WtCosmc质粒转染对Tn~+肿瘤细胞恶性行为的影响

目的:以Tn~+肿瘤细胞为靶标,研究野生型Cosmc(WtCosmc)质粒转染对其恶性行为的影响,以期为肿瘤的诊断及治疗提供新思路。方法:流式细胞术检测肿瘤细胞表面Tn抗原阳性率,免疫磁珠分选Tn~+与Tn-肿瘤细胞;分选后的Tn~+肿瘤细胞经Fugene 6转染WtCosmc质粒,并用免疫荧光检测细胞表面Tn抗原表达状况。分别采用荧光法、CCK-8和Transwell检测转染前后Tn~+肿瘤细胞的T-synthase活性、细胞增殖、迁移能力及对Apo2L/TRAIL的敏感性。结果:与Tn~-细胞相比,Tn~+肿瘤细胞T-synthase活性及对Apo2L/TRAIL诱导凋亡的敏感性较低;细胞增殖、迁移能力较强。经WtCosmc质粒转染后,Tn细胞T-synthase活性及其对Apo2L/TRAIL诱导凋亡的敏感性明显增高;Tn抗原表达受抑;细胞增殖及迁移能力明显下降。结论:转染WtCosmc可恢复Tn~+细胞T-synthse活性,抑制Tn抗原的表达,进而增强其对Apo2L/TRAL诱导凋亡的敏感性,降低细胞增殖、迁移能力,有效抑制Tn~+肿瘤细胞的恶性行为。     

CD107a/b分子用于评价SARS-CoV/S抗原特异性免疫应答

目的 观察抗原刺激以后CD4+和CD8+T细胞表面CD107a/b分子的表达与效应性细胞因子的产生之间的关系.方法 正常Balb/c小鼠脾细胞经多克隆刺激剂刺激或者SARS-CoV/S DNA疫苗免疫小鼠脾细胞经S抗原多肽刺激以后,使用流式细胞仪检测CD4+和CD8+T细胞表面CD107a/b分子的表达与IFN-γ、INF-α、IL-2等细胞因子的表达之间的关系.结果 经抗原多肽刺激后,抗原特异性CD4+ 和CD8+T细胞表面均表达CD107a/b.约80%的CD8+IFN-γ+细胞同时表达CD107a/b;约25%～40%的CD8+TNF-α+细胞表达CD107a/b;而大部分分泌IL-2的抗原特异性T细胞均不表达CD107a/b.结论 可以通过对抗原特异性T细胞表面CD107a/b分子的检测来鉴定抗原特异性T细胞.同时检测效应性细胞因子,可以提高检测的准确性和灵敏度.     

抗原肽浓度对初始T细胞向Th1/Tc1、Th2/Tc2分化影响的研究

目的 研究不同浓度抗原肽(OVA)对初始CD4~+T细胞向Thl/Th2、初始CD8~+T细胞向Tc1/Tc2分化偏向性的影响.方法 采用系列浓度TCR特异性抗原肽与小鼠脾脏树突状细胞联合培养,然后刺激小鼠初始CD4~+或CD8~+T细胞.流式细胞术检测细胞内因子IFN-γ和IL-4的表达水平,同时用CFSE检测活化T细胞的增殖水平.结果 低浓度抗原肽刺激初始CD4~+T向Th2、初始CD8~+T向Tc2分化;而高浓度抗原肽刺激初始CD4~+T向Th1、初始CD8~+T向Tc1分化.Th细胞比Tc细胞受影响程度要明显.结论 抗原肽在很大浓度范围内均可以刺激初始T细胞的活化,但随着浓度的升高,分化方向从Th2、Tc2逐渐向Th1、Tc1倾斜.这一结论为动物实验中疫苗免疫剂量的控制提供了重要的参考价值.     

