CPP-SOM整合cDNA基因芯片平台的建立

目的　在转录组水平全面迅速地了解疾病发生和药物作用的分子机理 ,以及寻找潜在的治疗靶标。方法　通过建立基因芯片平台 ,用全反式维甲酸诱导急性早幼粒细胞白血病来源的 NB4细胞分化作为模型 ,并应用自主开发的自组织图结合成分平面展示 (componentplane presentation integrated self-organizing map,CPP- SOM)的方法对数据进行初步分析。结果　建立的 c DNA芯片共有 12 6 30条克隆 ,其中已知基因 94 36条。应用该芯片进行实验 ,结果重复性好、准确性高。CPP- SOM不仅可以将功能相关的基因进行聚类 ,而且可以动态性地从全基因组水平观察药物作用过程中基因的表达变化。结论　我们建立的芯片平台是稳定、可靠的技术平台 ,CPP- SOM是一种新型有效的芯片数据的处理方法。     

Rational approaches to immune regulation

Our studies are mainly focused on developing strategies of immune regulation. In the case of infectious and neoplastic disease, our approach is to upregulate cell-mediated immunity to viral of tumor antigens using an intracellular bacterium as a vector for targeting these antigens to the major histocompatibility complex (MHC) class I and class II pathways of antigen processing, in addition to exploiting the adjuvant properties of the vector to stimulate innate immunity. In the area of autoimmunity, we are attempting to downregulate the immune response by specific immune intervention directed against autoreactive T cells. In these studies we use murine models for multiple sclerosis. Our approach is to use both rationally designed T cell receptor (TCR) peptide analogs and recombinant viral vectors that express TCR components to regulate the disease.     

Rational approaches to immune regulation

Our laboratory is interested in the properties of proteins that render them immunogenic, and how such immunogenicity may be modulated in vivo. We are attempting to enhance the immune response in the design of more effective vaccines against viral diseases, such as HIV, and against tumor antigens expressed on breast, ovarian, and cervical cancer and B cell lymphomas. Our main approach is to use a facultative intracellular bacterium, Listeria monocytogenes, which has the unusual ability to live and grow in the cytoplasm of the cell and is thus an excellent vector for targeting passenger antigens to the major histocompatibility complex (MHC) class I pathway of antigen processing with the generation of authentic cytotoxic T lymphocytes (CTL) epitopes. In the field of tumor immunotherapy, we are also developing nonliving vaccine vectors for tumor antigens.     

Complement-dependent synergistic effects of rat monoclonal IgG antibodies in vivo

We describe studies aimed at maximizing the effector mechanisms responsible for eliminating target erythrocytes from the circulation in a fully homologous opsonization system in vivo. The effects on the subsequent fate of target erythrocytes were examined in both normal and decomplemented rats preinjected with a variety of rat IgG monoclonal antibodies (mAb) directed against different epitopes on the RTlA^a, the classical class I major histocompatibiliy complex antigen of the DA rat. In general, the clearance of both DA and (DA × PVG)F₁ erythrocytes in normal rats preinjected with various pairs of noncompetitive mAb was very rapid when compared with the overall clearance patterns seen with individual antibodies. With all mAb combinations containing IgG2b or IgG2a, an intact complement system was an essential requirement for augmenting the initial clearance and promoting hepatic sequestration of these target cells. The removal of (DA × PVG)F₁ erythrocytes, expressing half as much antigen, was considerably slower than the DA cells for each antibody pair tested although a notable degree of heterogeneity was observed in the overall behavior of both types of target cells with different mAb combinations. Our results suggest that the limiting effects of low antigen density on the target cells combined with the use of mAb of an isotype like the rat IgG2a can be overcome using pairs of mAb that recognize different epitopes on the same target antigen.     

