人乳头瘤病毒16型E6E7 ORF转化作用的研究

构建了含人乳头瘤病毒１６型（ＨＰＶ１６）－Ｅ６Ｅ７ＯＲＦｓ（ｎｔ８３－８５５）片段和ＨＰＶ１６长控制区（ＬＣＲ）加Ｅ６Ｅ７ＯＲＦｓ片段（ｎｔ７００７－７９０４／０－８７９）的逆转录病毒载体ｐＨ２１和ｐＨ１８质粒，利用Ｌｉｐｏｆｅｃｔｉｎ分别将它们导入病毒包装细胞ｐＡ３１７中，经过筛选获得Ｇ４１８抗性的病毒包装细胞，产生的重组病毒Ｈ２１和Ｈ１８感染的ＮＩＨ３Ｔ３细胞都具有恶性细胞的形态学特征，并能在裸鼠体内形成肿瘤。Ｓｏｕｔｈｅｒｎ杂交结果证明，上述两基因片段都整合到细胞基因组中。本实验结果说明ＨＰＶ１６－Ｅ６Ｅ７基因片段是ＨＰＶ１６转化ＮＩＨ３Ｔ３细胞的关键早期区，其自身ＬＣＲ区在该转化过程中没有显示出重要作用。     

CD4-positive and CD8-positive cytotoxic T lymphocytes contribute to human papillomavirus type 16 E6 and E7 responses.

Cytotoxic T-lymphocyte (CTL) responses to E6 and E7 were previously shown to be more commonly detectable in human papillomavirus type 16 (HPV-16)-positive women without squamous intraepithelial neoplasia (SIL) than in HPV-16-positive women with SIL (M. Nakagawa, D. P. Stites, S. Farhat, J. R. Sisler, B. Moss, F. Kong, A. B. Moscicki, and J. M. Palefsky, J. Infect. Dis. 175:927-931, 1997). The objective of this study was to characterize the phenotype(s) of the effector cell population responsible for HPV-16 E6- and E7-specific cytotoxic responses. Peripheral blood mononuclear cells were stimulated with HPV-16 E6 or E7 fusion protein. Cells from an autologous B-lymphoblastoid cell line, infected with vaccinia virus expressing E6 or E7, served as target cells. The effector cells were characterized by using natural-killer-cell removal, antibody blocking, and T-cell subset separation. Our results suggest that both CD4 and CD8 T lymphocytes contribute to HPV-16 E6- and E7-specific CTL responses although their relative contributions vary from individual to individual. On the other hand, natural killer cells in the effector cell population contribute to background activities but not to HPV-specific responses in this assay system.     

Different Methods of Identifying New Antigenic Epitopes of Human Papillomavirus Type 16 E6 and E7 Proteins

Human papillomavirus (HPV) infection is the most common cause of sexually transmitted viral infection and is the main cause of cervical cancer. Identification of HPV T-cell epitopes would be instrumental not only in our understanding of the protective immune response but also in the development of vaccines and immunotherapies. In contrast to viruses which cause systemic infection, identification of HPV epitopes is technically challenging because HPV causes a localized mucosal infection and the frequency of pathogen-specific T lymphocytes in peripheral blood is expected to be low. Here we describe three new antigenic epitopes (E7 7-15 [TLHEYMLDL], E6 52-61 [FAFRDLCIVY], and E7 79-87 [LEDLLMGTL]) of HPV 16 E6 and E7 proteins which have oncogenic activities. E7 7-15 was identified among peptides previously shown to bind to human leukocyte antigen (HLA)-A2.1 molecule, but it was found likely to be restricted by the HLA-B48 molecule. E6 52-61 (likely to be restricted by HLA-B57) and E7 79-87 (likely to be restricted by HLA-B60) were detected, based on the magnitude of the T-cell immune responses, in another individual. In particular, T-cell clones specific for the E6 52-61 epitope were isolated effectively by magnetically selecting them based on gamma interferon secretion. This is an efficient method of identifying new epitopes of antigens for which the number of specific T lymphocytes in the circulation is expected to be small, and it should be widely applicable in identifying new T-cell epitopes.     

