鼻咽癌组织中EB病毒相关性DNA酶的研究

60年代后期已有大量血清学材料显示了鼻咽癌与EB病毒(EBV)密切相关,1973年Wolf等直接在鼻咽癌活检组织中发现存在EB病毒DNA,EB病毒与鼻咽癌之间的相关性更引人注目。虽然长期以来人们作了各种尝试,但始终未能直接在鼻咽癌组织中找到EB病毒颗粒,早期抗原(EA)和壳抗原(VCA),因而认为存在于鼻咽癌细胞中的EB病毒DNA是处于一种潜伏状态。弄清楚存在于鼻咽癌     

鼻咽癌

Ⅸ中国人鼻咽癌患者抗 EB 病毒核抗原的抗体及与其他 EB 病毒抗体的关系本文报道了各期鼻咽癌病人血清的EB 病毒核抗原(EBNA)的抗体反应,并与其他肿瘤病人和健康人血清抗体做了比较,讨论了 EB 病毒核抗原的抗体与其他EB 病毒的抗体的关系,这些抗原包括病毒的壳膜抗原(VCA)、早期抗原(EA)和可     

抗EBV—EA（R）单克隆抗体的制备及特性

在用正丁酸或IudR诱导过的Raji细胞(n—Raji和IudR—Raji)内能测定到表达EB病毒早期抗原复合物(EA+)的细胞。本文以n-Raji细胞为免疫原制备出四种抗EA单抗BAE4、5、6、7,它们均属小鼠IgM。该组单抗对丙酮固定的EA(+)细胞有反应,抗体定位于胞质,但对甲醇固定的EA(+)细胞呈阴性反应。免疫萤光染色显示,其中BAE5抗体能与检测过的15例鼻咽癌冰冻切片中的癌细胞起阳性反应。上述结果提示,该组单抗识别的抗原决定簇存在于EBV—EA(R)型抗原中,其中之一能被鼻咽癌细胞表达。     

血清EB病毒VCA-IgA抗体测定对鼻咽癌诊断及监护病情的价值

鼻咽癌细胞中存在EB病毒核抗原和EB病毒基因组,提示鼻咽癌与EB病毒密切相关。研究证明,鼻咽癌患者血清中有抗EB病毒相关抗原的抗体升高,其中尤以抗EB病毒壳抗原(VCA)-IgA抗体升高最为特征。我们采用间接免疫荧光法测定了鼻咽癌患者及对照者血清中VCA-IgA抗体,观察了对鼻咽癌诊断及监护病情的价值。     

用抗原斑点试验法测定EB病毒核抗原的抗体

EB病毒的核抗原(EBNA)出现于所有EB病毒转化的类淋巴母细胞株和鼻咽癌细胞中,是细胞感染病毒后的早期产物并持续地存在于细胞核内。近年来,EBNA已被提纯,并被认为和其他DNA肿瘤病毒如SV40与腺病毒等的肿瘤抗原(T抗原)相似。后者是一种和细胞转化有关并维持细胞转化状态的重要产物。EBNA在细胞核内的含量很少,必须用高度敏感的抗补体免疫荧光法或抗补体免疫酶法才能检出。从EBNA阳性细胞中提取的可溶性抗原,可用于补体结合试验(CF),其用耐热的补体结合抗原成份即为EBNA。上述各种方法不能用于测定IgA抗体。由于鼻咽癌患者血清中抗EB病毒IgA抗体的出现和水平的升高是一种相当特异的     

血清EB病毒VCA-IgA抗体测定对鼻咽癌诊断及监护病情的价值

鼻咽癌细胞中存在EB病毒核抗原和EB病毒基因组,提示鼻咽癌与EB病毒密切相关。研究证明,鼻咽癌患者血清中有抗EB病毒相关抗原的抗体升高,其中尤以抗EB病毒壳抗原(VCA)-IgA抗体升高最为特征。我们采用间接免疫荧光法测定了鼻咽癌患者及对照者血清中VCA—IgA抗体,观察了对鼻咽癌诊断及监护病情的价值。材料与方法一、检测对象经病理证实的鼻咽癌患者730例(疗前患者463例,疗后患者267例)、头颈部其它恶性肿瘤33例、其它部位恶性肿瘤(肺癌、乳腺     

