions, currently without treatments. The common underlying feature of these diseases is the accumulation of an abnormal isoform (PrP^Sc) of a host-encoded neuronal cell surface sialoglycoprotein (PrP^C). This accumulation and the tendency for resultant amyloid formation may be the primary pathological event in TSEs and the production, accumulation and deposition of PrP^Schave become the major focus of therapeutic interest. This particular patent concerns the possible therapeutic usefulness of tetrapyrroles. Various tetrapyrroles have been evaluated in cell-free, cell culture and whole animal experiments, using a hamster-adapted strain of scrapie. Significant benefit has been demonstrated in these experimental models, suggesting therapeutic promise for these drugs. However, no evidence is provided for effectiveness with other prion strains, in particular, for strains related to human diseases. There is also no evidence concerning effectiveness in treating clinically established disease and no evidence concerning long-term safety in humans. The patent makes unsupported extensions of these claims to untested members of the same group of compounds and to their use in other neurodegenerative conditions such as Alzheimer’s disease. Nevertheless, this patent includes data indicating that tetrapyrroles are leading anti-amyloidogenic candidates for the treatment of TSE.