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Autophagic flux without a block differentiates varicella from herpes simplex virus infection 无阻断的自噬通量可区分水痘与单纯疱疹病毒感染
Pub Date : 2015-05-01 DOI: 10.1016/j.nhtm.2015.07.066
Charles Grose

Varicella-zoster virus (VZV) is a herpesvirus that causes a characteristic vesicular exanthem in humans with primary infection (varicella) or reactivation (zoster). We have previously observed that vesicular cells are filled with autophagosomes that are easily detectable by confocal microscopy after immunolabeling for the LC3 protein. Through a 3D imaging software program called Imaris we have quantitated autophagosomes as greater than 100 per cell. Similarly, we have assessed autophagy in VZV-infected monolayers after inoculation by the traditional method with infected cells at a ratio of one infected to 8 uninfected cells. Again, autophagosomes are easily detected, but their count is lower than that observed in human skin cells. As an additional control, we enumerated the autophagosomes in the Severe Combined Immuno-Deficient (SCID) Mouse model of VZV infection. In this model, human skin is inserted under the skin of the mouse and subsequently inoculated with VZV-infected cells. Again, autophagy was abundant in the VZV-infected skin and minimal in the mock-infected skin sample. Subsequently, we investigated autophagy following infection with sonically prepared cell free virus in cultured cells. After cell free virus inoculation, autophagy was detected in a majority of infected cells at all time points, but was less than that seen after an infected-cell inoculum. Finally, we investigated VZV-induced autophagic flux by two different methods (radiolabeling proteins and a dual-colored LC3 plasmid); both showed no evidence of a block in autophagy. Overall, therefore, autophagy within a VZV-infected cell was remarkably different from autophagy within an HSV-infected cell, whose genome contains two modifiers of autophagy, ICP34.5 and US11, not present in VZV.

水痘-带状疱疹病毒(VZV)是一种疱疹病毒,可引起原发感染(水痘)或再激活(带状疱疹)的人出现特征性水疱性渗漏。我们之前观察到,囊泡细胞充满自噬体,在LC3蛋白免疫标记后,通过共聚焦显微镜很容易检测到自噬体。通过一个名为Imaris的3D成像软件程序,我们对每个细胞超过100个的自噬体进行了定量。同样,我们用传统的方法,用1个感染细胞比8个未感染细胞接种vzv感染单层后,评估了自噬情况。同样,自噬体很容易被检测到,但它们的数量低于在人类皮肤细胞中观察到的数量。作为额外的对照,我们在VZV感染的严重联合免疫缺陷(SCID)小鼠模型中列举了自噬体。在该模型中,将人皮肤插入小鼠皮肤下,随后用vzv感染的细胞接种。同样,自噬在vzv感染的皮肤中丰富,而在模拟感染的皮肤样本中很少。随后,我们研究了用声音制备的无细胞病毒感染培养细胞后的自噬现象。在无细胞病毒接种后,大多数感染细胞在所有时间点都检测到自噬,但比感染细胞接种后观察到的自噬少。最后,我们用两种不同的方法(放射性标记蛋白和双色LC3质粒)研究了vzv诱导的自噬通量;两者均未显示自噬阻滞的证据。因此,总的来说,VZV感染细胞内的自噬与单纯疱疹病毒感染细胞内的自噬有显著差异,单纯疱疹病毒的基因组包含两个自噬修饰因子ICP34.5和US11,而在VZV中不存在。
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引用次数: 0
Animation of VZV DNA VZV DNA动画
Pub Date : 2015-05-01 DOI: 10.1016/j.nhtm.2015.07.064
Randall. J. Cohrs , J. Rovnak

Varicella zoster virus (VZV) is a ubiquitous neurotropic alphaherpesvirus that typically causes childhood varicella (chickenpox) on primary infection and zoster (shingles) after reactivation. During latency most of the ~70 virus genes are transcriptionally silent; however, analysis of latent VZV gene transcription in its natural setting requires analysis of human ganglia removed at autopsy. Recognizing the problems associated with such samples, we have observed that as the post-mortem time interval increases, so do the number of VZV genes transcribed. Based on our data and recent similar findings concerning reactivation of HSV-1, we propose an interesting testable model to describe epigenetic control of neurotropic alphaherpesvirus gene transcription during latency and early reactivation.

