摘要
黑龙江立克次体(Rickettsia heilongjiangensis)是在我国首先分得的斑点热群立克次体新种,是远东蜱传斑点热(Far Eastern tick-borne spotted fever)的病原体。黑龙江立克次体为专性细胞内寄生的革兰氏阴性菌,血管内皮细胞是其主要靶细胞,外膜蛋白B (OmpB)是黑龙江立克次体最主要的表面蛋白抗原。
我们依据文献建立体外分离、原代培养人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)的方法,以传代培养第3代的HUVEC作为黑龙江立克次体与宿主细胞相互作用的实验细胞。用Vero细胞培养并用泛影葡胺密度梯度纯化的黑龙江立克次体感染HUVEC。采用间接免疫荧光检测和扫描电镜观察不同时相宿主细胞内黑龙江立克次体,发现黑龙江立克次体感染HUVEC的24h内,在第6h及第24h各出现一个感染高峰,与文献报告立氏立克次体感染HUVEC的高峰出现时间基本一致。在黑龙江立克次体感染HUVEC的12d内,早期立克次体无显著增殖,感染后第5~9d,立克次体在宿主细胞内快速增殖并播散感染相邻细胞,感染后第10~12d,宿主细胞胞质内弥漫生长黑龙江立克次体,宿主细胞明显病变并脱落。结果表明黑龙江立克次体能够感染血管内皮细胞,在血管内皮细胞内不断增殖而使宿主细胞损伤和死亡。
通过计算机抗原表位预测,本研究采用PCR将黑龙江立克次体外膜蛋白B基因(ompB,4 875bp)分4段扩增,将4个ompB基因片段分别做原核表达,成功制备出4个重组OmpB蛋白(OmpB-P1、OmpB-P2、OmpB-P3、OmpB-P4)。免疫印迹分析证明4个重组OmpB蛋白均能与黑龙江立克次体感染血清特异性反应。用纯化的4个重组蛋白分别免疫C3H/HeN小鼠,IFA检测血清抗体效价表明4个OmpB蛋白均能有效地诱导机体产生特异性体液免疫应答(抗体滴度≥5 120),显示4个重组OmpB蛋白具有良好的免疫原性。
树突状细胞(dendritic cells,DC)是专职捕获和处理抗原并将抗原提呈给淋巴细胞的免疫细胞,可使机体产生免疫或耐受。本研究将4个重组OmpB蛋白分别刺激体外诱导培养的小鼠骨髓源DC,24h后用流式细胞仪分析抗原刺激的DC表型,结果发现4个重组OmpB蛋白刺激树突状细胞的表型分子(CD40, CD80, CD86和MHC-II)的表达均显著高于阴性对照,使DC成熟。将4个重组OmpB蛋白刺激DC分别经腹腔转移至正常C3H/HeN小鼠,14d后用黑龙江立克次体毒株攻击DC受体小鼠。攻击后第7d用实时荧光定量PCR检测小鼠脾、肺、肝、脑等脏器黑龙江立克次体载量。结果显示接受黑龙江立克次体全菌抗原、OmpB-P2、OmpB-P3和OmpB-P4激活DC的小鼠立克次体载量显著低于非抗原刺激DC受体小鼠(阴性对照),而接受OmpB-P1或OmpB的融合蛋白(TrxA)激活DC的小鼠立克次体载量与阴性对照相比无显著性差异。说明OmpB-P2、OmpB-P3和OmpB-P4能够诱导特异性免疫保护,为保护性抗原。
将4个重组OmpB蛋白刺激的树突状细胞分别与磁珠分选纯化的小鼠脾脏CD4~+T细胞和CD8~+T细胞在体外共培养,结果显示与OmpB-P2、OmpB-P3或OmpB-P4激活DC相互作用的CD4~+T细胞和CD8~+T细胞的IFN-γ表达水平显著高于与OmpB-P1激活DC相互作用的CD4~+T细胞和CD8~+T细胞,提示树突状细胞介导的抗黑龙江立克次体的免疫保护作用与抗原激活的CD4~+T细胞和CD8~+T细胞分别向Th1细胞分化和细胞毒性T细胞(CTL)分化及其高效表达的IFN-γ密切相关。
Rickettsia heilongjiangensis is an obligate, intracellular, gram-negative bacterium that causes Far Eastern tick-borne spotted fever. It is a new species of spotted fever group rickettsia firstly isolated from Dermacentor silvarum ticks collected in Heilongjiang Province of China. Vascular endothelial cells are the main target cells of R. heilongjiangensis in infection and outer membrane protein B (OmpB) is it’s abundant surface-exposed protein.
