贵州省2010-2015年克-雅病病例监测结果分析

目的 分析2010-2015年贵州省克-雅病(Creutzfeldt-Jakob disease,CJD)监测病例流行病学、临床特征、以及病例转归情况.方法 对贵州省克-雅病监测网络发现的疑似病例的流行病学、临床特征以及随访资料进行分析,同时结合病例脑脊液、血液标本的实验室检测结果.结果 2010-2015年贵州省报告的23例CJD疑似病例中发现CJD病例11例,其中sCJD临床诊断病例8例,sCJD临床疑似病例2例,gCJD确诊诊断病例1例.11例病例中,首发症状以快速进行性痴呆为主要表现,其次是精神症状、锥体外系症状、小脑症状和皮质性失明;临床症状中进行性痴呆为主要症状,其次是视觉或小脑障碍、肌阵挛、锥体系/锥体外系功能异常、无动性缄默;辅助检查以头颅核磁共振(MRI)异常为主(45.45％);实验室检测脑脊液14-3-3蛋白阳性率较高(70％),血液标本中朊蛋白基因(PRNP)检测129位氨基酸多态性均为M/M型,除1例gCJD确诊诊断病例PRNP基因检测发现D178N突变外,均未发现其它位点突变.11例CJD病例无季节、地区聚集性和职业倾向,以男性为主,年龄中位数在65岁,主要为汉族.流行病学史无特殊.对所有CJD病例在报告当年进行随访,失访率27％,大多数病例均在1年内死亡.结论 2010-2015年贵州省CJD病例中以sCJD为主,其流行病学特征与同期全国监测情况相符.首次在贵州省发现1例gCJD病例,其PRNP基因突变与2011-2014年全国CJD监测网络发现的gCJD突变位点均不相同,与河南2011-2013年间报告的2例遗传型朊蛋白病病例在临床表现及PRNP基因突变位点相似.     

朊病毒研究进展:朊蛋白变构与辅助因子(Cofactor)

传染性海绵状脑病（transmitted spongiforrn encephalopathy）或称朊病毒病（pfion diseases）是一种致命的脑病。该病严重威胁着流行国家家畜的健康。大量证据显示，朊病毒病的传染因子是一种特异的、不含核酸，但具传染性的蛋白质，即朊蛋白（PrionProtein，PrP）。目前广为接受的朊病毒假说认为，当正常朊蛋白PrP^c（对蛋白酶敏感）构象异常变化时可形成致病朊病毒PrP^Se（对蛋白酶不敏感）。虽然，大量研究结果都支持朊病毒病的主要致病机理是由PrPc变构成PrP^Se，但是否还有其它因子或成分参与，一直备受科学家的关注。最近，Deleault等发现PrP^c大量转变为抗蛋白酶的PrP^Se样蛋白（PrP^res）的过程需要某种特异性RNA的参与，这是近年来朊病毒研究的重大突破；而Garcion等则提出遗传性朊病毒病与RNA突变有关，是RNA与朊蛋白关系的一个重要假说。由于疯牛病的广泛流行及其严重的危害性已普遍引起各国政府的高度关注，作为疯牛病（mad cow disease，MCD）和其他相关的海绵状脑病的致病因子，朊病毒一直是世界一流实验室的竞相研究的对象之一。     

异常朊蛋白检测研究进展

Prion病是一组致死性神经变性疾病。其病因主要是在人或动物脑组织内出现并沉积大量异常的Prion蛋白。而导致神经细胞死亡。临床表现为迅速进展的痴呆。诊断Prion病的金标准是检测到异常朊蛋白。但是,因为正常人体内也存在朊蛋白(normal cellular prion protein,PrP^ac),与致病的异常朊蛋白(PrP^Sc或PrP^CJD)相比,仅在空间构象上不同。     