水痘带状疱疹病毒(VZV)是一种普遍存在的嗜神经性甲疱疹病毒,通常引起儿童水痘(水痘)初次感染和带状疱疹(带状疱疹)后再激活。在潜伏期,约70个病毒基因中的大多数处于转录沉默状态;然而,在自然环境中分析潜伏的VZV基因转录需要分析尸检中切除的人类神经节。认识到与这些样本相关的问题,我们观察到,随着死后时间间隔的增加,VZV基因转录的数量也在增加。基于我们的数据和最近关于HSV-1再激活的类似发现,我们提出了一个有趣的可测试模型来描述潜伏和早期再激活期间嗜神经性α疱疹病毒基因转录的表观遗传控制。
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引用次数: 0
Predicting Prion Propensity of Human Proteins 预测人类蛋白质的朊病毒倾向
Pub Date : 2015-05-01 DOI: 10.1016/j.nhtm.2015.07.042
Cascarina S, Ross E.

In humans only a single prion-forming protein named PrPc (for “cellular prion protein”) is currently known, yet many more neurodegenerative disorders involve aberrant protein aggregation. The classical model for these diseases has involved cell-autonomous aggregation, assuming that aggregation occurs independently in each cell within a diseased patient. However, more recent models have proposed a non-cell-autonomous progression of disease in which aggregates formed in one cell may be transmitted to neighboring cells. These aggregate seeds then cause aggregation of the soluble protein in the “infected” cells, similar to the prion diseases. Within the past few years, a number of proteins that exhibit prion-like aggregation and spread to neighboring tissues have been discovered in patients with Amyotrophic Lateral Sclerosis (ALS). Although ALS has been studied for a number of decades, these proteins were only recently linked to ALS by chance. This demonstrates a clear need for an accurate method to systematically identify additional proteins that may play a pathological role in neurodegenerative disorders. Taking advantage of the compositional similarity of these proteins to the known yeast prions, I plan to use the prion prediction methodology that our lab has pioneered to develop an entirely new algorithm specifically suited for this class of neuronal proteins.

在人类中,目前只知道一种名为PrPc的朊病毒形成蛋白(即“细胞朊病毒蛋白”),然而更多的神经退行性疾病涉及异常的蛋白质聚集。这些疾病的经典模型涉及细胞自主聚集,假设聚集在患病患者的每个细胞中独立发生。然而,最近的模型提出了一种非细胞自主的疾病进展,其中在一个细胞中形成的聚集体可能会传递给邻近的细胞。然后,这些聚集的种子导致“感染”细胞中的可溶性蛋白质聚集,类似于朊病毒疾病。在过去的几年中,在肌萎缩性侧索硬化症(ALS)患者中发现了一些表现出朊病毒样聚集并扩散到邻近组织的蛋白质。尽管ALS已经被研究了几十年,但这些蛋白质直到最近才偶然地与ALS联系起来。这表明需要一种准确的方法来系统地识别可能在神经退行性疾病中起病理作用的其他蛋白质。利用这些蛋白质与已知酵母朊病毒的组成相似性,我计划使用我们实验室首创的朊病毒预测方法来开发一种专门适用于这类神经元蛋白质的全新算法。
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引用次数: 0
Transcriptome markers of viral persistence in naturally-infected Andes Hantavirus (Bunyaviridae) seropositive rice rats 自然感染安第斯汉坦病毒(布尼亚病毒科)血清阳性水稻大鼠中病毒持久性的转录组标记物
Pub Date : 2015-05-01 DOI: 10.1016/j.nhtm.2015.07.063
Corey L. Campbell , Fernando Torres-Perez , Mariana Acuna-Retamar , Tony Schountz

The long-tailed pygmy rice rat (Oligoryzomys longicaudatus) is the reservoir host of Andes (ANDV) and Oran hantaviruses (Bunyaviridae). To examine transcriptome features of persistently infected rice rats, spleens from ANDV sero-positive wild-caught rice rats were assessed. RNA-seq analysis, de novo reference-independent assembly and stringent orthology assignments produced 17,756 unique coding and non-coding RNAs. Differential expression analysis of persistently-infected seropositive rice rat spleens revealed 18 differentially expressed transcripts from 16 unique genes. A three-pronged effect on the immune response were observed in 1) suppression of the JAK-STAT pathway at Stat5b and Ccnot1, as well as 2) a bias toward a TH2 response in the enrichment of caspase-1 and 3) stimulation of RIG-I pathway factors Ppp1cc and MFF. Two of these differentially expressed transcripts, caspase-1 and STAT5b, code for proteins expected to stimulate T helper follicular (TFH) cell development, a phenomenon that has also been described for hantavirus-infected P. maniculatus. Differential expression of a single seropositive rice rat with a higher viral load revealed a robust response of 243 differentially expressed transcripts, suggesting an acute infection. Together, these data help define the fundamental features of the immune response in a hantavirus reservoir host.