Human umbilical vein endothelial cells (HUVEC) were isolated and cultured in vitro. R. heilongjiangensis organisms cultured in Vero cells and purified by renografin gradient centrifugation were applied to infect HUVEC and rickettsiae in HUVEC were examined by both indirect immune fluorescence assay (IFA) and scanning electron micrograph (SEM) after infection. During the first 24 hours of infection, two infection peaks were found at 6 and 24 hours post-infection (pi), respectively, which is similar to R. rickettaii infection of HUVEC. Multiplication of rickettsiae and pathological changes in the host cells were observed at 5 days pi, while the amount of intracellular rickettsiae was found to markedly increase in the host cells and rickettsial dissemination between neighbor cells was observed within 5 to 9 days pi. A few of rickettsiae were observed in the nucleus of host cells that appeared severe pathological changes within 8 to 9 days pi. Due to the overwhelming rickettsial infection, most of the host cells shed 12 days pi. Our results demonstrated that R. heilongjiangensis has a capability to infect vascular endothelial cells and cause vascular injury.
Outer membrane protein B gene (ompB, 4 875bp) of R. heilongjiangensis was divided into 4 fragments based on immunogenic epitope prediction of OmpB by computer analysis. Four fragments of ompB were amplified from the genomic DNA of R. heilongjiangensis, and subsequently 4 recombinant OmpB proteins (OmpB-P1, OmpB-P2, OmpB-P3, and OmpB-P4) were highly expressed in Escherichia coli cells transformed by ompB-p1-, ompB-p2-, ompB-p3-, ompB-p4-insered plasmids (pET32a), respectively. In western blotting analysis, all of the 4 recombinant OmpB proteins reacted with sera from mice infected with R. heilongjiangensis. The 4 OmpB proteins were used to immune C3H/HeN mice, respectively, and high levels of specific antibody titers (≧1 :5 120) were determined in mouse sera by IFA. The results suggest the OmpB proteins have good immunogenicity.
Dendritic cells (DC) are antigen presenting cells that are specialized to capture, process, and present antigens to T lymphocytes to cause immunity or tolerance to the antigens. To investigate their role in Far Eastern tick-borne spotted fever, we analyzed the responses of murine bone marrow–derived dendritic cells (BMDCs) stimulated by whole cell antigen of R. heilongjiangensis and the 4 OmpB proteins in vitro and their protective roles of the antigen-pulsed BMDCs were evaluated. BMDCs pulsed by whole cell antigen, OmpB-P1, OmpB-P2, OmpB-P3, and OmpB-P4 were intraperitoneally transferred to C3H/HeN mice, respectively. On day 14 post-transfer of BMDCs, mice were intraperitoneally challenged with R. heilongjiangensis, and the challenged mice were sacrificed and their spleen, lung, liver, and brain were harvested on day 7 pi for detection of R. heilongjiangensis load by a quantitative PCR analysis. Compared with mice receiving unpulsed BMDCs (negative control), mice receiving BMDCs pulsed with whole cell antigen, OmpB-P2, OmpB-P3, or OmpB-P4 exhibited significantly lower R. heilongjiangensis load, while mice receiving BMDCs pulsed with OmpB-P1 or TrxA encoded by pET32a displayed high levels of R. heilongjiangensis load similar to that of negative control. This result suggests that OmpB-P2, OmpB-P3, and OmpB-P4 are protective antigens, but OmpB-P1 is not.
CD4~+ and CD8~+ T cells from spleens of C3H/HeN mice immunized with antigen-pulsed BMDCs were isolated with T Cell Isolation Kit, respectively. The antigens-pulsed BMDCs were cocultured with the CD4~+ and CD8~+ T cells. Phenotypic molecules and intra-cellular cytokines of CD4~+ and CD8~+ T cells were analyzed on a FACScalibur flow cytometer. CD4~+/CD8~+ T cells from mice immunized with whole cell antigen-, OmpB-P2-, OmpB-P3-, or OmpB-P4-pulsed BMDCs were efficiently activated with high expression of IFN-γthat were significantly higher than that from mice immunized with OmpB-P1-pulsed BMDCs. This result suggests that protection against R. heilongjiangensis offered by BMDCs activated with the protective antigens were associated with proliferation and activation of Th1-type CD4~+ T cells and of Cytotoxic T lymphocyte (CTL) that are more potent to produce IFN-γagainst this intracellular bacterium.
引文
1. Biological and chemical terrorism: strategic plan for preparedness and response. Recommendations of the CDC Strategic Planning Workgroup. MMWR Recomm Rep 2000, 49(RR-4):1-14.
2. Office of the Assistant Secretary for Preparedness and Response; HHS Public Health Emergency Medical Countermeasures Enterprise implementation plan for chemical, biological, radiological and nuclear threats. Notice. Fed Regist 2007, 72(77):20117-20128.
3.俞树荣,陈香蕊:立克次体与立克次体病.北京:军事医学科学出版社1999: 33-59.
4.范明远:立克次体病.北京:人民卫生出版社(第二版) 2004, 336-342.
5. Walker DH: Rickettsiae and rickettsial infections: the current state of knowledge. Clin Infect Dis 2007, 45 Suppl 1:S39-44.
6. Walker DH, Ismail N: Emerging and re-emerging rickettsioses: endothelial cell infection and early disease events. Nat Rev Microbiol 2008, 6(5):375-386.
