HLA—DR抗原在肝细胞癌组织中的表达及其意义

为了探讨ＨＬＡ－ＤＲ在原发性肝细胞癌中的表达及其意义，本文采用免疫组化ＡＢＣ法检测了ＨＬＡ－ＤＲ在４６例肝细胞癌组织中的表达情况，并分析了其表达水平与肝细胞癌病理分级的关系。结果表明，３９．１％（１８／４６）的肝细胞癌癌细胞表达ＨＬＡ－ＤＲ，而在癌旁非肿瘤细胞中ＨＬＡ－ＤＲ的表达为阴性。其在肝细胞癌中表达的阳性率随肝癌分化程度的下降而降低，在不同病理分级之间的阳性率具有显著性差异（Ｐ＜０．０５）。本研究说明ＨＬＡ－ＤＲ可能作为肝细胞癌发生、发展不同时期的一个标志物，但其在参与抗肿瘤免疫应答中的作用有待进一步探讨。     

HCMV感染对单核细胞HLA－DR表达的影响

本文采用细胞ＥＬＩＳＡ法，研究发现人巨细胞病毒（ＨＣＭＶ）感染对单核细胞ＨＬＡ－ＤＲ的影响。结果表明ＨＣＭＶ感染后１ｄ，单核细胞ＨＬＡ－ＤＲ表达显著增高（Ｐ＜０．０１），以后逐渐降低，ｄ５降至对照水平；ＩＦＮγ（５００Ｕ／ｍｌ）．ＴＮＦ（２５０Ｕ／ｍｌ）、ＩＬ－６（５００／ｍｌ）、ＩＬ－１（５００／ｍｌ）均能不同程度地刺激单核细胞ＨＬＡ－ＤＲ表达；ＨＣＭＶ感染后，细胞因子刺激ＨＬＡ－ＤＲ表达的水平在感染后ｄ５，较对照组均显著降低（Ｐ＜０．０１）；ＩＬ－１＋ＩＦＮ－γ及ＴＮＦ＋ＩＦＮ－γ在刺激单核细胞ＨＬＡ－ＤＲ表达时有协同作用；ＨＣＭＶ感染后，ＩＦＮ－γ＋ＩＬ－１及ＴＮＦ＋ＩＦＮ协同刺激单核细胞ＨＬＡ－ＤＲ表达水平较对照组显著降低（Ｐ＜０．０１）。结果提示：在ＨＣＭＶ感染引起免疫抑制过程中，其引起单核细胞ＨＬＡ－ＤＲ表达降低是一重要机制。     

HCMV感染对单核细胞HLA－DR表达的影响

本文采用细胞ＥＬＩＳＡ法，研究发现人巨细胞病毒（ＨＣＭＶ）感染对单核细胞ＨＬＡ－ＤＲ的影响。结果表明ＨＣＭＶ感染后１ｄ，单核细胞ＨＬＡ－ＤＲ表达显著增高（Ｐ＜０．０１），以后逐渐降低，ｄ５降至对照水平；ＩＦＮγ（５００Ｕ／ｍｌ）．ＴＮＦ（２５０Ｕ／ｍｌ）、ＩＬ－６（５００／ｍｌ）、ＩＬ－１（５００／ｍｌ）均能不同程度地刺激单核细胞ＨＬＡ－ＤＲ表达；ＨＣＭＶ感染后，细胞因子刺激ＨＬＡ－ＤＲ表达的水平在感染后ｄ５，较对照组均显著降低（Ｐ＜０．０１）；ＩＬ－１＋ＩＦＮ－γ及ＴＮＦ＋ＩＦＮ－γ在刺激单核细胞ＨＬＡ－ＤＲ表达时有协同作用；ＨＣＭＶ感染后，ＩＦＮ－γ＋ＩＬ－１及ＴＮＦ＋ＩＦＮ协同刺激单核细胞ＨＬＡ－ＤＲ表达水平较对照组显著降低（Ｐ＜０．０１）。结果提示：在ＨＣＭＶ感染引起免疫抑制过程中，其引起单核细胞ＨＬＡ－ＤＲ表达降低是一重要机制。     

