固有免疫标志物MIF与TLR5在监测同种移植排斥中的意义
|
摘要
研究目的
器官移植是目前治疗各种脏器终末期功能衰竭的最有效手段。但是移植后的急性排斥反应(AR)仍然是移植术后的主要并发症,也是导致慢性排斥反应(CR)和移植器官功能丧失的最重要的危险因素。如何快速诊断和及时治疗急性排斥反应一直是临床器官移植研究的热点。目前临床上诊断急性排斥反应的主要方法包括移植受者临床表现、血液相关指标检测、移植器官病理学活检和影像学辅助检查等,其中活检组织病理学检查仍然是诊断移植排斥反应的最可靠手段,但其侵入性、潜在并发症及取样误差,限制了其临床应用。器官移植排斥反应涉及范围广泛,免疫学机制复杂,迄今为止仍未发现公认的、无创伤性高敏感性和特异性的监测指标。因此,寻找新的非创伤性侵入性监测指标,早期、有效监测移植排斥反应,在移植器官功能发生损害之前及时做出诊断就成为移植免疫学领域亟待解决的问题。
分子影像学(molecular imaging)是将分子生物学技术和现代医学影像学相结合而产生的一门新兴的边缘学科,是运用影像学手段显示组织水平、细胞和亚细胞水平的特定分子,反映活体状态下分子水平变化,对其生物学行为在影像方面进行定性和定量研究的科学。经典的影像诊断(CT、MRI等)主要显示的是一些分子改变的终效应,具有解剖学改变的疾病；而分子影像学通过发展新的工具、试剂及方法,探查疾病过程中细胞和分子水平的异常,在尚无解剖学改变前检出异常,为探索疾病的发生、发展和转归,评价药物的疗效,为分子水平疾病的治疗奠定实验和理论基础。应用分子影像学技术研究器官及组织移植术后的免疫学细胞及分子水平的变化,有可能为临床监测移植排斥反应提供重要手段。急性排斥反应的分子影像学信息对于移植术后免疫抑制剂的个体化应用、功效评价、危险因素监测与评估,具有极其重要的指导意义。
免疫应答可以分为固有免疫和适应性免疫两大类。前者是指与生俱来的机体天然抵抗力。后者是指接触抗原后机体产生的特异性免疫力。移植排斥反应是一个复杂的免疫学过程,众多免疫分子参与其中。固有免疫是机体免疫系统的重要组成部分,随着对移植免疫机理的深入研究,人们逐渐意识到在移植排斥反应过程中,固有免疫并不仅仅发挥非特异吞噬、清除作用,它与适应性免疫一样能够正确区分“自己”和“非己”,并进一步调控适应性免疫。在器官移植的不同阶段,固有免疫与适应性免疫系统之间都存在着复杂的密不可分的联系。因此选择在固有免疫和适应性免疫中均发挥作用的标志分子,研究其在同种移植排斥过程中的表达状态,探索其能否成为监测移植排斥反应的靶点分子,对于深入阐明移植排斥分子机理,指导临床监测排异反应,更有效地防治移植排斥具有重要的理论和实际意义。
移植物进入机体后,移植物抗原致敏机体免疫细胞,被激活的免疫细胞进而产生多种细胞因子,引起免疫排斥反应,由于细胞因子通常在排斥反应早期即被释放,因此可作为移植排斥反应分子影像学的靶分子。已有研究者尝试将放射性核素123I或111In标记的MHCII类分子,99mTc标记的MCP-1 (Monocyte Chamotactic Protein)用于移植排斥反应的分子影像学诊断,但由于所选显像剂特异性不高且在体内清除缓慢,影响了其在临床上的应用。
巨噬细胞移动抑制因子(macrophage migration inhibitory factor, MIF)是具有多种生物活性的可溶性淋巴因子,由活化的T细胞、巨噬细胞和其它多种细胞分泌,是免疫和内分泌系统的重要介质,具有激活巨噬细胞、抑制其游走、激活T细胞并增强其功能的作用,在急性炎症和迟发型超敏反应中有重要意义。近年来已有研究表明MIF是同种移植排斥过程中的重要介质,MIF表达的上调与巨噬细胞、T细胞浸润及同种肾移植排斥反应的程度密切相关。此外,Toubai等研究发现在大鼠同种异体干细胞移植模型中MIF对移植物抗宿主病(graft-versus-host disease, GVHD)的发生、发展也起着关键的作用。鉴于MIF在同种移植排斥反应中的重要作用,本研究第一部分选择MIF作为同种移植排斥分子影像学的新靶点,阐明MIF的表达与移植排斥反应程度的相关性,并在此基础上利用放射性核素131I标记的MIF单克隆抗体作为分子显像剂,动态监测同种移植排斥反应的全过程,旨在为无创伤性监测移植排斥反应探索新的途径。
准确评价移植术后免疫抑制治疗的效能,监测受者的免疫状态,根据受者的免疫状态来调整免疫抑制剂的用量是移植监测的另一关键目标。通过移植监测可以指导移植后免疫抑制剂的合理使用,实现药物应用的个体化。由于免疫抑制剂可以抑制T细胞、B细胞等靶细胞的增殖及细胞因子合成,因此应用免疫抑制剂之后如仍以移植排斥早期释放的细胞因子作为分子影像学的成像对象显然不能客观地反应移植物的免疫状态。寻找移植排斥分子显像新靶点,从而准确地评价移植后免疫抑制治疗后的效能,是移植监测领域的又一关注热点。
Toll样受体家族(Toll-like Receptor, TLRs)作为一种模式识别受体(pattern recognition receptor, PRR)可特异性识别病原相关分子模式(pathogen-associated molecular patterns, PAMPs),不仅在激活固有免疫中发挥重要作用,而且还在诱导和调节适应性免疫中具有重要意义,是连接固有免疫和适应性免疫的重要桥梁。TLRs信号可以在自身抗原,同种抗原和异种抗原的刺激下激活。2008年研究发现小鼠心脏皮肤同种移植物的长期生存需要天然Tregs的存在,TLR9的特异激活剂CpG可以通过抑制Treg的功能和促进Thl效应性T细胞的分化,抑制同种移植物的存活。TLR7的激活可以促进皮肤同种移植物的排斥；TLR4的激活阻断了利用抗CD154单抗建立的心脏同种移植物的长期存活,由此可见TLRs信号与移植排斥关系密切。
TLR5是TLRs家族中唯一具有免疫负调节作用的分子,可直接间接地参与自身免疫病的发病过程。细菌鞭毛蛋白(Flagellin)是TLR5的唯一配体,Flagellin可特异识别结合TLR5,通过激活核转录因子NF-κB刺激TNF-α、IL-1β、IL-8等前炎症细胞因子的产生,介导机体天然免疫。最新研究表明,TLR5可选择性高表达Treg细胞表面,TLR5的特异激活剂Flagellin可增强CD4+CD25+调节性T细胞(Treg)对效应性T细胞的抑制作用。2008年有研究报告体内给予TLR5特异激活剂Flagellin,可以保护机体免受化学、细菌病毒和辐射等损害。鉴于Treg细胞在诱导同种移植物耐受过程具有重要作用,TLR5可以调控Treg细胞免疫抑制效应,提示TLR5可能是同种移植排斥过程中又一关键免疫分子。本课题第二部分在研究同种皮肤移植急性排斥过程中TLR5动态表达状况和探讨了免疫抑制剂雷帕霉素对TLR5表达的影响的基础上,纯化提取TLR5特异激活剂Flagellin,利用放射性核素13’I标记Flagellin,并进行了初步鉴定,旨在阐明TLR5在移植排斥过程中的动态表达,初步探讨其作为同种移植排斥标志分子的可行性。
研究方法
1.MIF在同种移植模型中的表达状况：以BABL/c小鼠为受体,分别以B6小鼠、BABL/c小鼠为供体,建立小鼠同种、同系皮肤移植模型,于移植后第1、7、14、天,分别取移植部位的皮片,逆转录聚合酶链反应(RT-PCR)检测MIF mRNA的相对表达。同时利用免疫组织化学分析法测定移植后不同时间移植皮片内MIF蛋白表达状况。
2.小鼠MIF单克隆抗体(anti-MIFMcAb)的制备及生物学活性鉴定：采用杂交瘤技术制备小鼠MIF单克隆抗体,蛋白质印迹技术(Western blotting)及酶联免疫吸附试验(ELISA)鉴定其特异性,巨噬细胞移动抑制试验(MM I)鉴定其生物学活性。
3.131I-anti MIF McAb在小鼠同种皮肤移植模型中的分子显像研究：建立小鼠同种及同系皮肤移植模型,Iodogen法制备131I-anti-MIF McAb、对照抗体131I-IgG。各实验组分别于移植后第1、7、14天尾静脉注射131I-anti-MIF McAb或131I-gc185 kBq/只,24小时后处死,收集血液、移植部位皮片、对侧正常皮片及各组织、器官,观察131I-anti-MIF McAb在小鼠同种皮肤移植模型中的生物学分布状况,计算靶组织与非靶组织的放射性分布比值(T/NT)。同时于移植后各时间点尾静脉注射131I-anti-MIF McAb或131I-IgG3.7MBq,24小时后放射性自显影下观察131I-anti-MIF McAb及其对照抗体在移植皮片部位的放射性浓聚状况。
4. TLR5在同种移植模型中的表达状况：建立小鼠同种及同系皮肤移植模型,于移植后第1、7、14、21天,分别取同种移植及同系移植小鼠移植部位的皮片,RT-PCR法检测TLR5 mRNA的相对表达量；免疫组织化学分析法测定移植皮片内TLR5的蛋白表达状况。
5.免疫抑制剂雷帕霉素(Rapa)对移植受体TLR5表达及IL-10分泌的影响：建立小鼠同种皮肤移植模型,于移植后第1天腹腔注射Rapa 1.5mg/kg诱导移植耐受,观察Rapa对TLR5 mRNA表达的影响。ELISA检测Rapa对同种移植排斥高峰期活化T细胞IL-10分泌的影响。
6. TLR5特异性激活剂Flagellin的制备：体外扩增培养甲型副伤寒杆菌,分离、纯化Flagellin,鉴定Flagellin的纯度及特异性。
5. Flagellin体内外处理对同种移植排斥的影响：建立小鼠同种皮肤移植模型,分别给予生理盐水、Flagellin处理后,观察Flagellin对TLR5 mRNA的表达丰度、移植皮片生存状况的影响及两者间的相关性；同时收集同种皮肤移植术后第14天小鼠脾T细胞,经体外分别给予不同浓度的Flagellin处理不同时间后,观察各组TLR5 mRNA的表达状况。
6. Iodogen法制备131I-Flagellin,鉴定其标记率、放射化学纯度及体外稳定性。
结果
1.移植后第1天同种移植与同系移植组移植皮片MIF mRNA的表达量与对侧正常皮片无明显差异。随着移植排斥程度的加剧,同种移植组MIF的mRNA表达量在移植后第7天明显增加,并于移植后第14天达到峰值,而同系移植组MIF的mRNA表达在移植后各时间点无明显变化。免疫组织化学分析显示在移植排斥过程中MIF蛋白的表达呈现与MIF mRNA表达一致的结果：即MIF的高表达仅出现在同种移植排斥模型中,且与移植排斥程度呈正相关。
2.成功建立3株稳定高效价分泌抗anti-MIF McAb的杂交瘤细胞株,分别命名为5G2D7,5G2C7,5G2E3。经Western blotting、ELISA及MMI鉴定证明该抗anti-MIF McAb具有良好的抗原特异性和生物学活性。
3.生物学分布结果显示：与对侧正常皮片相比,131I-anti-MIF McAb在同种移植皮片部位可呈现较高的放射性,而同系移植皮片部位的放射性与对侧正常皮片无明显差异。131I-anti-MIF McAb在各组织器官的放射性分布结果提示该显像剂主要通过肝脏与肾脏代谢。在同种移植排斥早期131I-anti-MIF McAb的T/NT比值与对照显像剂131I-mIgG相比无明显差异,随着移植排斥程度的加剧,131I-anti-MIF McAb组的T/NT比值逐渐增加,在移植后第14天达到峰值17.13,而对照显像剂的T/NT比值在整个移植排斥过程中呈现逐渐下降的趋势。放射性自显影结果验证了上述结果：即同种移植后第1天131I-anti-MIF McAb与131I-IgG均可在移植皮片部位呈现少量的放射性浓聚,随着移植排斥反应的加剧,131I-IgG在移植皮片部位的非特异性浓聚逐渐消退,而131I-anti-MIF McAb则呈现出较为清晰的影像。
4.同种移植组TLR5的mRNA表达量在移植后逐渐增加,并于移植后第14天达到峰值,而同系移植组TLR5的mRNA表达在移植后各时间点无明显变化。
5.体内给予Rapa处理后,移植皮片部位TLR5 mRNA的表达量在移植排斥高峰期显著增加。体外实验结果显示,同种移植排斥高峰期T细胞经Rapa (100nmol/L)处理后,培养上清中的IL-10分泌逐渐增加,并于培养后72h达到峰值。
6.从甲型副伤寒杆菌中成功分离、纯化出Flagellin, Western Blotting证实所制备Flagellin具有高纯度和高特异性。
7.与生理盐水处理组相比,同种皮肤移植模型小鼠经体内给予Flagellin处理后,TLR5 mRNA明显高表达(p<0.05),且可显著延长移植皮片生存时间(21.57±1.4d), TLR5mRNA表达量与移植皮片生存时间的变化呈明显正相关趋势；经不同浓度Flagellin体外处理急性排斥期小鼠T细胞不同时间后发现,TLR5 mRNA在Flagellin浓度100ng/ml,培养6h组表达量最高。
8.成功制备131I-Flagellin,其标记率为91.6%,比活度为35.27 GBq/μmol;室温放置72小时后仍具有较好的稳定性与免疫学活性。
结论
1.在同种皮肤移植模型中,MIF的表达量与移植排斥程度密切正相关。
2.成功制备了抗小鼠MIF单克隆抗体。
3.131I-anti-MIF McAb在同种移植皮片部位可呈现特异性浓聚,作为分子显像剂可较为清晰地显示移植排斥全过程。
4.在同种皮肤移植模型中,TLR5的表达量与移植排斥程度密切相关。免疫抑制剂Rapa可上调移植皮片部位TLR5 mRNA的表达,促进同种移植排斥高峰期活化T细胞IL-10的分泌。
5.成功制备TLR5特异性激活剂Flagellin。体内外实验证实Flagellin可显著增加移植部位TLR5 mRNA的表达,并延长同种移植皮片的生存时间。
6.成功制备了131I-Flagellin,131I-Flagellin具有较好的体外稳定性。
1.本研究以MIF作为同种移植排斥监测的新靶点,首次以131I-anti-MIF McAb作为同种移植排斥的分子显像剂,证实了该显像剂在同种移植排斥监测中的高敏感与高特异性。
2.首次利用磷屏放射性自显影技术,证实利用131I-anti-MIF McAb作为分子显像剂,可以直观、清晰地动态监测同种移植排斥全部进程。
3.首次研究了TLR5与移植排斥进程的相关性及免疫抑制剂Rapa对TLR5表达的上调作用；首次制备了放射性碘131标记的TLR5特异性配体Flagellin;初步探讨了将131I-Flagellin用于评价免疫抑制治疗过程中移植排斥监测的意义。
Background and Objectives
Organ transplantation is an important means of managing end-organ failure. However, acute allograft rejection remains a major complication after transplantation. Thus how to diagnose the acute rejection timely is still one of the hot topics of in this field. Traditional techniques to predict rejection include clinical symptoms, hematological indexes, immunopathology, genomics and imaging techniques et al. Biopsies were considered to be the gold standard for monitoring allograft well-being after transplantation. However, biopsies are invasive, associated with morbidity if performed on a routine basis and can potentially miss focal rejection. Because of the widely-influence of the transplantation rejection and the complex immunological mechanism, now it is hard to find an accepted, no invasive immunological indexe with high sensitivity and specificity. Therefore, how to find a new noninvasive index that can monitor the transplant rejection effectively and make the timely diagnosis and differential diagnosis before the transplant organ dysfunction has become the hot topic in transplantation immunology.