7. Jordan JM, Woods ME, Olano J, Walker DH: The absence of Toll-like receptor 4 signaling in C3H/HeJ mice predisposes them to overwhelming rickettsial infection and decreased protective Th1 responses. Infect Immun 2008, 76(8):3717-3724.
8. Wu JJ, Huang DB, Pang KR, Tyring SK: Rickettsial infections around the world, part 2: rickettsialpox, the typhus group, and bioterrorism. J Cutan Med Surg 2005, 9(3):105-115.
9. Fan MY, Walker DH, Liu QH, Han L, Bai HC, Zhang JK, Lenz B, Hong C: Rickettsial and serologic evidence for prevalent spotted fever rickettsiosis in inner Mongolia. Am J Trop Med Hyg 1987, 36(3):615-620.
10. Fan MY, Walker DH, Yu SR, Liu QH: Epidemiology and ecology of rickettsial diseases in the People's Republic of China. Rev Infect Dis 1987, 9(4):823-840.
11. Fan MY, Wang JG, Jiang YX, Zong DG, Lenz B, Walker DH: Isolation of a spotted fever group rickettsia from a patient and related ecologic investigations in Xinjiang Uygur Autonomous Region of China. J Clin Microbiol 1987, 25(4):628-632.
12. Fan MY, Yu XJ, Walker DH: Antigenic analysis of Chinese strains of spotted fever group rickettsiae by protein immunoblotting. Am J Trop Med Hyg 1988, 39(5):497-501.
13.娄丹,吴益民,王冰,刘国栋,丁桂林,张兴旺,等:黑龙江立克次体的一个新成员——黑龙江立克次体的分离和鉴定.中华微生物学和免疫学杂志1985, 5(4): 250-253.
14.吴益民,魏安明,胡玲美,杨青,刘昕昕,张志强,鲁志新:从患者血液分离的斑点热群立克次体HLJ—H5株的鉴定.中国人兽共患病学杂志1998, 14(6): 23-26.
15. Chen M, Fan MY, Bi DZ, Zhang JZ, Chen XR: Sequence analysis of a fragment of rOmpA gene of several isolates of spotted fever group rickettsiae from China. Acta Virol 1998, 42(2):91-93.
16. Zhang JZ, Fan MY, Wu YM, Fournier PE, Roux V, Raoult D: Genetic classification of“Rickettsia heilongjiangii”and“Rickettsia hulinii,”two Chinese spotted fever group rickettsiae. J Clin Microbiol 2000, 38(9):3498-3501.
17. Fournier PE, Dumler JS, Greub G, Zhang J, Wu Y, Raoult D: Gene sequence-based criteria for identification of new rickettsia isolates and description of Rickettsia heilongjiangensis sp. nov. J Clin Microbiol 2003, 41(12):5456-5465.
18. Mediannikov OY, Sidelnikov Y, Ivanov L, Mokretsova E, Fournier PE, Tarasevich I, Raoult D: Acute tick-borne rickettsiosis caused by Rickettsia heilongjiangensis in Russian Far East. Emerg Infect Dis 2004, 10(5):810-817.
19. Ando S, Kurosawa M, Sakata A, Fujita H, Sakai K, Sekine M, Katsumi M, Saitou W, Yano Y, Takada N et al: Human Rickettsia heilongjiangensis infection, Japan. Emerg Infect Dis 2010, 16(8):1306-1308.
20. Mediannikov O, Makarova VA: Far-eastern spotted fever: description of new infectious disease. Vestn Ross Akad Med Nauk 2008(7):41-44.
21. Schoeler GB, Moron C, Richards A, Blair PJ, Olson JG: Human spotted fever rickettsial infections. Emerg Infect Dis 2005, 11(4):622-624.
22. Rydkina E, Sahni A, Baggs RB, Silverman DJ, Sahni SK: Infection of human endothelial cells with spotted Fever group rickettsiae stimulates cyclooxygenase 2 expression and release of vasoactive prostaglandins. Infect Immun 2006, 74(9):5067-5074.
23. Walker TS: Rickettsial interactions with human endothelial cells in vitro: adherence and entry. Infect Immun 1984, 44(2):205-210.
24. Martinez JJ, Seveau S, Veiga E, Matsuyama S, Cossart P: Ku70, a component of DNA-dependent protein kinase, is a mammalian receptor for Rickettsia conorii. Cell 2005, 123(6):1013-1023.
25. Wu JJ, Huang DB, Pang KR, Tyring SK: Rickettsial infections around the world, part 1: pathophysiology and the spotted fever group. J Cutan Med Surg 2005, 9(2):54-62.
26. Blanc G, Ngwamidiba M, Ogata H, Fournier PE, Claverie JM, Raoult D: Molecular evolution of rickettsia surface antigens: evidence of positive selection. Mol Biol Evol 2005, 22(10):2073-2083.
27. Anacker RL, List RH, Mann RE, Hayes SF, Thomas LA: Characterization of monoclonal antibodies protecting mice against Rickettsia rickettsii. J Infect Dis 1985, 151(6):1052-1060.