2009-2018年安徽省克-雅病病例监测特征分析

目的 分析2009-2018年安徽省克-雅病(Creutzfeldt-Jackob Disease,CJD)病例流行病学、临床特征、以及病例转归情况。方法 结合病例脑脊液、血液标本的实验室检测结果,对安徽省克-雅病监测的疑似病例的流行病学、临床特征以及随访资料进行分析。结果 2009-2018年安徽省报告的29例CJD病例中临床诊断CJD病例17例,疑似病例CJD病例9例,遗传或家族型CJD病例3例。病例报告无季节聚集性,长久居住地呈散在分布,职业分布广泛。临床资料分析,进行性痴呆为最常见的首发症状,占全部的65.52%(19/29);散发型CJD临床诊断病例发病年龄中位数为61.11岁,男女比例1.23∶1,均是汉族。辅助检查以头颅核磁共振(MRI)异常为主(80.00%)。实验室检测脑脊液14-3-3蛋白阳性10例,阳性率37.93%;血标本中PRNP检测129位氨基酸多态性均为M/M型;PRNP基因检测与遗传型相关的基因突变有3例,基因突变位点T188K、D178N和R208H各1例。结论 2009-2018年安徽省CJD 病例流行病学特征与同期全国监测情况相符合。首次在安徽发现第一例以R208H为基因突变位少见的遗传型CJD病例。     

阿魏酸抑制朊病毒复制作用的研究

目的本试验通过已建立的ScN2a细胞模型,探讨阿魏酸对朊病毒复制的影响。方法通过Western blot法检测阿魏酸对朊病毒复制作用的影响。结果0．5、1和5μg／mL的阿魏酸能够极显著的抑制PrPsc的表达水平（P〈0.01）。结论由于中药单体阿魏酸可以抑制ScN2a中PrPsc蛋白的复制,所以中药单体阿魏酸对朊病毒病可能起到一定的治疗作用,这为我们以后对于研究朊病毒的治疗提供了新的思路。     

新疆奇台县某中学一起流行性脑脊髓膜炎疫情调查

目的对新疆奇台县某中学一起暴发性流行性脑脊髓膜炎疫情的现场处置方法进行科学分析，为今后疫情的处置提供科学依据。方法通过对患者就诊病案的收集、现况调查、实验室荧光定量PCR检测技术等方法，对疫情处置进行分析；实验室检测及病例的诊断参照《流行性脑脊髓膜炎诊断标准》（WS295—2008）。结果4例患者大多以四肢、臀部出现散在瘀点、瘀斑为主要症状，采集患者的血液、脑脊液标本，细菌培养均未检出脑膜炎奈瑟菌；荧光定量PCR检测3例患者的脑脊液和2例患者的血液，检出A群脑膜炎奈瑟菌。结论此次疫情的暴发由A群脑膜炎奈瑟菌引起，流行性脑脊髓膜炎发病急，如果不能迅速诊断、及时用药会给患者留下严重的后遗症，甚至威胁生命；因此要加强流行性脑脊髓膜炎高发季节的甄别诊断，在对疫区进行应急接种的同时，重点开展教育宣传，加强居室通风是控制疫情暴发的有效措施。     

转录因子FoxO3a对PC12细胞朊蛋白管家基因表达调控的影响

目的探讨叉头转录因子O亚型(FoxO)3a对PC12细胞朊蛋白管家基因(PRNP)表达调控的影响。方法以PC12细胞为研究对象,采用siRNAs沉默基因实验、实时荧光聚合酶链反应(RT-PCR)及Western印迹检测PRNP转录及蛋白表达水平。通过双荧光素酶(Luc)报告基因检测试验检测Luc活性,进而评估启动子的活性。结果 FoxO3a基因沉默后,S1探针(S1)组PRNP基因表达明显上升(P<0.05);S1组FoxO3a基因被沉默后,朊蛋白(PrP)蛋白表达显著升高,与基因水平一致;双荧光素酶报告基因检测结果显示,与基础对照组(pGL3-Basic+pRL-Tk)相比,阳性对照组(pGL3-Control+pRL-TK)FLuc/Rluc值明显提高(P<0.01),实验组Ⅰ(pGL3-PNRP+pRL-TK)FLuc/Rluc也显著高于基础对照组(P<0.01);当pGL3-PNRP质粒、pcDNA3.1-FoxO3a高表达质粒与海肾荧光素酶pRL系列(pRL-TK)质粒共转入细胞内,48 h后,与实验组Ⅰ相比,实验组Ⅱ(pGL3-PNRP+pcDNA3.1-FoxO3a+pRL-TK)的FLuc/RLuc值明显下降(P<0.01)。结论 FoxO3a可以负调控PRNP基因的表达,这种调控是通过结合PRNP基因启动子区域而抑制其转录来实现的。     