Molecular imaging is a newly emerging science; it integrates the principles of cell and molecular biology, immunology, nuclear medicine and diagnostic imaging. The application of molecular imaging technology in studying the transplantation immunological phenomenon will become one of the important methods to monitor the transplant rejection. In addition, the molecular imaging informations are important for the individualized application, efficacy and the risk factors evaluation of immunosuppressant after transplantation.
Transplantation rejection is a complicated process.Numerous immune molecules participate in it. The innate immune system is an important part of the immune system. The innate immune system can not only play the nonspecific devouring and scavenging effect, but also can regulate the adaptive immune system. At different stages of organ transplantation, there is a complex and close connection between the innate immune system and the adaptive immune system.
Recently, cytokines infiltrated in the grafts and their specific antibodies are becoming the targets of molecular imaging for transplantation rejection, and nuclear medicine imaging with radioisotope-labeled cytokine or a related antibody for detection of transplantation rejection has been reported, such as 123I or 111In labeled major MHC classⅡmolecules and 99mTc labeled MCP-1. However, non-specificity, a side effect or slow clearance affected these agents' application in clinic.
Macrophage migration inhibitory factor (MIF) was originally identified as a kind of lymphokine that recruits the macrophages to inflamed sites and was involved in cell-mediated immunity and delayed type hypersensitivity (DTH). Recently, MIF was found to be one of the major cytokines involved in the allograft rejection. In addition, the important role of MIF in the development of acute GVHD in a mouse model of allogeneic stem cell transplantation has also been reported. In "Part one" of this dissertation, we choose MIF as a new target of molecular imaging in allograft rejection.The aim of this study was to better understand the relationship between MIF and acute skin allograft rejection, then clarify whether 131I-anti-MIF mAb may enable non-invasive evaluation of acute rejection specifically.
Besides of this, accurately evaluation of the immunosuppressive curative effect and monitoring the recipients' immune status is another key objective for transplant monitoring. Under the guidance of transplant monitoring, we can realize the individualized application of the immunosuppressant. However, since the immunosuppressant could inhibit the proliferation of T cells, B cell and the secretion of cytokines, so if we still use the cytokine as the molecular imaging agent, it can not evaluate the receptor's immunity state accurately. Thus finding a new immune molecule will be useful for monitoring the immunosuppressive curative effect after transplantation in clinic.
TLRs can specifically identify the pathogen-associated molecular patterns (PAMPs), and play important roles not only in the activation of innate immunity, but also in the induction and regulation of adaptive immunity. So the TLRs can also be regard as an important bridge between innate immunity and adaptive immunity. TLRs can be activated by autologous antigens, allogenic antigens and heterologous antigens. The latest study in 2008 showed that existence of natural Tregs was necessary for the long-term survival of heart and skin allograft in mice, the CpG-specific activator of TLR9 can inhibit the function of Tregs and promote the differentiation of Thl effective T cells to inhibit the survival of the allograft. Activation of TLR7 can promote the allograft rejection; activation of TLR4 can inhibit the long-term graft of heart survival, prevented the accumulation of the CD4+/Foxp3+Treg within grafts. So we propose that the activation of TLRs may be critical in the allograft rejection.
It was found recently that TLR5 is the only negative regulatory TLRs which may be directly or indirectly involved in the process of autoimmune diseases. TLR5 can be activated by bacterial Flagellin, a specific ligand to TLR5, and mediate the immune response by stimulating the production of TNF-a, IL-Iβ, IL-8 and other pro-inflammatory cytokine through the activation of NF-κB (nuclear transcription factor-KB). Previous studies showed that TLR5 was found selectively high-expressed on Tregs, and Flagellin can enhance the inhibition function of Treg's on effective T cells. Recently a paper in 2008 reported that Flagellin can protect the body from damage of chemicals, bacteria, viruses and radiation through binding to TLR5.
In view of the important role of TLR5 in induction immune tolerance and its regulatory effect on the Treg, we propose that TLR5 may be another important immune molecule in allograft rejection. In "Part two" of this dissertation, we studied the expression of TLR5 dynamically during the skin allograft rejection progress; detected the effect of Flagellin on the survival time and the expression of TLR5, and then revealed the regulatory role of the immunosuppressant Rapamycin (Rapa) on the expression of TLR5. The aim of this study was to clarify the relationship between TLR5 and allograft rejection process, then evaluated whether TLR5 can be a new noninvasive index for monitoring the allograft rejection.
Methods
1. The expression of MIF in allograft model:The skin allotransplantation and isotransplantation mice were respectively sacrificed at 1,7 and 14 days after transplantation. The expression of MIF mRNA in grafts was quantified by RT-PCR. Meaningwhile the protein expression was analyzed by immunohistochemical staining after transplantation.
2. The preparation and the biology active identification of anti-MIF McAb:anti-MIF McAb was made with hybridoma technology whose specificity and biological activities were identified with Western blotting, ELISA and MMI.
3. The molecular imaging of 131I-anti-MIF McAb in skin allotransplantation mice: Radioiodination of the anti-MIF McAb and control IgG was performed by the Iodogen method, The skin allotrantinsplantation and isotransplantation mice were injected intravenously via the tail vein with a PBS solution (100μL) of I31I-anti-MIF McAb (185 kBq) respectively. At each point of time (1,7 and 14 days after transplantation), five mice of each group were sacrificed at 24 h after injection, blood, grafts, the recipient normal skins of opposite side and the organs were weighed and counted in the gamma counter. Then the%ID/g and the T/NT (target/nontarget) ratios of each group were calculated. The skin allotransplantation mice were injected intravenously via the tail vein with a PBS solution (100μL) of 131I-anti-MIF mAb or 131I-IgG (3.7 MBq) respectively. At each point of time (1,7 and 14 days after transplantation), five mice of each group were imaged by whole-body autoradiography. Images were performed at 24 h after injection.
4. The expression of TLR5 in allograft model:The skin allotransplantation and isotransplantation mice were respectively sacrificed at 1,7,14and 21 days after transplantation. The expression of TLR5 mRNA in grafts was quantified by RT-PCR. Then the protein expression was analyzed by immunohistochemical staining.
5. The regulatory role of Rapa on the expression of TLR5:The skin allotransplantation mice were intraperitoneally injected with Rapa (2.5mg/kg-d) 1 time/day (Rapa Group) or NS (Control Group) respectively. The grafts from two groups were collected at 1,7,14 and 21 days after transplantation. The expression of TLR5 mRNA in grafts was quantified by RT-PCR. The T cells collected on day 14 after transplantation were treated with different concentrations of Rapa in different times to study the effect of Rapa on the secretion of IL-10 by ELISA.
6. The extraction and purification of Flagellin:Flagellin was extracted and purified from the Salmonella Paratyphoid A.Then the purity and the specificity of purified flagellin was identified by Western Blotting.
7. The regulatory role of Flagellin on the expression of TLR5:The recipient BALB/c mice were injected with NS or Flagellin respectively to observe the function of Flagellin on the expression of TLR5 mRNA and the survival time of the grafts.Then the correlation between them was clarified. The T cells collected on day 14 after transplantation were treated with different concentrations of Flagellin in different times to study the effect of Flagellin on the expression of TLR5.
8. Radioiodination of the Flagellin was performed by the Iodogen method.Then we identified its labeling efficiency, radiochemical purity and stability in vitro.
Results
1. The expression of MIF in allograft model:The distribution of MIF mRNA expression within grafts was assessed by RT-PCR. There was no change in MIF gene expression in day 1 isografts or allografts compared with the normal skin of the opposite side. However, there was a marked increase of MIF mRNA expression in day 7 allografts. With the degree of the allograft rejection intensified, the expression of MIF mRNA kept on increasing, at day 14, it reached its peak. In contrast, MIF mRNA levels in isotransplantation mice barely changed through the experiment. Consistent with the result of RT-PCR, immunohistochemistry staining showed the isografts showing little change in MIF expression compared with the normal skin. In contrast, the allografts showed a marked up-regulation in MIF immunostaining. The expression of MIF was closely relevant to the degree of the rejection.
2. Three stable cell lines were obtained and named as 5G2D7,5G2C7 and 5G2 E3 with high specificity and biological activity to MIF.
3. The biodistribution of 131I-anti-MIF mAb was studied at each point of time (day 1, 7, and day 14 after transplantation) in the skin allotransplantation and isotransplantation mice.131I-anti-MIF mAb showed a considerably higher uptake and a lower decrease in the allografts compared with the normal skin of the opposite side. There was no statistically significant difference in the radioactivity of 131I-anti-MIF mAb between the isografts and the normal skin. The biodistribution of 131I-anti-MIF mAb in organs indicated this imaging agents were excreted from the kidney or swallowed by reticuloendothelium of liver. There was no difference between the 131I-anti-MIF mAb group and the 131I-IgG group in the early stage of allograft rejection. With the development of the allograft rejection, the T/NT ratio of the 131I-anti-MIF mAb group increased continually, at day 14 it was 17.13, which is the highest. The T/NT ratio of the 131I-IgG group decreased during the progression of rejection. Both of the imaging agents showed a few uptakes in the grafts at day 1. Nevertheless, with the development of the allograft rejection, the 'i-anti-MIF mAb group showed much more clear images than the 131I-IgG, which is in accordance with its high T/NT ratio.
4. The expression of TLR5 in allograft model increased gradually after transplantation, and on the day 14 reached its peak. In contrast, TLR5 mRNA levels in isotransplantation mice barely changed through the experiment.