28. Anacker RL, List RH, Mann RE, Wiedbrauk DL: Antigenic heterogeneity in high- and low-virulence strains of Rickettsia rickettsii revealed by monoclonal antibodies. Infect Immun 1986, 51(2):653-660.
29. Walker DH, Liu QH, Yu XJ, Li H, Taylor C, Feng HM: Antigenic diversity of Rickettsia conorii. Am J Trop Med Hyg 1992, 47(1):78-86.
30. Hackstadt T, Messer R, Cieplak W, Peacock MG: Evidence for proteolytic cleavage of the 120-kilodalton outer membrane protein of rickettsiae: identification of an avirulent mutant deficient in processing. Infect Immun 1992, 60(1):159-165.
31. Walker DH, Valbuena GA, Olano JP: Pathogenic mechanisms of diseases caused by Rickettsia. Ann N Y Acad Sci 2003, 990:1-11.
32. Olano JP: Rickettsial infections. Ann N Y Acad Sci 2005, 1063:187-196.
33. Uchiyama T, Kawano H, Kusuhara Y: The major outer membrane protein rOmpB of spotted fever group rickettsiae functions in the rickettsial adherence to and invasion of Vero cells. Microbes Infect 2006, 8(3):801-809.
34. Renesto P, Samson L, Ogata H, Azza S, Fourquet P, Gorvel JP, Heinzen RA, Raoult D: Identification of two putative rickettsial adhesins by proteomic analysis. Res Microbiol 2006, 157(7):605-612.
35. Gilmore RD, Jr., Hackstadt T: DNA polymorphism in the conserved 190 kDa antigen gene repeat region among spotted fever group Rickettsiae. Biochim Biophys Acta 1991, 1097(1):77-80.
36. Feng HM, Whitworth T, Popov V, Walker DH: Effect of antibody on the rickettsia-host cell interaction. Infect Immun 2004, 72(6):3524-3530.
37. Stenos J, Walker DH: The rickettsial outer-membrane protein A and B genes of Rickettsia australis, the most divergent rickettsia of the spotted fever group. Int J Syst Evol Microbiol 2000, 50 Pt 5:1775-1779.
38. Feng HM, Popov VL, Walker DH: Depletion of gamma interferon and tumor necrosis factor alpha in mice with Rickettsia conorii-infected endothelium: impairment of rickettsicidal nitric oxide production resulting in fatal, overwhelming rickettsial disease. Infect Immun 1994, 62(5):1952-1960.
39. Walker DH, Olano JP, Feng HM: Critical role of cytotoxic T lymphocytes in immune clearance of rickettsial infection. Infect Immun 2001, 69(3):1841-1846.
40. de Sousa R, Ismail N, Nobrega SD, Franca A, Amaro M, Anes M, Pocas J, Coelho R, Torgal J, Bacellar F et al: Intralesional expression of mRNA of interferon- gamma , tumor necrosis factor- alpha , interleukin-10, nitric oxide synthase, indoleamine-2,3-dioxygenase, and RANTES is a major immune effector in Mediterranean spotted fever rickettsiosis. J Infect Dis 2007, 196(5):770-781.
41. Bechah Y, Capo C, Mege JL, Raoult D: Rickettsial diseases: from Rickettsia-arthropod relationships to pathophysiology and animal models. Future Microbiol 2008, 3:223-236.
42. Fang R, Ismail N, Soong L, Popov VL, Whitworth T, Bouyer DH, Walker DH: Differential interaction of dendritic cells with Rickettsia conorii: impact on host susceptibility to murine spotted fever rickettsiosis. Infect Immun 2007, 75(6):3112-3123.
43. Feng H, Popov VL, Yuoh G, Walker DH: Role of T lymphocyte subsets in immunity to spotted fever group Rickettsiae. J Immunol 1997, 158(11): 5314-5320.
44. Herrero Herrero JI, Ruiz Beltran R, Perez A: Immunohistochemical diagnosis of Mediterranean spotted fever: detection of rickettsial antigens in the initial skin lesions. Enferm Infecc Microbiol Clin 1990, 8(6):344-349.
45. Feng HM, Whitworth T, Olano JP, Popov VL, Walker DH: Fc-dependent polyclonal antibodies and antibodies to outer membrane proteins A and B, but not to lipopolysaccharide, protect SCID mice against fatal Rickettsia conorii infection. Infect Immun 2004, 72(4):2222-2228.
46. Jordan JM, Woods ME, Soong L, Walker DH: Rickettsiae stimulate dendritic cells through toll-like receptor 4, leading to enhanced NK cell activation in vivo. J Infect Dis 2009, 199(2):236-242.
47. Billings AN, Feng HM, Olano JP, Walker DH: Rickettsial infection in murine models activates an early anti-rickettsial effect mediated by NK cells and associated with production of gamma interferon. Am J Trop Med Hyg 2001, 65(1):52-56.
