广州管圆线虫感染免疫中ICOS-ICOSL通路及发育期蛋白的分析和研究
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摘要
研究背景：广州管圆线虫是一种人兽共患寄生虫。其幼虫阶段可侵入非正常宿主——人，引起中枢神经系统的病变。
在感染免疫中抗原由抗原提呈细胞(APC)处理成多肽，它与MHC结合并移至APC表面，产生活化TCR信号；而APC或靶细胞表面的共刺激分子与T淋巴细胞表面的有关受体结合就产生第二活化信号，即协同刺激信号。在双信号刺激下，T淋巴细胞被激活后转化为淋巴母细胞，并迅速增殖、分化。CD28-B7共刺激途径是研究最早最深入的共刺激途径之一,但CD28并不是惟一的共刺激分子。可诱导共刺激因子（inducible co-stimulator, ICOS）为CD28家族的新成员，主要表达于活化的Ｔ细胞；其配体ICOSL主要表达于B细胞、单核细胞、树突状细胞和T细胞。
此外，寄生虫需要通过改变宿主来完成其生活史，因此，他们有根据宿主环境来调节基因的差异表达的能力。广州管圆线虫生活史的完成需要几个不同的宿主。Ⅲ期幼虫（L3）感染大鼠后，穿过血脑屏障发展成Ⅴ期幼虫（L5），再移行至心、肺部血管内发育成成虫。
本研究首先应用ICOSL-KO和C57BL/6J两种小鼠建立广州管圆线虫感染的模型，观察ICOS-ICOSL信号通路在广州管圆线虫感染免疫调节中的意义；其次，运用双向荧光差异凝胶电泳和质谱相结合的蛋白质组学技术分析鉴定广州管圆线虫发育期特异性蛋白质并分析他们在宿主-寄生虫相互作用中的意义。本研究结果可为广州管圆线虫感染的发展和消退研究提供新的思路，还为广州管圆线虫病的治疗提供可能性靶标。
目的：通过观察两种广州管圆线虫感染宿主模型（ICOSL-KO和C57BL/6J）血清抗体、脾淋巴细胞表面协同刺激因子及脾淋巴细胞分泌细胞因子的动态变化，探索ICOS-ICOSL信号在广州管圆线虫感染免疫调节中的意义。
方法：实验室条件下构建广州管圆线虫感染的ICOSL-KO和C57BL/6J小鼠模型。分别于感染前、感染后1周、2周、3周、4周，分批处死小鼠每批每组各取5只小鼠，眼眶取血，收集血清，ELISA法测定血清中IgG、IgG1和IgG2a的水平。同时分离不同感染时期小鼠脾淋巴细胞悬液，流式细胞仪测定CD4+T淋巴细胞上CD28、CD154和ICOS的表达，其结果用SPSS17.0采用t检验和F检验分析其统计学意义。体外培养脾CD4+T淋巴细胞。加入广州管圆线虫L5可溶性抗原干预共刺激信号。培养72小时后，收集细胞上清。ELISA法测定上清中IFN-γ、IL-12、IL-4、IL-5和IL-13等细胞因子表达水平。
结果：ICOSL-KO组与C57BL/6J组在感染后均表现为IgG抗体水平的升高；前者IgG1水平在感染后升高不显著，但后者的IgG1水平相对在感染后3W开始出现升高，4W时仍维持升高；同时C57BL/6J小鼠的IgG2a水平在感染后升高不显著，并于3W开始出现下降，反观ICOSL-KO小鼠IgG2a水平在感染后3W开始出现升高，4W时仍保持升高状态。ICOSL-KO组与C57BL/6J组相比IgG1动态趋势及IgG2a水平升高趋势，显示ICOS-ICOSL信号通路的阻断通过抑制Th2的极化使宿主无法组织产生有效的体液保护性免疫，ICOSL-KO小鼠表现为Th1的的免疫类型占优势。感染后ICOSL-KO组的CD4+T淋巴细胞上协同刺激分子产生也明显滞后，感染后3W才出现显著性差异；C57BL/6组针对广州管圆线虫急性感染，感染后1W即出现显著性差异。C57BL/6J组感染早期出现IFN-γ、IL-12增高现象，而后下降，提示感染2W内Th1免疫应答水平占优势，随之逐渐被Th2免疫应答水平取代；而ICOSL-KO组Th1型细胞因子IFN-γ、IL-12的早期不增高，表明ICOS-ICOSL信号通路也参与到感染免疫早期的Th1型免疫的活化过程。在感染早期Th2型细胞因子IL-4和IL-5受ICOS-ICOSL信号通路影响不大。IL-13在C57BL/6J小鼠感染后3W后出现增高，IL-13在ICOSL-KO小鼠出现延后升高，两种动物感染模型动态检测的显著性差异，也说明了ICOS-ICOSL通路阻断对Th2型免疫应答水平的调控作用。