5. At 10,14 and 21 days after transpantation, there was a markedly increase of TLR5 mRNA expression within grafts after treated with Rapa. The secretion of IL-10 was increased gradually when treated with Rapa (100 nmol/L) and reached its peak after 72h incubarition.
6. Flagellin was successfully extracted and purified from Salmonella Paratyphoid A. It can provide properties of high specificity and high purity.
7. Compared with normal saline treatment group, the expression of TLR5 mRNA in Flagellin treatment group increased obviously (p< 0.05) and the survival time of the grafts was extended to 21.57±1.4 days. There was positive relationship between the expression of TLR5 and the survival time of the grafts. In vitro experiment, the expression of TLR5 mRNA markedly increased when treated with Flagellin (100ng/ml) and reached its peak after 6h incubarition.
8. The preparation 131I-Flagellin was successfully. The labeling efficiency of 131I-Flagellin was 91.6%. The specific activity of 131I-Flagellin was 35.27 GBq/μmol.When placed in room temperature after 72 hours, it still has good stability and immunological activity.
Conclusions and Potential Impact
1. MIF expression correlated closely with the gradually increasing allograft rejection.
2. We successfully construct the anti-MIF McAb with high specificity and biological activity to MIF.
3. The higher T/NT ratio of 131I-anti-MIF mAb and its specificity targeting the grafts provide a possible method for molecular imaging of allograft rejection.
4. There was positive relationship between the expression of TLR5 and the degree of the allograft rejection.
5. The immunosuppressant Rapa could increase the expression of TLR5 mRNA and up-regulate the secretion of IL-10.
6. Successfully extracted and purified Flagellin.
7. TLR5's specific ligands Flagellin could increase the expression of TLR5 mRNA and extend the survival time of the grafts.
8. The preparation 131I-Flagellin was successfully and it can provide propertie of good stability in vitro.
Points of Innovation
1. For the first time, MIF was choosed to be a new molecular imaging agent of the allograft rejection monitoring. It can provide properties of high sensitivity and high specificity.
2. For the first time the phosphor screen autoradiography technique was used to dynamically monitor the whole processes of allograft rejection with clear and visualized imaging data.
3. For the first time we claryfied the expression of TLR5 was closely relevant to the degree of the rejection and prepared the radioiodinated Flagellin (the specific ligand of TLR5). These results will provide basises for taking TLR5 as a new index for accurately evaluation of the immunosuppressive curative effect after transplantation.
器官移植是目前治疗各种脏器终末期功能衰竭的最有效手段。但是移植后的急性排斥反应(AR)仍然是移植术后的主要并发症,也是导致慢性排斥反应(CR)和移植器官功能丧失的最重要的危险因素。如何快速诊断和及时治疗急性排斥反应一直是临床器官移植研究的热点。目前临床上诊断急性排斥反应的主要方法包括移植受者临床表现、血液相关指标检测、移植器官病理学活检和影像学辅助检查等,其中活检组织病理学检查仍然是诊断移植排斥反应的最可靠手段,但其侵入性、潜在并发症及取样误差,限制了其临床应用。器官移植排斥反应涉及范围广泛,免疫学机制复杂,迄今为止仍未发现公认的、无创伤性高敏感性和特异性的监测指标。因此,寻找新的非创伤性侵入性监测指标,早期、有效监测移植排斥反应,在移植器官功能发生损害之前及时做出诊断就成为移植免疫学领域亟待解决的问题。
分子影像学(molecular imaging)是将分子生物学技术和现代医学影像学相结合而产生的一门新兴的边缘学科,是运用影像学手段显示组织水平、细胞和亚细胞水平的特定分子,反映活体状态下分子水平变化,对其生物学行为在影像方面进行定性和定量研究的科学。经典的影像诊断(CT、MRI等)主要显示的是一些分子改变的终效应,具有解剖学改变的疾病；而分子影像学通过发展新的工具、试剂及方法,探查疾病过程中细胞和分子水平的异常,在尚无解剖学改变前检出异常,为探索疾病的发生、发展和转归,评价药物的疗效,为分子水平疾病的治疗奠定实验和理论基础。应用分子影像学技术研究器官及组织移植术后的免疫学细胞及分子水平的变化,有可能为临床监测移植排斥反应提供重要手段。急性排斥反应的分子影像学信息对于移植术后免疫抑制剂的个体化应用、功效评价、危险因素监测与评估,具有极其重要的指导意义。
免疫应答可以分为固有免疫和适应性免疫两大类。前者是指与生俱来的机体天然抵抗力。后者是指接触抗原后机体产生的特异性免疫力。移植排斥反应是一个复杂的免疫学过程,众多免疫分子参与其中。固有免疫是机体免疫系统的重要组成部分,随着对移植免疫机理的深入研究,人们逐渐意识到在移植排斥反应过程中,固有免疫并不仅仅发挥非特异吞噬、清除作用,它与适应性免疫一样能够正确区分“自己”和“非己”,并进一步调控适应性免疫。在器官移植的不同阶段,固有免疫与适应性免疫系统之间都存在着复杂的密不可分的联系。因此选择在固有免疫和适应性免疫中均发挥作用的标志分子,研究其在同种移植排斥过程中的表达状态,探索其能否成为监测移植排斥反应的靶点分子,对于深入阐明移植排斥分子机理,指导临床监测排异反应,更有效地防治移植排斥具有重要的理论和实际意义。
移植物进入机体后,移植物抗原致敏机体免疫细胞,被激活的免疫细胞进而产生多种细胞因子,引起免疫排斥反应,由于细胞因子通常在排斥反应早期即被释放,因此可作为移植排斥反应分子影像学的靶分子。已有研究者尝试将放射性核素123I或111In标记的MHCII类分子,99mTc标记的MCP-1 (Monocyte Chamotactic Protein)用于移植排斥反应的分子影像学诊断,但由于所选显像剂特异性不高且在体内清除缓慢,影响了其在临床上的应用。
巨噬细胞移动抑制因子(macrophage migration inhibitory factor, MIF)是具有多种生物活性的可溶性淋巴因子,由活化的T细胞、巨噬细胞和其它多种细胞分泌,是免疫和内分泌系统的重要介质,具有激活巨噬细胞、抑制其游走、激活T细胞并增强其功能的作用,在急性炎症和迟发型超敏反应中有重要意义。近年来已有研究表明MIF是同种移植排斥过程中的重要介质,MIF表达的上调与巨噬细胞、T细胞浸润及同种肾移植排斥反应的程度密切相关。此外,Toubai等研究发现在大鼠同种异体干细胞移植模型中MIF对移植物抗宿主病(graft-versus-host disease, GVHD)的发生、发展也起着关键的作用。鉴于MIF在同种移植排斥反应中的重要作用,本研究第一部分选择MIF作为同种移植排斥分子影像学的新靶点,阐明MIF的表达与移植排斥反应程度的相关性,并在此基础上利用放射性核素131I标记的MIF单克隆抗体作为分子显像剂,动态监测同种移植排斥反应的全过程,旨在为无创伤性监测移植排斥反应探索新的途径。
准确评价移植术后免疫抑制治疗的效能,监测受者的免疫状态,根据受者的免疫状态来调整免疫抑制剂的用量是移植监测的另一关键目标。通过移植监测可以指导移植后免疫抑制剂的合理使用,实现药物应用的个体化。由于免疫抑制剂可以抑制T细胞、B细胞等靶细胞的增殖及细胞因子合成,因此应用免疫抑制剂之后如仍以移植排斥早期释放的细胞因子作为分子影像学的成像对象显然不能客观地反应移植物的免疫状态。寻找移植排斥分子显像新靶点,从而准确地评价移植后免疫抑制治疗后的效能,是移植监测领域的又一关注热点。
Toll样受体家族(Toll-like Receptor, TLRs)作为一种模式识别受体(pattern recognition receptor, PRR)可特异性识别病原相关分子模式(pathogen-associated molecular patterns, PAMPs),不仅在激活固有免疫中发挥重要作用,而且还在诱导和调节适应性免疫中具有重要意义,是连接固有免疫和适应性免疫的重要桥梁。TLRs信号可以在自身抗原,同种抗原和异种抗原的刺激下激活。2008年研究发现小鼠心脏皮肤同种移植物的长期生存需要天然Tregs的存在,TLR9的特异激活剂CpG可以通过抑制Treg的功能和促进Thl效应性T细胞的分化,抑制同种移植物的存活。TLR7的激活可以促进皮肤同种移植物的排斥；TLR4的激活阻断了利用抗CD154单抗建立的心脏同种移植物的长期存活,由此可见TLRs信号与移植排斥关系密切。
TLR5是TLRs家族中唯一具有免疫负调节作用的分子,可直接间接地参与自身免疫病的发病过程。细菌鞭毛蛋白(Flagellin)是TLR5的唯一配体,Flagellin可特异识别结合TLR5,通过激活核转录因子NF-κB刺激TNF-α、IL-1β、IL-8等前炎症细胞因子的产生,介导机体天然免疫。最新研究表明,TLR5可选择性高表达Treg细胞表面,TLR5的特异激活剂Flagellin可增强CD4+CD25+调节性T细胞(Treg)对效应性T细胞的抑制作用。2008年有研究报告体内给予TLR5特异激活剂Flagellin,可以保护机体免受化学、细菌病毒和辐射等损害。鉴于Treg细胞在诱导同种移植物耐受过程具有重要作用,TLR5可以调控Treg细胞免疫抑制效应,提示TLR5可能是同种移植排斥过程中又一关键免疫分子。本课题第二部分在研究同种皮肤移植急性排斥过程中TLR5动态表达状况和探讨了免疫抑制剂雷帕霉素对TLR5表达的影响的基础上,纯化提取TLR5特异激活剂Flagellin,利用放射性核素13’I标记Flagellin,并进行了初步鉴定,旨在阐明TLR5在移植排斥过程中的动态表达,初步探讨其作为同种移植排斥标志分子的可行性。
研究方法
1.MIF在同种移植模型中的表达状况：以BABL/c小鼠为受体,分别以B6小鼠、BABL/c小鼠为供体,建立小鼠同种、同系皮肤移植模型,于移植后第1、7、14、天,分别取移植部位的皮片,逆转录聚合酶链反应(RT-PCR)检测MIF mRNA的相对表达。同时利用免疫组织化学分析法测定移植后不同时间移植皮片内MIF蛋白表达状况。
2.小鼠MIF单克隆抗体(anti-MIFMcAb)的制备及生物学活性鉴定：采用杂交瘤技术制备小鼠MIF单克隆抗体,蛋白质印迹技术(Western blotting)及酶联免疫吸附试验(ELISA)鉴定其特异性,巨噬细胞移动抑制试验(MM I)鉴定其生物学活性。
3.131I-anti MIF McAb在小鼠同种皮肤移植模型中的分子显像研究：建立小鼠同种及同系皮肤移植模型,Iodogen法制备131I-anti-MIF McAb、对照抗体131I-IgG。各实验组分别于移植后第1、7、14天尾静脉注射131I-anti-MIF McAb或131I-gc185 kBq/只,24小时后处死,收集血液、移植部位皮片、对侧正常皮片及各组织、器官,观察131I-anti-MIF McAb在小鼠同种皮肤移植模型中的生物学分布状况,计算靶组织与非靶组织的放射性分布比值(T/NT)。同时于移植后各时间点尾静脉注射131I-anti-MIF McAb或131I-IgG3.7MBq,24小时后放射性自显影下观察131I-anti-MIF McAb及其对照抗体在移植皮片部位的放射性浓聚状况。
4. TLR5在同种移植模型中的表达状况：建立小鼠同种及同系皮肤移植模型,于移植后第1、7、14、21天,分别取同种移植及同系移植小鼠移植部位的皮片,RT-PCR法检测TLR5 mRNA的相对表达量；免疫组织化学分析法测定移植皮片内TLR5的蛋白表达状况。
5.免疫抑制剂雷帕霉素(Rapa)对移植受体TLR5表达及IL-10分泌的影响：建立小鼠同种皮肤移植模型,于移植后第1天腹腔注射Rapa 1.5mg/kg诱导移植耐受,观察Rapa对TLR5 mRNA表达的影响。ELISA检测Rapa对同种移植排斥高峰期活化T细胞IL-10分泌的影响。
6. TLR5特异性激活剂Flagellin的制备：体外扩增培养甲型副伤寒杆菌,分离、纯化Flagellin,鉴定Flagellin的纯度及特异性。
5. Flagellin体内外处理对同种移植排斥的影响：建立小鼠同种皮肤移植模型,分别给予生理盐水、Flagellin处理后,观察Flagellin对TLR5 mRNA的表达丰度、移植皮片生存状况的影响及两者间的相关性；同时收集同种皮肤移植术后第14天小鼠脾T细胞,经体外分别给予不同浓度的Flagellin处理不同时间后,观察各组TLR5 mRNA的表达状况。
6. Iodogen法制备131I-Flagellin,鉴定其标记率、放射化学纯度及体外稳定性。
结果
1.移植后第1天同种移植与同系移植组移植皮片MIF mRNA的表达量与对侧正常皮片无明显差异。随着移植排斥程度的加剧,同种移植组MIF的mRNA表达量在移植后第7天明显增加,并于移植后第14天达到峰值,而同系移植组MIF的mRNA表达在移植后各时间点无明显变化。免疫组织化学分析显示在移植排斥过程中MIF蛋白的表达呈现与MIF mRNA表达一致的结果：即MIF的高表达仅出现在同种移植排斥模型中,且与移植排斥程度呈正相关。
2.成功建立3株稳定高效价分泌抗anti-MIF McAb的杂交瘤细胞株,分别命名为5G2D7,5G2C7,5G2E3。经Western blotting、ELISA及MMI鉴定证明该抗anti-MIF McAb具有良好的抗原特异性和生物学活性。
3.生物学分布结果显示：与对侧正常皮片相比,131I-anti-MIF McAb在同种移植皮片部位可呈现较高的放射性,而同系移植皮片部位的放射性与对侧正常皮片无明显差异。131I-anti-MIF McAb在各组织器官的放射性分布结果提示该显像剂主要通过肝脏与肾脏代谢。在同种移植排斥早期131I-anti-MIF McAb的T/NT比值与对照显像剂131I-mIgG相比无明显差异,随着移植排斥程度的加剧,131I-anti-MIF McAb组的T/NT比值逐渐增加,在移植后第14天达到峰值17.13,而对照显像剂的T/NT比值在整个移植排斥过程中呈现逐渐下降的趋势。放射性自显影结果验证了上述结果：即同种移植后第1天131I-anti-MIF McAb与131I-IgG均可在移植皮片部位呈现少量的放射性浓聚,随着移植排斥反应的加剧,131I-IgG在移植皮片部位的非特异性浓聚逐渐消退,而131I-anti-MIF McAb则呈现出较为清晰的影像。
4.同种移植组TLR5的mRNA表达量在移植后逐渐增加,并于移植后第14天达到峰值,而同系移植组TLR5的mRNA表达在移植后各时间点无明显变化。
5.体内给予Rapa处理后,移植皮片部位TLR5 mRNA的表达量在移植排斥高峰期显著增加。体外实验结果显示,同种移植排斥高峰期T细胞经Rapa (100nmol/L)处理后,培养上清中的IL-10分泌逐渐增加,并于培养后72h达到峰值。
6.从甲型副伤寒杆菌中成功分离、纯化出Flagellin, Western Blotting证实所制备Flagellin具有高纯度和高特异性。
7.与生理盐水处理组相比,同种皮肤移植模型小鼠经体内给予Flagellin处理后,TLR5 mRNA明显高表达(p<0.05),且可显著延长移植皮片生存时间(21.57±1.4d), TLR5mRNA表达量与移植皮片生存时间的变化呈明显正相关趋势；经不同浓度Flagellin体外处理急性排斥期小鼠T细胞不同时间后发现,TLR5 mRNA在Flagellin浓度100ng/ml,培养6h组表达量最高。
8.成功制备131I-Flagellin,其标记率为91.6%,比活度为35.27 GBq/μmol;室温放置72小时后仍具有较好的稳定性与免疫学活性。
结论
1.在同种皮肤移植模型中,MIF的表达量与移植排斥程度密切正相关。
2.成功制备了抗小鼠MIF单克隆抗体。
3.131I-anti-MIF McAb在同种移植皮片部位可呈现特异性浓聚,作为分子显像剂可较为清晰地显示移植排斥全过程。
4.在同种皮肤移植模型中,TLR5的表达量与移植排斥程度密切相关。免疫抑制剂Rapa可上调移植皮片部位TLR5 mRNA的表达,促进同种移植排斥高峰期活化T细胞IL-10的分泌。
5.成功制备TLR5特异性激活剂Flagellin。体内外实验证实Flagellin可显著增加移植部位TLR5 mRNA的表达,并延长同种移植皮片的生存时间。
6.成功制备了131I-Flagellin,131I-Flagellin具有较好的体外稳定性。
1.本研究以MIF作为同种移植排斥监测的新靶点,首次以131I-anti-MIF McAb作为同种移植排斥的分子显像剂,证实了该显像剂在同种移植排斥监测中的高敏感与高特异性。
2.首次利用磷屏放射性自显影技术,证实利用131I-anti-MIF McAb作为分子显像剂,可以直观、清晰地动态监测同种移植排斥全部进程。
3.首次研究了TLR5与移植排斥进程的相关性及免疫抑制剂Rapa对TLR5表达的上调作用；首次制备了放射性碘131标记的TLR5特异性配体Flagellin;初步探讨了将131I-Flagellin用于评价免疫抑制治疗过程中移植排斥监测的意义。
Background and Objectives