48. Fang R, Ismail N, Shelite T, Walker DH: CD4+ CD25+ Foxp3- T-regulatory cells produce both gamma interferon and interleukin-10 during acute severe murine spotted fever rickettsiosis. Infect Immun 2009, 77(9):3838-3849.
49. Iinuma H, Okinaga K, Fukushima R, Inaba T, Iwasaki K, Arai T, Tamura J, Kumagai H: Reduction of immunosuppression and shift to Th1 response by tumor-DC (dendritic cells) fusion vaccine. Gan To Kagaku Ryoho 2004, 31(11):1640-1642.
50. Mayer ML, Phillips CM, Stadnyk AW, Halperin SA, Lee SF: Synergistic BM-DC activation and immune induction by the oral vaccine vector Streptococcus gordonii and exogenous tumor necrosis factor. Mol Immunol 2009, 46(8-9):1883-1891.
51. Richards AL: Rickettsial vaccines: the old and the new. Expert Rev Vaccines 2004, 3(5):541-555.
52. Walker DH: Targeting rickettsia. N Engl J Med 2006, 354(13):1418-1420.
53. Ammerman N, Beier-Sexton M, Azad A: Laboratory maintenance of Rickettsia rickettsii. Curr Protoc Microbiol 2008, Chapter 3:Unit 3A.5.
54. Wike DA, Tallent G, Peacock MG, Ormsbee RA: Studies of the rickettsial plaque assay technique. Infect Immun 1972, 5(5):715-722.
55. McDade JE, Stakebake JR, Gerone PJ: Plaque assay system for several species of Rickettsia. J Bacteriol 1969, 99(3):910-912.
56. Weinberg EH, Stakebake JR, Gerone PJ: Plaque assay for Rickettsia rickettsii. J Bacteriol 1969, 98(2):398-402.
57. Jaffe E, Nachman R, Becker C, Minick C: Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973, 52(11):2745-2756.
58.李丽莉,温博海,张晶波,邱玲,陈梅玲,牛东升:贝氏柯克斯体被单核细胞吞噬及其在单核细胞内生长的初步研究.中国人兽共患病杂志2005, 21(11): 940-944.
59. Balraj P, Nappez C, Raoult D, Renesto P: Western-blot detection of RickA within spotted fever group rickettsiae using a specific monoclonal antibody. FEMS Microbiol Lett 2008, 286(2):257-262.
60. Martinez JJ, Cossart P: Early signaling events involved in the entry of Rickettsia conorii into mammalian cells. J Cell Sci 2004, 117(Pt 21):5097-5106.
61. Silverman DJ, Wisseman CL, Jr.: In vitro studies of rickettsia-host cell interactions: ultrastructural changes induced by Rickettsia rickettsii infection of chicken embryo fibroblasts. Infect Immun 1979, 26(2):714-727.
62. Wisseman CL, Jr., Edlinger EA, Waddell AD, Jones MR: Infection cycle of Rickettsia rickettsii in chicken embryo and L-929 cells in culture. Infect Immun 1976, 14(4):1052-1064.
63. Sporn LA, Haidaris PJ, Shi RJ, Nemerson Y, Silverman DJ, Marder VJ: Rickettsia rickettsii infection of cultured human endothelial cells induces tissue factor expression. Blood 1994, 83(6):1527-1534.
64. Sporn LA, Sahni SK, Lerner NB, Marder VJ, Silverman DJ, Turpin LC, Schwab AL: Rickettsia rickettsii infection of cultured human endothelial cells induces NF-kappaB activation. Infect Immun 1997, 65(7):2786-2791.
65.张丽娟,范明远:黑龙江蜱传斑点热研究进展.中国人兽共患病2005, 10(5):810-817.
66. Morrison DA: Transformation in Escherichia coli: cryogenic preservation of competent cells. J Bacteriol 1977, 132(1):349-351.
67.金东雁,黎孟枫,侯云德:分子克隆实验指南.第2版.北京:科学出版社, 1992, 882.
68. McAllister LA, Hixon MS, Schwartz R, Kubitz DS, Janda KD: Synthesis and application of a novel ligand for affinity chromatography based removal of endotoxin from antibodies. Bioconjug Chem 2007, 18(2):559-566.
69. Bhor VM, Thomas CJ, Surolia N, Surolia A: Polymyxin B: an ode to an old antidote for endotoxic shock. Mol Biosyst 2005, 1(3):213-222.
70. Iwanaga S, Miyata T, Tokunaga F, Muta T: Molecular mechanism of hemolymph clotting system in Limulus. Thromb Res 1992, 68(1):1-32.
71. Jiang Z, Hong Z, Guo W, Xiaoyun G, Gengfa L, Yongning L, Guangxia X: A synthetic peptide derived from bactericidal/permeability-increasing protein neutralizes endotoxin in vitro and in vivo. Int Immunopharmacol 2004, 4(4):527-537.
72. Roux V, Raoult D: Phylogenetic analysis of the genus Rickettsia by 16S rDNA sequencing. Res Microbiol 1995, 146(5):385-396.