肿瘤细胞HLA分子的表达及IFN—γ的调控作用

目的 研究肿瘤细胞表达HLA分子及IFN－γ的调控作用。检测TIL细胞中T细胞亚群的变化,并与PBMC比较。方法 采用FACS检测细胞表达的CD抗原,采用ABC免疫组化染色检测肿瘤细胞HLA－Ⅰ,Ⅱ类分子的表达。以IFN－γ诱导建系的卵巢癌细胞表达HLA分子。结果 明细胞癌患者TIL细胞中的T细胞明显多于大肠癌患者。肿瘤患者PBMC中的T细胞数少于正常对照;肿瘤组织HLA－Ⅰ类抗原的表达明显低于正常组织,HLA－Ⅱ类抗原泊表达高于正常组织。与正常组织相比较,大肠癌患者比肾细胞癌患者HLAⅠ类抗原的表达更低。IFN－γ可诱导建系卵巢癌细胞表达HLA－Ⅰ类抗原,但不能诱导HLA－Ⅱ类抗原的表达。结论 肿瘤组织HLA表达的变化与肿瘤组织中TIL的数有关;IFN－γ能选择性地诱导卵巢癌细胞表达HLA－Ⅰ类分子,在抗肿瘤免疫中具有重要的意义。     

分析胃癌组织淋巴细胞亚群及其表达细胞因子比例的变化特征

目的探讨胃癌组织淋巴细胞亚群及其表达细胞因子比例的变化特征。方法胃癌患者110例,取手术切除的胃癌样本,采用流式细胞术比较胃癌组织与正常组织之间NK细胞、B细胞、CD4+T细胞、CD8+T细胞的比例,分析γδT细胞亚群表达TNF-α、IL-1β和IL-10的γδT细胞数量及其在胃癌不同分期组织的比例变化。结果在胃癌组织中,B细胞和NK细胞的比例与正常胃部组织相比无明显变化;CD4+T细胞在胃癌组织中的比例显著大于在正常组织(P0.05);CD8+T细胞在胃癌组织中的比例低于正常组织(P0.05)。表达TNF-α和IL-1β的γδT细胞在胃癌部位的比例低于正常组织中的比例(P0.05);而表达IL-10的γδT细胞在胃癌组织中的比例高于正常组织(P0.05)。对胃癌不同分期组织进行检测,结果发现,在Ⅲ/Ⅳ期胃癌组织中,表达TNF-α和IL-1β的γδT细胞的比例均低于Ⅰ/Ⅱ期胃癌组织(P0.05);而表达IL-10的γδT细胞的比例则高于Ⅰ/Ⅱ期胃癌组织(P0.05)。结论在胃癌组织中CD4+T细胞数量增加,CD8+T细胞下降;表达TNF-α和IL-1β的γδT细胞随着胃癌的进展而不断减少;表达IL-10的γδT细胞随着胃癌的进展而不断增加。通过对胃癌组织淋巴细胞亚群的分析检测,说明淋巴细胞亚群与胃癌组织的发生发展密切相关。     

建立猪MHCⅠ类抗原特异性人T细胞系的探讨

目的　建立间接识别中国版纳猪SLAⅠ类P1分子的特异性人T细胞系的方法 ,以进一步研究SLAⅠ类分子的间接识别在猪→人异种细胞性排斥中的作用。方法　以E·coli中表达并纯化的SLAⅠ类分子P1为抗原 ,体外反复刺激健康人PBMC ,3H TdR掺入法筛选特异性增殖的T细胞 ,FACS作表型初步分析。结果　初步测定健康人外周血版纳猪SLAⅠ类P1分子特异性T细胞反应频率约 6 .67× 1 0 - 7,所建 4个T细胞系表型均为TCRαβ+ ,其中 3株以CD4+ 为主 ,1株以CD8+ 为主。结论　利用E .coli表达的纯蛋白抗原可建立间接识别版纳猪SLAⅠ类P1分子的特异性人T细胞系。     

T细胞直接活化与交叉活化研究进展

T细胞介导的免疫应答在机体排斥肿瘤的过程中起核心主导作用 ,原始的CD8+ T细胞分化成效应细胞 ,在自身MHCⅠ类分子存在的条件下能识别特异性抗原肽 ,CD8+ CTL可直接识别、杀伤表达特异性抗原的肿瘤细胞。多数抗原加工通道能产生有效的CD8+ T细胞介导的免疫反应     