联合检测EB病毒不同抗体及EB病毒DNA在鼻咽癌血清学诊断中的价值

目的:评价EB病毒(EBV)抗衣壳抗原VCA-IgA抗体、抗早期抗原EA-IgA抗体、抗立即早期抗原Rta-IgG抗体和EBV-DNA检测在鼻咽癌诊断中的价值。方法:收集160例初治鼻咽癌,133例症状相似的非鼻咽癌患者和163名健康体检者的血清和血浆。采用酶联免疫法检测血清VCA-IgA、EA-IgA和Rta-IgG抗体的水平,用实时荧光定量PCR检测血浆EBV-DNA的相对含量。按鼻咽癌2008临床TNM分期法进行分期,计算不同分组、各临床分期以及鼻咽癌治疗前后的各抗体阳性率、抗体水平以及EB-DNA的检测结果并进行数据分析。结果:鼻咽癌组VCA-IgA、EA-IgA、Rta-IgG和EBV-DNA阳性率均高于鼻咽相关疾病组及健康对照组（均P<0.05）。血清VCA-IgA和EA-IgA结果相对A值（即rA或S/CO值）在Ⅰ、Ⅱ期低于Ⅲ、Ⅳ期,差异有统计学意义(P<0.05),但阳性率在各期间比较差异无统计学意义(P>0.05);Ⅰ、Ⅱ期患者Rta-IgG的rA值和阳性率明显低于Ⅲ、Ⅳ期患者(P<0.05);血浆中EBV-DNA阳性率及EBV-DNA中位数水平随着临床分期的升高而增高,Ⅰ、Ⅱ期与Ⅲ、Ⅳ期比较差异有统计学意义（P<0.05）。治疗有效（CR+ PR）患者的EBV-DNA含量明显低于治疗前水平（P<0.05）。结论:VCA-IgA、EA-IgA、Rta-IgG、EBV-DNA检测有助于鼻咽癌的辅助诊断、临床分期预测及疗效评估。     

鼻咽癌EB病毒血清学检测的临床应用

1964年Epstein和Barr首先在非洲儿童恶性淋巴瘤组织中发现带有疱疹型病毒颗粒的嗜入类淋巴细胞的疱疹病毒,后定名为EB病毒。1966年Old等首次报告在鼻咽癌患者血清中存在着EB病毒抗体。继后的许多研究发现,鼻咽癌患者血清中出现针对多种EB病毒特异性抗原—EBNA、EA、VCA、DNA酶等的抗体,其含量远高于其它癌瘤患者和健康人水平,且这些抗体水平大都随鼻咽癌病情的发展而变化。现将各有关抗体的检测意义阐述如下: 一、EBNA(EB病毒核抗原)是在EB病毒侵染细胞的早期出现,是一种EB病毒的决定性抗原。可用抗补体复层免疫荧光法     

EB病毒DNA检测在鼻咽癌中的应用及研究进展

鼻咽癌是一种发生于鼻咽部黏膜上皮的恶性肿瘤,以我国南方及东南亚地区最为常见。研究证实EB病毒（Epstein-Barr virus,EBV)感染与鼻咽癌的发生发展密切相关,并且EB病毒DNA（Epstein-Barr virus DNA,EBV-DNA）定量水平在鼻咽癌疾病进程、治疗及转归的过程中均会发生相应的规律性改变。因此,EBV-DNA检测在鼻咽癌筛查、早期诊断、个体化治疗及预后评估等方面具有较大的价值。本文就EBV-DNA检测在鼻咽癌中的应用价值及研究进展作简要综述。     