结论：在广州管圆线虫的急性感染期，Th2型的免疫方式在免疫应答过程中占优势；ICOS-ICOSL通路调节作用既能延迟Th2免疫反应的进行，也具有调节Th1型免疫活化的能力。
目的：通过研究广州管圆线虫期特异性差异蛋白，可以为宿主环境在寄生虫发育过程中的调控或宿主-寄生虫相互作用中的关键蛋白质提供实验依据。
方法：实验室条件下建立广州管圆线虫生活史，分别于感染的不同时期收集广州管圆线虫L3、FL5、ML5、FA和MA。将收集的虫体分别使用裂解液裂解、超声裂解、离心，获得的上清，使用Bio-Rad protein assay reagent测定蛋白质浓度。分别用Cy3或Cy5标记样品，Cy2标记内标。进行双向荧光差异凝胶电泳，并利用UMax Powerlook2110XL和Typhoon FLA9000对电泳后的胶板图像扫描，得到不同颜色的扫描图。采用DeCyder7.0图像分析筛选差异表达蛋白谱，继而对候选蛋白点进行MALDI-TOF-MS鉴定、数据库检索。用Real-time PCR方法进行验证。
结果：双向荧光差异凝胶电泳得到了清晰的蛋白质表达谱。蛋白表达差异值经统计学分析(t检验)，筛选出在24张图谱中均出现的点共183个蛋白点。5点在L5中上调，而在成虫期下调且没有表现出显著的性别差异；与L3比，89个蛋白点在成虫和L5中上调，而13个蛋白点在L3中出现明显上调；另一方面，实验中还发现有些性别差异表达的蛋白，雌雄成虫比对中，12个蛋白点在MA中表达上调而7个蛋白点在FA中表达上调；L5中各有3个蛋白在FL5、ML5中表达上调，还有点1496在MA和ML5均表达增加，表现为性别特异性。
质谱鉴定80个点，成功鉴定了其中37个蛋白质点，29个点是功能蛋白，从细胞骨架相关蛋白到参与代谢过程、免疫反应和转录调控相关等相关过程，8个点是未命名蛋白。此外，还有12个蛋白点与6个广州管圆线虫EST登录序列有较高可信度。定量PCR验证实验扩增出的两个蛋白点的基因609和1004，并发现结果与DIGE研究结果一致。
结论：这些生活史期特异性蛋白质有助于提高对寄生虫在宿主调控或宿主-寄生虫相互作用关系中的理解，也能为广州管圆线虫感染的发展和消退提供新的思路，还为广州管圆线虫病的治疗提供靶标。
Angiostrongyliasis cantonesis is a human zoonotic parasitic disease, results frominfections with the nematodes that A.cantonensis accidentally infects humans andmigrates to the central nervous system.
Specifically, infections with A.cantonensis cause eosinophilic meningitis andeosinophilic enteritis. T cells require two signals to become fully activated. A first signal,which is antigen-specific, is provided through the T cell receptor which produced byA.cantonensis with peptide-MHC molecules on the membrane of antigen presenting cells(APC). A second signal, the co-stimulatory signal, is antigen provided by theA.cantonensis between co-stimulatory molecules expressed on the membrane of APCand the T cell. One of the best costimulatory molecules expressed by T cells is CD28,which interacts with CD80(B7.1) and CD86(B7.2) on the membrane of APC. Anothercostimulatory receptor expressed by T cells is ICOS, which interacts with ICOSL.