广东汉族人类风湿关节炎易感性与HLA-DRB1基因相关性研究

目的探讨ＨＬＡ－ＤＲＢ１基因与类风湿关节炎（ＲＡ）相关性。方法采用ＰＣＲ－ＳＳＰ方法对４７例广东汉族人ＲＡ患者进行ＨＬＡ－ＤＲＢ１基因分型，并与相应人群健康者１０２例结果比较。结果ＨＬＡ－ＤＲ４基因在ＲＡ组显著增高（３５．１％，ＲＲ＝３．５５，Ｐ＜０．００５，ＥＦ＝０．２５２），ＤＲ１６在ＲＡ组也高于正常（ＲＲ＝２．５７，Ｐ＜０．０５）；而ＤＲ９基因在ＲＡ组显著减少（Ｐ＜０．００５）。３１例ＤＲ４＋患者患病年龄较早，病情较重（类风湿因子阳性率和Ⅱ期ＲＡ骨关节Ｘ线改变者显著高于ＤＲ４－患者，Ｐ值分别＜０．０５和０．０２５）。结论广东汉族人ＲＡ易感性与宿主ＤＲ４基因密切相关，ＨＬＡ－ＤＲ４可能是一个对判断病情和预后有价值的实验指标。     

IFN_γ对健康和肿瘤患者单核细胞HLA－DR表达的影响

本文采用ＢＳＡ－ＥＬＩＳＡ方法，研究ｒＨｕＩＦＮ－γ（ＩＦＮγ）对体外培养的健康人及恶性肿瘤患者外周血单核细胞表达ＨＬＡ－ＤＲ抗原的影响。结果表明：ＩＦＮγ能促进两者单核细胞ＨＬＡ－ＤＲ抗原的表达，但ＩＦＮγ对健康人单核细胞ＨＬＡ－ＤＲ表达促进程度较恶性肿瘤患者强（Ｐ＜０．０５）。     

IFNγ对健康和肿瘤患者单核细胞HLA—DR表达的影响

本文采用ＢＳＡ－ＥＬＩＳＡ方法，研究ｒＨｕＩＦＮ－γ（ＩＦＮγ）对体外培养的健康人及恶性肿瘤患者外周血单核细胞表达ＨＬＡ－ＤＲ抗原的影响。结果表明：ＩＦＮγ能促进两者单核细胞ＨＬＡ－ＤＲ抗原的表达，但ＩＦＮγ对健康人单核细胞ＨＬＡ－ＤＲ表达促进程度较恶性肿瘤患者强（Ｐ＜０．０５）。     

北方汉族过敏性紫癜与HLA相关性研究

为了研究过敏性紫癜（ＡＰ）的发病机理中是否有免疫遗传因素参与，采用国际通用的ＮＩＨ标准微量淋巴细胞毒试验方法检测４０例ＡＰ患者的ＨＬＡ－Ⅰ类抗原，并与１００例北方汉族正常人ＨＬＡ－Ⅰ类抗原频率进行比较。利用聚合酶链反应－序列特异性引物技术（ＰＣＲ－ＳＳＰ）对其中３０例ＡＰ患者进行ＨＬＡ－Ⅱ类基因分型，并与１０４例北方汉族正常人的ＨＬＡ－Ⅱ类基因频率进行了比较。发现ＡＰ患者ＨＬＡ－Ａ３０＋３１、Ｂ１３、Ｂ３５、Ｂ４０抗原频率较对照组明显增高（Ａ３０＋３１：Ｐｃ＜０．０１，ＲＲ＝７．９７；Ｂ１３∶ＰＣ＜０．０１，ＲＲ＝６．００，Ｂ３５∶Ｐｃ＜１０－５，ＲＲ＝１０．４０；Ｂ４０∶Ｐｃ＜０．０５，ＲＲ＝３．８５）。ＨＬＡ－ＤＲ１０基因频率在ＡＰ患者较正常对照组明显增高（ＤＲ１０∶Ｐｃ＜１０－５，ＲＲ＝２１．８８），而ＨＬＡ－ＤＱ３、ＤＱ６基因频率较正常对照组明显降低（ＤＱ３∶Ｐｃ＜１０－５，ＲＲ＝０．１３；ＤＱ６∶Ｐｃ＜０．０５，ＲＲ＝０．２３）。提示ＡＰ与ＨＬＡ－Ａ３０＋３１、Ｂ１３、Ｂ３５、Ｂ４０、ＤＲ１０正相关，与ＨＬＡ－ＤＱ３ＤＱ６负相关。     