Epstein-Barr病毒致癌潜能的证据

EB病毒与非洲儿童Burkitt淋巴瘤(BL)有密切关系,也可能与鼻咽癌(NPG)有关。在淋巴瘤(RL)活检组织中还未能发现 EB 颗粒,免疫萤光检查虽偶见病毒外壳抗原(VGA)与早期抗原(EA),阳性细胞也只有十万分之一。但淋巴瘤(BL)活检组织经培养后往往在3天内就可看到10％的细胞产生早期抗原(BA),     

DETECTION OF EB VIRUS EARLY ANTIGEN IN NASOPHARYNGEAL CARCINOMA (NPC) USING A NEW MONOCLONAL ANTIBODY--BAE-5

A mouse monoclonal antibody against EB virus EA-R named BAE- 5 was prepared applying hybridoma technique by use of n-butyric add and croton oil induced Raji cells as immunogen.And then, 34 NPCs and 29 mm-NPC neoplasms were detected by the BAE-5 using APAAP immunohistochemical staining method.The results Indicated that all of the 34 NPCs and 9 of the 29 non-NPC neoplasms could reacted with the BAE-5 in spite of the columnar epithelium reserve cells showing positivity also. It is reasonable to come to the conclusion that most of the NPC cells can not only harbour EBV DNA but some of them be able to express EA-R. Some non-NPC neoplasms being demonstrated BAE-5 posttlvlty may be due to the presence of EBV EA- R antigenic epitope or polypeptides simulating the structure of EA-R. The authors think that these findings have theoretical significance for understanding the role of EBV- encoded poiypeptides, especially EA complex, In the development and progression of NPC as well as some non- NPC neoplasms harbouri     

Noninvasive diagnosis of nasopharyngeal carcinoma: nasopharyngeal brushings reveal high Epstein-Barr virus DNA load and carcinoma-specific viral BARF1 mRNA

Nasopharyngeal carcinoma (NPC) is the most prevalent ENT-tumour in Indonesia. We investigated the primary diagnostic value of Epstein-Barr virus (EBV) DNA load and mRNA detection in noninvasive nasopharyngeal (NP) brushings, obtained prospectively from consecutive Indonesian ENT-patients with suspected NPC (N=106) and controls. A subsequent routine NP biopsy was taken for pathological examination and EBER-RISH, yielding 85 confirmed NPC and 21 non-NPC tumour patients. EBV DNA and human DNA load were quantified by real-time PCR. NP brushings from NPC patients contained extremely high EBV DNA loads compared to the 88 non-NPC controls (p<0.0001). Using mean EBV DNA load in controls plus 3 SD as cut-off value, specificity, sensitivity, positive and negative predictive values were 98, 90, 97 and 91%, respectively. Epstein-Barr nuclear antigen 1 (EBNA1) and the carcinoma-specific BARF1 mRNA were detected by nucleic acid sequence based amplification and found in 86 and 74% of NP brushings, confirming NPC tumour cell presence. EBV RNA positivity was even higher in fresh samples stored at -80 degrees C until RNA expression analyses (88% for both EBNA1 and BARF1). EBV RNA-negative NP brushings from proven NPC cases had the lowest EBV DNA loads, indicating erroneous sampling. No EBV mRNA was detected in NP brushings from healthy donors and non-NPC patients. In conclusion, EBV DNA load measurement combined with detection of BARF1 mRNA in simple NP brushings allows noninvasive NPC diagnosis. It reflects carcinoma-specific EBV involvement at the anatomical site of tumour development and reduces the need for invasive biopsies. This procedure may be useful for confirmatory diagnosis in large serological NPC screening programs and has potential as prognostic tool.     