In addition, when parasites changed their hosts to finish their life cycle, theyrequire the ability to respond to the host environments and regulate differential geneexpression. In the life cycle of A.cantonensis, adult worms reside in normal host（rats），which discharging the L1after infection. Widely disseminated via water, L1enter theintermediate host (such as Pomacantonensisea canaliculata), and develop into L3. L3infects rats, penetrates blood-brain barrier, and then develop into L4and L5, anddevelop into adults in the blood vessels of heart and lungs.
This study established ICOSL-KO and C57BL/6J mice as experimentalAngiostrongyliasis model and further explained ICOS-ICOSL interaction contributes tothe immune respond in mice infected with A.cantonensis. Secondly, the stage-specificproteins of L3, FL5，ML5, FA and MA of A.cantonensis were also analysed by2D-DIGE binding mass spectrometry. The finding provided a new way for controlingeffectively the progress and regression of inflammatory reaction and provided the newstrategy of treatment of Angiostrongyliasis cantonesis.
Objective: Establish the model of ICOSL-KO mice (CD275knockout mice） andC57BL/6J mice infection with A.cantonensis, observe the dynamic change of IgG、IgG1、IgG2a in serum, the co-stimulatory factor expression of CD4+T lymphocytesand the expression of cytokine spleen lymphocytes in different period of infection, thenexplore the immunological mechanisms that may be related to ICOS-ICOSL signalingpathway and the related regulatory function of hosts.
Methods: Establish the model of ICOSL-KO mice and C57BL/6J mice infectionwith A.cantonensis. Collect blood from the mice eye socket, and obtain serum.Determinate the expression levels of IgG, IgG1and IgG2by ELISA in different periodof infection. Separate and observe the co-stimulatory signal of cell from mice infectedA.cantonensise in CD4+T lymphocytes. Measure the fluorescence of cells by flowcytometry (FCM), analysis the positive expression ratio with EXP30ADC software.Furthermore, culture spleen CD4+T lymphocytes of mouse, add soluble antigen of theL5of A.cantonensis and ConA, then collect cell supernatant after72hours ofincubation. Determinate the expression levels of cytokine such as IFN-γ, IL-12, IL-4,IL-5and IL-13by ELISA. Statistically significant was determined by Student’s t testor F test with SPSS17.0.
Results: The level of IgG increased in both groups of C57BL/6J mice and thegroup of ICOSL-KO mice. The subtypes IgG1increased at3weeks postinfection andIgG2a reduceded at3week postinfection in the group of C57BL/6J mice，however, thelevels of IgG1was stable postinfection and IgG2a increased at3weeks postinfectionin the group of ICOSL-KO, shows that the signaling pathways are blocked in theprocess of the generation of protective immunity, so it is unable to quickly organize theeffective dominant response of Th2. The produce of co-stimulatory molecule oflymphocytes has significant hysteresis in the group of ICOSL-KO and has significantdifference after3weeks post infection; however, those of lymphocytes have significantdifference after1week post infection in C57BL/6J. It indicates that it has a closerelationship between the immune response of CD4+T lymphocytes and ICOSco-stimulatory molecules in different period of infection of A.cantonensis. The IFN-γand IL-12were increased in the early period and then gradually decreased along with infection in C57BL/6J; it indicates that the immune response level of Th1takesadvantage within2weeks post infection, and then replaced by the immune responselevel of Th2in the group of C57BL/6J. However, the stable of Th1cytokines such asIFN-γ and IL-12in the early infection and the expression of Th2cytokines IL-13wererelatively lower at3weeks post infection in ICOSL-KO mice. Above all, ICOS-ICOSLsignaling pathway is an important co-stimulatory molecule for infection-inducedexpansion of Th1and Th2responses.
Conclusion: It indicated that Th2responses are predominant in mice infectedwith A.cantonensis. The ICOS-ICOSL signaling pathway plays a key role on theoccurrence and development of infection-induced expansion of Th1and Th2responses.
Objective: Identify the stage-specific proteins between L3, FL5, ML5, FA, andMA of A. cantonensis. The results might prove evidence on key parasite proteins thatare involved in the host regulation or host-parasite interaction.