73. Leclerque A: Whole genome-based assessment of the taxonomic position of the arthropod pathogenic bacterium Rickettsiella grylli. FEMS Microbiol Lett 2008, 283(1):117-127.
74. Moron CG, Bouyer DH, Yu XJ, Foil LD, Crocquet-Valdes P, Walker DH: Phylogenetic analysis of the rompB genes of Rickettsia felis and Rickettsia prowazekii European-human and North American flying-squirrel strains. Am J Trop Med Hyg 2000, 62(5):598-603.
75. Steinman RM, Cohn ZA: Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med 1973, 137(5):1142-1162.
76. Steinman RM: The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991, 9:271-296.
77. Banchereau J, Steinman RM: Dendritic cells and the control of immunity. Nature 1998, 392(6673):245-252.
78. Steinman RM: Dendritic cells and the control of immunity: enhancing the efficiency of antigen presentation. Mt Sinai J Med 2001, 68(3):160-166.
79. Lee HK, Iwasaki A: Innate control of adaptive immunity: dendritic cells and beyond. Semin Immunol 2007, 19(1):48-55.
80. Rodrigue-Gervais IG, Lamarre D: Hepatitis C virus subverts pattern recognition receptors-mediated control of adaptative immunity orchestrated by dendritic cells. Med Sci (Paris) 2010, 26(10):869-874.
81. Masurier C, Pioche-Durieu C, Colombo BM, Lacave R, Lemoine FM, Klatzmann D, Guigon M: Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti-tumoral cell therapy. Immunology 1999, 96(4):569-577.
82. Hart DN: Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 1997, 90(9):3245-3287.
83. Talmor M, Mirza A, Turley S, Mellman I, Hoffman LA, Steinman RM: Generation or large numbers of immature and mature dendritic cells from rat bone marrow cultures. Eur J Immunol 1998, 28(3):811-817.
84. Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, Romani N, Schuler G: An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods 1999, 223(1):77-92.
85. Inaba K, Inaba M, Deguchi M, Hagi K, Yasumizu R, Ikehara S, Muramatsu S, Steinman RM: Granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class II-negative progenitor in mouse bone marrow. Proc Natl Acad Sci U S A 1993, 90(7):3038-3042.
86. Rydkina E, Sahni SK, Santucci LA, Turpin LC, Baggs RB, Silverman DJ: Selective modulation of antioxidant enzyme activities in host tissues during Rickettsia conorii infection. Microb Pathog 2004, 36(6):293-301.
87. Steinman RM, Inaba K: Myeloid dendritic cells. J Leukoc Biol 1999, 66(2):205-208.
88. Bell EJ, Stoenner HG: Spotted fever vaccine; potency assay by direct challenge of vaccinated mice with toxin of Rickettsia rickettsii. J Immunol 1961, 87:737-746.
89. Kenyon RH, Acree WM, Wright GG, Melchior FW, Jr.: Preparation of vaccines for Rocky Mountain spotted fever from rickettsiae propagated in cell culture. J Infect Dis 1972, 125(2):146-152.
90. Kenyon RH, Sammons LS, Pedersen CE, Jr.: Comparison of three rocky mountain spotted fever vaccines. J Clin Microbiol 1975, 2(4):300-304.
91. Vishwanath S, McDonald GA, Watkins NG: A recombinant Rickettsia conorii vaccine protects guinea pigs from experimental boutonneuse fever and Rocky Mountain spotted fever. Infect Immun 1990, 58(3):646-653.
92. Jordan JM, Woods ME, Feng HM, Soong L, Walker DH: Rickettsiae-stimulated dendritic cells mediate protection against lethal rickettsial challenge in an animal model of spotted fever rickettsiosis. J Infect Dis 2007, 196(4):629-638.
93. Stanciu LA, Djukanovic R: Isolation of T-cell subsets by magnetic cell sorting (MACS). Methods Mol Biol 2000, 134:133-141.
94. Wixler V, Klarmann B, Begemann U, Gahn G, Lorenz R, Hempel K: Isolation and quantification of class 1 MHC gene mutants in mouse T cells by immunoselection with a magnetic cell sorter (MACS). J Immunol Methods 1994, 171(1):121-130.
95. Psaroulaki A, Ragiadakou D, Kouris G, Papadopoulos B, Chaniotis B, Tselentis Y: Ticks, tick-borne rickettsiae, and Coxiella burnetii in the Greek Island of Cephalonia. Ann N Y Acad Sci 2006, 1078:389-399.
96. Kim TG, Kim CH, Won EH, Bae SM, Ahn WS, Park JB, Sin JI: CpG-ODN-stimulated dendritic cells act as a potent adjuvant for E7 protein delivery to induce antigen-specific antitumour immunity in a HPV 16 E7-associated animal tumour model. Immunology 2004, 112(1):117-125.