长尾侏儒稻鼠(Oligoryzomys longicaudatus)是安第斯病毒(ANDV)和奥兰汉坦病毒(布尼亚病毒科)的宿主。为了研究持续感染水稻大鼠的转录组特征,对野生捕获的ANDV血清阳性水稻大鼠的脾脏进行了评估。RNA-seq分析,从头开始独立于参考的组装和严格的同源性分配产生了17,756个独特的编码和非编码rna。对持续感染的血清阳性水稻大鼠脾脏进行差异表达分析,发现来自16个独特基因的18个差异表达转录本。我们观察到对免疫应答的三管齐下作用:1)抑制JAK-STAT通路Stat5b和Ccnot1; 2)富集caspase-1时偏向TH2应答;3)刺激RIG-I通路因子Ppp1cc和MFF。其中两种差异表达转录物,caspase-1和STAT5b,编码刺激T辅助滤泡(TFH)细胞发育的蛋白质,这种现象也被描述为汉坦病毒感染的P. maniculatus。单个血清阳性水稻大鼠的差异表达与较高的病毒载量显示243个差异表达转录物的强烈反应,表明急性感染。总之,这些数据有助于确定汉坦病毒宿主体内免疫反应的基本特征。
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引用次数: 0
Further Characterization of Rio Grande Virus and Potential for Serological Cross Reactivity with other Phleboviruses 格兰德河病毒的进一步鉴定及其与其他静脉病毒的血清学交叉反应潜力
Pub Date : 2015-05-01 DOI: 10.1016/j.nhtm.2015.07.059
Szymczak M , Reeves W , Miller M

Members of the genus Phlebovirus (family Bunyaviridae) are new and emerging disease pathogens of humans and animals. Newly identified viruses include Heartland virus (HRTV), Lone Star virus in the USA, and Severe Fever with Thrombocytopenia Syndrome virus in Asia. Assays to support surveillance, epidemiologic studies, and diagnosis of these viruses may also detect related viruses within the genus, confounding interpretation. Rio Grande virus (RGV) was isolated in 1973 from southern plains woodrats (Neotoma micropus) in the United States and has been preliminarily identified as a phlebovirus transmitted by the sand fly Lutzomyia anthophora. RGV is not known to cause disease in humans, but it could be detected by assays designed for HRTV or other phleboviruses. The goal of this study was to determine antigenic cross-reaction between RGV and other phleboviruses. A commercially available ELISA based sand fly fever antigen detection kit was tested for the ability to detect RGV and other New and Old-World phleboviruses, including attenuated Rift valley fever virus (RVFV) strain MP12, Punta Toro virus (PTV), Toscana virus, Aguacate virus, Anhanga virus, Arumowot virus, and Chagres virus. Immunocytochemistry and Western blotting were used to detect cross reactions between RGV, MP12, and PTV using rabbit anti-RVFV nucleocapsid protein and glycoproteins GC and GN, mouse monoclonal anti-PTV, and sheep polyclonal anti-MP12. The ELISA test detected cross reactivity for all phleboviruses excluding RGV, but Western blotting detected the presumed RGV nucleocapsid protein (N) using rabbit anti-RVFV-N serum, RGV-infected cells were also identified when labeled with this antibody. Our findings demonstrate assay specific antigenic cross reactivity between these phleboviruses, thus further characterization of the molecular targets of the cross-reaction is required for proper interpretation of serological assays.