EB病毒潜在膜蛋白对鼻咽癌细胞系CNE1生长及HLA表达的影响

目的研究ＥＢＶ－ＬＭＰ对高分化鼻咽癌细胞株ＣＮＥ１生长及ＨＬＡ表达的影响。方法以人高分化鼻咽癌细胞株（ＣＮＥ１）为对象，采用电穿孔基因转染技术，将重组ＥＢＶ－ＬＭＰ表达质粒ｐＣＭＶａ－ＬＭＰ转染ＣＮＥ１细胞。用细胞体外增殖试验、免疫组化和流式细胞术等方法，观察细胞生长及ＨＬＡ表达的变化。结果ＥＢＶ－ＬＭＰ在体外可明显促进ＣＮＥ１细胞的增殖，增殖吸光度（Ａ）比值试验组与空白组及阴性对照组比较有显著性差异（Ｐ＜０．０１）；实验组细胞软琼脂克隆形成率显著高于空白及阴性对照组（Ｐ＜０．０１）；细胞ＤＮＡ含量明显增高（Ｐ＜０．０１／０．０５）；ＦＣＭ法测定细胞角蛋白表达，实验组比空白及阴性对照组阳性率显著降低（Ｐ＜０．０５）；ＨＬＡ免疫组化检测结果表明，ＬＭＰ表达细胞系ＨＬＡⅠ类及Ⅱ类抗原的表达都明显下降（Ｐ＜０．０１）。结论ＥＢＶ－ＬＭＰ对ＣＮＥ１细胞生长有明显的促进作用且可明显抑制细胞分化，ＬＭＰ可致鼻咽癌细胞ＨＬＡ抗原的表达改变。     

中国汉人HLA—DQA1基因对类风湿关节炎的遗传易感性研究

用ＰＣＲ－ＰＡＧＥ方法，结合高灵敏的银染色作ＨＬＡ－ＤＱＡ１等位基因分型，研究ＤＱＡ１基因对类风湿关节炎（ＲＡ）的遗传易感性。选择无亲缘关系的广东籍汉族健康者１０６例和５０例ＲＡ患者。发现该方法测的６种ＨＬＡ－ＤＱＡ１等位基因中，ＲＡ组ＤＱＡ１＊０１０１（２７％，ＲＲ＝２．３３４，Ｐ＜０．００５，ＥＦ＝０．１５４）等位基因明显增高；而ＤＱＡ１＊０１０２（１％，ＲＲ＝０．０６８，Ｐ＜０．０１，ＰＦ＝０．５７７）明显下降；ＤＱＡ１的２种纯合子基因型（０１０１／０１０１和０３０１／０３０１）在ＲＡ组明显增高（Ｐ值分别小于０．０２５和０．００５）。上述结果显示：ＨＬＡ－ＤＱＡ１＊０１０１对ＲＡ有遗传易感作用，ＤＱＡ１＊０１０２等位基因有遗传抵抗作用；ＤＱＡ１基因型的检测对预测ＲＡ易感者和判断预后及疗效可能提供理论依据。     

IL－4对多发性骨髓瘤患者外周血细胞CD20及HLA－DR表达的影响

本文报道用流式细胞分析技术，检测了２８例多发性骨髓瘤（ＭＭ）患者和２０名健康对照者外周血Ｂ细胞（ＣＤ２０＋）以及Ｂ、Ｔ和单核细胞中ＨＬＡ－ＤＲ＋细胞比率。结果发现，ＭＭ患者ＣＤ２０＋以及Ｂ、Ｔ及单核细胞中ＨＬＡ－ＤＲ＋细胞比率与正常对照组差异非常显著（Ｐ＜０．０１）。加入重组ＩＬ－４，可使８名ＭＭ患者ＣＤ２０＋、ＨＬＡ－ＤＲ＋ＣＤ２０＋、ＨＬＡ－ＤＲ＋单核细胞比率提高非常显著（Ｐ＜０．０１）。而在Ｔ细胞，Ｐ值则＞０．０５。我们认为由于ＩＬ－４分泌减少，一方面使多克隆Ｂ细胞激活及增殖受抑，另一方面使单核细胞ＨＬＡ－ＤＲ抗原表达减少，抗原提呈能力下降可能是ＭＭ发生多克隆免疫球蛋白抑制的重要原因。     

Major histocompatibility complex status in breast carcinogenesis and relationship to apoptosis