Organ transplantation is an important means of managing end-organ failure. However, acute allograft rejection remains a major complication after transplantation. Thus how to diagnose the acute rejection timely is still one of the hot topics of in this field. Traditional techniques to predict rejection include clinical symptoms, hematological indexes, immunopathology, genomics and imaging techniques et al. Biopsies were considered to be the gold standard for monitoring allograft well-being after transplantation. However, biopsies are invasive, associated with morbidity if performed on a routine basis and can potentially miss focal rejection. Because of the widely-influence of the transplantation rejection and the complex immunological mechanism, now it is hard to find an accepted, no invasive immunological indexe with high sensitivity and specificity. Therefore, how to find a new noninvasive index that can monitor the transplant rejection effectively and make the timely diagnosis and differential diagnosis before the transplant organ dysfunction has become the hot topic in transplantation immunology.
Molecular imaging is a newly emerging science; it integrates the principles of cell and molecular biology, immunology, nuclear medicine and diagnostic imaging. The application of molecular imaging technology in studying the transplantation immunological phenomenon will become one of the important methods to monitor the transplant rejection. In addition, the molecular imaging informations are important for the individualized application, efficacy and the risk factors evaluation of immunosuppressant after transplantation.
Transplantation rejection is a complicated process.Numerous immune molecules participate in it. The innate immune system is an important part of the immune system. The innate immune system can not only play the nonspecific devouring and scavenging effect, but also can regulate the adaptive immune system. At different stages of organ transplantation, there is a complex and close connection between the innate immune system and the adaptive immune system.
Recently, cytokines infiltrated in the grafts and their specific antibodies are becoming the targets of molecular imaging for transplantation rejection, and nuclear medicine imaging with radioisotope-labeled cytokine or a related antibody for detection of transplantation rejection has been reported, such as 123I or 111In labeled major MHC classⅡmolecules and 99mTc labeled MCP-1. However, non-specificity, a side effect or slow clearance affected these agents' application in clinic.
Macrophage migration inhibitory factor (MIF) was originally identified as a kind of lymphokine that recruits the macrophages to inflamed sites and was involved in cell-mediated immunity and delayed type hypersensitivity (DTH). Recently, MIF was found to be one of the major cytokines involved in the allograft rejection. In addition, the important role of MIF in the development of acute GVHD in a mouse model of allogeneic stem cell transplantation has also been reported. In "Part one" of this dissertation, we choose MIF as a new target of molecular imaging in allograft rejection.The aim of this study was to better understand the relationship between MIF and acute skin allograft rejection, then clarify whether 131I-anti-MIF mAb may enable non-invasive evaluation of acute rejection specifically.
Besides of this, accurately evaluation of the immunosuppressive curative effect and monitoring the recipients' immune status is another key objective for transplant monitoring. Under the guidance of transplant monitoring, we can realize the individualized application of the immunosuppressant. However, since the immunosuppressant could inhibit the proliferation of T cells, B cell and the secretion of cytokines, so if we still use the cytokine as the molecular imaging agent, it can not evaluate the receptor's immunity state accurately. Thus finding a new immune molecule will be useful for monitoring the immunosuppressive curative effect after transplantation in clinic.
TLRs can specifically identify the pathogen-associated molecular patterns (PAMPs), and play important roles not only in the activation of innate immunity, but also in the induction and regulation of adaptive immunity. So the TLRs can also be regard as an important bridge between innate immunity and adaptive immunity. TLRs can be activated by autologous antigens, allogenic antigens and heterologous antigens. The latest study in 2008 showed that existence of natural Tregs was necessary for the long-term survival of heart and skin allograft in mice, the CpG-specific activator of TLR9 can inhibit the function of Tregs and promote the differentiation of Thl effective T cells to inhibit the survival of the allograft. Activation of TLR7 can promote the allograft rejection; activation of TLR4 can inhibit the long-term graft of heart survival, prevented the accumulation of the CD4+/Foxp3+Treg within grafts. So we propose that the activation of TLRs may be critical in the allograft rejection.
It was found recently that TLR5 is the only negative regulatory TLRs which may be directly or indirectly involved in the process of autoimmune diseases. TLR5 can be activated by bacterial Flagellin, a specific ligand to TLR5, and mediate the immune response by stimulating the production of TNF-a, IL-Iβ, IL-8 and other pro-inflammatory cytokine through the activation of NF-κB (nuclear transcription factor-KB). Previous studies showed that TLR5 was found selectively high-expressed on Tregs, and Flagellin can enhance the inhibition function of Treg's on effective T cells. Recently a paper in 2008 reported that Flagellin can protect the body from damage of chemicals, bacteria, viruses and radiation through binding to TLR5.
In view of the important role of TLR5 in induction immune tolerance and its regulatory effect on the Treg, we propose that TLR5 may be another important immune molecule in allograft rejection. In "Part two" of this dissertation, we studied the expression of TLR5 dynamically during the skin allograft rejection progress; detected the effect of Flagellin on the survival time and the expression of TLR5, and then revealed the regulatory role of the immunosuppressant Rapamycin (Rapa) on the expression of TLR5. The aim of this study was to clarify the relationship between TLR5 and allograft rejection process, then evaluated whether TLR5 can be a new noninvasive index for monitoring the allograft rejection.
Methods
1. The expression of MIF in allograft model:The skin allotransplantation and isotransplantation mice were respectively sacrificed at 1,7 and 14 days after transplantation. The expression of MIF mRNA in grafts was quantified by RT-PCR. Meaningwhile the protein expression was analyzed by immunohistochemical staining after transplantation.
2. The preparation and the biology active identification of anti-MIF McAb:anti-MIF McAb was made with hybridoma technology whose specificity and biological activities were identified with Western blotting, ELISA and MMI.
3. The molecular imaging of 131I-anti-MIF McAb in skin allotransplantation mice: Radioiodination of the anti-MIF McAb and control IgG was performed by the Iodogen method, The skin allotrantinsplantation and isotransplantation mice were injected intravenously via the tail vein with a PBS solution (100μL) of I31I-anti-MIF McAb (185 kBq) respectively. At each point of time (1,7 and 14 days after transplantation), five mice of each group were sacrificed at 24 h after injection, blood, grafts, the recipient normal skins of opposite side and the organs were weighed and counted in the gamma counter. Then the%ID/g and the T/NT (target/nontarget) ratios of each group were calculated. The skin allotransplantation mice were injected intravenously via the tail vein with a PBS solution (100μL) of 131I-anti-MIF mAb or 131I-IgG (3.7 MBq) respectively. At each point of time (1,7 and 14 days after transplantation), five mice of each group were imaged by whole-body autoradiography. Images were performed at 24 h after injection.
4. The expression of TLR5 in allograft model:The skin allotransplantation and isotransplantation mice were respectively sacrificed at 1,7,14and 21 days after transplantation. The expression of TLR5 mRNA in grafts was quantified by RT-PCR. Then the protein expression was analyzed by immunohistochemical staining.
5. The regulatory role of Rapa on the expression of TLR5:The skin allotransplantation mice were intraperitoneally injected with Rapa (2.5mg/kg-d) 1 time/day (Rapa Group) or NS (Control Group) respectively. The grafts from two groups were collected at 1,7,14 and 21 days after transplantation. The expression of TLR5 mRNA in grafts was quantified by RT-PCR. The T cells collected on day 14 after transplantation were treated with different concentrations of Rapa in different times to study the effect of Rapa on the secretion of IL-10 by ELISA.
6. The extraction and purification of Flagellin:Flagellin was extracted and purified from the Salmonella Paratyphoid A.Then the purity and the specificity of purified flagellin was identified by Western Blotting.
7. The regulatory role of Flagellin on the expression of TLR5:The recipient BALB/c mice were injected with NS or Flagellin respectively to observe the function of Flagellin on the expression of TLR5 mRNA and the survival time of the grafts.Then the correlation between them was clarified. The T cells collected on day 14 after transplantation were treated with different concentrations of Flagellin in different times to study the effect of Flagellin on the expression of TLR5.
8. Radioiodination of the Flagellin was performed by the Iodogen method.Then we identified its labeling efficiency, radiochemical purity and stability in vitro.