白蛉病毒属(布尼亚病毒科)的成员是人类和动物新的和正在出现的疾病病原体。新发现的病毒包括美国的心脏地带病毒(HRTV)、孤星病毒和亚洲的发热伴血小板减少综合征病毒。支持这些病毒的监测、流行病学研究和诊断的检测也可能检测到属内的相关病毒,从而混淆解释。格兰德河病毒(Rio Grande virus, RGV)于1973年从美国南部平原木鼠(Neotoma micropus)中分离得到,初步鉴定为一种由沙蝇(luzomyia anthophora)传播的静脉病毒。目前还不知道RGV会在人类中引起疾病,但可以通过为HRTV或其他静脉病毒设计的检测方法检测到RGV。本研究的目的是确定RGV与其他静脉病毒之间的抗原交叉反应。对市售的基于ELISA的沙蝇热抗原检测试剂盒检测RGV和其他新、旧世界白蛉病毒(包括减毒裂谷热病毒(RVFV)毒株MP12、蓬塔托罗病毒(PTV)、托斯卡纳病毒、Aguacate病毒、Anhanga病毒、Arumowot病毒和Chagres病毒)的能力进行了测试。免疫细胞化学和Western blotting检测兔抗rvfv核衣壳蛋白和糖蛋白GC、GN、小鼠单克隆抗PTV和绵羊多克隆抗MP12与RGV、MP12和PTV的交叉反应。ELISA检测除RGV外的所有静脉病毒的交叉反应性,但Western blotting检测兔抗RGV- N血清中推测的RGV核衣壳蛋白(N),当标记该抗体时,RGV感染细胞也被鉴定出来。我们的研究结果证明了这些静脉病毒之间的特异性抗原交叉反应,因此需要进一步表征交叉反应的分子目标,以正确解释血清学分析。
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引用次数: 0
The Intrathecal Antibody Response in Multiple Sclerosis Brain Does Not React Against Measles Virus 多发性硬化症脑鞘内抗体反应对麻疹病毒没有反应
Pub Date : 2015-05-01 DOI: 10.1016/j.nhtm.2015.07.060
Deandra L Walker, Mark P Burgoon

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) and is the most common disabling neurological disease of young adults. Although the cause of MS is unknown, genetic and epidemiological studies indicate that MS may be triggered by an environmental agent. The presence of intrathecally produced antibodies, which produce oligoclonal Ig bands in the CNS of MS patients, provides tools for investigating the target of the inflammatory response. In most of the CNS conditions with oligoclonal bands the target is known and the antibody is directed against an infectious, causative agent. For example, in subacute sclerosing panencephalitis, a measles virus (MV) infection of the brain, the oligoclonal bands and intrathecal antibodies are primarily directed against MV. In earlier studies of MS, we demonstrated that the intrathecal antibody response in MS brain does not react to varicella zoster or Epstein-Barr virus. The current study investigates the reactivity of the intrathecal antibody response in MS brain to MV. We isolated individual CD38(+) plasma cells from MS brain to produce recombinant antibodies (rAbs). These rAbs likely represent oligoclonal bands and were used to immunostain MV-infected or uninfected monkey kidney (Vero) cells. Although many of the rAbs from MS brain reacted against auto-antigens in several mouse and human tissues, none of fifteen MS rAbs reacted against MV-infected cells. These results indicate that measles virus is not a disease-relevant antigen in MS.

多发性硬化症(MS)是一种中枢神经系统(CNS)的慢性炎症性脱髓鞘疾病,是年轻人最常见的致残神经系统疾病。虽然MS的病因尚不清楚,但遗传学和流行病学研究表明,MS可能是由环境因素引发的。鞘内产生的抗体在MS患者的中枢神经系统中产生寡克隆Ig带,为研究炎症反应的目标提供了工具。在大多数具有寡克隆带的中枢神经系统条件下,目标是已知的,抗体是针对传染性病原体的。例如,在亚急性硬化性全脑炎(一种脑麻疹病毒感染)中,寡克隆带和鞘内抗体主要针对麻疹病毒。在MS的早期研究中,我们证明MS脑鞘内抗体反应对水痘带状疱疹或爱泼斯坦-巴尔病毒没有反应。本研究探讨MS脑鞘内抗体对MV的反应性。我们从MS脑中分离出单个CD38(+)浆细胞来产生重组抗体(rAbs)。这些rAbs可能代表寡克隆带,并用于对mv感染或未感染的猴肾(Vero)细胞进行免疫染色。尽管来自多发性硬化症大脑的许多rAbs对几种小鼠和人类组织中的自身抗原起反应,但15种MS rAbs中没有一种对感染mv的细胞起反应。这些结果表明麻疹病毒在多发性硬化症中不是疾病相关抗原。
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引用次数: 0
Ivermectin for the Control of West Nile Virus Transmission 控制西尼罗病毒传播的伊维菌素
Pub Date : 2015-05-01 DOI: 10.1016/j.nhtm.2015.07.043
Nguyen C, Burton T, Kuklinski W, Gray M, Foy BD