胃癌患者外周血CD4+CD25+调节性T细胞的检测及临床意义

目的研究胃癌患者外周血CD4+CD25+调节性T细胞频数的变化,并探讨该群细胞频数的变化与肿瘤的病理类型及临床分期的相关性.方法应用流式细胞仪分析胃癌患者和健康对照组外周血中单个核细胞(pBMCs)的CD4+CD25+调节性T细胞频数,并探讨该种细胞频数的变化与胃癌的相关性.结果32例胃癌患者CD4+CD25+T细胞占CD4+T细胞19.40%±8.76%,高于25例健康对照组的10.68%±3.57%(P＜0.01).分化较好的胃癌患者CD4+CD25+T细胞占CD4+T细胞16.79%±4.21%,低分化胃癌患者CD4+CD25+T细胞占CD4+T细胞20.02%±10.42%,两者之间差异显著(P＞0.05).Ⅲ、Ⅳ期的胃癌患者CD4+CD25+T细胞占CD4+T细胞22.43%±8.36%,明显高于Ⅰ、Ⅱ期的胃癌患者(CD4+CD25+T细胞占CD4+T细胞的15.27%±6.85%,P＜0.05).结论CD4+CD25+T细胞在胃癌患者中比例升高,可能是产生肿瘤免疫抑制的重要机制,CD4+CD25+T细胞的高表达可作为胃癌患者肿瘤进展的重要标志.     

胃癌患者外周血nuocytes相关因子的检测及其免疫病理意义

目的检测转录因子维甲酸相关孤核受体α(retinoid acid receptor related orphan receptorα,RORα)、细胞表面受体T1/ST2和IL-17RB以及IL-5和IL-13等细胞因子的表达水平;了解胃癌患者外周血中nuocytes细胞的极化状态,探讨其与胃癌发生发展的关系。方法常规Ficoll-泛影葡胺密度梯度离心法分离外周血单个核细胞;实时荧光定量PCR检测30例胃癌患者PBMC中RORα、IL-17RB、T1/ST2、IL-5、IL-13 mRNA表达水平。结果胃癌患者外周血PBMC中RORα、IL-17RB、T1/ST2、IL-5 mRNA表达水平明显高于健康对照组,而IL-13的表达水平则明显低于健康对照组。相关性分析结果表明,胃癌患者RORαmRNA水平与IL-17RB、T1/ST2、IL-5、IL-13 mRNA水平呈正相关。结论胃癌患者机体存在着nuocytes极化状态,可能是构成胃癌患者Th1/Th2细胞平衡失调的基础,也与MDSC、M2巨噬细胞参与的免疫抑制微环境有着密切的关系。     

Differential expression of the cancer associated antigens T (Thomsen-Friedenreich) and Tn to the skin in primary and metastatic carcinomas.

AIM: To study the immunohistochemical expression of the Thomsen-Friedenreich antigen (T) and its precursor, Tn, in the skin in various cancers. METHODS: T and Tn antigens were studied with monoclonal antibodies in 91 primary premalignant and malignant lesions, 13 cases of Paget's disease, and 26 carcinomas metastatic to the skin. The material had been collected over a 10 year period, formalin fixed, and paraffin embedded. Diagnoses had been made after examination of standard histological sections, supplemented when needed by appropriate immunohistochemical staining. RESULTS: 21% and 29% of the primary cutaneous premalignant and malignant epithelial tumours expressed the Tn and T antigens, respectively. By contrast, 81% of metastatic carcinomas to the skin were Tn positive, while only 23% of them expressed the T antigen. All cases of Paget's disease were Tn positive but only 15% of them expressed the T antigen. The 21 nonepithelial tumours (including melanomas) were as a rule unreactive. CONCLUSIONS: The accumulation of the precursor (Tn) antigen in tumours metastasising to the skin highlights the incomplete glycosylation of carbohydrate antigens occurring in these tumours. The predominant Tn versus T antigen expression appears to be a useful immunohistochemical feature which may aid in the differentiation of primary cutaneous carcinomas from metastatic tumours.     

Expression of Tn and sialyl-Tn antigens in tumor tissues of the ovary.

