监测后腹腔感染的免疫细胞贩运荧光朊病毒棒时间
Summary
在这里，我们描述了一种新的分析监测朊蛋白的吸收和免疫细胞的活动，通过净化后立即腹腔注射和荧光标记从被感染的大脑，然后监测其吸收和运动从注射部位和调解这些事件的细胞特征的材料汇总朊病毒棒。
Abstract
异常的形式存在的主机编码朊蛋白（PrPC）是蛋白酶耐，病理和传染性的朊病毒疾病，如慢性消耗病（CWD）鹿和羊痒病的特征。断言，这种不正常的构象构成的大部分或全部的传染性朊病毒，朊病毒假说。的免疫系统在外围朊病毒的发病机制中的早期事件中的作用已令人信服地证明 CWD和痒病1-3。在保留和复制朊病毒感染后4-6年初，在小鼠的基因和药理学研究发现补体系统的重要作用 。7-10树突状细胞在体外和体内研究也观察到朊病毒保留，尽管他们在贩卖的作用仍然不清楚11-16。巨噬细胞也同样被牵连在早期朊病毒的发病机制，但这些研究都集中在事件发生后感染3,11,17周。这些以前的研究也受到来自内源性的PrP C和传染性朊病毒之间的区别问题。在这里，我们描述了一个半定量的评估接种部位招募免疫细胞朊蛋白的吸收和贩运，不带偏见的方法。从被感染的脑组织匀浆洗涤剂溶解非聚合蛋白，通过蔗糖垫层离心纯化聚合朊病毒棒。聚丙烯酰胺凝胶电泳，考马斯亮蓝染色和免疫印迹证实颗粒分数高浓缩铀的朊病毒棒的回收。朊病毒棒荧光标记，然后注射到小鼠腹腔内。两个小时后测定腹腔灌洗液，脾，纵隔，肠系膜淋巴结的免疫细胞朊病毒杆保留和多色流量使用单核细胞，中性粒细胞，树突状细胞，巨噬细胞和B细胞和T细胞的标记流式细胞仪确定的细胞亚群。此试验允许首次直接监测的免疫收购细胞，感染后数小时内贩运朊病毒在体内。此实验也明确区分汇总朊病毒的传染性，从PrPC通常在宿主细胞中所表达的，可困难，并导致其他检测系统中的数据解释问题。可以适应其他接种途径（口服，静脉，intranervous和皮下，如）和抗原（共轭珠，细菌，病毒和寄生虫病原体和蛋白质，鸡蛋），以及该协议。
Protocol
1。净化和标签朊病毒棒该协议是改编自一个以前出版了18本 在900毫升的冰冷的均质缓冲朊病毒感染的脑组织匀浆100克（HB，1X PBS含320毫米蔗糖，氯化钠150毫米和4毫米EDTA）1分钟，最大速度在商业搅拌机，然后在冰上2分钟。重复三次。 在3000 XG 10分钟和4 ° C离心匀浆删除，并保存在冰上清。重新悬浮颗粒在1L HB和重复步骤1.1和1.2。 池上清液和?…
Discussion
在这里，我们展示了一个标记和跟踪朊病毒的协议 ，在体内，大大方便了监控周边朊病毒感染的早期事件。该协议大大提高了监测朊病毒吸收过去的尝试标签前的高浓缩铀朊病毒接种 9体外和 体内 3,12明确区分内源性的PrP彗星。由于传染性朊病毒PrP的彗星共享相同的主要的氨基酸序列，产生朊蛋白的特异性抗体一直是个问题。我们已经执行了?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢史蒂夫McBryant和杰夫汉森鼠标操作的帮助与超速离心和帕蒂凯泽帮助。授予5R01NS056379 – 02在美国国立卫生研究院，美国国家神经疾病和中风研究所资助的这项工作。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| CWD-infected elk brain | Private elk farm in Colorado | Use any non-human prion-infected brain | ||
| Blender | Oster | 6694-015 | Use any commercial blender | |
| Centrifuge | Sorvall | SS34 rotor | Use any centrifuge /rotor that can reach 3000 x g and hold ≥ 500 ml volumes | |
| Ultracentrifuge | Beckman | 50.2 Ti rotor | Use any ultracentrifuge /rotor that can reach 100,000 x g and hold ≥ 500 ml volumes | |
| Bradford Reagent | Sigma-Aldrich | B6916 | ||
| Complete mini protease inhibitor cocktail | Roche | 11 836 170 001 | ||
| Sonicator | Misonix | MP4000X | Use any horn or probe sonicator set to ~70% max power | |
| DyLight antibody Labeling kit | Thermo Scientific | 53050 | ||
| microcentrifuge | Eppendorf | 55430R | Use any refrigerated microcentrifuge that can achieve 13,000x g | |
| centrifugal filter columns | Millipore | Microcon YM-100 | Use any filter or dialysis membrane with 100 Kd molecular weight cutoff | |
| 8-40 week-old FVB mice | Charles River | 207 | Use any inbred mouse strain | |
| 1 μm red fluorescent beads | Phosphorex | 2307 | Use any fluorescent bead ≤ 10 μm | |
| RPMI 1640 medium | Invitrogen | 11875-093 | ||
| 40 μm cell strainer | Falcon | 352340 | ||
| fluorescent antibodies | BD pharmingen | Various | Use any fluorescent antibody appropriate for your application. | |
| flow cytometer | Dakocytomation | CyanADP | Use any flow cytometer capable of multicolor fluorescence detection |
Referências
- Klein, M. A. A crucial role for B cells in neuroinvasive scrapie. Nature. 390, 687-687 (1997).