Hemodialysis Affects Phenotype and Proliferation of CD4-Positive T Lymphocytes

CD4⁺ T lymphocytes of patients with chronic kidney disease (CKD) are characterized by reduced levels of crucial surface antigens and changes in the cell cycle parameters. Recombinant human erythropoietin (rhEPO) normalizes their altered phenotype and proliferative capacity. Mechanisms leading to the deficient responses of T lymphocytes are still not clear but it is postulated that immunological changes are deepened by hemodialysis (HD). Study of activation parameters of CD4⁺ T lymphocytes in hemodialyzed and predialysis CKD patients could bring insight into this problem. Two groups of patients, treated conservatively (predialysis, PD) and hemodialyzed (HD), as well as healthy controls, were included into the study; neither had received rhEPO. Proportions of main CD4⁺CD28⁺, CD4⁺CD25⁺, CD4⁺CD69⁺, CD4⁺CD95⁺, and CD4⁺HLA-DR⁺ lymphocyte subpopulations and proliferation kinetic parameters were measured with flow cytometry, both ex vivo and in vitro. No differences were seen in the proportions of main CD4⁺ lymphocyte subpopulations (CD4⁺CD28⁺, CD4⁺CD25⁺, CD4⁺HLA-DR⁺, CD4⁺CD69⁺, CD4⁺CD95⁺) between all examined groups ex vivo. CD4⁺ T lymphocytes of HD patients exhibited significantly decreased expression of co-stimulatory molecule CD28 and activation markers CD25 and CD69 after stimulation in vitro when compared with PD patients and healthy controls. HD patients showed also decreased percentage of CD4⁺CD28⁺ lymphocytes proliferating in vitro; these cells presented decreased numbers of finished divisions after 72 h of stimulation in vitro and had longer G0→G1 time when compared to healthy controls. CD4⁺ T lymphocytes of PD patients and healthy controls were characterized by similar cell cycle parameters. Our study shows that repeated hemodialysis procedure influences phenotype and proliferation parameters of CD4⁺ T lymphocytes.     

含人乳头瘤病毒16型E6E7反意基因的假型逆转录病毒对人乳头瘤病毒16型转化细胞生长的抑制作用

构建了一个含人乳头瘤病毒１６型Ｅ６Ｅ７反意基因片断的逆转录病毒载体ｐＨ１９。用Ｌｉｐｏｆｅｃｔｉｎ将ｐＨ１９导入病毒包装细胞ｐＡ３１７，使之产生假型逆转录病毒Ｈ１９。Ｈ１９病毒感染人乳头瘤病毒１６型转化的细胞后，使转化细胞的生长速率和裸鼠体内形成肿瘤的能力均明显下降。     

Patterns of CD8 T-Cell Epitopes within the Human Papillomavirus Type 16 (HPV 16) E6 Protein among Young Women Whose HPV 16 Infection Has Become Undetectable

The patterns of CD8 T-cell epitopes recognized within the E6 protein in women who had cleared their human papillomavirus 16 infection were examined. T-cell lines were established using autologous dendritic cells infected with a recombinant vaccinia virus. Evidence of potential antigenic epitopes was shown in 8 of 23 (34.8%) women.     

人乳头瘤病毒16型转换基因的序列分析

为了解中国地区宫颈癌病人中人乳头瘤病毒１６型Ｅ６Ｅ７基因结构特点,从中国山东地区宫颈癌活检组织中提取组织ＤＮＡ,经ＨＰＶ多重引物ＰＣＲ法鉴定标本中感染ＨＰＶ型别,选单纯感染ＨＰＶ１６两例标本ＤＮＡ为模板进行ＰＣＲ扩增,获得ＨＰＶ１６Ｅ６Ｅ７基因后,重组入ｐＡＬＴＥＲ－１载体,进行双向测序、分析。ＤＮＡ序列分析表明：两例标本的ＨＰＶ１６Ｅ６Ｅ７序列全长均为７７６ｂｐ,与已发表的德国标准株长度相等,两     

人乳头瘤病毒16型变异株E7蛋白在HeLa细胞中的表达及亚细胞定位

目的构建增强绿色荧光蛋白(EGFP)-人乳头瘤病毒16型变异株E7(HPV16-HBE7)重组质粒pEGFP-HBE7,研究HPV16-HBE7蛋白亚细胞定位,为进一步了解其生物学功能奠定基础。方法采用分子克隆技术,将HPV16-HBE7基因克隆在pEGFP-C1表达载体上,用脂质体法导入宫颈癌细胞中;West-ern印迹检测HBE7蛋白的表达;同时借助免疫荧光技术和EGFP-融合蛋白技术,采用激光共聚焦显微镜观察HBE7蛋白的亚细胞定位。结果重组质粒经PCR、酶切和测序鉴定,其目的片段大小、插入位点和核苷酸序列完全正确;结果表明,转染细胞HBE7蛋白的相对表达量其胞浆明显多于胞核;各个时间段HBE7蛋白均以胞浆分布为主,绿色荧光密集点状分布于细胞浆内,而野生株E7(WE7)蛋白分布在核内。结论人乳头瘤病毒16型变异株E7蛋白主要分布在细胞浆内,以胞浆为主的分布可能和HBE7基因发生突变后丢失核定位信号有关。     

