Influence of Bacterial Superantigen TSST-1 Against the Anti-Proliferative Efficacy of Immunosuppressive Drugs and Interleukin 2 Production in Peripheral Blood Mononuclear Cells of Hemodialysis Patients and Healthy Subjects

We investigated the influence of bacterial superantigen on the efficacies of immunosuppressive drugs on the blastogenesis of peripheral-blood mononuclear cells of 27 hemodialysis patients awaiting renal transplantation. The IC₅₀ values for prednisolone, methylprednisolone, cyclosporine, and tacrolimus evaluated in the superantigen-stimulated cells were significantly higher than those evaluated in concanavalin A-stimulated cells (p = 0.0002–0.018). Interleukin-2 amounts produced from superantigen-stimulated cells were significantly larger than those from concanavalin A-stimulated cells (p = 0.0363). These results suggest that superantigen attenuates the suppressive efficacies of glucocorticoids and calcineurin inhibitors by stimulating lymphocytes of hemodialysis patients awaiting transplantation to overproduce interleukin-2.     

肝癌靶向性超抗原基因疫苗的设计和预测

目的：探讨AFP顺式作用元件调控的肝癌特异性减毒超抗原疫苗(SEA(D227A)—Linker—CD80tm)设计的合理性。方法：应用核酸和蛋白质分析软件Cene Construction Kit2.0、ANTHERPRO5.0、JAMBW1.1和www.expasy.com网站提供的方案，分析重组体的开放读框以及融合蛋白的可及性、柔性、抗原性和疏水性，并作了跨膜区和二级结构模拟。结果：重组体的转录受AFP顺式作用元件调控，Linker所在部位柔性高，可及性弱，不改变两侧蛋白分子的抗原性和疏水性。融合蛋白表达后，被CD80tm锚定于肝癌细胞表面，超抗原部分(SEA)表达在细胞膜外，二级结构预测Linker不改变蛋白结构，完全符合作者设计疫苗的初衷。结论：重组疫苗设计合理，特异性高，融合蛋白有很大可能保留了SEA和CD80tm的理化特性，为进一步的实践操作提供了可靠的理论基础。     

DC联合SEA激活TIL体外杀伤小鼠肝癌细胞活性研究

目的探讨H22细胞全细胞性抗原致敏的DC联合SEA激活的TIL体外抗小鼠肝癌活性作用。方法从荷瘤小鼠四肢长骨骨髓中获取DC,应用粒/巨噬细胞集落刺激因子(GM-CSF)、白介素-4(IL-4)和H22细胞全细胞性抗原致敏DC,然后用已致敏的DC联合SEA激活从荷瘤小鼠中提取的TIL和脾淋巴细胞,观察受激活后的TIL和脾淋巴细胞作为效应细胞在体外对H22细胞的杀伤活性,并与对照组进行比较。结果TIL对H22细胞的杀伤活性[杀伤率为(50.63±2.52)%]高于脾淋巴细胞对H22细胞的杀伤活性[杀伤率为(12.26±1.28)%],用不同方式激活的TIL对H22细胞的杀伤活性均高于各自对应方式激活的脾淋巴细胞对H22细胞的杀伤活性;SEA激活的TIL对H22细胞的杀伤活性[杀伤率为(64.35±2.82)%]高于TIL对H22细胞的杀伤活性[杀伤率为(50.63±2.52)%],已致敏DC激活的TIL对H22细胞的杀伤活性更高[杀伤率为(75.23±3.21)%],而已致敏DC联合SEA激活的TIL对H22细胞的杀伤活性最高[杀伤率为(86.29±3.83)%]。结论用H22细胞全细胞性抗原致敏的DC联合SEA能显著激活TIL产生高效的对H22细胞的杀伤活性。     

SEC对淋巴细胞分泌TNF-α的诱导作用

目的探讨超抗原SEC的抗癌作用机制。方法采用ELISA法测定SEC在不同浓度及作用时间促淋巴细胞分泌TNF-α的作用;采用RT-PCR法检测SEC在不同作用时间促进淋巴细胞表达TNF-α的mRNA。结果ELISA法显示:淋巴细胞在未经SEC激活之前,培养上清液中TNF-α较低,经SEC激活后的第1天,培养上清液中TNF-α迅速上升,到作用的第3天,TNF-α上升至峰值。RT-PCR显示:经SEC活化的淋巴细胞有大量TNF-αmRNA的表达,高峰出现于第3天。结论SEC能促进淋巴细胞大量分泌TNF-α。     

SEA和喉癌MAGE-A3构建的真核质粒pMAGEA3-IRES-SEA经过电穿孔转染在小鼠体内的转录

目的检测真核质粒pMAGEA3-IRES-SEA经过电穿孔转染在小鼠中的转录。方法将葡萄球菌肠毒素A(staphylococcal endotoxin A,SEA)和喉癌来源的黑色素瘤抗原A3(melanomaassociated antigen A3,MAGE-A3),构建成基因共表达的真核质粒pMAGEA3-IRES-SEA。将pMAGEA3-IRES-SEA用电转染的方法免疫BALB/C小鼠单侧股四头肌,每2周1次,每次注射50μg,共免疫3次。末次免疫2周后,取注射部位组织,用荧光定量PCR(qRT-PCR)法检测目的基因在注射部位肌肉中的转录情况。结果在实验小鼠注射部位的骨骼肌内检测到SEA、MAGE-A3基因转录mRNA。结论所构建的pMAGEA3-IRES-SEA真核表达质粒,可通过电转染的方式,在小鼠体内能有效地转录。这为研究该质粒通过小鼠的体内转录蛋白的表达,诱导机体细胞免疫、体液免疫来清除喉癌,奠定了理论基础。     