Results
1. The expression of MIF in allograft model:The distribution of MIF mRNA expression within grafts was assessed by RT-PCR. There was no change in MIF gene expression in day 1 isografts or allografts compared with the normal skin of the opposite side. However, there was a marked increase of MIF mRNA expression in day 7 allografts. With the degree of the allograft rejection intensified, the expression of MIF mRNA kept on increasing, at day 14, it reached its peak. In contrast, MIF mRNA levels in isotransplantation mice barely changed through the experiment. Consistent with the result of RT-PCR, immunohistochemistry staining showed the isografts showing little change in MIF expression compared with the normal skin. In contrast, the allografts showed a marked up-regulation in MIF immunostaining. The expression of MIF was closely relevant to the degree of the rejection.
2. Three stable cell lines were obtained and named as 5G2D7,5G2C7 and 5G2 E3 with high specificity and biological activity to MIF.
3. The biodistribution of 131I-anti-MIF mAb was studied at each point of time (day 1, 7, and day 14 after transplantation) in the skin allotransplantation and isotransplantation mice.131I-anti-MIF mAb showed a considerably higher uptake and a lower decrease in the allografts compared with the normal skin of the opposite side. There was no statistically significant difference in the radioactivity of 131I-anti-MIF mAb between the isografts and the normal skin. The biodistribution of 131I-anti-MIF mAb in organs indicated this imaging agents were excreted from the kidney or swallowed by reticuloendothelium of liver. There was no difference between the 131I-anti-MIF mAb group and the 131I-IgG group in the early stage of allograft rejection. With the development of the allograft rejection, the T/NT ratio of the 131I-anti-MIF mAb group increased continually, at day 14 it was 17.13, which is the highest. The T/NT ratio of the 131I-IgG group decreased during the progression of rejection. Both of the imaging agents showed a few uptakes in the grafts at day 1. Nevertheless, with the development of the allograft rejection, the 'i-anti-MIF mAb group showed much more clear images than the 131I-IgG, which is in accordance with its high T/NT ratio.
4. The expression of TLR5 in allograft model increased gradually after transplantation, and on the day 14 reached its peak. In contrast, TLR5 mRNA levels in isotransplantation mice barely changed through the experiment.
5. At 10,14 and 21 days after transpantation, there was a markedly increase of TLR5 mRNA expression within grafts after treated with Rapa. The secretion of IL-10 was increased gradually when treated with Rapa (100 nmol/L) and reached its peak after 72h incubarition.
6. Flagellin was successfully extracted and purified from Salmonella Paratyphoid A. It can provide properties of high specificity and high purity.
7. Compared with normal saline treatment group, the expression of TLR5 mRNA in Flagellin treatment group increased obviously (p< 0.05) and the survival time of the grafts was extended to 21.57±1.4 days. There was positive relationship between the expression of TLR5 and the survival time of the grafts. In vitro experiment, the expression of TLR5 mRNA markedly increased when treated with Flagellin (100ng/ml) and reached its peak after 6h incubarition.
8. The preparation 131I-Flagellin was successfully. The labeling efficiency of 131I-Flagellin was 91.6%. The specific activity of 131I-Flagellin was 35.27 GBq/μmol.When placed in room temperature after 72 hours, it still has good stability and immunological activity.
Conclusions and Potential Impact
1. MIF expression correlated closely with the gradually increasing allograft rejection.
2. We successfully construct the anti-MIF McAb with high specificity and biological activity to MIF.
3. The higher T/NT ratio of 131I-anti-MIF mAb and its specificity targeting the grafts provide a possible method for molecular imaging of allograft rejection.
4. There was positive relationship between the expression of TLR5 and the degree of the allograft rejection.
5. The immunosuppressant Rapa could increase the expression of TLR5 mRNA and up-regulate the secretion of IL-10.
6. Successfully extracted and purified Flagellin.
7. TLR5's specific ligands Flagellin could increase the expression of TLR5 mRNA and extend the survival time of the grafts.
8. The preparation 131I-Flagellin was successfully and it can provide propertie of good stability in vitro.
Points of Innovation
1. For the first time, MIF was choosed to be a new molecular imaging agent of the allograft rejection monitoring. It can provide properties of high sensitivity and high specificity.
2. For the first time the phosphor screen autoradiography technique was used to dynamically monitor the whole processes of allograft rejection with clear and visualized imaging data.
3. For the first time we claryfied the expression of TLR5 was closely relevant to the degree of the rejection and prepared the radioiodinated Flagellin (the specific ligand of TLR5). These results will provide basises for taking TLR5 as a new index for accurately evaluation of the immunosuppressive curative effect after transplantation.
引文
[1]Elkhammas EA, Henry ML, Ferguson RM, et al. Simultaneous pancreas kidney transplantation:overview of the Ohio state experience [J]. YonseiMed J,2004,45 (6): 1095-1100.
[2]Andrew BA, Christian PL, Thomas CP. Costimulation blockade and tolerance. Curr Opin Organ Transplantation,2002,7 (1):7-12.
[3]Jun ichi S, Mitsuaki I, Atsushi I, et al. Differential Thl and Th2 cell regulation of marine cardiac allogaft acceptance by blocking cell adhesion of ICAM-1/LFA-1 andVCANt-1/I/LA-4. Transpl Immunol,1999,7 (1):65-72.
[4]Steiniger B, Stehling O, Scriba A, et al. Monocytes in the rat:phenotype and function during acute allograft rejection. Immunol Rev.2001 Dec; 184:38-44.
[5]Weissleder R. Molecular imaging:exploring the new frontiers. Radiology,1999; 212(3):609-614
[6]M. Isobe, J.F. Southern, Y. Yazaki, Scintigraphic detection of early cardiac rejection by iodine 123-labeled monoclonal antibody directed against monomorphic determinant of major histocompatibility complex class Ⅱ antigens, Am. Heart J.127 (1994)1309-1317.
[7]M. Isobe, E. Haber, B.A. Khaw, Early detection of rejection and assessment of cyclosporine therapy by 111In antimyosin imaging in mouse heart allografts, Circulation 84 (1991) 1246-1255.
[8]A.I. McGhie, B. Radovancevic, P. Capek, W.H. Moore, L. Kasi, L. Lamki, F.J. Clubb Jr., O.H. Frazier, J.T. Willerson, Major histocompatibility complex class Ⅱ antigen expression in rejecting cardiac allografts:detection using in vivo imaging with radiolabeled monoclonal antibody, Circulation 96 (1997) 1605-1611.
[9]M.H. Kown, C.L. Jahncke, M.A. Lijkwan, M.L. Koransky, C. Mari, G.J. Berry, F.G. Blankenberg, H.W. Strauss, R.C. Robbins, The use of (99 m) technetium-labeled MCP-1 to assess graft coronary artery disease in rat cardiac allografts, J. Heart Lung Transplant.21 (2002) 1009-1015.
[10]T. Calandra, J. Bernhagen, C.N. Metz, et al. MIF as a glucocorticoidinduced modulator of cytokine production, Nature 377 (1995) 68-71.
[11]Calendra T, Bernhagen J, Mitchell RA, et al. The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J Exp Med,1994,79:1895
[12]Christine N, Richard Bucala. Role of macrophage migration inhibitory factor in the regulation of the immune response. Advances in Immunology 1997; 66:197-223.
[13]M. Bacher, C.N. Metz, T. Calandra, K. Mayer, J. Chesney, M. Lohoff, D. Gemsa, T. Donnelly, R. Bucala, An essential regulatory role for macrophage migration inhibitory factor in T-cell activation, Proc. Natl Acad. Sci. USA 93 (1996) 7849-7854.
[14]Lue H, Kleemann R, Calandra T, et al. Macrophage migration inhibitory factor MIF:mechanisms of action and role in disease. Microbes Infect 2002 Apr;(4): 449-460.
[15]Brown FC, Nikolic-Paterson DJ, Metz C, Bucala R, Atkins RC. Up-regulation of macrophage migration inhibitory factor in acute renal allograft rejection in the rat. Clin Exp Immunol 1999; 118(2):329.
[16]Lan HY, Yang N, Brown FG, et al. Macrophage migration inhibitory factor expression in human renal allograft rejection. Transplantation 1998; 66(11):1465
[17]Lo JW, Leung AY, Huang XR, Macrophage migratory inhibitory factor (MIF) expression in acute graft-versus-host disease (GVHD) in allergenic hemopoietic stem cell transplant recipients. Bone Marrow Transplant.2002 Sep; 30(6):375-80.
[18]Brown FG, Nikolic-Paterson DJ, Chadban SJ, et al. Urine macrophage migration inhibitory factor concentrations as a diagnostic tool in human renal allograft rejection. Transplantation.2001,71(12):1777-83.
[19]GH Hou, Anna Valujskikh, Jorg Bayer, et al. In vivo blockade of macrophage migration inhibitory factor prevents skin graft destruction after indirect allorecognition. Transplantation 2001; 72:1890-1897.
[20]Krenskey AM, Weiss A, Crabtree G, et al. T lymphocyte antigen interactions in transplant rejection. N Engl J Med,1990,322(8):510-517.
[21]A. Valujskikh, D. Matesic, A. Gilliam, D. Anthony, T.M. Haqqi, P.S. Heeger, T cells reactive to a single immunodominant selfrestricted allopeptide, J. Clin. Invest. 101 (1998)1398-1407.
[22]A. Valujskikh, D. Matesic, P.S. Heeger, Characterization and manipulation of T cell immunity to skin grafts expressing a transgenic minor antigen, Transplantation 68 (1999) 1029-1036.
[23]A. Valujskikh, P.S. Heeger, CD4+T cells responsive through the indirect pathway can mediate skin graft rejection in the absence of interferongamma, Transplantation 69 (2000) 1016-1019.
[24]Dominique D, Michael S, PastoralM, et al. Molecular diagnosis of renal allograft rejection:correlation with histopathology evaluation and anti-rejection therapy resistance. Transplantation,2004,78(8):647-653.
[25]Corns PA, Kirby JA. A role for cytokine measurement in therapeutic monitoring of immunosuppressive drugs following lung transplantation. Clin Exp Immunol,2005, 131(2):176-178.
[26]Vu MD, Li XC. The common gammac cytokines and transplantation tolerance. Arch Immunol Ther Exp (Warsz),2004,52(4):267-273.
[27]Sayegh M, Watschinger B, Carpenter C. Mechanisms of T cell recognition of alloantigen:the role of peptides. Transplantation.1994; 57:1295.
[28]Luque J, Torres MI, Aumente MD, et al. sHLA-G levels in the monitoring of immunosuppressive therapy and rejection following heart transplantation. Transpl Immunol.2006 Dec; 17 (I):70-73.
[29]Pirenne J. Kitade H, Kawai M, Koshiba T, et al. Regulatory cells, TH1/TH2 unbalance, and antibody-induced chronic rejection in operational tolerance induced by donor-specific blood transfusion. Transplantation.2005 Feb 15:79(3 Suppl):S25 — 7.
[30]Tung TH, Mohanakumar T, Mackinnon SE. TH1/TH2 cytokine profile of the immune response in limb component transplantation. Plast Reconstr Surg.2005 Aug; 116(2):557-566.
[31]Han FH, Zhang ZD, Li YC, et al. The monitoring of Thl/Th2 cytokine expression and its significance to the diagnosis of acute rejection after pancreatic transplantation. Sichuan Da Xue Xue Bao Yi Xue Ban.2006 Jul; 37(4):587-91.
[32]Amirzargar A, Lessanpezeshki M, Fathi A, et al. TH1/TH2 cytokine analysis in Iranian renal transplant recipients. Transplant Proc.2005 Sep; 37(7):2985-7
[33]lakulppi NS,Kyllonen LE, Partanen J, et al. Diagnosis of acute renal allograft rejection by analyzing whole blood mRNA expression of lymphocyte marker molecules. Transplantation.2007 Mar 27;83(6):791-798.
[34]Hu WM, Zhang ZD, Han FH, et al. The experimental research on the perforin and granzyme B expressions to the early diagnostic value of acute rejection response after pancreatic transplantation. Sichuan Da Xue Xue Bao Yi Xue Ban.2007 Jan; 38(1):88-92.
[35]Veale JL, Liang LW. Zhang Q, et al. Noninvasive diagnosis of cellular and antibody mediated rejection by perform and granzyme B in renal allografts. Hum Immunol.2006 Oct;67(10):777-786.
[36]Kosmaczewska A, Magott-Procelewska M, Frydecka I, et al. CD40L, CD28 and CTLA-4 expression on CD4+T cells in kidney graft recipients:a relationship with post-transplantation clinical course. Transpl Immunol.2006 Jun;16(1):32-40. Epub 2006Apr 19.
[37]Graziotto R, Del Prete D, Rigotti P, et al. Perforin Granzyme B, and fas ligand for molecular diagnosis of acute renal-allograft rejection:analyses on serial biopsies suggest methodological issues. Transplantation,2006 Apr 7;81(8):1125—32.