子宫颈癌中人乳头瘤病毒16型E7基因检测及变异分析

用聚合酶链反庆法检测宫颈癌中人乳头瘤病毒１６型转化基因Ｅ７，检出阳性率为１０／２２，其中４例进行ＰＣＲ－ＳＳＣＰ分析发现该基因与标准对照株存在明显变异。核苷酸序列分析证实有两处发生碱基突变。研究提示，ＰＣＲ方法能快速检测宫颈癌中人乳头瘤 转化基因，可作为宫颈癌的辅助诊断方法，ＳＳＣＰ分析能发现病毒株间小变异的基因片段。     

人乳头瘤病毒16 E6蛋白单克隆抗体的研制

运用杂交瘤技术，我们成功地建立了两株能稳定分泌小鼠抗人乳头瘤病毒１６Ｅ６蛋白单克隆抗体的杂交瘤细胞。经鉴定单克隆抗体均属ＩｇＧ１ｋ。试验结果表明，所得单克隆抗体仅与ＨＰＶ１６Ｅ６融合蛋白反应，不与ＨＰＶ１６Ｅ７、Ｌ１、Ｌ２融合蛋白以及Ｌ１－Ｌ２真核表达蛋白反应，也只与Ｃａｓｋｉ细胞反应，不与Ｈｅｌａ细胞反应。初步结果说明，该抗体是ＨＰＶ１６Ｅ１６蛋白特异性的ＭｃＡｂ。     

Time Course of Humoral and Cell-Mediated Immune Responses to Human Papillomavirus Type 16 in Infected Women

The time course of cell-mediated and humoral immune responses was elucidated in eight women with human papillomavirus type 16 (HPV-16) infection by performing serial HPV-16 E6 and E7 cytotoxic T-lymphocyte (CTL) assays and HPV-16 virus-like particle (VLP) antibody analyses. Four subjects had a single incident of HPV-16 DNA detection, and four subjects had two periods of HPV-16 DNA detection. In two of the women in the latter group, the second episode of HPV-16 detection occurred in the presence of high titers of HPV-16 VLP antibody, bringing into question the protective role of humoral immunity in preventing repeated infection. However, all four subjects rapidly became HPV-16 DNA negative following the second detection of HPV-16 DNA, suggesting the presence of immunological memory. In addition, one subject rapidly became negative for HPV-16 DNA despite having no evidence of CTL or VLP antibody response prior to the second HPV-16 DNA detection, suggesting the presence of immunological responses at an undetectable level. Overall, seven of eight subjects (88%) had detectable HPV-16 E6 and/or E7 CTL responses and seven of eight women (88%) had detectable HPV-16 VLP antibody responses.     

The Role of the CD8-Positive Subset of T Cells in Proliferative Responses to Soluble Antigens

The role of CD8 (T8, Leu 2)-positive T lymphocytes in the proliferative T-lymphocyte response to mumps and Coxsackie B4 viral antigens in vitro was investigated. The frequency among enriched T-lymphocyte blasts of antigen-reactive T lymphocytes (ARTL) restricted by different DR-associated elements was investigated, using antigenic restimulation with allogeneic antigen-presenting cells in a limiting dilution assay. A decreased frequency of DR3-restricted and an increased frequency of DR4-restricted mumps and Coxsackie B4 ARTL were seen in the limiting dilution assay, whether or not HLA class I determinants were shared in the antigenic restimulation. Removal of CD8-positive cells did not increase the primary in vitro responsiveness to mumps and Coxsackie B4 viral antigens, and did not change the DR-associated differences in the frequency of ARTL seen in the limiting dilution assay.     

The Role of the CD8-Positive Subset of T Cells in Proliferative Responses to Soluble Antigens

Using magnetic monosized polymer particles (M 450) coated with a monoclonal mouse IgM anti-CD8 (ITI 5C2) antibody, we were able to selectively remove and isolate functionally active CD8+ T cells from human peripheral blood mononuclear cells. Isolated CD8+ cells did not respond by proliferation to soluble antigens, but proliferated in response to phytohaemagglutinin. However, in the presence of CD4+ T cells, CD8+ cells were able to mount a substantial proliferation when stimulated with soluble antigens. Depletion of CD8+ cells decreased rather than increased the T-cell responses to the antigens glyc-gli, Coxsackie B4, and mumps in healthy individuals. We therefore found no indication of involvement of functionally-active CD8+ suppressor cells in vitro. The T-cell responsiveness to these antigens has previously been shown to be influenced by HLA-DR-associated restriction elements, but the tendency for decreased responsiveness to these antigens by CD8 depletion seemed independent of the DR type of the cell donors. As in healthy subjects, CD8 depletion resulted in a decreased responsiveness to the gluten antigen glyc-gli in untreated and treated coeliac disease patients and to Coxsackie B4 and mumps antigens in Type 1 diabetics.     