Presently there are limited options for controlling the transmission of West Nile virus (WNV), including the use of larvicides and adulticides to target the mosquito vector. However, these methods are poorly-targeted, restricted to wealthy semi-urban and urban areas that are able to fund the efforts, and opposed in some communities due to toxicity concerns. This study evaluated the use of endectocide-treated bird feed to control WNV transmission by targeting the primary vector in Colorado, Culex tarsalis. Ivermectin susceptibility in C. tarsalis was first measured through ivermectin-spiked bloodmeals fed using membrane feeders, and the LC50 was determined to be 49.94 ng/ml (39.71-59.93 95% CI, n=988). Chickens were then fed ivermectin-treated feed to examine its safety and palatability, and mosquitoes were blood fed directly on the chickens to assess in vivo effects. Finally, ivermectin pharmokinetics were analyzed using vein blood from chickens as well the C. tarsalis that bloodfed on the chickens. A mixture of 200 mg ivermectin/kg of bird feed was determined to be a palatable and safe dose on which chickens would feed while also being effective in killing C. tarsalis in bioassays. Pharmacokinetic data from the in vivo tests produced conflicting results compared to in vitro blood feeds but drug was detected in chicken blood at concentrations that may be expected to affect C. tarsalis. Dosing, safety, and bioassays are currently being conducted in doves and sparrows. Additional studies are currently determining the effect of ivermectin on mortality in WNV-infected mosquitoes, as well as if ivermectin reduces WNV replication and transmission. Our study indicates that the use of ivermectin-treated bird feed could be a novel method of controlling WNV transmission.

目前控制西尼罗河病毒(WNV)传播的选择有限,包括使用杀幼虫剂和杀成虫剂来瞄准蚊子媒介。然而,这些方法的针对性很差,仅限于富裕的半城市和城市地区,这些地区有能力资助这些努力,并且由于毒性问题在一些社区遭到反对。本研究通过针对科罗拉多州的主要媒介库蚊(库蚊),评估了使用杀虫剂处理过的鸟饲料来控制西尼罗河病毒传播的效果。首次采用膜喂食器加伊维菌素血粉检测tarsalis对伊维菌素的敏感性,LC50为49.94 ng/ml (95% CI为39.71 ~ 59.93,n=988)。然后用伊维菌素处理过的饲料喂养鸡,以检验其安全性和适口性,并直接用血喂蚊子,以评估对鸡的体内影响。最后,利用鸡的静脉血液和以鸡为食的tarsalis对伊维菌素的药动学进行了分析。在生物测定中,每公斤禽鸟饲料中加入200毫克伊维菌素的混合物被确定为一种美味和安全的鸡饲料剂量,同时也能有效地杀死tarsalis。与体外血液饲料相比,体内试验的药代动力学数据产生了相互矛盾的结果,但在鸡血液中检测到的药物浓度可能会影响tarsalis。目前正在鸽子和麻雀中进行给药、安全性和生物测定。其他研究目前正在确定伊维菌素对西尼罗河病毒感染蚊子死亡率的影响,以及伊维菌素是否能减少西尼罗河病毒的复制和传播。我们的研究表明,使用伊维菌素处理的鸟类饲料可能是控制西尼罗河病毒传播的一种新方法。
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引用次数: 3
Roche Research Portfolio: Trusted Performance, Efficient Workflow Solutions 罗氏研究组合:可靠的性能,高效的工作流程解决方案
Pub Date : 2015-05-01 DOI: 10.1016/j.nhtm.2015.07.068
Konet D

Along with Roche Pharmaceuticals, Roche Diagnostics is an important part of the foundation that modern healthcare builds upon. Our broad range of innovative diagnostic tests and systems play a pivotal role in the groundbreaking area of integrated healthcare solutions and cover the early detection, targeted screening, evaluation and monitoring of disease. Roche Diagnostics is active in all market segments, from scientific research and clinical laboratory systems to patient self-monitoring.