The expression of sialyl-Tn and Tn antigens in various benign, borderline, and malignant ovarian tumors was examined immunohistochemically using newly developed antibodies specific for sialyl-Tn and Tn antigens. Sialyl-Tn antigen was detected in only one benign tumor, a mucinous adenoma that showed faint cytoplasmic staining in a few cells. However, sialyl-Tn was present in 5 of 12 serous borderline tumors, 10 of 19 mucinous borderline tumors, 10 of 13 serous adenocarcinomas, 15 of 16 mucinous adenocarcinomas, 14 of 15 endometrioid adenocarcinomas, and 7 of 7 clear cell carcinomas of the ovary. The antigen expression was observed throughout the cytoplasm of cancer cells and in the apical cytoplasm and luminal contents of some glands. The incidence and intensity of staining for sialyl-Tn antigen were higher in malignant tumors than in borderline tumors, but these results did not correlate with the histologic classification or differentiation. Coexpression of sialyl-Tn antigen and Tn antigen was observed in two serous adenocarcinomas, six mucinous borderline tumors, five mucinous adenocarcinomas, eight endometrioid, and seven clear cell carcinomas. In no case was Tn antigen expressed without concomitant sialyl-Tn antigen expression. Accumulation of sialyl-Tn antigen seems to be an early event of carcinogenesis of the ovary.     

Expression of the Tn antigen on erythroid cells from a patient with Tn syndrome

Summary In order to examine expression of the Tn antigen on erythroid cells from a patient with Tn syndrome, we applied a selective two phase liquid culture system for human erythroid progenitors in peripheral blood. The cells were analyzed with flow cytometry employing an anti-Tn antibody and a lectin ofVicia villosa which recognizes only the Tn determinant. In the second phase, the Tn antigen was expressed on the cultured cells from the patient on day 3 and Tn-positive cells reached 62.7% on day 9. On the other hand, Tn-positive cells were not detected in the volunteer's cultured cells. When the patient's cells were co-cultured with the cells from a healthy voluteer, the percentage of Tn-positive cells was much lower than the expected value, suggesting that the normal cells suppressed the expression of Tn antigen on the patient's cells.     

Aberrant Cosmc genes result in Tn antigen expression in human colorectal carcinoma cell line HT-29

The Tn antigen, which arises from mutation in the Cosmc gene is one of the most common tumor associated carbohydrate antigens. Cosmc resides in X24 encoded by a single gene and functions as a specific molecular chaperone for T-synthase. While the Tn antigen cannot be detected in normal cells, Cosmc mutations inactivate T-synthase and consequently result in Tn antigen expression within certain cancers. In addition to this Cosmc mutation-induced expression, the Tn antigen is also expressed in such cell lines as Jurkat T, LSC and LS174T. Whether the Cosmc mutation is present in the colon cancer cell line HT-29 is still unclear. Here, we isolate HT-29-Tn⁺ cells from HT-29 cells derived from a female colon cancer patient. These HT-29-Tn⁺ cells show a loss of the Cosmc gene coding sequence (CDS) leading to an absence of T-synthase activity and Tn antigen expression. Additionally, almost no methylation of Cosmc CpG islands was detected in HT-29-Tn⁺ as well as in HT-29-Tn^- and Tn^- tumor cells from male patients. In contrast, the methylation frequency of CpG island of Cosmc in normal female cells was ~50%. Only one active allele of Cosmc existed in HT-29-Tn⁺ and HT-29-Tn^- cells as based upon detection of SNP sites. These results indicate that Tn antigens expression and T-synthase inactivity in HT-29-Tn⁺ cells can be related to the absence of CDS in Cosmc active alleles, while an inactive allele deletion of Cosmc in HT-29 cells has no influence on Cosmc function.     

Expression of MUC1 and MUC2 and carbohydrate antigen Tn change during malignant transformation of biliary papillomatosis