97. Huang XL, Fan Z, Borowski L, Rinaldo CR: Multiple T-cell responses to human immunodeficiency virus type 1 are enhanced by dendritic cells. Clin Vaccine Immunol 2009, 16(10):1504-1516.
98. Wei Y, Wang X, Xiong X, Wen B: Coxiella burnetii antigen-stimulated dendritic cells mediated protection against Coxiella burnetii in BALB/c mice. J Infect Dis 2011, 203(2):283-291.
99. Testi R, Phillips JH, Lanier LL: T cell activation via Leu-23 (CD69). J Immunol 1989, 143(4):1123-1128.
100. Moretta A, Poggi A, Pende D, Tripodi G, Orengo AM, Pella N, Augugliaro R, Bottino C, Ciccone E, Moretta L: CD69-mediated pathway of lymphocyte activation: anti-CD69 monoclonal antibodies trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolytic T lymphocytes expressing T cell receptor alpha/beta. J Exp Med 1991, 174(6):1393-1398.
101. Lindsey WB, Lowdell MW, Marti GE, Abbasi F, Zenger V, King KM, Lamb LS, Jr.: CD69 expression as an index of T-cell function: assay standardization, validation and use in monitoring immune recovery. Cytotherapy 2007, 9(2):123-132.
1. Blanc G, Ogata H, Robert C, Audic S, Claverie JM, Raoult D: Lateral gene transfer between obligate intracellular bacteria: evidence from the Rickettsia massiliae genome. Genome Res 2007, 17(11):1657-1664.
2. Bernabeu-Wittel M, Segura-Porta F: Rickettsioses. Enferm Infecc Microbiol Clin 2005, 23(3):163-172.
3. Bassetti S: Rickettsioses of the spotted fever-group. Internist (Berl) 2004, 45(6):669-676.
4. Walker DH, Paddock CD, Dumler JS: Emerging and re-emerging tick-transmitted rickettsial and ehrlichial infections. Med Clin North Am 2008, 92(6):1345-1361, x.
5. Walker DH: Rickettsiae and rickettsial infections: the current state of knowledge. Clin Infect Dis 2007, 45 Suppl 1:S39-44.
6. Biological and chemical terrorism: strategic plan for preparedness and response. Recommendations of the CDC Strategic Planning Workgroup. MMWR Recomm Rep 2000, 49(RR-4):1-14.
7. Office of the Assistant Secretary for Preparedness and Response; HHS Public Health Emergency Medical Countermeasures Enterprise implementation plan for chemical, biological, radiological and nuclear threats. Notice. Fed Regist 2007, 72(77):20117-20128.
8. Billings AN, Feng HM, Olano JP, Walker DH: Rickettsial infection in murine models activates an early anti-rickettsial effect mediated by NK cells and associated with production of gamma interferon. Am J Trop Med Hyg 2001, 65(1):52-56.
9. Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K, Akira S: Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999, 162(7):3749-3752.
10. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C et al: Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998, 282(5396):2085-2088.
11. Jordan JM, Woods ME, Soong L, Walker DH: Rickettsiae stimulate dendritic cells through toll-like receptor 4, leading to enhanced NK cell activation in vivo. J Infect Dis 2009, 199(2):236-242.
12. Jordan JM, Woods ME, Olano J, Walker DH: The absence of Toll-like receptor 4 signaling in C3H/HeJ mice predisposes them to overwhelming rickettsial infection and decreased protective Th1 responses. Infect Immun 2008, 76(8):3717-3724.
13. Jordan JM, Woods ME, Feng HM, Soong L, Walker DH: Rickettsiae-stimulated dendritic cells mediate protection against lethal rickettsial challenge in an animal model of spotted fever rickettsiosis. J Infect Dis 2007, 196(4):629-638.
14. Fang R, Ismail N, Soong L, Popov VL, Whitworth T, Bouyer DH, Walker DH: Differential interaction of dendritic cells with Rickettsia conorii: impact on host susceptibility to murine spotted fever rickettsiosis. Infect Immun 2007, 75(6):3112-3123.
15. Bechah Y, Capo C, Mege JL, Raoult D: Rickettsial diseases: from Rickettsia-arthropod relationships to pathophysiology and animal models. Future Microbiol 2008, 3:223-236.
16. Herrero-Herrero JI, Walker DH, Ruiz-Beltran R: Immunohistochemical evaluation of the cellular immune response to Rickettsia conorii in taches noires. J Infect Dis 1987, 155(4):802-805.
17. Constant SL, Bottomly K: Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu Rev Immunol 1997, 15:297-322.
18. Belkaid Y, Piccirillo CA, Mendez S, Shevach EM, Sacks DL: CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 2002, 420(6915):502-507.
19. Graca L, Thompson S, Lin CY, Adams E, Cobbold SP, Waldmann H: Both CD4(+)CD25(+) and CD4(+)CD25(-) regulatory cells mediate dominant transplantation tolerance. J Immunol 2002, 168(11):5558-5565.