献血者血液核酸检测的关键及控制点思考

随着我国无偿献血事业的迅速发展,群众的思想觉悟越来越高,越来越多献血者自愿献血,这对临床抢救和治疗具有重大意义。献血者血液核酸检测对控制血液质量起到关键的把关作用,测得值应在试剂说明书规定范围之内。检测的过程中,必须核对可疑标本,对标本进行复查,必要时进行双孔复查,检测质量控制是保证检测质量的关键所在。为了确保血站实验室的检测结果能够记录完整并得到妥善的保留,提高血液标本检测过程中的规范性,卫生部颁发了相关质量管理制度,明确了质量控制的主要范围,明确了用血的规范,为了保证血液的质量,必须对检验过程的关键控制点进行检查,确保批号和有效期限的准确性。为此本文重点探讨献血者血液核酸检测的关键及控制。     

美国《Emerging infectious diseases》2013年第10期有关人兽共患病论文摘译

P1938—1947在转基因小鼠中自发生成的传染性朊病毒病／／Juan-MariaTorres，Joaquin Castilla。等我们在转基因小鼠身上表达113位密码子为亮氨酸（113L突变）的牛朊病毒蛋白（PrPC）。该蛋白质与102I。突变的人蛋白质同源，102L突变已证实与GSS有遗传学的联系。这种PrPC突变可导致完全渗透的、致命的海绵状脑病。该遗传疾病可通过表达突变牛PrP的病鼠的脑匀浆颅内接种到表达野生型牛PrP的小鼠而传导，这表明重新合成传染性朊病毒。研究结果证实，在PrP。     

Cells release prions in association with exosomes

Prion diseases are infectious neurodegenerative disorders linked to the accumulation in the central nervous system of the abnormally folded prion protein (PrP) scrapie (PrPsc), which is thought to be the infectious agent. Once present, PrPsc catalyzes the conversion of naturally occurring cellular PrP (PrPc) to PrPsc. Prion infection is usually initiated in peripheral organs, but the mechanisms involved in infectious spread to the brain are unclear. We found that both PrPc and PrPsc were actively released into the extracellular environment by PrP-expressing cells before and after infection with sheep prions, respectively. Based on Western blot with specific markers, MS, and morphological analysis, our data revealed that PrPc and PrPsc in the medium are associated with exosomes, membranous vesicles that are secreted upon fusion of multivesicular endosomes with the plasma membrane. Furthermore, we found that exosomes bearing PrPsc are infectious. Our data suggest that exosomes may contribute to intercellular membrane exchange and the spread of prions throughout the organism.     

Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient

We report a case of preclinical variant Creutzfeldt-Jakob disease (vCJD) in a patient who died from a non-neurological disorder 5 years after receiving a blood transfusion from a donor who subsequently developed vCJD. Protease-resistant prion protein (PrP(res)) was detected by western blot, paraffin-embedded tissue blot, and immunohistochemistry in the spleen, but not in the brain. Immunohistochemistry for prion protein was also positive in a cervical lymph node. The patient was a heterozygote at codon 129 of PRNP, suggesting that susceptibility to vCJD infection is not confined to the methionine homozygous PRNP genotype. These findings have major implications for future estimates and surveillance of vCJD in the UK.     

Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets

A definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) relies on the detection of pathological prion protein (PrP(Sc)). However, no test for PrP(Sc) in cerebrospinal fluid (CSF) has been available thus far. Based on a setup for confocal dual-color fluorescence correlation spectroscopy, a technique suitable for single molecule detection, we developed a highly sensitive detection method for PrP(Sc). Pathological prion protein aggregates were labeled by specific antibody probes tagged with fluorescent dyes, resulting in intensely fluorescent targets, which were measured by dual-color fluorescence intensity distribution analysis in a confocal scanning setup. In a diagnostic model system, PrP(Sc) aggregates were detected down to a concentration of 2 pM PrP(Sc), corresponding to an aggregate concentration of approximately 2 fM, which was more than one order of magnitude more sensitive than Western blot analysis. A PrP(Sc)-specific signal could also be detected in a number of CSF samples from patients with CJD but not in control samples, providing the basis for a rapid and specific test for CJD and other prion diseases. Furthermore, this method could be adapted to the sensitive detection of other disease-associated amyloid aggregates such as in Alzheimer's disease.     