AIM: Biliary papillomatosis is characterized by papillary proliferations of biliary lining cells without invasion or metastasis. The neoplastic character and biological behaviour of this disease remain still speculative. These issues were examined in this study. METHODS AND RESULTS: Mucin core protein MUC1, MUC2, MUC3, MUC5AC and carbohydrate antigens (T, Tn and sialosyl Tn) were immunohistochemically examined, using 11 lesions of biliary papillomatosis from seven patients, and five lesions of biliary papillomatosis with foci of carcinoma from four patients. Five cases of papillary intrahepatic cholangiocarcinoma and 12 histologically normal livers were used as a control. Patients with biliary papillomatosis alone or with carcinoma were middle-aged or elderly (five men and six women). Microscopically, biliary papillomatosis showed a villous, papillo-tubular, papillary, or papillo-villous pattern with a thin fibrovascular core. Cytologically, they were classifiable into biliary epithelial or pyloric gland-like type. The former was frequent in the cases associated with carcinoma. Expression of MUC1, Tn antigen and sialosyl Tn antigen was frequent and marked in biliary papillomatosis alone and with carcinoma and also intrahepatic papillary carcinoma. In addition, marked expression of MUC1 and Tn antigen were rather frequent in biliary papillomatosis with carcinoma and intrahepatic biliary papillary carcinoma compared with biliary papillomatosis. MUC2 was rather frequent and marked in biliary papillomatosis alone compared to other two disease groups. Focal expression of MUC5AC and MUC2 was rather frequent and infrequent irrespective of disease group, respectively. Focal expression of T antigen was frequent in papillary ICC. CONCLUSION: Biliary papillomatosis could undergo overt malignant transformation along with altered phenotypic expression of MUC proteins and mucin carbohydrate antigens.     

Expression of sialyl-Tn, Tn and T antigens in primary liver cancer

Sialyl-Tn, Tn and T antigens are caused by aberrant or incomplete glycosylation of apomucins and are related to the aggressiveness of malignant neoplasms. Using 41 liver samples from patients with cholangiocarcinoma (including four with cirrhosis), 21 with combined hepatocellular-cholangiocellular carcinoma and 17 with hepatocellular carcinoma, the expression of sialyl-Tn, Tn and T antigens were characterized immunohistochemically and the correlation with apomucin profiles was evaluated. The prevalence of sialyl-Tn, Tn and T antigens expression was 89, 95 and 51% in cholangiocarcinoma without cirrhosis; 25, 75, and 0% in cholangiocarcinoma with cirrhosis; 29, 90, and 48% in combined hepatocellular-cholangiocellular carcinoma; and 0, 12 and 6% in hepatocellular carcinoma, respectively. Sialyl-Tn antigen was frequently expressed in cholangiocarcinoma without cirrhosis compared with cholangiocarcinoma with cirrhosis and combined hepatocellular-cholangiocellular carcinoma (P < 0.01). Although sialyl-Tn expression was associated with MUC1, MUC6 and MUC7 expression, the expression sites among them were not identical in the individual cases. These data suggest that the different expressions of sialyl-Tn antigen among cholangiocarcinoma without cirrhosis, cholangiocarcinoma with cirrhosis and combined hepatocellular-cholangiocellular carcinoma may reflect the biological features inherent to these tumors, such as the ability of invasion.     

Immunohistochemical distinction of malignant mesothelioma from pulmonary adenocarcinoma with anti-surfactant apoprotein, anti-Lewisa, and anti-Tn antibodies

Nine cases of malignant mesothelioma of pure epithelial and biphasic types (five pleural, three peritoneal, and one pericardial mesotheliomas), seven cases of benign adenomatoid tumor of the uterus, and 21 cases of peripheral pulmonary adenocarcinoma of non-mucus-producing type were examined immunohistochemically for expression of keratin, vimentin, carcinoembryonic antigen (CEA), surfactant apoprotein, Lewis blood group antigens, and Tn antigen. The majority (78%) of the malignant mesotheliomas expressed keratin, but CEA and surfactant apoprotein were not detected in any mesotheliomas. On the other hand, pulmonary adenocarcinomas expressed not only keratin (100%), but also CEA (62%) and surfactant apoprotein (62%). The expression of Lewisa blood group antigen and Tn antigen was detected in 76% and 62% of the pulmonary adenocarcinomas, respectively, but only one mesothelioma was stained for Lewisa antigen. This study reveals that the majority of malignant mesotheliomas can be distinguished from pulmonary adenocarcinomas by immunohistochemcial staining for CEA, surfactant apoprotein, Lewisa antigen, and Tn antigen. Immunohistochemically, adenomatoid tumors behaved similarly to malignant mesotheliomas.     