Major histocompatibility complex (MHC) molecules are of central importance in regulating the immune response against tumors. In this study we used immunohistochemistry to study human leukocyte antigen (HLA) class I and II antigen expression in normal breast tissues and benign, preneoplastic, primary, and metastatic breast lesions using antibodies against beta-2-microglobulin (beta2-m), heavy-chain, and HLA-DR antigens. Whereas all normal tissues and benign lesions were positive for beta2-m and HLA-A, -B, and -C antigens, total loss of HLA class I antigens was found in 37% (11 of 30) of in situ carcinomas, in 43% (56 of 131) of the primary tumors, and in 70% (31 of 45) of the lymph node metastases. HLA-DR was also underexpressed in breast cancer cells; thus 20% (6 of 30) of in situ carcinomas, 15% of invasive carcinomas (20 of 131), and only 1 metastatic case were positive for this antigen. Both HLA class I and II antigen expression were more frequently down-regulated in metastatic lesions than in primary breast lesions (P <0.05), and a tendency toward a simultaneous defective expression of HLA class I and II antigens was observed in primary carcinomas (P = 0.07). However, no correlation was found between the expression of any of the aforementioned molecules and pathological parameters or survival. Interestingly, HLA class I expression was expressed more frequently in tissues with high apoptotic activity and was significantly associated with the expression of the proapoptotic bax gene (P = 0.02), and was inversely associated with expression of the antiapoptotic bcl-2 gene (P = 0.03). We conclude that alterations in HLA class I and II antigen expression are early events in breast carcinogenesis and play significant roles in metastatic progression. In addition, their expression is correlated with apoptosis-regulating proteins, which may influence the cytotoxicity of T cells against HLA class I-specific tumor antigens.     

Tumor infiltrating lymphocytes in HLA+ and HLA- laryngeal cancer--quantitative approach.

In search of factors governing the accumulation of tumor infiltrating lymphocytes (TIL), frozen sections from fresh surgical specimens of laryngeal carcinoma (n = 36) were tested by alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry for monomorphic determinants of HLA class I and class II expression on tumor cells and for the distribution of lymphoid cells bearing CD differentiation antigens. Cell subsets were quantitated in two tumor compartments, tumor mass and tumor stroma, by computer-assisted image analysis. In a portion of examined samples lymphoid cell suspension was isolated from cancerous tissues and assessed by flow cytometry. It has been found that T cells, localized mostly in tumor stroma, were predominant cell population in the tumor microenvironment. Their ability to penetrate tumor mass but not tumor stroma, by CD8+ T cells in particular, but also by natural killer (NK) cells, was associated with HLA class I antigen expression on tumor cells. In flow cytometric analysis activated T lymphocytes (CD3+DR+) were abundant in HLA+ tumors as compared to HLA- ones. In 4 year follow up of 20 patients the mortality was higher in HLA- group but the data were not statistically significant. These results show that HLA class I expression on tumor cells favor penetration of cytotoxic lymphoid cells into tumor mass, at least in the laryngeal cancer.     

Gene products of the HLA-D region in normal and malignant tissues of nonlymphoid origin

Indirect immunofluorescence staining with monoclonal antibodies has shown a differential distribution of HLA-DR, DQ, and DP antigens in normal tissues of nonlymphoid origin. The distribution of HLA-DP antigens is similar to that of HLA-DR antigens, while that of HLA-DQ antigens is more restricted. Malignant transformation of cells of nonlymphoid origin may be associated with the appearance of the gene products of the HLA-D region. HLA-DR antigens appear more frequently than the other two types of HLA class II antigens and HLA-DP antigens more frequently than HLA-DQ antigens. Differential expression of the gene products of the HLA-D region was also found in autologous metastases removed from different anatomic sites from patients with melanoma. The HLA class II phenotype of surgically removed malignant lesions did not correlate with the degree of differentiation of tumor cells and/or with the expression and/or cellular distribution of HLA class I antigens. Furthermore, in melanoma lesions, no relationship was found between the HLA class II phenotype and the expression of 3 membrane bound and 1 cytoplasmic melanoma associated antigen recognized by monoclonal antibodies. The functional significance and the practical implications of the differential expression of the gene products of the HLA-D region by tumor cells are discussed.     

Expression of MHC class I and class II antigens in colonic carcinomas

Malignant and non-malignant ('normal') colonic tissues from patients with colonic carcinoma were examined for the expression of MHC class I and class II antigens by immunoenzymatic staining using monoclonal antibodies. The amount of class I antigen as detected by 2 monoclonal antibodies, FMC 16 or W6/32 was clearly diminished in 11 of 14 tumours when compared to the amount present on 'normal' colonic tissue from the same individual. The loss of class I antigen did not correlate with tumour stage or differentiation. The reactivities of FMC 16 and W6/32 with these tissues were not identical, which indicates that the 2 monoclonal antibodies may recognize different epitopes on the HLA class I molecule. Class II antigens were absent from 'normal' colonic epithelium but were present on 20 of 28 tumours, with DR being detected more often than DP, and DQ found only on 4 of 28 tumours. When present, staining for class II antigens was heterogeneous within the tumour, in that all tumour cells did not stain equally. DR and DP antigens were found more often on moderately or poorly differentiated adenocarcinomas and on stage B, C and D tumours in that order of frequency. Thus tumours with a better prognosis were less likely to express DR and DP. The expression of DQ was unrelated to staging or differentiation.     