Methods: Collect parasites from the life cycle of A. cantonensis in laboratoryconditions, separate L3, FL5, ML5, FA, and MA respectively. Pyrolysis the sampleswith lysis buffer and sonication, and obtain supernatant after centrifugation. The totalprotein concentration was determined with Bio-Rad protein assay reagent. Sampleslabeled with Cy3or Cy5, and the internal standard labeled with Cy2. After2D-DIGE,the gels were scanned by UMax Powerlook2110XL and Typhoon FLA9000, obtainedfluorescent images. Analysis images with DeCyder7.0software. Then the proteins wereidentificated by MALDI-TOF-MS and searched database. Two genes and the internalcontrol18s were chosen for quantitative real-time PCR (qPCR).
Results: Obtain clear protein expression profiles, after statistical analysis (t test)the differential expressed proteins, select183protein spots that appear in24proteinexpression profiles.5spots were up-regulated in L5compared with adults, but showno significantly different in gender difference;89spots such were up-regulated in L5and adults compared with L3, in contrast,13spots were up-regulated in L3.12spots were up-regulated and7spots were down-regulated in MA relative to FA.3spots wereup-regulated in FL5and3spots were up-regulated in ML5. Compared with female,spot1496increases in both MA and ML5, suggesting a difference between the sexes.37proteins were successfully identified with high confidence (>95%) scores byMS. Among the predicted proteins,29spots were cytoskeleton-associated proteins andfunctional proteins, such as actin, paramyosin, heat shock protein, and transformationdomain-associated protein (TRR-1).8spots represented unnamed proteins.12spotswere matched to the EST of different-stage larvae of A. cantonensis, they have not beenidentified. Two genes corresponding to protein spots designated609and1004and theinternal control18s were chosen for qPCR analysis to quantify their levels oftranscription. The qPCR results were consistent with those of the DIGE studies,suggesting that the proteins here identified as differentially expressed were regulated atthe transcriptional level.Conclusion: The findings will provide a new basis for understanding thecharacteristics of growth and development of A. cantonensis in environmentalregulation and the host–parasite relationship. They may also provide a new way forcontroling effectively the progress and regression of inflammatory reaction and as drugtargets for the control of eosinophilic meningitis induced by A. cantonensis.
在感染免疫中抗原由抗原提呈细胞(APC)处理成多肽，它与MHC结合并移至APC表面，产生活化TCR信号；而APC或靶细胞表面的共刺激分子与T淋巴细胞表面的有关受体结合就产生第二活化信号，即协同刺激信号。在双信号刺激下，T淋巴细胞被激活后转化为淋巴母细胞，并迅速增殖、分化。CD28-B7共刺激途径是研究最早最深入的共刺激途径之一,但CD28并不是惟一的共刺激分子。可诱导共刺激因子（inducible co-stimulator, ICOS）为CD28家族的新成员，主要表达于活化的Ｔ细胞；其配体ICOSL主要表达于B细胞、单核细胞、树突状细胞和T细胞。