[38]Kiyomoto T, Toyokawa H, Nakao A, Kaizu T, Demetris AJ, Starzl TE, Murase N. The difficulty of eliminating donor leukocyte microchimerism in rat recipients bearing established organ allografts. Transplantation,2006 Feb 15; 81(3):438-44.
[39]Colvin RB. Antibody-mediated renal allograft rejection:diagnosis and pathogenesis. J Am Soc Nephrol.2007 Apr; 18(4):1046-56. Epub 2007 Mar 14.
[40]Mizutani K. Terasaki P, Hamdam E, Esquenazi V, Rosen A, Miller J, Ozawa M. The importance of anti-HLA-Specific antibody strength in monitoring kidney transplant Patients. Am J Transplant.2007 Apr; 7(4):1027-31.
[41]Mao Q, Terasaki PI, Cai J et al. Analysis of HLA class Ⅰspecific antibodies in patients with failed allografts. Transplantation.2007 Jan 15; 83(1):54-61.
[42]Rodrigues FM, de Carvalho MF, Franchi MR, et al. In situ expression of mononuclear cell markers and interleukin-2 receptor in renal allograft biopsies of acute rejection and borderline cases. Ren Fail 2007; 29(l):29-35.
[43]Kaminrska D, Tyran B, Mazanowska O et al. Gene expression of INF-gamma, IL-10, IL-2, IL-6, PDGF-B,TGF-beta in kidney tissue after renal transplantation. Pol Merkur Lekarski.2006 Aug;21(122):148-50.
[44]Engstrand M, Larsson E, Naghibi M, et al. Lymphocyte propagation from biopsies of kidney allografts. Transpl Immunol.2006 Nov; 16(3-4):215-9.
[45]Brandacher G, Cakar F, Winkler C, et al. Non-invasive monitoring of kidney allograft rejection through IDO metabolism evaluation. Kidney Int.2007 Jan; 71(l):60-7.
[46]Kim JH, Eun flW, Lee HJ. Clinical use of renal perfusion imaging by means of harmonic sonography with a microbubble contrast agent in patients after renal transplantation. J Uhra-sound Med,2005,24(6):755-762.
[47]Fischer T, Muhler M, Kroncke TJ, etal. Early postoperative ultrasound of kidney transplants:evaluation of contrast medium dynamics using time-intensity curves. Rofo,2004,176(4):472-477
[48]Zhu W, Xia L, Qi J, et al. MRI and magnetic resonance angiography in evaluating simultaneous pancreas-kidney transplantation. Chin Med J(Eng1),2002, 115(11):1868-1872.
[49]Sadowski EA, Fain SB, Afford SK, et al. Assessment of acute renal transplant rejection with blood oxygen level dependent MR imaging:initial experienc-Radiology,2005,236(3):911-919.
[50]Yang D, Ye Q, Williams DS, et al.Normal and transplanted rat kidney:diffusion MR ima-ging at 7T. Radiology,2004,231(5):702-709.
[51]Johansson L, Johnsson C, Penno E, et al. Acute cardiac transplant rejection: detection and grading with MR imaging with a blood pool contrast agent experimental study in the rat. Radiology,2002,225(1):97-103.
[52]Weller GE, LuE, CsikariMM, et al. Ultrasound imaging of acute cardiac transplant rejection with microbubbles targeted to intercellular adhesion molecule-1. Circulation,2003,108(2):218-224.
[53]KondoI, Ohmori K, OshitaA, etal. Leukocyte-targeted myocardial contrast echocardiography can assess the degree of acute allograft reiection in a rat cardiac transplantation model. Circulation,2004,109(8):1056-1061.
[54]Shah K, Weissleder R. Molecular optical imaging:applications leading to the development of present day therapeutics. NeuroRx,2005,2(2):215-225.
[55]Maxwell D J, Bonde J, Hess D A, et al. Fluorophoreconjugated iron oxide nanoparticle labeling and analysis of engrafting human hematopoietic stem cells [J]. Stem Cells,2008,26(2):517-524.
[56]Rosen A B, Kelly D J, Schuldt A J, et al. Finding fluorescent needles in the cardiac haystack:tracking human mesenchymal stem cells labeled with quantum dots for quantitative in vivo three-dimensional fluorescence analysis [J]. Stem Cells,2007, 25(8):2128-2138.
[57]Modo M, Cash D, Mellodew K, et al. Tracking transplanted stem cell migration using bifunctional, contrast agent enhanced, magnetic resonance imaging. Neuroimage,2002,17(2):803-811.
[58]Kirik D, Breysse N, Bjorklund T, et al. Imaging in cell-based therapy for neurodegenerative diseases. Eur J Nucl Med Mol Imaging,2005,32 (Suppl 2): 417-434.
[59]Miller J C, Trall J H. Clinical molecular imaging. J Am Coll Radiol,2004,1(1 Suppl):4-23.
[60]Doyle B, Kemp B J, Chareonthaita wee P, et al. Dynamic tracking during intracoronary injection of 18F-FDG-labeled progenitor cell therapy for acute myocardial Infarction. J Nucl Med,2007,48(10):1708-1714.
[61]Hofmann M, Wollert K C, Meyer G P, et al. Monitoring of bone marrow cell homing into the infracted human myocardium. Circulation,2005,111 (17):2198-2202.
[62]Stelljes M, Hermann S, Albring J, et al. Clinical molecular imaging in intestinal graft-versus-host disease:mapping of disease activity, prediction and monitoring of treatment efficiency by positron emission tomography.Blood,2008,111:2909-2918.
[63]Jennifer Bell, Innate Immunity MIF:keeps macrophages on guard. Nature Reviews Immunology 2002; 2:70
[64]Bernhagen J, Bacher M, Calandra T, et al. An essential role for macrophage migration inhibitory factor in the tuberculin delayed type hypersensitivity reaction J ExpMed,1996,183; 277.
[65]A. Mikulowska, C.N. Metz, R. Bucala, R. Holmdahl, Macrophage migration inhibitory factor is involved in the pathogenesis of collagen type II-induced arthritis in mice, J. Immunol.158 (1997) 5514-5517.
[66]L.L. Santos, P. Hall, C.N. Metz, R. Bucala, E.F. Morand, Role of macrophage migration inhibitory factor (MIF) in murine antigen-induced arthritis:interaction with glucocorticoids, Clin. Exp. Immunol.123 (2001) 309-314.
[67]H. Ichiyama, S. Onodera, J. Nishihira, et al. Inhibition of joint inflammation and destruction induced by anti-type II collagen antibody/lipopolysaccharide (LPS)-induced arthritis in mice due to deletion of macrophage migration inhibitory factor (MIF), Cytokine 26 (2004) 187-194.
[68]H.Y. Lan, N. Yang, D.J. Nikolic-Paterson, et al. Expression of macrophage migration inhibitory factor in human glomerulonephritis, Kidney Int.57 (2000) 499-509.
[69]Shimizu T, Abe R, Ohkawara A, et al. Macrophage migration inhibitory factor is an essential immunoregulatory cytokine in atopic dermatitis. Biochem Biophys Res Commun,1997; 2409(1):173-178.
[73]T. Toubai, J. Tanaka, J. Nishihira, et al. Effect of macrophage migration inhibitory factor (MIF) on acute graft-versus-host disease in a murine model of allogeneic stem cell transplantation, Transpl. Immunol.16 (2006) 117-124.
[70]Demir Y, Chen Y, Metz C, et al. Cardiac allograft rejection in the absence of macrophage migration inhibitory factor. Transplantation.2003 Jul 15; 76(1):244-7.
[71]S. Fukuyama, I. Yoshino, M. Yamaguchi, A. Osoegawa, T. Kameyama, T. Tagawa, Y. Maehara, Blockage of themacrophage migration inhibitory factor expression by short interference.
[72]Y. Le Meur, M.D. Jose, W. Mu, et al. Macrophage colony-stimulating factor expression and macrophage accumulation in renal allograft rejection, Transplantation 73(2002)1318-1324.
[73]T. Toubai, J. Tanaka, J. Nishihira, et al. Effect of macrophage migration inhibitory factor (MIF) on acute graft-versus-host disease in a murine model of allogeneic stem cell transplantation, Transpl. Immunol.16 (2006) 117-124.
[74]Mather SJ, Ward BG. High efficiency iodination of monoclonalantibodeis for radiotherapy. J Nucl Med,1987,28:1034-1039.
[75]Rudolph NS. Biopharmaceutical production in transgenic live stock,1999; 17:367-374.
[76]Thrall JH and Ziessman HA, nuclear Medicine:The Requisites, Mosby-year Book, Inc, St. Louis, Missour,1994.
[77]Pickett C. Evaluation of storage phosphor imaging systems. Molecular Dynamics Technical.1992. Note#55, Sunnyvale, CA.
[78]A. Shigematsu, N. Motoji, A. Hatori, et al. Progressive application of autoradiography in pharmacokinetic and metabolic studies for the development of new drugs. Regul Toxicol Pharmacol.1995; 22:122-142.
[79]Bergstrom M, Awad R, Estrada S, et al. Autoradiography with positron emitting isotopes in positron emission tomography tracer discovery. Mol Imag Biol.2004; 5:390-396.
[80]Johnston RF, Pickett SC, Barker DL.Autoradiography using storage phosphor technology. Electrophoresis.1990; 11:355-360.
[81]Anderson KV. Toll signaling pathways in the innate immune response. Curr Opin Immunol,2000,12(1):13-19.
[82]Faure E, Thomas L, Xu H, et al. Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells:role of NF-kappa B activation. J Immunol,2001,166 (3):2018-2024.
[83]Medzhitov R, Preston HP, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature,1997,388(6 640):394-397.
[84]Akira S, Takeda K. Toll-like receptor signaling. Nat Rev Immunol,2004, 4(7):499-511.
[85]王海坤,韩代书.Toll样受体(TLRs)的信号转导与免疫调节术.生物化学与生物物理进展Progress in Biochemistry and hysics,2006,33(9):820-827.
[86]Gay, N. J., and M. Gangloff. Structure and Function of Toll Receptors and Their Ligands. Annus Rev Biochem.2007.
[87]Lee, M. S., and Y. J. Kim. Signaling Pathways Downstream of Pattern-Recognition Receptors and Their Cross Talk. Annu Rev Biochem.2007.
[88]Beutler, B.,Z. Jiang, P.Georgel,K Crozat,B. Croker, S. Rutschmann, X. Du, and K. Hoebe.2006. Genetic analysis of host resistance:Toll-like receptor signaling and immunity at large. Annu Rev Immunol,24:353-389.
[89]钱程,安华章. Toll样受体在适应性免疫中的作用与相关机制研究进展.《国外医学》免疫学分册,2005,28(2)：16-19.
[90]Muzio M, Bosisio D, Polentarutti N, et al. Differential expression and regulation of Toll-like receptors (TLR) in human leukocytes:Selective expression of TLR3 in dendritic cells. J Immunol,2000,164(11):5998-6004.
[92]Goldstein DR, Tesar BM, Akira S, et al. Critical role of the Tolllike receptor signal adaptor protein MyD88 in acute allograft rejection. J Clin Invest,2003,111 (10):1571-1578.
[91]Samstein B, Johnson GB, Platt JL, et al. Toll like receptor 4 and allograft responses. Transplantation,2004.77(3):475-477.
[93]Tesar BM, Zhang J, Li Q, et al. TH1 immune responses to fully MHC mismatched allografts are diminished in the absence of MyD88, a toll like receptor signal adaptor protein[J]. Am J Transplant,2004,4(9):1429-1439.
[94]Janeway CA Jr, Medzhitov R. Introduction:the role of innate immunity in the adaptive immune response. Semin Immunol,1998,10(5):349-350.
[95]Akira S., S. Uematsu, and O. Takeuchi. Pathogen recognition and innate immunity. Cell,2006; 24:783-801.
[96]Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat immunol,2001.2(10):947-950.
[97]O'Neill, How Toll-like receptors signal:what we know and what we don't know. Curr Opin Immunol,2006,18:3-9.
[98]Moynagh, P. N. TLR signaling and activation of IRFs:revisiting old friends from the NF-kappaB pathway. Trends Immunol.2005,26:469-476.
[99]Takeuchi O, Akira S. Toll-like receptors; their physiological role and signal transduction system. Inte Immunopharmacol,2001,1(4) 625-635.
[100]Kupiec Weglinski JW, Busuttil RW. Ischemia and reperfusion injury in liver transplantation. Transplant Proc,2005,37(4):1653-1656.
[101]Charron D. Immunogenomics of hematopoietic stem cell transplantation Pathol iol(Paris),2005,53(3):171-173.
