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Persistence of high cd40 and cd40L expression after restorative proctocolectomy for ulcerative colitis

World Journal of Gastroenterology 2005年第34期11卷 5303-5308页

作者： Lino Polese Imerio Angriman Giuseppe De Franchis Attilio Cecchetto Giacomo C.Sturniolo Renata D'Incà Marco Scarpa Cesare Ruffolo Lorenzo Norberto Mauro Frego Davide F D'Amico Department of Gastroenterological and Surgical Sciences. Surgical Unit University of Padova Italy Department of Laboratory Medicine University of Padova Italy Department of Pathology University of Padova Italy Department of Gastroenterological and Surgical Sciences Gastroenterological Unit University of Padova Italy

AIM： To focus on the role of cd40 and cd40L in their pathogenesis. METHODS： We analyzed by immunohistochemistry the cd40 and cd40L expression in the pouch mucosa of 28 patients who had undergone RPC for UC, in the terminal ileum of 6 patients with UC and 11 healthy subjects. We also examined by flow cytometry the expression of CEH0 by B lymphoo/tes and monocytes in the peripheral blood of 20 pouch patients, 15 UC patients and 11 healthy controls. RESULTS： Ileal pouch mucosa leukocytes presented a significantly higher expression of cd40 and cd40L as compared to controls. This alteration correlated with pouchitis, but was also present in the healthy pouch and in the terminal ileum of UC patients. cd40 expression of peripheral B lymphocytes was significantly higher in patients with UC and pouch, respect to controls. Increased cd40 levels in blood B cells of pouch patients correlated with the presence of spondyloarthropathy, but not with pouchitis, or inflammatory indices. CONCLUSION： High cd40 expression in the ileal pouch mucosa could be implied in the pathogenesis of pouchitis following proctocolectomy for UC, whereas its increased levels on peripheral blood B lymphocytes are associated with the presence of extraintestinal manifestations.

关键词： Ulcerative colitis Pouchitis Spondyloarthropathy cd40 cd4ol Arthritis

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A novel T cell-based vaccine capable of stimulating long-term functional CTL memory against B 16 melanoma via cd40L signaling

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Cellular & Molecular Immunology 2013年第1期10卷 72-77页

作者： Yufeng Xie Lu Wang Andrew Freywald Mabood Qureshi Yue Chen Jim Xiang Saskatchewan Cancer Agency Department of Oncology University of Saskatchewan Saskatoon Saskatchewan Canada Department of Pathology University ofSaskatchewan Saskatoon Saskatchewan Canada and Department of Epidemiology and Community Medicine University of Ottawa Ottawa Ont. Canada Xie and Wang contributed equally to this study.

The ultimate goal of antitumor vaccines is to develop memory cd8＋ cytotoxic T lymphocytes （CTLs）, which are critical mediators of antitumor immunity. We previously demonstrated that the ovalbumin （OVA）-specific cd4＋ T cell-based （OVA-TExo） vaccine generated using OVA-pulsed dendritic cell （DCovA）-released exosomes （EXOovA） stimulate CTL responses via IL-2 and costimulatory cd80 signaling. To assess the potential involvement of other costimulatory pathways and to define the key constituent of costimulation for memory CTL development, we first immunized wild-type （WT） C57BIJ6 and gene-knockout mice with WT cd4~ OVA-TExo cells or OVA-TExo cells with various molecular deficiencies. We then assessed OVA-specific primary and recall CTL responses using PE-H-2Kb/ 0VA257_264 tetramer and FITC-anti-cd8 antibody staining by flow cytometry. We also examined antitumor immunity against the OVA-expressing B16 melanoma cell line BL6-1OovA. We demonstrated that cd4＋ OVA-TExo cells stimulated more efficient CTL responses compared to DCovA. By assessing primary and recall CTL responses in mice immunized with OVA-TExo or with OVA-TExo lacking the costimulatory molecules cd4ol, 4-1BBL or OX4ol, we demonstrated that these costimulatory signals are dispensable for CTL priming by OVA-TExo. Interestingly, cd4ol, but not 4-1BBL or OX4ol, plays a crucial role in the development of functional memory CTLs against BL6-1OovA tumors. Overall, this work suggests that a novel cd4＋ T cell-based vaccine that is capable of stimulating long-term functional CTL memory via cd40L signaling may represent a novel, efficient approach to antitumor vaccination.