罗氏诊断与罗氏制药一起,是现代医疗保健基础的重要组成部分。我们广泛的创新诊断测试和系统在综合医疗解决方案的开创性领域发挥着关键作用,涵盖疾病的早期发现,有针对性的筛查,评估和监测。罗氏诊断活跃于所有细分市场,从科学研究和临床实验室系统到患者自我监测。
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引用次数: 0
Longitudinal analysis of blood-borne prion infection 血源性朊病毒感染的纵向分析
Pub Date : 2015-05-01 DOI: 10.1016/j.nhtm.2015.07.045
Alan M. Elder , Davin M. Henderson , Amy V. Nalls , Anthony E. Kincaid , Edward A. Hoover , Jason C. Bartz , Candace K. Mathiason

Transmissible spongiform encephalopathies (TSEs), or prion diseases, affecting human and animal species can be transmitted from TSE-infected individuals to naïve susceptible hosts during the long asymptomatic (years to decades) and symptomatic disease stages. The presence of infectious hematogenous prions in asymptomatic TSE-infected hosts demonstrates the highly infectious nature of blood-borne prions in hosts lacking overt clinical symptoms. It is currently unknown when and how infectious prions first enter the blood following initial exposure. We have previously shown that the whole-blood real-time quaking-induced conversion assay (wbRT-QuIC) possesses 100% specificity and >92% sensitivity, making it an ideal tool to address this question. Here, we use wbRT-QuIC to analyze whole blood collected from oral, extranasal or aerosol TSE-exposed hosts for blood-borne prions. Our results demonstrate that conversion competent prions in the inoculum are capable of crossing mucosal surfaces and entering the circulatory system within 30 min—no matter the route of exposure. Detection of the inoculum minutes post exposure is followed by a steady decline in the detection of blood-borne prions up to 3 days which is followed by a progressive increase in the detection of nascent conversion competent prions between 1 and 17 months post exposure. These data provide the first evidence for the facile transport of mucosally acquired prions into the circulatory system, providing evidence for multiple routes of inter- and intra- host prion trafficking and shedding.

传染性海绵状脑病(tse)或朊病毒疾病,影响人类和动物物种,可在长期无症状(数年至数十年)和有症状的疾病阶段从tse感染个体传播给naïve易感宿主。感染性血源性朊病毒存在于无症状的tse感染宿主中,表明血源性朊病毒在没有明显临床症状的宿主中具有高度传染性。目前尚不清楚感染性朊病毒在初次接触后何时以及如何首次进入血液。我们之前的研究表明,全血实时震动诱导转化试验(wbRT-QuIC)具有100%的特异性和92%的灵敏度,使其成为解决这一问题的理想工具。在这里,我们使用wbRT-QuIC分析从口腔、鼻外或气溶胶暴露于tse的宿主收集的全血,以检测血源性朊病毒。我们的研究结果表明,无论接触途径如何,接种物中的转化能力朊病毒都能够在30分钟内穿过粘膜表面进入循环系统。在暴露后接种数分钟的检测之后,血源性朊病毒的检测持续下降至3天,随后在暴露后1至17个月期间,新生转化能力朊病毒的检测逐渐增加。这些数据为粘膜获得的朊病毒进入循环系统的便捷运输提供了第一个证据,为宿主间和宿主内朊病毒运输和脱落的多种途径提供了证据。
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引用次数: 1
Signal regulatory protein alpha (SIRPA) and kinase domain receptor (KDR) are key expression markers in cardiac specific precursor selection from hADSCs 信号调节蛋白α (SIRPA)和激酶结构域受体(KDR)是hascs中心脏特异性前体选择的关键表达标记
Pub Date : 2015-05-01 DOI: 10.1016/j.nhtm.2015.02.001
Vinod Kumar Verma , Syed Sultan Beevi , Tanya Debnath , Usha Shalini , Suguna Ratnakar Kamaraju , Lakshmi Kumari Kona , Yamuna Mohanram , Lakshmi Kiran Chelluri

Background

Cardiomyocyte enrichment strategies so far have not yielded scalable cardiac specific cell type. More so, the current data is restricted to embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs), wherein the use of viral vectors is fraught with increased risk during clinical use. Herein, we profiled time-dependent gene/protein expression patterns across the cardiac ectoderm, endoderm, and mesoderm for isolating cardiac precursors from human adipose derived stem cells (hADSC).