[102]Lorenz E, Schwartz DA, Martin PJ, et al. Association of TLR4 mutations and the risk for acute GVHD after HLA matched sibling hematopoietic stem cell transplantation. Biol Blood Marrow Transplant,2001,7(7):384-387.
[103]Porrett PM, Yuan X, LaRosa DF, et al. Mechanisms underlying blockade of allograft acceptance by TLR ligands. J Immunol.2008 Aug 1;181(3):1692-9.
[104]Methe H, Zimmer E, Grimm C, et al. Evidence for a role of Toll like receptor 4 in development of chronic allograft rejection after cardiac transplantation. Transplantation,2004,78(9):1324-1331.
[105]Roger P.M, Mary E, Mihai G. Toll-like receptors on regulatory T cells: expanding immune regulation. Trends in immunology,2006,27(8):387-393.
[106]Natasha K, Rosa V, Omeed H, et al. Human CD4+T Cells Express TLR5 and Its Ligand Flagellin Enhances the Suppressive Capacity and Expression of FOXP3 in CD4+CD25+T Regulatory Cells. J Immunol,2005,175:8051-8059.
[107]Morris R E, Meiser BM. Identification of a new pharmacologic actin for an old compound. Med Sci Res,1989,17:609-610.
[108]Wiederrecht GJ, Sabers CJ, Brunn GJ, et al. Mechansim of action of rapamycin: new insights into the regulation of Gl-hase progression in eukaryotic cells. Prog Cell Cycle Res,1995; 1:53-71.
[109]Manuela B, Angela S, Barbara M, et al. Rapamycin promotes expansion of functional CD4+CD25+FOXP3+regulatory T cells of both healthy subjects and type 1 diabetic patients. J Immunol,2006,177:8338-47.
[110]Zheng YX, Hou GH, Song J, et al. Effect of Rapamycin on CD4(+)CD25(+)regulatory T cells in allo-transplantation tolerance model. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi.2007 Apr; 23(4):327-30.
[111]Hayashi F, Smith K D, Ozinshky A O, et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature,2001,410 (6832):1099-1103.
[112]Andersen Nissen E, H awn TR, Smith KD, et al. Cutting edge:TLR5-/-mice are more susceptible to Escherichia coliurinary tract infection. J Immunol,2007,178: 4717-4720.
[113]Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity[J]. Cell,2006,124(4):783-801.
[114]Sun J, Fegan PE, Desai AS, et al. Flagellin induced tolerance of the Toll like receptor 5 signaling pathway in polarized intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol,2007,292:G767-778.
[2]Andrew BA, Christian PL, Thomas CP. Costimulation blockade and tolerance. Curr Opin Organ Transplantation,2002,7 (1):7-12.
[3]Jun ichi S, Mitsuaki I, Atsushi I, et al. Differential Thl and Th2 cell regulation of marine cardiac allogaft acceptance by blocking cell adhesion of ICAM-1/LFA-1 andVCANt-1/I/LA-4. Transpl Immunol,1999,7 (1):65-72.
[4]Steiniger B, Stehling O, Scriba A, et al. Monocytes in the rat:phenotype and function during acute allograft rejection. Immunol Rev.2001 Dec; 184:38-44.
[5]Weissleder R. Molecular imaging:exploring the new frontiers. Radiology,1999; 212(3):609-614
[6]M. Isobe, J.F. Southern, Y. Yazaki, Scintigraphic detection of early cardiac rejection by iodine 123-labeled monoclonal antibody directed against monomorphic determinant of major histocompatibility complex class Ⅱ antigens, Am. Heart J.127 (1994)1309-1317.
[7]M. Isobe, E. Haber, B.A. Khaw, Early detection of rejection and assessment of cyclosporine therapy by 111In antimyosin imaging in mouse heart allografts, Circulation 84 (1991) 1246-1255.
[8]A.I. McGhie, B. Radovancevic, P. Capek, W.H. Moore, L. Kasi, L. Lamki, F.J. Clubb Jr., O.H. Frazier, J.T. Willerson, Major histocompatibility complex class Ⅱ antigen expression in rejecting cardiac allografts:detection using in vivo imaging with radiolabeled monoclonal antibody, Circulation 96 (1997) 1605-1611.
[9]M.H. Kown, C.L. Jahncke, M.A. Lijkwan, M.L. Koransky, C. Mari, G.J. Berry, F.G. Blankenberg, H.W. Strauss, R.C. Robbins, The use of (99 m) technetium-labeled MCP-1 to assess graft coronary artery disease in rat cardiac allografts, J. Heart Lung Transplant.21 (2002) 1009-1015.
[10]T. Calandra, J. Bernhagen, C.N. Metz, et al. MIF as a glucocorticoidinduced modulator of cytokine production, Nature 377 (1995) 68-71.
[11]Calendra T, Bernhagen J, Mitchell RA, et al. The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J Exp Med,1994,79:1895
[12]Christine N, Richard Bucala. Role of macrophage migration inhibitory factor in the regulation of the immune response. Advances in Immunology 1997; 66:197-223.
[13]M. Bacher, C.N. Metz, T. Calandra, K. Mayer, J. Chesney, M. Lohoff, D. Gemsa, T. Donnelly, R. Bucala, An essential regulatory role for macrophage migration inhibitory factor in T-cell activation, Proc. Natl Acad. Sci. USA 93 (1996) 7849-7854.
[14]Lue H, Kleemann R, Calandra T, et al. Macrophage migration inhibitory factor MIF:mechanisms of action and role in disease. Microbes Infect 2002 Apr;(4): 449-460.
[15]Brown FC, Nikolic-Paterson DJ, Metz C, Bucala R, Atkins RC. Up-regulation of macrophage migration inhibitory factor in acute renal allograft rejection in the rat. Clin Exp Immunol 1999; 118(2):329.
[16]Lan HY, Yang N, Brown FG, et al. Macrophage migration inhibitory factor expression in human renal allograft rejection. Transplantation 1998; 66(11):1465
[17]Lo JW, Leung AY, Huang XR, Macrophage migratory inhibitory factor (MIF) expression in acute graft-versus-host disease (GVHD) in allergenic hemopoietic stem cell transplant recipients. Bone Marrow Transplant.2002 Sep; 30(6):375-80.
[18]Brown FG, Nikolic-Paterson DJ, Chadban SJ, et al. Urine macrophage migration inhibitory factor concentrations as a diagnostic tool in human renal allograft rejection. Transplantation.2001,71(12):1777-83.
[19]GH Hou, Anna Valujskikh, Jorg Bayer, et al. In vivo blockade of macrophage migration inhibitory factor prevents skin graft destruction after indirect allorecognition. Transplantation 2001; 72:1890-1897.
[20]Krenskey AM, Weiss A, Crabtree G, et al. T lymphocyte antigen interactions in transplant rejection. N Engl J Med,1990,322(8):510-517.
[21]A. Valujskikh, D. Matesic, A. Gilliam, D. Anthony, T.M. Haqqi, P.S. Heeger, T cells reactive to a single immunodominant selfrestricted allopeptide, J. Clin. Invest. 101 (1998)1398-1407.
[22]A. Valujskikh, D. Matesic, P.S. Heeger, Characterization and manipulation of T cell immunity to skin grafts expressing a transgenic minor antigen, Transplantation 68 (1999) 1029-1036.
[23]A. Valujskikh, P.S. Heeger, CD4+T cells responsive through the indirect pathway can mediate skin graft rejection in the absence of interferongamma, Transplantation 69 (2000) 1016-1019.
[24]Dominique D, Michael S, PastoralM, et al. Molecular diagnosis of renal allograft rejection:correlation with histopathology evaluation and anti-rejection therapy resistance. Transplantation,2004,78(8):647-653.
[25]Corns PA, Kirby JA. A role for cytokine measurement in therapeutic monitoring of immunosuppressive drugs following lung transplantation. Clin Exp Immunol,2005, 131(2):176-178.
[26]Vu MD, Li XC. The common gammac cytokines and transplantation tolerance. Arch Immunol Ther Exp (Warsz),2004,52(4):267-273.
[27]Sayegh M, Watschinger B, Carpenter C. Mechanisms of T cell recognition of alloantigen:the role of peptides. Transplantation.1994; 57:1295.
[28]Luque J, Torres MI, Aumente MD, et al. sHLA-G levels in the monitoring of immunosuppressive therapy and rejection following heart transplantation. Transpl Immunol.2006 Dec; 17 (I):70-73.
[29]Pirenne J. Kitade H, Kawai M, Koshiba T, et al. Regulatory cells, TH1/TH2 unbalance, and antibody-induced chronic rejection in operational tolerance induced by donor-specific blood transfusion. Transplantation.2005 Feb 15:79(3 Suppl):S25 — 7.
[30]Tung TH, Mohanakumar T, Mackinnon SE. TH1/TH2 cytokine profile of the immune response in limb component transplantation. Plast Reconstr Surg.2005 Aug; 116(2):557-566.
[31]Han FH, Zhang ZD, Li YC, et al. The monitoring of Thl/Th2 cytokine expression and its significance to the diagnosis of acute rejection after pancreatic transplantation. Sichuan Da Xue Xue Bao Yi Xue Ban.2006 Jul; 37(4):587-91.
[32]Amirzargar A, Lessanpezeshki M, Fathi A, et al. TH1/TH2 cytokine analysis in Iranian renal transplant recipients. Transplant Proc.2005 Sep; 37(7):2985-7
[33]lakulppi NS,Kyllonen LE, Partanen J, et al. Diagnosis of acute renal allograft rejection by analyzing whole blood mRNA expression of lymphocyte marker molecules. Transplantation.2007 Mar 27;83(6):791-798.
[34]Hu WM, Zhang ZD, Han FH, et al. The experimental research on the perforin and granzyme B expressions to the early diagnostic value of acute rejection response after pancreatic transplantation. Sichuan Da Xue Xue Bao Yi Xue Ban.2007 Jan; 38(1):88-92.
[35]Veale JL, Liang LW. Zhang Q, et al. Noninvasive diagnosis of cellular and antibody mediated rejection by perform and granzyme B in renal allografts. Hum Immunol.2006 Oct;67(10):777-786.
[36]Kosmaczewska A, Magott-Procelewska M, Frydecka I, et al. CD40L, CD28 and CTLA-4 expression on CD4+T cells in kidney graft recipients:a relationship with post-transplantation clinical course. Transpl Immunol.2006 Jun;16(1):32-40. Epub 2006Apr 19.
[37]Graziotto R, Del Prete D, Rigotti P, et al. Perforin Granzyme B, and fas ligand for molecular diagnosis of acute renal-allograft rejection:analyses on serial biopsies suggest methodological issues. Transplantation,2006 Apr 7;81(8):1125—32.
[38]Kiyomoto T, Toyokawa H, Nakao A, Kaizu T, Demetris AJ, Starzl TE, Murase N. The difficulty of eliminating donor leukocyte microchimerism in rat recipients bearing established organ allografts. Transplantation,2006 Feb 15; 81(3):438-44.
[39]Colvin RB. Antibody-mediated renal allograft rejection:diagnosis and pathogenesis. J Am Soc Nephrol.2007 Apr; 18(4):1046-56. Epub 2007 Mar 14.
[40]Mizutani K. Terasaki P, Hamdam E, Esquenazi V, Rosen A, Miller J, Ozawa M. The importance of anti-HLA-Specific antibody strength in monitoring kidney transplant Patients. Am J Transplant.2007 Apr; 7(4):1027-31.
[41]Mao Q, Terasaki PI, Cai J et al. Analysis of HLA class Ⅰspecific antibodies in patients with failed allografts. Transplantation.2007 Jan 15; 83(1):54-61.
[42]Rodrigues FM, de Carvalho MF, Franchi MR, et al. In situ expression of mononuclear cell markers and interleukin-2 receptor in renal allograft biopsies of acute rejection and borderline cases. Ren Fail 2007; 29(l):29-35.
[43]Kaminrska D, Tyran B, Mazanowska O et al. Gene expression of INF-gamma, IL-10, IL-2, IL-6, PDGF-B,TGF-beta in kidney tissue after renal transplantation. Pol Merkur Lekarski.2006 Aug;21(122):148-50.
[44]Engstrand M, Larsson E, Naghibi M, et al. Lymphocyte propagation from biopsies of kidney allografts. Transpl Immunol.2006 Nov; 16(3-4):215-9.
[45]Brandacher G, Cakar F, Winkler C, et al. Non-invasive monitoring of kidney allograft rejection through IDO metabolism evaluation. Kidney Int.2007 Jan; 71(l):60-7.
[46]Kim JH, Eun flW, Lee HJ. Clinical use of renal perfusion imaging by means of harmonic sonography with a microbubble contrast agent in patients after renal transplantation. J Uhra-sound Med,2005,24(6):755-762.