携带超抗原SEA基因的特异性溶瘤腺病毒载体的构建和鉴定

目的 构建携带超抗原SEA基因的肿瘤特异性溶瘤腺病毒表达载体.方法 采用聚合酶链反应(PCR)技术,从产SEA的葡萄球菌标准菌株ATCC13565基因组DNA中获得SEA全长基因序列,酶切后克隆入pCA13质粒,构建重组病毒质粒pCA13-SEA.将鉴定正确的pCA13-SEA与含有腺病毒右臂的质粒pBHGE3通过Lipofectamine 2000共转染HEK293细胞,经同源重组产生重组腺病毒Ad-SEA.Ad-SEA在293细胞中大量扩增并通过氯化铯密度梯度离心法纯化、测定其滴度.结果 克隆得到771bpSEA全长基因.经PCR扩增、酶切鉴定、序列测定证实,SEA基因成功克隆到溶瘤腺病毒载体中,可实现SEA基因的表达,且病毒滴度达6.5×109pfu/ml.结论 成功构建表达超抗原SEA基因的溶瘤腺病毒载体.     

表达金黄色葡萄球菌肠毒素A基因的高靶向、双调控溶瘤腺病毒载体的构建、鉴定及其抗膀胱肿瘤作用

目的 构建表达超抗原金黄色葡萄球菌肠毒素A基因的高靶向、双调控选择增殖型溶瘤腺病毒SG502-SEA及对照组携带超抗原SEA基因的非增殖溶瘤腺病毒DC318-SEA,并探讨其潜在的抗膀胱肿瘤作用.方法 将超抗原SEA基因片段经SpeⅠ和SalⅠ双酶切后,克隆入经同样酶切后的非增殖溶瘤腺病毒载体pSG218中,将鉴定正确的腺病毒载体命名为pDC318-SEA.同样方法将超抗原SEA基因片段克隆入双调控特异性增殖溶瘤腺病毒载体pSG502中,将鉴定正确的腺病毒载体命名为pSG502-SEA.将以上两种携带SEA基因的病毒载体与病毒骨架质粒PPE3-ccdB共转染293细胞,9～14 d出现病毒空斑,经过3次病毒空斑纯化,提取腺病毒DNA,应用PCR进行鉴定,经鉴定正确的腺病毒分别命名为DC318-SEA和SG502-SEA.大量扩增后,氯化铯梯度离心纯化腺病毒,测病毒滴度.结果 经PCR及酶切鉴定,SEA基因成功克隆到两病毒载体中,可以表达SEA基因,且病毒滴度为2.5 × 1010pfu/ml.结论 成功构建表达超抗原SEA基因的双调控、高靶向选择增殖型溶瘤腺病毒SG502-SEA及对照组携带超抗原SEA基因的非增殖溶瘤腺病毒DC318-SEA.     

The SCID-hu xenogeneic transplantation model: complex but telling

Abstract Animal models based on knock-out or transgenic technology are widely used in basic and applied biomedical research. An alternative to these approaches is the generation of xenogeneic transplantation models allowing the in-vivo investigation of cell types and organs. In the field of dermatology transplantation of human skin onto mice lacking functional B and T cells (SCID mice) and subsequent manipulation of these grafts yielded new insights in many different aspects of skin biology. This review highlights some of the applications of this versatile model focussing on phenomena relevant for the subject of dermatology. Received: 5 March 1999 / Accepted: 18 March 1999     

A role for antigen-presenting cells and bacterial superantigens in reversal of human T lymphocyte anergy

The induction of anergy in T lymphocytes generates T cells incapable of proliferation in response to a conventional antigenic stimulus. To investigate the induction and maintenance of anergy in human T cells, we used T cell-T cell presentation of myelin basic protein (MBP) or MBP synthetic peptides to induce anergy in vitro. Although anergic T cells responded normally to interleukin-2 (IL-2), these T cells did not produce IL-2 or IL-4 when peripheral blood mononuclear cells presented MBP or MBP peptides. Proliferation of anergic T cells was reduced by greater than 95% compared to nonanergic, control T cells. However, when autologous B cell lines were used to present MBP, anergy was partially reversed with a proliferation response about 50% of nonanergic levels. Bacterial superantigens also partially restored proliferation in anergic T cells following presentation by either B cell lines or macrophage isolated from peripheral blood mononuclear cells. Anergic, MBP-reactive T cells fully retained antigen-specific cytolytic activity against both B cell and T cell targets presenting MBP. These results suggest that T cell proliferative anergy may be reversible with both the type of antigen-presenting cell and superantigens potentially contributing to the initiation or maintenance of an autoimmune response.