Quantitative assessment of Tn antigen in breast tissue micro-arrays using CdSe aqueous quantum dots

In this study, we examined the use of CdSe aqueous quantum dots (AQDs) each conjugated to three streptavidin as a fluorescent label to image Tn antigen expression in various breast tissues via a sandwich staining procedure where the primary monoclonal anti-Tn antibody was bound to the Tn antigen on the tissue, a biotin-labeled secondary antibody was bound to the primary anti-Tn antibody, and finally the streptavidin-conjugated AQDs were bound to the biotin on the secondary antibody. We evaluated the AQD staining of Tn antigen on tissue microarrays consisting of 395 cores from 115 cases including three tumor cores and one normal-tissue core from each breast cancer case and three tumor cores from each benign case. The results indicated AQD-Tn staining was positive in more than 90% of the cells in the cancer cores but not the cells in the normal-tissue cores and the benign tumor cores. As a result, AQD-Tn staining exhibited 95% sensitivity and 90% specificity in differentiating breast cancer against normal breast tissues and benign breast conditions. These results were better than the 90% sensitivity and 80% specificity exhibited by the corresponding horse radish peroxidase (HRP) staining using the same antibodies on the same tissues and those of previous studies that used different fluorescent labels to image Tn antigen. In addition to sensitivity and specificity, the current AQD-Tn staining with a definitive threshold was quantitative.     

Carbohydrate antigen expression in primary tumors, metastatic lesions, and serous effusions from patients diagnosed with epithelial ovarian carcinoma: evidence of up-regulated Tn and Sialyl Tn antigen expression in effusions

The object of this study was the investigation of carbohydrate antigen expression in malignant epithelial cells and benign mesothelial cells in serous effusions from patients diagnosed with epithelial ovarian carcinomas. In addition, to compare antigen expression in carcinoma cells in effusions with those of corresponding primary tumors and metastatic lesions. Sections from 63 malignant effusions from ovarian carcinoma patients and 15 reactive effusions were immunohistochemically stained, using 5 monoclonal antibodies for Lewis(y), Sialyl Lewis(x), Tn, and Sialyl Tn antigens. Tissue sections (n = 97) from corresponding primary ovarian carcinomas and metastatic lesions, as well as from 12 malignant mesotheliomas, were additionally stained using the above panel. Staining for the 4 antigens was seen in carcinoma cells in serous effusions in the majority of cases (range = 71% to 85%). In contrast, immunoreactivity was detected in mesothelial cells in only 6% to 23% of the specimens studied (P < .001 for all 5 markers). With the exception of B3 antibody against Lewis(y) antigen, malignant mesotheliomas stained negative, infrequently showing focal immunoreactivity. An up-regulation of Tn and Sialyl Tn expression was detected in carcinoma cells in effusions when compared with both primary tumors (P < .003 and P < .007, respectively) and metastatic lesions (P < .034 and .041, respectively). Cancer-associated carbohydrate antigens can thus be used as an adjunct in the differentiation between malignant epithelial and reactive mesothelial cells. Ovarian carcinoma cells in effusions show up-regulation of Tn and Sialyl Tn, possibly representing a transient phenotypic alteration facilitating metastasis.     

Innate immune defense in the inner ear – mucines are expressed by the human endolymphatic sac

The human endolymphatic sac has been shown recently to have immunological capacities and has thus been proposed as the main entity protecting the inner ear from pathogen invasion, equivalent to mucosa‐associated lymphoid tissue (MALT). Although the sac expresses molecules of the innate immune system, the potential expression of members of the important mucin family has not been detailed. Thus, this paper explores endolymphatic sac expression of a number of mucins and mucin precursors. Twelve fresh tissue samples from the human endolymphatic sac were obtained during translabyrinthine surgery. The expression of Mucin 1, 2, 5B/AC and 16, as well as the core structure elements (mucin precursors) T‐antigen, Tn‐antigen and Sialyl‐Tn‐antigen was investigated by immunohistochemistry. The endolymphatic sac epithelium expressed MUC1 (both apically towards the endolymphatic sac (ES) lumen and basally towards the capillary network), MUC 16 and Tn‐antigen. There was no labeling after incubation with antibodies against T‐antigen, sialyl‐Tn‐antigen, MUC2 and MUC5B/AC. We conclude that the human endolymphatic sac epithelium expresses a number of mucin molecules, which supports the hypothesis of the sac as the primary immunological tissue structure of the inner ear, equivalent to MALT in other organs. The mucins may also play a role in the formation and continuous homeostasis of the inner ear fluids, as well as the pathogenesis of Meniere's disease.