Cyfra21-1、CA125 测定、B超检查对卵巢癌诊断及监测价值比较

目的：评价细胞角蛋白19片段抗原21-1(Cyfra21-1)、CA125测定和B超检查在卵巢癌诊断和监测中的价值。方法：对23例卵巢良性病变及25例卵巢癌患者进行血清Cyfra21-1、CA125免疫放射测定和B超检查，并进行比较。结果：Cyfra21-1、CA125测定和B超检查鉴别卵巢良性病变和卵巢癌的敏感性分别为84．0％，80．0％，84．0％。特异性分别为100．0％，78．5％，82．6％。3种方法联合应用，对卵巢癌总的阳性检出率为100．0％，对工期卵巢癌检出率为80．0％(4／5)。对18例卵巢癌患者动态观察结果显示，Cyfra21-1和CA125水平与病情进展一致。结论：联合应用Cyfra21-1、CA125测定和B超检查对诊断卵巢癌具有重要价值，Cyfra21-1和CA125可作为诊断早期复发卵巢癌的监测指标。     

供者抗原门静脉输注对大鼠皮肤移植存活的影响

目的：探讨特异性抗原诱导免疫耐受的作用。方法：通过门静脉途径输注脾细胞、骨髓细胞,观察皮肤移植模型。结果：受体接受供体脾细胞（DSC）、骨髓细胞（DBMC）及同时接受（DSC）和（DBMC）后能诱导受体产生免疫耐受。结论：肝脏特殊的免疫功能在免疫耐受产生中起重要作用。     

Differential processing of influenza nucleoprotein in human and mouse cells

To investigate how early events in antigen processing affect the repertoire of peptides presented by MHC class I molecules, we compared the presentation of the influenza A nucleoprotein epitope 265 – 273 by HLA-A3 class I molecules in human and mouse cells. Mouse cells that express HLA-A3 failed to present the NP265 – 273 peptide when contained within the full-length nucleoprotein, to HLA-A3-restricted human cytotoxic T lymphocytes. However, when the epitope was generated directly in the cytosol using a recombinant vaccinia virus that expressed the nonamer peptide, mouse cells were recognized by HLA-A3-restricted CTL. Poor transport of the peptide by mouse TAP was not responsible for the defect as co-infection of mouse cells with recombinant vaccinia viruses encoding the full-length nucleoprotein and the human TAP1 and TAP2 peptide transporter complex failed to restore presentation. These results therefore demonstrate a differential processing of the influenza nucleoprotein in mouse and human cells. This polymorphism influences the repertoire of peptides presented by MHC class I molecules at the cell surface.     

IL-10 prevents the differentiation of monocytes to dendritic cells but promotes their maturation to macrophages

Human monocytes cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-13 for 7 days differentiate into cells with the morphology and function of dendritic cells (DC). We have investigated the effect of IL-10 on this differentiation pathway. In the presence of IL-10 cells did not develop DC morphology, did not express CD1a and had lower levels of MHC class II. IL-10 promoted the differentiation of large cells with the morphology, cytochemistry and membrane phenotype of macrophages, including staining for nonspecific esterase and high levels of CD14, CD16 and CD68. The effect of IL-10 was dose dependent and was best appreciated when the cytokine was added at the initiation of the culture, as addition on day 3 was less inhibitory. When added to already differentiated DC on day 6, IL-10 caused only a modest reduction of MHC class II and CD1a expression, and no acquisition of the macrophage markers CD14, CD16 and CD68. Prolonged incubation up to 5 days with IL-10 did not induce a shift of differentiated DC to macrophages. On the other hand, the macrophages obtained by culturing for 7 days with GM-CSF+IL-13+IL-10 did not shift to DC upon removal of IL-10 for up to 3 days. Thus, the effect of IL-10 on monocyte differentiation, occurs only at the precursor level and confers an irreversible phenotype. From a functional point of view, cells cultured in the presence of IL-10 were poor stimulators of allogeneic cord blood T cells in mixed lymphocyte reaction (MLR) and presented tetanus toxin (TT) to specific T cell lines with much less efficiency than control DC. In contrast, IL-10-cultured DC showed 7 times greater endocytosis of FITC-dextran. This increased endocytosis was mostly mediated via the mannose receptor, as demonstrated by blocking with unlabeled mannose. In conclusion, IL-10 inhibits DC differentiation from monocytes and, in a substantial proportion of the cells, promotes the differentiation to mature macrophages. Intriguingly, IL-10 inhibits antigen presentation while it stimulates endocytic activity.     