20. Jarboe DL, Eisemann CS, Jerrells TR: Production and characterization of cloned T-cell hybridomas that are responsive to Rickettsia conorii antigens. Infect Immun 1986, 52(1):326-330.
21. Feng H, Popov VL, Yuoh G, Walker DH: Role of T lymphocyte subsets in immunity to spotted fever group Rickettsiae. J Immunol 1997, 158(11):5314-5320.
22. Walker DH, Olano JP, Feng HM: Critical role of cytotoxic T lymphocytes in immune clearance of rickettsial infection. Infect Immun 2001, 69(3):1841-1846.
23. Valbuena G, Walker DH: The endothelium as a target for infections. Annu Rev Pathol 2006, 1:171-198.
24. Fang R, Ismail N, Shelite T, Walker DH: CD4+ CD25+ Foxp3- T-regulatory cells produce both gamma interferon and interleukin-10 during acute severe murine spotted fever rickettsiosis. Infect Immun 2009, 77(9):3838-3849.
25. Feng HM, Popov VL, Walker DH: Depletion of gamma interferon and tumor necrosis factor alpha in mice with Rickettsia conorii-infected endothelium: impairment of rickettsicidal nitric oxide production resulting in fatal, overwhelming rickettsial disease. Infect Immun 1994, 62(5):1952-1960.
26. Jerrells TR, Li H, Walker DH: In vivo and in vitro role of gamma interferon in immune clearance of Rickettsia species. Adv Exp Med Biol 1988, 239:193-200.
27. Walker DH, Popov VL, Crocquet-Valdes PA, Welsh CJ, Feng HM: Cytokine-induced, nitric oxide-dependent, intracellular antirickettsial activity of mouse endothelial cells. Lab Invest 1997, 76(1):129-138.
28. Sahni SK: Endothelial cell infection and hemostasis. Thromb Res 2007, 119(5):531-549.
29. Pober JS, Sessa WC: Evolving functions of endothelial cells in inflammation. Nat Rev Immunol 2007, 7(10):803-815.
30. Epperson DE, Pober JS: Antigen-presenting function of human endothelial cells. Direct activation of resting CD8 T cells. J Immunol 1994, 153(12):5402-5412.
31. Clifton DR, Rydkina E, Freeman RS, Sahni SK: NF-kappaB activation during Rickettsia rickettsii infection of endothelial cells involves the activation of catalytic IkappaB kinases IKKalpha and IKKbeta and phosphorylation-proteolysis of the inhibitor protein IkappaBalpha. Infect Immun 2005, 73(1):155-165.
32. Mutunga M, Preston PM, Sumption KJ: Nitric oxide is produced by Cowdria ruminantium-infected bovine pulmonary endothelial cells in vitro and is stimulated by gamma interferon. Infect Immun 1998, 66(5):2115-2121.
33. Mwangi DM, Mahan SM, Nyanjui JK, Taracha EL, McKeever DJ: Immunization of cattle by infection with Cowdria ruminantium elicits T lymphocytes that recognize autologous, infected endothelial cells and monocytes. Infect Immun 1998, 66(5):1855-1860.
34. Turco J, Winkler HH: Gamma-interferon-induced inhibition of the growth of Rickettsia prowazekii in fibroblasts cannot be explained by the degradation of tryptophan or other amino acids. Infect Immun 1986, 53(1):38-46.
35. Li H, Jerrells TR, Spitalny GL, Walker DH: Gamma interferon as a crucial host defense against Rickettsia conorii in vivo. Infect Immun 1987, 55(5):1252-1255.
36. Nacy CA, Belosevic M, Crawford RM, Healy AT, Schreiber RD, Meltzer MS: Lymphokine regulation of macrophage effector activities. Adv Exp Med Biol 1988, 239:1-11.
37. Feng HM, Walker DH: Interferon-gamma and tumor necrosis factor-alpha exert their antirickettsial effect via induction of synthesis of nitric oxide. Am J Pathol 1993, 143(4):1016-1023.
38. Hong JE, Santucci LA, Tian X, Silverman DJ: Superoxide dismutase-dependent, catalase-sensitive peroxides in human endothelial cells infected by Rickettsia rickettsii. Infect Immun 1998, 66(4):1293-1298.
39. Feng HM, Walker DH: Mechanisms of intracellular killing of Rickettsia conorii in infected human endothelial cells, hepatocytes, and macrophages. Infect Immun 2000, 68(12):6729-6736.
40. Sahni SK, Rydkina E: Host-cell interactions with pathogenic Rickettsia species. Future Microbiol 2009, 4:323-339.
41. Valbuena G, Walker DH: Expression of CX3CL1 (fractalkine) in mice with endothelial-target rickettsial infection of the spotted-fever group. Virchows Arch 2005, 446(1):21-27.
42. Kreisel D, Krupnick AS, Balsara KR, Riha M, Gelman AE, Popma SH, Szeto WY, Turka LA, Rosengard BR: Mouse vascular endothelium activates CD8+ T lymphocytes in a B7-dependent fashion. J Immunol 2002, 169(11):6154-6161.