此外，寄生虫需要通过改变宿主来完成其生活史，因此，他们有根据宿主环境来调节基因的差异表达的能力。广州管圆线虫生活史的完成需要几个不同的宿主。Ⅲ期幼虫（L3）感染大鼠后，穿过血脑屏障发展成Ⅴ期幼虫（L5），再移行至心、肺部血管内发育成成虫。
本研究首先应用ICOSL-KO和C57BL/6J两种小鼠建立广州管圆线虫感染的模型，观察ICOS-ICOSL信号通路在广州管圆线虫感染免疫调节中的意义；其次，运用双向荧光差异凝胶电泳和质谱相结合的蛋白质组学技术分析鉴定广州管圆线虫发育期特异性蛋白质并分析他们在宿主-寄生虫相互作用中的意义。本研究结果可为广州管圆线虫感染的发展和消退研究提供新的思路，还为广州管圆线虫病的治疗提供可能性靶标。
目的：通过观察两种广州管圆线虫感染宿主模型（ICOSL-KO和C57BL/6J）血清抗体、脾淋巴细胞表面协同刺激因子及脾淋巴细胞分泌细胞因子的动态变化，探索ICOS-ICOSL信号在广州管圆线虫感染免疫调节中的意义。
方法：实验室条件下构建广州管圆线虫感染的ICOSL-KO和C57BL/6J小鼠模型。分别于感染前、感染后1周、2周、3周、4周，分批处死小鼠每批每组各取5只小鼠，眼眶取血，收集血清，ELISA法测定血清中IgG、IgG1和IgG2a的水平。同时分离不同感染时期小鼠脾淋巴细胞悬液，流式细胞仪测定CD4+T淋巴细胞上CD28、CD154和ICOS的表达，其结果用SPSS17.0采用t检验和F检验分析其统计学意义。体外培养脾CD4+T淋巴细胞。加入广州管圆线虫L5可溶性抗原干预共刺激信号。培养72小时后，收集细胞上清。ELISA法测定上清中IFN-γ、IL-12、IL-4、IL-5和IL-13等细胞因子表达水平。
结果：ICOSL-KO组与C57BL/6J组在感染后均表现为IgG抗体水平的升高；前者IgG1水平在感染后升高不显著，但后者的IgG1水平相对在感染后3W开始出现升高，4W时仍维持升高；同时C57BL/6J小鼠的IgG2a水平在感染后升高不显著，并于3W开始出现下降，反观ICOSL-KO小鼠IgG2a水平在感染后3W开始出现升高，4W时仍保持升高状态。ICOSL-KO组与C57BL/6J组相比IgG1动态趋势及IgG2a水平升高趋势，显示ICOS-ICOSL信号通路的阻断通过抑制Th2的极化使宿主无法组织产生有效的体液保护性免疫，ICOSL-KO小鼠表现为Th1的的免疫类型占优势。感染后ICOSL-KO组的CD4+T淋巴细胞上协同刺激分子产生也明显滞后，感染后3W才出现显著性差异；C57BL/6组针对广州管圆线虫急性感染，感染后1W即出现显著性差异。C57BL/6J组感染早期出现IFN-γ、IL-12增高现象，而后下降，提示感染2W内Th1免疫应答水平占优势，随之逐渐被Th2免疫应答水平取代；而ICOSL-KO组Th1型细胞因子IFN-γ、IL-12的早期不增高，表明ICOS-ICOSL信号通路也参与到感染免疫早期的Th1型免疫的活化过程。在感染早期Th2型细胞因子IL-4和IL-5受ICOS-ICOSL信号通路影响不大。IL-13在C57BL/6J小鼠感染后3W后出现增高，IL-13在ICOSL-KO小鼠出现延后升高，两种动物感染模型动态检测的显著性差异，也说明了ICOS-ICOSL通路阻断对Th2型免疫应答水平的调控作用。
结论：在广州管圆线虫的急性感染期，Th2型的免疫方式在免疫应答过程中占优势；ICOS-ICOSL通路调节作用既能延迟Th2免疫反应的进行，也具有调节Th1型免疫活化的能力。
目的：通过研究广州管圆线虫期特异性差异蛋白，可以为宿主环境在寄生虫发育过程中的调控或宿主-寄生虫相互作用中的关键蛋白质提供实验依据。
方法：实验室条件下建立广州管圆线虫生活史，分别于感染的不同时期收集广州管圆线虫L3、FL5、ML5、FA和MA。将收集的虫体分别使用裂解液裂解、超声裂解、离心，获得的上清，使用Bio-Rad protein assay reagent测定蛋白质浓度。分别用Cy3或Cy5标记样品，Cy2标记内标。进行双向荧光差异凝胶电泳，并利用UMax Powerlook2110XL和Typhoon FLA9000对电泳后的胶板图像扫描，得到不同颜色的扫描图。采用DeCyder7.0图像分析筛选差异表达蛋白谱，继而对候选蛋白点进行MALDI-TOF-MS鉴定、数据库检索。用Real-time PCR方法进行验证。
结果：双向荧光差异凝胶电泳得到了清晰的蛋白质表达谱。蛋白表达差异值经统计学分析(t检验)，筛选出在24张图谱中均出现的点共183个蛋白点。5点在L5中上调，而在成虫期下调且没有表现出显著的性别差异；与L3比，89个蛋白点在成虫和L5中上调，而13个蛋白点在L3中出现明显上调；另一方面，实验中还发现有些性别差异表达的蛋白，雌雄成虫比对中，12个蛋白点在MA中表达上调而7个蛋白点在FA中表达上调；L5中各有3个蛋白在FL5、ML5中表达上调，还有点1496在MA和ML5均表达增加，表现为性别特异性。
质谱鉴定80个点，成功鉴定了其中37个蛋白质点，29个点是功能蛋白，从细胞骨架相关蛋白到参与代谢过程、免疫反应和转录调控相关等相关过程，8个点是未命名蛋白。此外，还有12个蛋白点与6个广州管圆线虫EST登录序列有较高可信度。定量PCR验证实验扩增出的两个蛋白点的基因609和1004，并发现结果与DIGE研究结果一致。
结论：这些生活史期特异性蛋白质有助于提高对寄生虫在宿主调控或宿主-寄生虫相互作用关系中的理解，也能为广州管圆线虫感染的发展和消退提供新的思路，还为广州管圆线虫病的治疗提供靶标。
Angiostrongyliasis cantonesis is a human zoonotic parasitic disease, results frominfections with the nematodes that A.cantonensis accidentally infects humans andmigrates to the central nervous system.