Laboratory markers of tumor burden in nasopharyngeal carcinoma: a comparison of viral load and serologic tests for Epstein-Barr virus

Epstein-Barr virus (EBV) is present within the tumor cells of most cases of nasopharyngeal carcinoma (NPC). Recent studies suggest that tumor burden is proportional to the level of EBV DNA in blood and that rapid blood testing can be used to guide therapeutic intervention. The relative utility of viral load vs. serology has been insufficiently studied. In our study, EBV viral load was measured by quantitative PCR using either real-time or end-point detection systems in serum samples from 124 NPC patients (93 pretreatment, 13 relapsed, 18 in remission) and 40 controls. Serologic titers against EBV early antigen were measured in the same serum samples. EBV DNA was detectable in 64 of 93 untreated NPC patients (69%; mean viral load 11,211 copies/ml), 11 of 13 relapsed NPC patients (85%; mean 53,039 copies/ml) and 0 of 18 remission patients. EBV DNA was detectable in only 1 of 40 non-NPC controls (3%). In 34 instances where paired plasma and serum samples were available for testing, both were effective sample types, and there was no significant difference between end-point and real-time methods for measuring viral load. Early antigen (EA) IgA and IgG titers were elevated in most NPC patients regardless of whether their disease was active or in remission. EBV viral load was more informative than was EA serology for distinguishing remission from relapsed disease. EBV DNA measurement appears to be a noninvasive way to monitor tumor burden after therapy.     

Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma

Expression of the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNA 1 to 6) and membrane-associated protein (LMP) was investigated by immunoblotting in 83 nasopharyngeal carcinoma (NPC) biopsies and 25 other tumor and normal tissue specimens from the head and neck region. Fifty-eight of the 83 NPC biopsies were large enough to yield parallel data on virus DNA and viral expression. All 16 cases of clinically diagnosed and histologically confirmed NPCs from North Africa contained EBV DNA and expressed EBNA-1. Of 31 clinically diagnosed NPCs from China, 29 contained EBV DNA and 25 of these expressed EBNA-1. One control tissue biopsy from the oropharynx of NPC patients contained EBV DNA, but none expressed EBNA-1. The latent membrane protein (LMP) was detected in 22/31 of the Chinese and in 10/16 of the North African NPC biopsies. None of the NPC biopsies or control tissues expressed detectable amounts of EBNA 2 or any of the other 4 nuclear antigens which are invariably expressed in EBV-transformed B cells. A smaller number of tumors from Malaysia and East Africa exhibited a similar pattern of expression. EBV was rescued from a nude-mouse-passaged North African NPC tumor by co-cultivation of the tumor cells with umbilical cord blood lymphocytes. The tumor expressed EBNA 1 and LMP, but not EBNA 2 or the other 4 EBNAs. The resulting LCLs expressed all 6 nuclear antigens, EBNA 1 to 6 and LMP. Our data suggest that expression of the EBV genome is regulated in a tissue-specific fashion.     

Frequency of Epstein Barr Virus Type 1 Among Nasopharyngeal Carcinomas in Iranian Patients

Background: Around 95% of the world’s population are infected with the Epstein-Barr virus (EBV), which can persist latent in B lymphocytes and epithelial cells life-long. EBV has been linked with lymphoid and epithelial cancers and persistence of EBV infection in lymphoid or epithelial cells may result in virus-associated B-cell tumors or nasopharyngeal carcinomas (NPC). This study was conducted to determine the frequency of EBV DNA in nasopharyngeal carcinoma tissue of Iranian patients. Materials and methods: A total of 50 blocks of formalin-fixed paraffin-embedded tissue of NPCs from 38 (76 %) male and 12 (24%) female patients were collected from archives of Ahvaz hospitals. Sections were cut at 5 μm and DNA was extracted for detection of EBV DNA and EBV typing by mested PCR. DNA sequencing was performed to confirm PCR results. The distribution of EBV DNA was compared among WHO histological subtypes of NPC. Results: Some 3 female and 11 (22%) male NPC samples showed positive for EBV DNA type 1, 2/14(22.2%)WHO histological type II and 12/41(29.3%) WHO histological type III. Conclusions: The frequency of EBV DNA among NPCs in Iranian patients was found to be 28%, EBV type I predominating. Both WHO histological type II and III NPC subtypes demonstrated approximately the same detection prevalence.     