Generation of the vesicular stomatitis virus nucleoprotein cytotoxic T lymphocyte epitope requires proteasome-dependent and -independent proteolytic activities

The proteasome is involved in the generation of most of the MHC class I antigenic epitopes. However, it is not known if the proteasome generates the exact cytotoxic T lymphocyte (CTL) epitope or only epitope precursors which require further modification by additional proteases. Digestion of the extended vesicular stomatitis virus nucleoprotein epitope 52 – 59 (RGYVYQGL) by the 20S proteasome in vitro shows that the proteasome is capable of generating the correct C terminus but not the exact N terminus of the CTL epitope. This finding suggests that proteolytic activity in addition to the proteasome is required for generation of the CTL epitope. By using the proteasome inhibitor lactacystin we were able to confirm this finding in vivo. Lactacystin prevented the processing of N- and C-terminally extended epitopes, whereas the processing of only N-terminally extended epitopes was unaffected. Thus, the proteasome is necessary and sufficient for the generation of the exact C terminus of this CTL epitope, whereas the exact N terminus seems to be generated by a different protease.     

Promiscuous liberation of MHC-class I-binding peptides from the C termini of membrane and soluble proteins in the secretory pathway

TAP can efficiently transport peptides up to twice as long as those bound to MHC class I molecules, suggesting a role for endoplasmic reticulum (ER) proteases in the trimming of TAP-transported peptides. To better define ER processing of antigenic peptides, we examined the capacity of TAP-deficient cells to present determinants derived from ER-targeted proteins encoded by recombinant vaccinia viruses. TAP-deficient cells failed to present antigenic peptides from internal locations in secreted proteins to MHC class I-restricted T lymphocytes. The same peptides were liberated from the C termini of a secreted protein and the lumenal domains of two membrane proteins delivered to the ER via different routes. These findings suggest that proteases in the secretory compartment can liberate C-terminal antigenic peptides from virtually any context. We propose that this activity often participates in the removal of N-terminal extensions from TAP-transported peptides, thereby creating optimally sized products for MHC class I binding. We further demonstrate that ER trimming of C termini can occur if we express an appropriate carboxypeptidase in the secretory pathway. The absence of such trimming under normal circumstances suggests that carboxypeptidase activity is generally deficient in the ER, consistent with the concordance between the specificity of TAP and MHC class I molecules for the same types of C-terminal residues.     

The making of the dominant MHC class I ligand SYFPEITHI

The proteasome contributes to the generation of most of the peptide ligands of MHC class I molecules. To compare the identity of the peptides generated by the proteasome with those finally presented by MHC class I molecules, we generated a monoclonal antibody recognizing the C-terminal part of the dominant H2-K ^d ligand SYFPEITHI derived from the JAK1 tyrosine kinase. Immunoprecipitations of lysates from H2-K ^d -expressing or non-expressing cells revealed that only in the presence of H2-K ^d SYFPEITHI could be isolated. No longer potential precursor peptide containing SYFPEITHI could be detected. Surprisingly, a peptide lacking the first two amino acids, FPEITHI, was isolated independently of the presence of H2-K ^d molecules. The detection of only SYFPEITHI and FPEITHI in cell lysates corresponded with the strong generation of these two peptides in in vitro digests of elongated SYFPEITHI-containing peptides with purified 20S proteasomes. Our results indicate that MHC ligands can be generated directly by the proteasome in vivo and that at least for SYF PEITHI the expression of the corresponding MHC molecule is critical for protection of the ligand in vivo.     