[47]Fischer T, Muhler M, Kroncke TJ, etal. Early postoperative ultrasound of kidney transplants:evaluation of contrast medium dynamics using time-intensity curves. Rofo,2004,176(4):472-477
[48]Zhu W, Xia L, Qi J, et al. MRI and magnetic resonance angiography in evaluating simultaneous pancreas-kidney transplantation. Chin Med J(Eng1),2002, 115(11):1868-1872.
[49]Sadowski EA, Fain SB, Afford SK, et al. Assessment of acute renal transplant rejection with blood oxygen level dependent MR imaging:initial experienc-Radiology,2005,236(3):911-919.
[50]Yang D, Ye Q, Williams DS, et al.Normal and transplanted rat kidney:diffusion MR ima-ging at 7T. Radiology,2004,231(5):702-709.
[51]Johansson L, Johnsson C, Penno E, et al. Acute cardiac transplant rejection: detection and grading with MR imaging with a blood pool contrast agent experimental study in the rat. Radiology,2002,225(1):97-103.
[52]Weller GE, LuE, CsikariMM, et al. Ultrasound imaging of acute cardiac transplant rejection with microbubbles targeted to intercellular adhesion molecule-1. Circulation,2003,108(2):218-224.
[53]KondoI, Ohmori K, OshitaA, etal. Leukocyte-targeted myocardial contrast echocardiography can assess the degree of acute allograft reiection in a rat cardiac transplantation model. Circulation,2004,109(8):1056-1061.
[54]Shah K, Weissleder R. Molecular optical imaging:applications leading to the development of present day therapeutics. NeuroRx,2005,2(2):215-225.
[55]Maxwell D J, Bonde J, Hess D A, et al. Fluorophoreconjugated iron oxide nanoparticle labeling and analysis of engrafting human hematopoietic stem cells [J]. Stem Cells,2008,26(2):517-524.
[56]Rosen A B, Kelly D J, Schuldt A J, et al. Finding fluorescent needles in the cardiac haystack:tracking human mesenchymal stem cells labeled with quantum dots for quantitative in vivo three-dimensional fluorescence analysis [J]. Stem Cells,2007, 25(8):2128-2138.
[57]Modo M, Cash D, Mellodew K, et al. Tracking transplanted stem cell migration using bifunctional, contrast agent enhanced, magnetic resonance imaging. Neuroimage,2002,17(2):803-811.
[58]Kirik D, Breysse N, Bjorklund T, et al. Imaging in cell-based therapy for neurodegenerative diseases. Eur J Nucl Med Mol Imaging,2005,32 (Suppl 2): 417-434.
[59]Miller J C, Trall J H. Clinical molecular imaging. J Am Coll Radiol,2004,1(1 Suppl):4-23.
[60]Doyle B, Kemp B J, Chareonthaita wee P, et al. Dynamic tracking during intracoronary injection of 18F-FDG-labeled progenitor cell therapy for acute myocardial Infarction. J Nucl Med,2007,48(10):1708-1714.
[61]Hofmann M, Wollert K C, Meyer G P, et al. Monitoring of bone marrow cell homing into the infracted human myocardium. Circulation,2005,111 (17):2198-2202.
[62]Stelljes M, Hermann S, Albring J, et al. Clinical molecular imaging in intestinal graft-versus-host disease:mapping of disease activity, prediction and monitoring of treatment efficiency by positron emission tomography.Blood,2008,111:2909-2918.
[63]Jennifer Bell, Innate Immunity MIF:keeps macrophages on guard. Nature Reviews Immunology 2002; 2:70
[64]Bernhagen J, Bacher M, Calandra T, et al. An essential role for macrophage migration inhibitory factor in the tuberculin delayed type hypersensitivity reaction J ExpMed,1996,183; 277.
[65]A. Mikulowska, C.N. Metz, R. Bucala, R. Holmdahl, Macrophage migration inhibitory factor is involved in the pathogenesis of collagen type II-induced arthritis in mice, J. Immunol.158 (1997) 5514-5517.
[66]L.L. Santos, P. Hall, C.N. Metz, R. Bucala, E.F. Morand, Role of macrophage migration inhibitory factor (MIF) in murine antigen-induced arthritis:interaction with glucocorticoids, Clin. Exp. Immunol.123 (2001) 309-314.
[67]H. Ichiyama, S. Onodera, J. Nishihira, et al. Inhibition of joint inflammation and destruction induced by anti-type II collagen antibody/lipopolysaccharide (LPS)-induced arthritis in mice due to deletion of macrophage migration inhibitory factor (MIF), Cytokine 26 (2004) 187-194.
[68]H.Y. Lan, N. Yang, D.J. Nikolic-Paterson, et al. Expression of macrophage migration inhibitory factor in human glomerulonephritis, Kidney Int.57 (2000) 499-509.
[69]Shimizu T, Abe R, Ohkawara A, et al. Macrophage migration inhibitory factor is an essential immunoregulatory cytokine in atopic dermatitis. Biochem Biophys Res Commun,1997; 2409(1):173-178.
[73]T. Toubai, J. Tanaka, J. Nishihira, et al. Effect of macrophage migration inhibitory factor (MIF) on acute graft-versus-host disease in a murine model of allogeneic stem cell transplantation, Transpl. Immunol.16 (2006) 117-124.
[70]Demir Y, Chen Y, Metz C, et al. Cardiac allograft rejection in the absence of macrophage migration inhibitory factor. Transplantation.2003 Jul 15; 76(1):244-7.
[71]S. Fukuyama, I. Yoshino, M. Yamaguchi, A. Osoegawa, T. Kameyama, T. Tagawa, Y. Maehara, Blockage of themacrophage migration inhibitory factor expression by short interference.
[72]Y. Le Meur, M.D. Jose, W. Mu, et al. Macrophage colony-stimulating factor expression and macrophage accumulation in renal allograft rejection, Transplantation 73(2002)1318-1324.
[73]T. Toubai, J. Tanaka, J. Nishihira, et al. Effect of macrophage migration inhibitory factor (MIF) on acute graft-versus-host disease in a murine model of allogeneic stem cell transplantation, Transpl. Immunol.16 (2006) 117-124.
[74]Mather SJ, Ward BG. High efficiency iodination of monoclonalantibodeis for radiotherapy. J Nucl Med,1987,28:1034-1039.
[75]Rudolph NS. Biopharmaceutical production in transgenic live stock,1999; 17:367-374.
[76]Thrall JH and Ziessman HA, nuclear Medicine:The Requisites, Mosby-year Book, Inc, St. Louis, Missour,1994.
[77]Pickett C. Evaluation of storage phosphor imaging systems. Molecular Dynamics Technical.1992. Note#55, Sunnyvale, CA.
[78]A. Shigematsu, N. Motoji, A. Hatori, et al. Progressive application of autoradiography in pharmacokinetic and metabolic studies for the development of new drugs. Regul Toxicol Pharmacol.1995; 22:122-142.
[79]Bergstrom M, Awad R, Estrada S, et al. Autoradiography with positron emitting isotopes in positron emission tomography tracer discovery. Mol Imag Biol.2004; 5:390-396.
[80]Johnston RF, Pickett SC, Barker DL.Autoradiography using storage phosphor technology. Electrophoresis.1990; 11:355-360.
[81]Anderson KV. Toll signaling pathways in the innate immune response. Curr Opin Immunol,2000,12(1):13-19.
[82]Faure E, Thomas L, Xu H, et al. Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells:role of NF-kappa B activation. J Immunol,2001,166 (3):2018-2024.
[83]Medzhitov R, Preston HP, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature,1997,388(6 640):394-397.
[84]Akira S, Takeda K. Toll-like receptor signaling. Nat Rev Immunol,2004, 4(7):499-511.
[85]王海坤,韩代书.Toll样受体(TLRs)的信号转导与免疫调节术.生物化学与生物物理进展Progress in Biochemistry and hysics,2006,33(9):820-827.
[86]Gay, N. J., and M. Gangloff. Structure and Function of Toll Receptors and Their Ligands. Annus Rev Biochem.2007.
[87]Lee, M. S., and Y. J. Kim. Signaling Pathways Downstream of Pattern-Recognition Receptors and Their Cross Talk. Annu Rev Biochem.2007.
[88]Beutler, B.,Z. Jiang, P.Georgel,K Crozat,B. Croker, S. Rutschmann, X. Du, and K. Hoebe.2006. Genetic analysis of host resistance:Toll-like receptor signaling and immunity at large. Annu Rev Immunol,24:353-389.
[89]钱程,安华章. Toll样受体在适应性免疫中的作用与相关机制研究进展.《国外医学》免疫学分册,2005,28(2)：16-19.
[90]Muzio M, Bosisio D, Polentarutti N, et al. Differential expression and regulation of Toll-like receptors (TLR) in human leukocytes:Selective expression of TLR3 in dendritic cells. J Immunol,2000,164(11):5998-6004.
[92]Goldstein DR, Tesar BM, Akira S, et al. Critical role of the Tolllike receptor signal adaptor protein MyD88 in acute allograft rejection. J Clin Invest,2003,111 (10):1571-1578.
[91]Samstein B, Johnson GB, Platt JL, et al. Toll like receptor 4 and allograft responses. Transplantation,2004.77(3):475-477.
[93]Tesar BM, Zhang J, Li Q, et al. TH1 immune responses to fully MHC mismatched allografts are diminished in the absence of MyD88, a toll like receptor signal adaptor protein[J]. Am J Transplant,2004,4(9):1429-1439.
[94]Janeway CA Jr, Medzhitov R. Introduction:the role of innate immunity in the adaptive immune response. Semin Immunol,1998,10(5):349-350.
[95]Akira S., S. Uematsu, and O. Takeuchi. Pathogen recognition and innate immunity. Cell,2006; 24:783-801.
[96]Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat immunol,2001.2(10):947-950.
[97]O'Neill, How Toll-like receptors signal:what we know and what we don't know. Curr Opin Immunol,2006,18:3-9.
[98]Moynagh, P. N. TLR signaling and activation of IRFs:revisiting old friends from the NF-kappaB pathway. Trends Immunol.2005,26:469-476.
[99]Takeuchi O, Akira S. Toll-like receptors; their physiological role and signal transduction system. Inte Immunopharmacol,2001,1(4) 625-635.
[100]Kupiec Weglinski JW, Busuttil RW. Ischemia and reperfusion injury in liver transplantation. Transplant Proc,2005,37(4):1653-1656.
[101]Charron D. Immunogenomics of hematopoietic stem cell transplantation Pathol iol(Paris),2005,53(3):171-173.
[102]Lorenz E, Schwartz DA, Martin PJ, et al. Association of TLR4 mutations and the risk for acute GVHD after HLA matched sibling hematopoietic stem cell transplantation. Biol Blood Marrow Transplant,2001,7(7):384-387.
[103]Porrett PM, Yuan X, LaRosa DF, et al. Mechanisms underlying blockade of allograft acceptance by TLR ligands. J Immunol.2008 Aug 1;181(3):1692-9.
[104]Methe H, Zimmer E, Grimm C, et al. Evidence for a role of Toll like receptor 4 in development of chronic allograft rejection after cardiac transplantation. Transplantation,2004,78(9):1324-1331.
[105]Roger P.M, Mary E, Mihai G. Toll-like receptors on regulatory T cells: expanding immune regulation. Trends in immunology,2006,27(8):387-393.
[106]Natasha K, Rosa V, Omeed H, et al. Human CD4+T Cells Express TLR5 and Its Ligand Flagellin Enhances the Suppressive Capacity and Expression of FOXP3 in CD4+CD25+T Regulatory Cells. J Immunol,2005,175:8051-8059.
[107]Morris R E, Meiser BM. Identification of a new pharmacologic actin for an old compound. Med Sci Res,1989,17:609-610.
[108]Wiederrecht GJ, Sabers CJ, Brunn GJ, et al. Mechansim of action of rapamycin: new insights into the regulation of Gl-hase progression in eukaryotic cells. Prog Cell Cycle Res,1995; 1:53-71.
[109]Manuela B, Angela S, Barbara M, et al. Rapamycin promotes expansion of functional CD4+CD25+FOXP3+regulatory T cells of both healthy subjects and type 1 diabetic patients. J Immunol,2006,177:8338-47.
[110]Zheng YX, Hou GH, Song J, et al. Effect of Rapamycin on CD4(+)CD25(+)regulatory T cells in allo-transplantation tolerance model. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi.2007 Apr; 23(4):327-30.
[111]Hayashi F, Smith K D, Ozinshky A O, et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature,2001,410 (6832):1099-1103.
[112]Andersen Nissen E, H awn TR, Smith KD, et al. Cutting edge:TLR5-/-mice are more susceptible to Escherichia coliurinary tract infection. J Immunol,2007,178: 4717-4720.
[113]Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity[J]. Cell,2006,124(4):783-801.
[114]Sun J, Fegan PE, Desai AS, et al. Flagellin induced tolerance of the Toll like receptor 5 signaling pathway in polarized intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol,2007,292:G767-778.