- Mabbott, N. A., Farquhar, C. F., Brown, K. L., Bruce, M. E. Involvement of the immune system in TSE pathogenesis. Immunol Today. 19, 201-201 (1998).
- Sigurdson, C. J. PrP(CWD) lymphoid cell targets in early and advanced chronic wasting disease of mule deer. J Gen Virol. 83, 2617-2617 (2002).
- Klein, M. A. Complement facilitates early prion pathogenesis. Nat Med. 7, 488-488 (2001).
- Mabbott, N. A. Temporary depletion of complement component C3 or genetic deficiency of C1q significantly delays onset of scrapie. Nat Med. 7, 485-485 (2001).
- Zabel, M. D. Stromal Complement Receptor CD21/35 Facilitates Lymphoid Prion Colonization and Pathogenesis. J Immunol. 179, 6144-6144 (2007).
- Cordier-Dirikoc, S., Chabry, J. Temporary depletion of CD11c+ dendritic cells delays lymphoinvasion after intraperitonal scrapie infection. J Virol. 82, 8933-8933 (2008).
- Dorban, G. Oral scrapie infection modifies the homeostasis of Peyer’s patches’ dendritic cells. Histochem Cell Biol. 128, 243-243 (2007).
- Flores-Langarica, A. Scrapie pathogenesis: the role of complement C1q in scrapie agent uptake by conventional dendritic cells. J Immunol. 182, 1305-1305 (2009).
- Huang, F. P., MacPherson, G. G. Dendritic cells and oral transmission of prion diseases. Adv Drug Deliv Rev. 56, 901-901 (2004).
- Ano, Y., Sakudo, A., Nakayama, H., Onodera, T. Uptake and dynamics of infectious prion protein in the intestine. Protein Pept Lett. 16, 247-247 (2009).
- Aucouturier, P. Infected splenic dendritic cells are sufficient for prion transmission to the CNS in mouse scrapie. J Clin Invest. 108, 703-703 (2001).
- Huang, F. P. Migrating intestinal dendritic cells transport PrP(Sc) from the gut. J Gen Virol. 83, 267-267 (2002).
- Jeffrey, M. Transportation of prion protein across the intestinal mucosa of scrapiesusceptible and scrapie-resistant sheep. J Pathol. 209, 4-4 (2006).
- Raymond, C. R., Mabbott, N. A. Assessing the involvement of migratory dendritic cells in the transfer of the scrapie agent from the immune to peripheral nervous systems. J Neuroimmunol. 187, 114-114 (2007).
- Mohan, J., Hopkins, J., Mabbott, N. A. Skin-derived dendritic cells acquire and degrade the scrapie agent following in vitro exposure. Immunology. 116, 122-122 (2005).
- Gilch, S. CpG and LPS can interfere negatively with prion clearance in macrophage and microglial cells. FEBS J. 274, 5834-5834 (2007).
- Safar, J. Molecular mass, biochemical composition, and physicochemical behavior of the infectious form of the scrapie precursor protein monomer. Proc.Natl.Acad.Sci.U.S.A. 87, 6373-6373 (1990).
- Büeler, H. R. Mice devoid of PrP are resistant to scrapie. Cell. 73, 1339-1339 (1993).
Tags
Immune CellsTraffickingFluorescent Prion RodsIntraperitoneal InfectionAbnormal FormHost-encoded Prion Protein (PrPC)Protease ResistantPathologicInfectiousPrion DiseasesChronic Wasting Disease (CWD)ScrapiePrion HypothesisImmune SystemPeripheral Prion PathogenesisComplement SystemDendritic CellsMacrophagesPrion UptakeTraffickingInoculation Site
Citar este artigo
Johnson, T. E., Michel, B. A., Meyerett, C., Duffy, A., Avery, A., Dow, S., Zabel, M. D. Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection. J. Vis. Exp. (45), e2349, doi:10.3791/2349 (2010).