Down-regulation of the MHC class I antigen-processing machinery after oncogenic transformation of murine fibroblasts

Malignant transformation is often associated with genetic alterations providing tumor cells with mechanisms for escape from immune surveillance. Human and murine tumors of various origin as well as in vitro models of viral and oncogenic transformation express reduced levels of major histocompatibility complex (MHC) class I antigens resulting in decreased sensitivity to MHC class I-restricted cytotoxic T lymphocyte (CTL)-mediated lysis. We here investigate whether the suppressed MHC class I surface expression of ras-transformed fibroblasts is due to dysregulation of the genes of the antigen-processing machinery, the peptide transporters TAP-1 and TAP-2 and the proteasome subunits LMP-2 and LMP-7, and whether it can be restored by gene transfer. In comparison to parental NIH3T3 cells, the ras oncogenic transformants revealed reduced TAP and LMP mRNA expression and impaired function of these genes, leading to deficient peptide transport and peptide loading of MHC class I molecules resulting in instable expression of the MHC class I complex on the cell surface. Enhanced H-2 surface expression due to stabilization of the MHC class I complex could be achieved by culturing ras transformants at low, unphysiological temperature (26 °C) or by loading these cells with either exogenous human β2-microglobulin or MHC class I-binding peptide alone or in combination. Furthermore, interferon-γ treatment was capable to enhance the expression of TAP, LMP and MHC class I molecules in both parental as well as ras-transformed fibroblasts. Stable transfection of the human TAP-1 cDNA into ras transformants caused a partial reconstitution of the peptide transport and an enhancement of the MHC class I surface expression, whereas the level of MHC class I biosynthesis was not affected by TAP-1 overexpression in parental cells. Together these results point to the existence of an association between oncogenic transformation and deficiencies in the MHC class I antigen-restricted immunosurveillance, suggesting intervention strategies involving specific MHC class I-binding peptides or transfection of the LMP and/or TAP genes to overcome the expression of the immune escape phenotype.     

Generation of human tumor-reactive cytotoxic T cells against peptides presented by non-self HLA class I molecules

The cyclin-D1 protein, which was found to be overexpressed in various human tumors, promotes cell cycle progression from the G1 into the S phase. It is normally expressed at low levels in several tissues and is likely to induce immunological tolerance. We have recently shown in a murine system that T cell tolerance to a widely expressed protein was circumvented by raising cytotoxic T lymphocytes (CTL) from major histocompatability complex mismatched donors. In this study, we tested whether it is possible to raise human allo-restricted CTL against the cyclin-D1 protein. The human cell line T2 is deficient in the genes encoding the transporter associated with antigen processing (TAP), resulting in inefficient loading of HLA-A2 class I molecules with endogenous peptides. Thus, a large number of A2 molecules can bind exogenously supplied synthetic peptides. Peripheral blood mononu clear cells from HLA-A2-negative donors were stimulated with T2 cells presenting cyclin-D1-derived synthetic peptides. Cloning of bulk cultures revealed that a large proportion of CTL clones were peptide specific. One peptide induced CTL which lysed cyclin-D1-expressing breast cancer cells, but not control Epstein-Barr virus-transformed B lymphoid cells. The results show that HLA-A2-negative donors can be used to isolate tumor-reactive CTL spe cific for cyclin-D1 peptides presented by HLA-A2 class I molecules.     

检测鼠疫抗原金标层析试剂盒的研制及应用

目的建立一种敏感、快速、简便的检测鼠疫抗原的诊断方法。方法以胶体金标记纯化的鼠疫单克隆抗体(M cAb),在硝酸纤维素膜上检测线处包被M cAb,组装成检测鼠疫抗原层析试纸条(盒)(G ICA)。在试剂盒的样品孔内滴加被检材料100μL,观察金标抗体释放后NC膜上检测线和质控线的反应情况,检测线和对照线同时出现红色条带判为阳性,仅对照线出现红色条带判为阴性,对照线未出现条带为无效试验。结果G ICA可检出鼠疫活菌10万菌体/mL,检出F1抗原1ng/mL,鼠疫实验动物脏器悬液检测阳性,鼠疫患者(死者)及自毙动物在收到检材后半小时内出现阳性定性结果。结论金标层析诊断试剂条达到检测鼠疫抗原敏感特异、使用简便、诊断快速的效果。