关键词： antitumor immunity cd4ol memory CTL T cell-based vaccine

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融合基因CCL21-cd40L对结肠癌主动免疫的诱导

融合基因CCL21-CD40L对结肠癌主动免疫的诱导

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作者：宫婷天津医科大学

学位级别：硕士

目的:树突状细胞是体内功能最强的抗原提呈细胞,能够捕获、加工、提呈抗原,启动免疫应答,在免疫抗肿瘤方面发挥举足轻重的作用。但是由于树突状细胞(Dendritic cells,DCs制备过程长、生存期短,体外难以模拟其生理成熟等因素,制约了DC瘤... 详细信息

目的:树突状细胞是体内功能最强的抗原提呈细胞,能够捕获、加工、提呈抗原,启动免疫应答,在免疫抗肿瘤方面发挥举足轻重的作用。但是由于树突状细胞(Dendritic cells,DCs制备过程长、生存期短,体外难以模拟其生理成熟等因素,制约了DC瘤苗的发展。本实验拟构建携带CCL21-linker-excd40L融合基因的真核表达载体,通过融合基因在体内肿瘤组织局部表达,趋化并活化DCs,发挥抗肿瘤作用。方法:首先,在GeneBank检索小鼠CCL21及cd40L基因编码序列,确定扩增区域,设计引物。以Trizol抽提小鼠脾脏总RNA,通过RT-PCR方法获得CCL21及excd40L基因。重叠PCR方法将CCL21分泌型信号肽(CCL21sig)与excd40L连接,获得CCL21sig-excd40L基因,使得cd40L能够以分泌形式表达,与CCL21基因分别作为融合基因的对照。重叠PCR构建CCL21-linker-excd40L融合基因,将其克隆入T载体进行测序,将测序正确的融合基因片段连接到真核表达载体pcdNA3.1。将pcdNA3.1/CCL21-linker-excd40L质粒转染CHO细胞,Western Blot法检测融合基因蛋白在细胞膜上的表达。通过趋化实验对融合基因进行活性检测。构建小鼠结肠癌肿瘤模型,体外转染CCL21-linker-excd40L真核表达载体,验证其抗肿瘤作用。结果:通过RT-PCR方法扩增约420bp大小的产物CCL21及约650bp大小的产物excd40L,二者均与预期目的片段大小一致,通过重叠PCR方法顺序连接CCL21与excd40L、CCL21sig与excd40L,分别得到产物大小约1100bp及720bp,与预期目的片段大小一致,将CCL21-linker-excd40L、CCL21、CCL21sig-excd40L进一步连接入T载体,经测序验证,序列正确。以上构建的3个重组T载体酶切后将目的片段与pcdNA3.1真核表达载体片段连接,构建真核表达载体pcdNA3.1/CCL21-linker-excd40L及对照组载体pcdNA3.1/CCL21、pcdNA3.1/CCL21sig-excd40L,酶切验证,分别得到与目的基因及pcdNA3.1载体片段(约5400bp)。Western blot法进一步验证次结果,分子量分别约为43KD,14KD及30KD,与目的蛋白大小一致。通过Millicell小室法验证融合蛋白对树突装细胞具有较强趋化功能,其每高倍镜视野穿膜细胞数是空载体对照组的14.95倍。用小鼠结肠癌细胞系C26成功构建小鼠肿瘤模型,肿瘤原位注射融合基因后,融合基因发挥抗肿瘤作用。结论:成功构建分别携带CCL21-linker-excd40L、CCL21、CCL21sig-excd40L基因的真核表达载体,将其转染真核细胞后均能够正常表达。体外实验证实转染融合基因的细胞载体对DCs具有趋化活性。瘤内注射小鼠后,融合基因发挥的抗肿瘤作用,且抗肿瘤作用较CCL21及CCL21sig-excd40L强。

关键词： CCL21 cd4ol 融合基因真核表达基因治疗

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