检测HSV等5种病原体抗体的微阵列技术

目的 建立抗原微阵列技术检测多种病原体抗体。方法 将病原体的基因工程抗原，以电脑操纵的机械手将其点在赖氨酸修饰的玻片上．制备抗原微阵列，与血清反应后随之与Cy3标记的二抗反应，用激光共聚焦扫描仪扫描玻片，检测血清中相应病原体的IgG，并进行了灵敏度和重复性的检测。结果 5种抗原的最低检测限度为HSV1-gG 1．56μg／ml，HSV2-gG 3．125μg／ml，CMV-p150 1．56μg／ml，RV-E1E2 3．125μg/ml，MT-EAM 31．26μg／ml；抗原微阵列与ELISA检测的结果相比有较高的符合率；检测IgG和IgM的灵敏度分别为50pg、10pg；不同批次2张玻片间总体变异系数为IgG 6．52，IgM 18．89。结论 抗原微阵列可用于平行检测血液中病原体特异性抗体。     

The rational design of TAP inhibitors using peptide substrate modifications and peptidomimetics

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. This step precedes the binding of peptides to MHC class I molecules and is essential for cell surface expression of the MHC class I/peptide complex. TAP has a broad sequence specificity and a preference for peptides of around 9 amino acids. To synthesize inhibitors for TAP, we studied various alterations of the peptide substrate. The results indicate that TAP is stereospecific and that peptide bonds engineered into isosteric structures can improve translocation of the peptide. Furthermore, TAP is able to translocate peptides with large side chains that correspond to a peptide of ～ 21 amino acids in extended conformation. Peptides with longer side chains compete for the peptide binding site of TAP but fail to be translocated. Therefore, they represent the first rationally designed inhibitors of TAP.     

Screening microarrays of novel monoclonal antibodies for binding to T-, B- and myeloid leukaemia cells

We have developed a microarray (DotScan) that enables rapid immunophenotyping and classification of leukaemias and lymphomas by measuring the capture of cells by immobilized dots of 82 CD antibodies [Belov, L., de la Vega, O., dos Remedios, C.G., Mulligan, S.P., 2001. Immunophenotyping of leukemia using a cluster of differentiation antibody microarray. Cancer Res. 61, 4483; Belov, L., Huang, P., Barber, N., Mulligan, S.P., Christopherson, R.I., 2003. Identification of repertoires of surface antigens on leukemias using an antibody microarray. Proteomics 3, 2147]. The DotScan technology has been used to investigate the properties of 498 new antibodies submitted to the HLDA8 Workshop. These antibodies have been applied as 10 nl dots to a film of nitrocellulose on a microscope slide to make an HLDA8 microarray. After blocking the remaining nitrocellulose surface, individual arrays were incubated with each of 7 cell types from a human leukaemia cell panel consisting of three cell lines, CCRF-CEM (a T-cell acute lymphocytic leukaemia), MEC-1 (derived from B-cell chronic lymphocytic leukaemia) and HL-60 (a promyelocytic leukaemia), and four leukaemias from patients: a T-cell prolymphocytic leukaemia, a B-cell chronic lymphocytic leukaemia, and two acute myeloid leukaemias. Leukaemia cells were captured by those immobilized antibodies for which they expressed the corresponding surface molecule. Unbound cells were gently washed off, bound cells were fixed to the arrays and dot patterns were recorded using a DotScan array reader and quantified using DotScan data analysis software. The data obtained show the unique expression profiles of the 7 cell types in the leukaemia cell panel obtained with the DotScan microarray, and the differential capture patterns for these 7 cell types screened against the 498 antibodies in the HLDA8 microarray constructed for this study.     

广东2003年末SARS患者冠状病毒抗原抗体动态特征分析

目的分析SARS-IgG、SARS-IgM、核壳蛋白-IgG抗体和核壳蛋白抗原的变化特点并探讨其意义。方法采用酶联免疫方法对2003年末至2004年初新发4例SARS病人的系列血清进行上述4项的滴度检测。结果在病人较早期的血清中可以检测到核壳蛋白抗原，随着抗体水平的升高，抗原含量迅速下降。抗体E升下降快，在最高水平维持时间短，并且除第1例外，其余3例的抗体滴度均小于1：100，核壳蛋白抗体变化规律与总的IgG抗体一致，但滴度更低。结论抗体变化规律与前次流行不同，抗体水平变化快，应注意及时采集标本。抗原检测可作为一种实验室诊断依据。核壳蛋白抗体可考虑用于早期诊断。     

抗体包被对红细胞变形性影响的研究

作者对抗体包被的红为性进行研究，发现抗体包被改变了红细胞的变形性，在我们研究的抗体量范围内，抗体量越多，红细胞的变形性越小，作者从血液流变学的角度对上述结果作了讨论，并提出了通过测定其对红细胞变形性的影响来比较准确地标定抗体效价的可能性。