Methods

Direct cardiac differentiation of hADSCs was carried out with 5-azacytidine and basic fibroblast growth factor (bFGF) in a one month long culture. The cells were periodically harvested, analyzed for unique persistent markers and their inherent regulation using quantitative polymerase chain reaction (qPCR), flow cytometry, immunoblot and immunocytochemistry assays. The identified markers were super paramagnetic iron oxide nanoparticle (SPION) tagged for segregation by magnetic activated cell sorting (MACS) and further evaluated their differentiation potential and checked for the purity by flow cytometry.

Results

The results demonstrated pronounced up-regulation of mesodermal and mature cardiac lineage markers at three weeks, while there was a down-regulation of pluripotent stem cell markers. This perhaps could be attributed to de-differentiation in maintaining the cardiac phenotype. However, signal regulatory protein alpha (SIRPA) and kinase domain receptor (KDR) persisted all through the culture period of one month, making them the most relevant and reliable cardiac specific markers. Dual labeling of these markers to SPION for cardiomyocyte enrichment by MACS column yielded cardiomyogenic-like cells in differentiation cultures with several functional positive markers.

Conclusions

Thus, SIRPA and KDR together provide cues in the enhancement and up-scaling of cardiomyocyte production in the cell replacement therapy.

Focal points

  • Benchside

    Identification of specific cell phenotypic markers to identify cardiac precursors in any tissue source with minimal cell manipulation is a novel process development tool in clinical translation.

  • Bedside

    A product developed in a closed system would minimize extraneous contaminants in long term cultures and development of such procedures minimizes culture failure rates from bench side.

  • Industry

    This unique identification of cell-specific marker would enable a tissue-specific translational plan and immensely help in the cardiac regeneration.

  • Government

    Financial inve

到目前为止,心肌细胞富集策略尚未产生可扩展的心脏特异性细胞类型。更重要的是,目前的数据仅限于胚胎干细胞(ESCs)/诱导多能干细胞(iPSCs),其中在临床使用中使用病毒载体充满了增加的风险。在此,我们分析了心脏外胚层、内胚层和中胚层的时间依赖性基因/蛋白表达模式,用于从人脂肪来源干细胞(hADSC)中分离心脏前体。方法采用5-氮杂胞苷和碱性成纤维细胞生长因子(bFGF)对hascs进行1个月的心脏直接分化培养。定期收集细胞,使用定量聚合酶链反应(qPCR)、流式细胞术、免疫印迹和免疫细胞化学分析独特的持久标记及其内在调节。鉴定的标记物为超顺磁性氧化铁纳米颗粒(SPION),经磁活化细胞分选(MACS)进行分离,并进一步通过流式细胞术检测其分化潜力和纯度。结果小鼠中胚层和成熟心脏谱系标记在3周时明显上调,而多能干细胞标记则下调。这可能归因于维持心脏表型的去分化。然而,信号调节蛋白α (SIRPA)和激酶结构域受体(KDR)在一个月的培养期间持续存在,使它们成为最相关和可靠的心脏特异性标志物。将这些标记物双重标记到SPION上,通过MACS柱富集心肌细胞,在分化培养物中产生具有几种功能阳性标记物的心肌样细胞。因此,在细胞替代疗法中,SIRPA和KDR共同为心肌细胞生成的增强和扩大提供了线索。特异性细胞表型标记的鉴定,以最小的细胞操作识别任何组织来源的心脏前体,是临床翻译中的一种新的过程开发工具。在封闭系统中开发的产品可以最大限度地减少长期培养中的外来污染物,并且开发此类程序可以最大限度地减少工作台侧的培养失败率。这种细胞特异性标记的独特识别将使组织特异性翻译计划成为可能,并极大地帮助心脏再生。•政府政府的财政投资和支持对于优化和验证更好的保健至关重要,并将有助于减轻疾病负担。•监管世界范围内对进入基于干细胞的临床试验的最小细胞操作的严格监管指南排除了在产品和工艺开发技术中开发替代方法的需要,这些方法可以很容易地转化为临床设置。
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引用次数: 4
期刊
New horizons in translational medicine
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