Specifically, infections with A.cantonensis cause eosinophilic meningitis andeosinophilic enteritis. T cells require two signals to become fully activated. A first signal,which is antigen-specific, is provided through the T cell receptor which produced byA.cantonensis with peptide-MHC molecules on the membrane of antigen presenting cells(APC). A second signal, the co-stimulatory signal, is antigen provided by theA.cantonensis between co-stimulatory molecules expressed on the membrane of APCand the T cell. One of the best costimulatory molecules expressed by T cells is CD28,which interacts with CD80(B7.1) and CD86(B7.2) on the membrane of APC. Anothercostimulatory receptor expressed by T cells is ICOS, which interacts with ICOSL.
In addition, when parasites changed their hosts to finish their life cycle, theyrequire the ability to respond to the host environments and regulate differential geneexpression. In the life cycle of A.cantonensis, adult worms reside in normal host（rats），which discharging the L1after infection. Widely disseminated via water, L1enter theintermediate host (such as Pomacantonensisea canaliculata), and develop into L3. L3infects rats, penetrates blood-brain barrier, and then develop into L4and L5, anddevelop into adults in the blood vessels of heart and lungs.
This study established ICOSL-KO and C57BL/6J mice as experimentalAngiostrongyliasis model and further explained ICOS-ICOSL interaction contributes tothe immune respond in mice infected with A.cantonensis. Secondly, the stage-specificproteins of L3, FL5，ML5, FA and MA of A.cantonensis were also analysed by2D-DIGE binding mass spectrometry. The finding provided a new way for controlingeffectively the progress and regression of inflammatory reaction and provided the newstrategy of treatment of Angiostrongyliasis cantonesis.
Objective: Establish the model of ICOSL-KO mice (CD275knockout mice） andC57BL/6J mice infection with A.cantonensis, observe the dynamic change of IgG、IgG1、IgG2a in serum, the co-stimulatory factor expression of CD4+T lymphocytesand the expression of cytokine spleen lymphocytes in different period of infection, thenexplore the immunological mechanisms that may be related to ICOS-ICOSL signalingpathway and the related regulatory function of hosts.
Methods: Establish the model of ICOSL-KO mice and C57BL/6J mice infectionwith A.cantonensis. Collect blood from the mice eye socket, and obtain serum.Determinate the expression levels of IgG, IgG1and IgG2by ELISA in different periodof infection. Separate and observe the co-stimulatory signal of cell from mice infectedA.cantonensise in CD4+T lymphocytes. Measure the fluorescence of cells by flowcytometry (FCM), analysis the positive expression ratio with EXP30ADC software.Furthermore, culture spleen CD4+T lymphocytes of mouse, add soluble antigen of theL5of A.cantonensis and ConA, then collect cell supernatant after72hours ofincubation. Determinate the expression levels of cytokine such as IFN-γ, IL-12, IL-4,IL-5and IL-13by ELISA. Statistically significant was determined by Student’s t testor F test with SPSS17.0.