Human papillomavirus types 16 E6 and E7 contribute differently to carcinogenesis

High-risk human papillomaviruses (HPVs) are etiologically implicated in human cervical cancer. Two viral genes, E6 and E7, are commonly found expressed in these cancer cells. We have previously shown that mice transgenic for the HPV-16 E6 gene or E7 gene, in which the E6 or E7 was expressed in the basal layer of epithelia, developed skin tumors. The spectrum of tumors derived from E6 and E7 mice differed, however; although most tumors derived from the E7-transgenic mice were benign, the majority of the tumors from the E6-transgenic mice were malignant. These findings led us to hypothesize that E6 and E7 play different roles in carcinogenesis. To assess at what stages in carcinogenesis E6 and E7 act, we treated the skin of K14E6- and K14E7-transgenic mice with chemical carcinogens known to contribute to distinct stages in carcinogenesis. Both E6 and E7 were found to synergize with chemical carcinogens in causing tumor formation. E6 was found to act weakly at the promotion stage of carcinogenesis in the formation of benign tumors but strongly at the progression stage which involves the malignant conversion of benign tumors. In contrast, E7 primarily affected the promotion stage of carcinogenesis. These results provide direct evidence that E6 and E7 contribute differently to carcinogenesis; E7 promotes the formation of benign tumors, and E6 acts primarily to accelerate progression of these benign tumors to the malignant stage. Consistent with this model, we found E6 and E7 to cooperate in inducing tumor formation in mice expressing both oncogenes.     

Viruses and Cytotoxic T Lymphocytes in Type 1 Diabetes

Histopathological studies on pancreas tissues from individuals with recent-onset type 1 diabetes (T1D) consistently find that CD8 T cells substantially contribute to the formation of islet lesions. CD8 T cells reactive against islet-associated antigens can also be found in blood samples from T1D patients. Mechanistic studies on the pathogenic role of this T cell subset have mostly focused on two animal models, i.e., the non-obese diabetic mouse and the virally induced rat insulin promoter–lymphocytic choriomeningitis virus model. Data were obtained in support of a role for viral infection in expanding a population of diabetogenic cytotoxic T lymphocytes. In view of the theorized association of viral infection with initiation of islet autoimmunity and progression to clinically overt disease, CD8 T cells thus represent an attractive target for immunotherapy. We will review here arguments in favor of a pivotal role for CD8 T cells in driving T1D development and speculate on etiologic agents that may provoke their aberrant activation.     

E Rosette Formation by Human T Lymphocytes: A Spontaneous Cell Mediated Cytotoxic Phencmenon

The nature and significance of spontaneous association between unsensitized human T lymphocytes and sheep erythrocytes has Seen studied in relation to a possible cytotoxic reaction. Human lymphocytes and 51Cr labelled sheep erythrocytes when mixed in ratios of 5 or 10:l released 70–100% of 51Cr into the supernatants. This suggests that E rosette formation may be the first step in a spontaneous T cell mediated cytotoxic reaction to sheep erythrocytes.     

E Rosette Formation by Human T Lymphocytes: A Spontaneous Cell Mediated Cytotoxic Phencmenon

The nature and significance of spontaneous association between unsensitized human T lymphocytes and sheep erythrocytes has Seen studied in relation to a possible cytotoxic reaction. Human lymphocytes and 51Cr labelled sheep erythrocytes when mixed in ratios of 5 or 10:l released 70-100% of 51Cr into the supernatants. This suggests that E rosette formation may be the first step in a spontaneous T cell mediated cytotoxic reaction to sheep erythrocytes.     

人乳头状瘤病毒16 E7 DNA在结直肠腺癌组织中的表达

结直肠癌与人类乳头状瘤病毒(Human papilloma virus,HPV)感染有一定的关系,但由于检测方法、标本选择及样本数量不同,各研究结果之间差异很大。为进一步明确结直肠癌变与HPV16感染的关系,采用多聚酶链反应(Polymerase chain reaction,PCR)对82例原发性结直肠腺癌患者手术切除的新鲜癌及癌旁正常粘膜组织进行了前瞻性对照研究,检测了组织中HPV16E7DNA的表达并进行测序鉴定。结果显示,结直肠腺癌组织HPV16E7的表达阳性率(42/82)明显高于癌旁正常粘膜组织(4/82);直肠癌组织中HPV16E7的表达(64.10%)明显高于升结肠癌(18.18%),即癌灶部位距肛门越近,感染率越高;HPV16阳性率与Dukes分期相关,Dukes分期越晚感染率越高;与癌组织分化程度无相关性。结果提示结直肠癌的发生、发展可能与HPV16感染有关。