遗传型朊蛋白病的研究进展

遗传型朊蛋白病（IPD）是由于编码朊蛋白（PrP）的PRNP基因发生突变，导致PrP发生空间构象改变而引起的神经系统退行性病变。与散发型克雅氏病（sCJD）患者相比，IPD患者的临床表型存在较大异质性，发病年龄往往较早，病程也相对较长，常规辅助检查（核磁、脑电图、14-3-3蛋白）通常无明显改变，且大多缺乏家族病史，临床上容易误诊漏诊。本文就IPD的流行病学特征、病因及发病机制、PRNP突变类型与疾病表型的相关性、3种IPD的具体临床特征、神经组织病理特征、辅助检查特点及相关研究进展等方面进行综述。     

A fatal familial insomnia patient newly diagnosed as having depression: A case report

Introduction:Fatal familial insomnia (FFI) is a rare clinical case. The study was mainly to report the clinical symptoms and imaging and genetic characteristics of a FFI case with depression, with relevant literature summarized.Patient concerns:A male, aged 57 years old, with mental disorders and progressive memory decline one year before admission.Diagnosis:Clinical manifestations: he had obvious abnormal mental behavior, rapidly progressing dementia symptoms, stubborn insomnia, abnormal movements and laryngeal stridor after falling asleep at night. Imaging and genetic test results: the cranial magnetic resonance imaging showed frontal temporal lobe atrophy; the polysomnography results showed no effective sleep; the 14-3-3 test result of cerebrospinal fluid was negative; the prion protein (PRNP) test showed that the D178N gene locus had mutations. And the patient was finally diagnosed as FFI.Interventions:There were no obvious effects in the treatment using medicines such as Risperidone, Olanzapine, Alprazolam, Clonazepam, and Deanxit.Outcomes:Mobility dysfunction of the patient was further aggravated. He was no longer able to move around on his own, and there were serious mental disorders.Conclusion:PRNP examination is of guiding significance for the diagnosis of the FFI of depression. Hence, it is very necessary to perform PRNP examination in clinical diagnosis of FFI of depression.     

Prion diseases and the gastrointestinal tract.

The gastrointestinal (GI) tract plays a central role in the pathogenesis of transmissible spongiform encephalopathies. These are human and animal diseases that include bovine spongiform encephalopathy, scrapie and Creutzfeldt-Jakob disease. They are uniformly fatal neurological diseases, which are characterized by ataxia and vacuolation in the central nervous system. Although they are known to be caused by the conversion of normal cellular prion protein to its infectious conformational isoform (PrPsc) the process by which this isoform is propagated and transported to the brain remains poorly understood. M cells, dendritic cells and possibly enteroendocrine cells are important in the movement of infectious prions across the GI epithelium. From there, PrPsc propagation requires B lymphocytes, dendritic cells and follicular dendritic cells of Peyer's patches. The early accumulation of the disease-causing agent in the plexuses of the enteric nervous system supports the contention that the autonomic nervous system is important in disease transmission. This is further supported by the presence of PrPsc in the ganglia of the parasympathetic and sympathetic nerves that innervate the GI tract. Additionally, the lymphoreticular system has been implicated as the route of transmission from the gut to the brain. Although normal cellular prion protein is found in the enteric nervous system, its role has not been characterized. Further research is required to understand how the cellular components of the gut wall interact to propagate and transmit infectious prions to develop potential therapies that may prevent the progression of transmissible spongiform encephalopathies.     

Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans

Mutation in the prion gene PRNP accounts for 10-15% of human prion diseases. However, little is known about the mechanisms by which mutant prion proteins (PrPs) cause disease. Here we investigated the effects of 10 different pathogenic mutations on the conformation and ligand-binding activity of recombinant human PrP (rPrP). We found that mutant rPrPs react more strongly with N terminus-specific antibodies, indicative of a more exposed N terminus. The N terminus of PrP contains a glycosaminoglycan (GAG)-binding motif. Binding of GAG is important in prion disease. Accordingly, all mutant rPrPs bind more GAG, and GAG promotes the aggregation of mutant rPrPs more efficiently than wild-type recombinant normal cellular PrP (rPrP(C)). Furthermore, point mutations in PRNP also cause conformational changes in the region between residues 109 and 136, resulting in the exposure of a second, normally buried, GAG-binding motif. Importantly, brain-derived PrP from transgenic mice, which express a pathogenic mutant with nine extra octapeptide repeats, also binds more strongly to GAG than wild-type PrP(C). Thus, several rPrPs with distinct pathogenic mutations have common conformational changes, which enhance binding to GAG. These changes may contribute to the pathogenesis of inherited prion diseases.     

Measurement and characterization of insulin-like growth factor binding protein-3 in human biological fluids: discrepancies between radioimmunoassay and ligand blotting.

The inability to detect insulin-like growth factor binding protein-3 (IGFBP-3) in some circumstances by Western ligand blot analysis has emphasized the need to characterize IGFBPs by both ligand binding and immunological techniques. In this study, we have: 1) characterized and quantified IGFBP-3 in nonpregnancy, pregnancy, and fetal cord serum, follicular, peritoneal, and amniotic fluid, seminal plasma, cerebrospinal fluid (CSF), and urine; 2) established a new IGFBP-3 RIA that detects both intact and fragments of IGFBP-3; 3) identified both intact and fragments of IGFBP-3 by Western immunoblot techniques; and 4) addressed the discordance between Western ligand blot analysis and RIA by assessing fluids for IGFBP proteolytic activity. All fluids examined, except pregnancy serum, CSF, and amniotic fluid, displayed a 44-34-kilodalton (kDa) IGFBP-3 doublet by Western ligand blot analysis. Western immunoblot analysis using specific IGFBP-3 antiserum showed a 44-34-kDa IGFBP-3 doublet and a 28-kDa fragment in nonpregnancy serum, fetal cord serum, follicular fluid, and peritoneal fluid. The immunoreactive 42-38-kDa doublet was faint in urine and seminal plasma. IGFBPs in CSF did not cross-react with IGFBP-3 antiserum. Pregnancy serum and amniotic fluid contained only the 28-kDa fragment when compared against equal volumes of nonpregnancy serum. With the development of an IGFBP-3 RIA, IGFBP-3 could be accurately measured; urine, CSF, and seminal plasma contained the lowest levels of IGFBP-3 at 27 +/- 3 ng/ml (mean +/- SEM), 110 +/- 26 ng/ml, and 209 +/- 56 ng/ml, respectively. In increasing concentration: fetal cord serum contained 753 +/- 101 ng/ml; peritoneal fluid, 1124 +/- 130 ng/ml; follicular fluid, 2356 +/- 211 ng/ml; nonpregnancy serum, 3556 +/- 508 ng/ml; pregnancy serum, 3718 +/- 842 ng/ml; and amniotic fluid, 5150 +/- 688 ng/ml. The measurable concentrations of IGFBP-3 in CSF and the high concentrations measured in pregnancy serum and amniotic fluid conflicted with Western blot analysis. Thus, fluids were assessed for IGFBP proteolytic activity by incubation with a source of IGFBP-3, either nonpregnancy serum or purified IGFBP-3. All fluids displayed some proteolytic activity with either assay. Fluids with little protease activity (nonpregnancy serum, follicular fluid, and urine) showed a close relationship between immunoassayable IGFBP-3 by RIA and IGFBP-3 band intensity by Western ligand blot. Fluids with high proteolytic activity (pregnancy serum, CSF, seminal plasma, peritoneal fluid, and amniotic fluid) gave discrepant IGFBP-3 values between RIA and Western ligand blot.(ABSTRACT TRUNCATED AT 400 WORDS)