Staining of interleukin-10 predicts clinical outcome in patients with nasopharyngeal carcinoma

BACKGROUND: Interleukin-10 (IL-10) has been implicated as an important modulator of lymphoid cells, and its sequence is homologous to an open reading frame in the Epstein-Barr virus (EBV) genome. Nasopharyngeal carcinoma (NPC) is a representative tumor related to EBV infection. METHODS: The authors investigated the expression of IL-10 in 21 primary NPCs by using an immunohistochemical approach to examine its prognostic significance. RESULTS: IL-10 staining was positive in 12 of 21 primary NPCs (57%). There was no association between IL-10 expression and gender, tumor size, the occurrence of lymph node metastases, clinical stage, or recurrence. However, there was a significant difference in overall survival between the negative expression and positive expression of IL-10 (P = 0.0348). Although 87.5% of the IL-10 negative group survived for 5 years, only 15.6% of IL-10 positive patients survived for that length of time by the Kaplan-Meier method. IL-10 expression was significant as an independent prognostic indicator of overall survival by multivariate analysis using the Cox proportional hazards model (odds ratio, 26.64; P = 0.0019). CONCLUSIONS: The results imply that expression of IL-10 is a prognostic factor in patients with NPC and may prove valuable in selecting patients with NPC who are candidates for aggressive therapy.     

Comparison of plasma Epstein-Barr virus (EBV) DNA levels and serum EBV immunoglobulin A/virus capsid antigen antibody titers in patients with nasopharyngeal carcinoma

BACKGROUND: Serologic measurement of antibodies to Epstein-Barr virus (EBV) immunoglobulin A/viral capsid antigen (IgA/VCA) and early antigen (IgA/EA) has been used widely to screen for nasopharyngeal carcinoma (NPC) in China. Recently, it was found that plasma EBV DNA concentration is an indicator for the staging and prognosis of patients with NPC. To determine whether there is a correlation between plasma EBV DNA levels and serum levels of IgA/VCA, the authors measured both in patients with NPC and in a control group. METHODS: Real-time polymerase chain reaction was used for quantitative analysis of plasma EBV DNA concentration, and enzyme-linked immunoadsorbent assay was used to measure EBV VCA/IgA in patients with primary NPC (n = 120 patients), locally recurrent NPC (n = 8 patients), and distant metastatic NPC (n = 21 patients) among 76 patients with NPC after the completion of radiotherapy, in 60 patients with NPC in clinical remission, in 38 patients with non-NPC tumors, and in 47 control individuals. RESULTS: The median plasma EBV DNA levels were 6200 copies/mL, 9200 copies/mL, and 2050 copies/mL in patients with primary, locally recurrent, and distant metastatic NPC, respectively, but declined to 0 copies/mL in patients with clinically remissive NPC, in patients who completed radiotherapy, in patients with non-NPC tumors, and in the control group. In contrast, EBV VCA/IgA titers and detection rates remained high in all NPC groups. Plasma EBV DNA levels were significantly higher in patients who had serum VCA/IgA titers > or = 1:640 (median, 83,450 copies/mL) compared with the levels in patients who had titers < or = 1:320 (median, 17,200 copies/mL). Patients with NPC who had advanced TNM stage (Stages III and IV; median, 8530 copies/mL) and T classification (T3 and T4 tumors; median, 8530 copies/mL) had significantly higher plasma EBV DNA levels compared with patients who had early TNM stage (Stages I and II; median, 930 copies/mL) and T classification (T1 and T2 tumors; median, 3700 copies). Patients who had advanced TNM stage NPC had significantly higher mean VCA/IgA titers (1:424) compared with patients who had early TNM stage NPC (1:246), but there was no correlation between IgA/VCA titer and T or N classification of NPC. CONCLUSIONS: The results suggest that plasma EBV DNA detection is a more sensitive and specific marker than the serum IgA/VCA titer for the diagnosis and monitoring of patients with NPC. These findings provide convincing evidence for the use of plasma EBV DNA measurements for the early diagnosis and staging of NPC as well as for monitoring recurrence and metastasis of this tumor.     

The 30-bp deletion variant: a polymorphism of latent membrane protein 1 prevalent in endemic and non-endemic areas of nasopharyngeal carcinomas in China.

