DEVELOPMENTAL EXPRESSION OF COXSACKIE ADENOVIRUS RECEPTOR

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。柯萨奇病毒B和腺病毒是与人类心肌炎发展有关的最常见病毒病原体，并通过科萨基腺病毒受体（CAR）进入细胞。先前的研究已经证明，CAR表达的鼠类似物的表达在年轻啮齿动物中比在老年啮齿动物中更高，并且腺病毒介导的基因转导在老年动物中效率较低。我们使用RT-PCR、免疫印迹和免疫荧光检查了非人灵长类动物中的CAR表达。RNA和蛋白质提取自15只不同年龄（2天-14岁）的恒河猴的心脏组织，所述恒河猴获自杜兰国家灵长类动物研究中心（TNPRC）。我们测定了恒河猴CAR（rhCAR）基因的核苷酸序列，并设计了一种引物探针组合，用于真实的PCR定量rhCAR。相对于GAPDH，rhCAR mRNA水平与恒河猴的年龄呈负相关;最高水平出现在不到一岁的灵长类动物中。定量相对蛋白质表达的努力正在进行中。这些数据表明，心肌炎和新生儿CVB感染的其他表现可能反映了较高的CAR表达。非人灵长类动物初步研究的资金是通过TNPRC的试点研究计划获得的。已分配动物，并获得基线ECG、柯萨奇病毒抗体水平和超声心动图。微创心脏活检技术的初步培训已经完成。拟议研究的第一阶段将于3月开始，将包括滴定病毒剂量，以引起疾病，而不会出现严重的急性并发症。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Coxsackievirus B and Adenoviruses are the most common viral pathogens implicated in the development of human myocarditis, and gain entry into cells via the Coxsackie Adenovirus Receptor (CAR). Previous studies have demonstrated that expression of the murine analogue of CAR expression is higher in young versus old rodents, and that adenovirus mediated gene transduction is less efficient in aged animals. We examined CAR expression in non-human primates, using RT- PCR, immunoblot, and immunofluorescence. RNA and protein was extracted from cardiac tissue from 15 Rhesus macaque monkey of various ages (2 days-14 yrs) obtained from the Tulane National Primate Research Center (TNPRC). We determined the nucleotide sequence of the rhesus CAR (rhCAR) gene, and designed a primer-probe combination for rhCAR quantitation by real time PCR. Relative to GAPDH, rhCAR mRNA levels were inversely correlated with age in Rhesus macaques; the highest levels were seen in primates less than one year of age. Efforts to quantify relative protein expression are underway. These data suggest indicate that myocarditis and other manifestations of CVB infection in newborns may reflect higher CAR expression. Funding for preliminary studies in nonhuman primates was obtained throught the Pilot Study Program at TNPRC. Animals have been assigned, and baseline ECG, antibody levels to coxsackievirus, and echocardiograms have been obtained. Initial training for minimally invasive cardiac biopsy techniques has been completed. The first phase of the proposed studies will begin in March and will include titration of viral dose to cause disease without severe acute complications.