Results: The level of IgG increased in both groups of C57BL/6J mice and thegroup of ICOSL-KO mice. The subtypes IgG1increased at3weeks postinfection andIgG2a reduceded at3week postinfection in the group of C57BL/6J mice，however, thelevels of IgG1was stable postinfection and IgG2a increased at3weeks postinfectionin the group of ICOSL-KO, shows that the signaling pathways are blocked in theprocess of the generation of protective immunity, so it is unable to quickly organize theeffective dominant response of Th2. The produce of co-stimulatory molecule oflymphocytes has significant hysteresis in the group of ICOSL-KO and has significantdifference after3weeks post infection; however, those of lymphocytes have significantdifference after1week post infection in C57BL/6J. It indicates that it has a closerelationship between the immune response of CD4+T lymphocytes and ICOSco-stimulatory molecules in different period of infection of A.cantonensis. The IFN-γand IL-12were increased in the early period and then gradually decreased along with infection in C57BL/6J; it indicates that the immune response level of Th1takesadvantage within2weeks post infection, and then replaced by the immune responselevel of Th2in the group of C57BL/6J. However, the stable of Th1cytokines such asIFN-γ and IL-12in the early infection and the expression of Th2cytokines IL-13wererelatively lower at3weeks post infection in ICOSL-KO mice. Above all, ICOS-ICOSLsignaling pathway is an important co-stimulatory molecule for infection-inducedexpansion of Th1and Th2responses.
Conclusion: It indicated that Th2responses are predominant in mice infectedwith A.cantonensis. The ICOS-ICOSL signaling pathway plays a key role on theoccurrence and development of infection-induced expansion of Th1and Th2responses.
Objective: Identify the stage-specific proteins between L3, FL5, ML5, FA, andMA of A. cantonensis. The results might prove evidence on key parasite proteins thatare involved in the host regulation or host-parasite interaction.
Methods: Collect parasites from the life cycle of A. cantonensis in laboratoryconditions, separate L3, FL5, ML5, FA, and MA respectively. Pyrolysis the sampleswith lysis buffer and sonication, and obtain supernatant after centrifugation. The totalprotein concentration was determined with Bio-Rad protein assay reagent. Sampleslabeled with Cy3or Cy5, and the internal standard labeled with Cy2. After2D-DIGE,the gels were scanned by UMax Powerlook2110XL and Typhoon FLA9000, obtainedfluorescent images. Analysis images with DeCyder7.0software. Then the proteins wereidentificated by MALDI-TOF-MS and searched database. Two genes and the internalcontrol18s were chosen for quantitative real-time PCR (qPCR).
Results: Obtain clear protein expression profiles, after statistical analysis (t test)the differential expressed proteins, select183protein spots that appear in24proteinexpression profiles.5spots were up-regulated in L5compared with adults, but showno significantly different in gender difference;89spots such were up-regulated in L5and adults compared with L3, in contrast,13spots were up-regulated in L3.12spots were up-regulated and7spots were down-regulated in MA relative to FA.3spots wereup-regulated in FL5and3spots were up-regulated in ML5. Compared with female,spot1496increases in both MA and ML5, suggesting a difference between the sexes.37proteins were successfully identified with high confidence (>95%) scores byMS. Among the predicted proteins,29spots were cytoskeleton-associated proteins andfunctional proteins, such as actin, paramyosin, heat shock protein, and transformationdomain-associated protein (TRR-1).8spots represented unnamed proteins.12spotswere matched to the EST of different-stage larvae of A. cantonensis, they have not beenidentified. Two genes corresponding to protein spots designated609and1004and theinternal control18s were chosen for qPCR analysis to quantify their levels oftranscription. The qPCR results were consistent with those of the DIGE studies,suggesting that the proteins here identified as differentially expressed were regulated atthe transcriptional level.Conclusion: The findings will provide a new basis for understanding thecharacteristics of growth and development of A. cantonensis in environmentalregulation and the host–parasite relationship. They may also provide a new way forcontroling effectively the progress and regression of inflammatory reaction and as drugtargets for the control of eosinophilic meningitis induced by A. cantonensis.
引文
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