Development of nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. However, NPC occurs with a marked geographic and racial distribution, whereas EBV infection is ubiquitous in the world. This leads to a question whether certain subtypes of EBV have a greater potential to induce cell transformation. Latent membrane protein 1 (LMP1) is an EBV-encoded oncogenic protein and its 30-bp deleted variant (del-LMP1) has been reported to be predominant in biopsies of NPC. We have assessed the polymorphism of LMP1 in 47 biopsies of NPC, 107 cases of throat washings (TWs) from NPC patients, and 106 cases of TWs from non-NPC patients in Guangzhou, an endemic area of NPC in southern China, as well as 103 cases of TWs from healthy donors in Haerbin, a non-endemic area of NPC in northern China. Our results found a similar extent of the LMP1 polymorphism between NPC patients and non-NPC patients in Guangzhou, with the del-LMP1 being predominant in both Guangzhou and Haerbin. Sequence analyses showed identical substitutions in other coding regions of the del-LMP1 isolated from Guangzhou and Haerbin. These results indicate that del-LMP1 represents a geographic or race-associated polymorphism rather than an NPC disease phenotype-associated polymorphism.     

Serologic diagnosis of nasopharyngeal carcinoma. A double-blind study of four EB virus antibodies with evaluation by sequential discrimination

It is generally known that a close relationship exists between Epstein-Barr Virus (EBV) and nasopharyngeal carcinoma (NPC). Recently, patients with early lesions of NPC have been detected in the general population by use of serologic mass survey. Using the double-blind method, we have studied the diagnostic value of the four EBV antibody titers, VCA-IgA, VCA-IgG, EA-IgA and EA-IgG, in four groups of subjects, each consisting of 50 persons: patients with nasopharyngeal carcinoma (NPC group), patients with cancers other than NPC in the head and neck regions (HNC group), patients with cancers outside of head and neck regions (OC group) and normal individuals (NS group). The results of these four antibodies were evaluated both singularly and together by multivariate sequential discrimination. Taking 1:10 as the criterion of being positive, in the NPC group, the positive rate of VCA-IgA is 88%, the VCA-IgG rate is 100%, the EA-IgA rate is 48% and the EA-IgG rate is 74%. In the non-NPC group, the positive rates of VCA-IgA are as high as 86%-92%, but those of the other antibodies are as low as 0-42%. The positive rates and the geometric mean titers of these four antibodies were all elevated as compared with those in the three non-NPC groups. These differences are statistically significant. VCA-IgG is unimportant in the diagnosis of NPC because of its low specificity. By treating the antibody titers of VCA-IgA, VCA-IgG, EA-IgA and EA-IgG with sequential discrimination, the correlation rate between the serology and pathology of NPC is 88% and the false positive rate is 7.3%.     

Elevated expression of ICAM1 (CD54) and minimal expression of LFA3 (CD58) in Epstein-Barr-virus-positive nasopharyngeal carcinoma cells.

Undifferentiated nasopharyngeal carcinoma (NPC) is a remarkable entity among human tumors because of its constant association with the Epstein-Barr virus (EBV). Malignant epithelial cells harbor the EBV genome and often express at least 2 species of latent EBV protein (EBNA1 and LMP1). Despite the massive presence of tumor-infiltrating lymphocytes, NPC cells obviously escape immune surveillance directed to EBV antigens. Previous investigations carried out on EBV-positive Burkitt lymphoma (BL) cells have shown that this fact may be partially accounted for by a lack of expression of ICAM1 (CD54) and LFA3 (CD58). ICAM1 and LFA3 have therefore been investigated in fresh NPC biopsies and transplanted NPCs. With only 1 exception out of 9 cases, NPC cells appear to express high levels of ICAM1 and low levels of LFA3. This is a complete inversion of the pattern observed in normal epithelial cells in vivo. Additional investigations will be required to determine to what extent these characteristics affect T-cell interactions with NPC cells, specially in the process of EBV-antigen recognition.