Expression of normal and tumor-associated antigens in human breast carcinoma

The expression of six cytoplasmic/membrane antigens (beta 2-microglobulin, HLA, HLA-DR, carcinoembryonic antigen, and two breast tumor-associated antigens (TAAs), B6.2 and B72.3) was investigated in serial sections of 28 human breast carcinomas using monoclonal antibodies and the avidin-biotin complex immunoperoxidase technique. The frequency of expression and linkage between these antigens was determined, and antigenic expression was related to patient age, morphologic differentiation, cytologic grade, and estrogen receptor/progesterone receptor content of the tumor. The expression of beta 2-microglobulin and HLA correlated with morphologic differentiation, well-differentiated and moderately well-differentiated tumors expressing these antigens more often than poorly differentiated tumors. Expression of the TAAs, however, was not related to differentiation. There was no linkage between beta 2-microglobulin/HLA and the TAAs. Carcinoembryonic antigen was found to be linked to the TAA, B6.2. Expression of the TAA, B72.3, correlated with patient age. Eighty percent (23 of 28) of the tumors were positive for carcinoembryonic antigen or at least one of the TAAs. The estrogen receptor/progesterone receptor status of the tumor was not statistically related to the expression of any of the antigens studied. Analysis of tumor antigen profiles may provide important information relevant to prognosis, therapy, and early detection of cancer, as well as insights into the nature of the neoplastic process.     

Establishment of a monoclonal anti-pan HLA class I antibody suitable for immunostaining of formalin-fixed tissue: unusually high frequency of down-regulation in breast cancer tissues

A novel monoclonal anti-pan human leukocyte antigen (HLA) class I heavy chain antibody, EMR8-5, was established. It could detect HLA-A, -B, and -C antigens in formalin-fixed paraffin embedded tissues. By immunohistochemical staining using the EMR8-5 antibody, various cancer tissues from 246 cases were examined for HLA class I expression. It was found that HLA class I expression was decreased in 20% to 42% of the cases of lung cancer, hepatocellular carcinoma, colon cancer, renal cell carcinoma, and urothelial carcinoma. In contrast, 85% of breast cancer cases had loss of or decreased HLA class I expression. Of the 35 breast cancer cases that had decreased HLA class I heavy chain expression, 33 (94%) also had decreased beta2-microglobulin expression detected by immunohistochemical staining. It was suggested that HLA class I down-regulation might be a common characteristic of breast cancer mostly caused by the down-regulation of beta2-microglobulin expression.     

MHC class I and II antigens on gastric carcinomas and autologous mucosa

The expression of HLA class I and II antigens was analysed in 30 primary gastric carcinomas, 27 autologous lymph node metastases and 25 autologous gastric mucosae. We used an immune alkaline phosphatase technique on cryostatic sections and mAbs directed against HLA class I monomorphic determinants, HLA-B locus-specific products and HLA-DR, -DP and -DQ molecules. In addition HLA class I genes were analysed in tumour tissue and compared by Southern blots with the RFLP from autologous mucosa using locus-specific HLA probes. Finally the infiltrating mononuclear cells were studied on gastric tumours and adjacent mucosa with mAbs defining CD4, CD8 and CD11b differentiation antigens. The results obtained showed that three out of 27 primary gastric carcinomas completely lack HLA-ABC antigens (10%). In addition, two primary tumours presented a variable expression. The remaining 22 tumours presented a homogeneous positive HLA class I expression. Interestingly, when the autologous mucosa was analysed, only 12 out 25 specimens were homogeneously stained with mAbs against HLA class I antigens, suggesting that this tissue may lack the expression of HLA antigens before becoming malignant. Indeed, the majority of the gastric carcinomas studied presented a higher HLA-ABC antigenic expression than autologous mucosa. Finally, the HLA expression observed in the primary tumour was similar to that observed in autologous metastases. As a second part of the study we have found a direct relationship between the expression of HLA-DR antigens in mucosa and the intensity of inflammatory infiltration. This relationship was not maintained in the tumour tissue. In the mucosa the CD4-positive T cell was the predominant lymphocyte, while it was CD8 in the HLA-DR-positive tumours. Finally the RFLP of class I genes did not show any differences in any of the cases when compared with autologous mucosa. We included in these studies DNAs from HLA class I-negative tumours, HLA positive and HLA-B-negative ones.