以双合成肽为抗原建立的ELISA试验在鼻咽癌血清学诊断中的应用价值

目的研究EBNA1和VCA-P18双多肽包被（EBNA1＋VCA-P18）的ELISA检测在鼻咽癌（NPC）血清学诊断中的价值。方法用合成肽EBNA1和VCA-P18建立ELISA法,检测37例病理确诊的鼻咽癌患者血清和33例健康人血清,并与国产EBVVCA/IgAELISA试剂盒比较。结果双多肽EBNA1＋VCA-P18/IgA灵敏度为91.9%,明显高于国产试剂盒的83.8%;二者特异度相同;EBNA1＋VCA-P18/IgA的阳性预测值、阴性预测值、阳性似然比及优势比均高于国产试剂盒。结论双多肽EBNA1＋VCA-P18/IgA相比商品化试剂盒在鼻咽癌血清学检测中有更高的灵敏度,适用于鼻咽癌早期诊断和人群筛查。     

五厂家EB病毒抗体检测试剂盒血清学诊断鼻咽癌的比较

目的 通过比较EB病毒抗体检测试剂盒血清学诊断鼻咽癌的准确性和检测结果的一致性,为试剂盒在临床上的使用选择和性能改进提供依据.方法 使用五厂家的EB病毒衣壳抗原IgA和IgG抗体检测试剂盒（VCA IgA和VCA IgG试剂盒）、核抗原I IgA和IgG抗体检测试剂盒（EBNA1 IgA和EBNA1IgG试剂盒）、早期抗原IgA和IgG抗体检测试剂盒（EA IgA和EA IgG试剂盒）以及Zta IgA抗体检测试剂盒,分别检测33例鼻咽癌患者（NPC）、30例健康体检者（HD）和41例非鼻咽癌的其他肿瘤患者（NNPC）血清或血浆样本.结果 A厂家的VCA IgA试剂盒灵敏度高于其他厂家同品种试剂盒,但对于NNPC特异度最低（36.6％）;而D厂家VCA IgA试剂盒的特异度最高（97.6％）,且对HD的特异度均大于90％.B和D厂家的EBNA1 IgA试剂盒间阳性、阴性符合率分别为92.1％和100.0％.A和E厂家的EA IgA试剂盒的灵敏度均较低而特异度高,试剂盒间阳性符合率低（39.4％）,阴性符合率高（98.6％）;而VCA IgG试剂盒的灵敏度高但特异度低.A和C厂家的EBNA1IgG试剂盒的灵敏度高（100.0％,97.0％）但特异度低（3.3％,13.3％）.C厂家EA IgG试剂盒检测所有样本结果均为阴性.结论 五个不同厂家VCA LgA、EA IgA试剂盒诊断鼻咽癌的准确性和检测结果的一致性存在差异,特别是A厂家和其他国内厂家同品种试剂间差异明显,需根据临床目的进行选择.三家国产VCA IgA试剂盒的灵敏度需进一步提高.相反,EBNA IgA试剂盒诊断鼻咽癌的准确性和结果一致性较好.单独使用VCA IgG和EBNA1 IgG试剂盒血清学诊断鼻咽癌的特异度差,其判读界值可能需根据检测且的进行调整.     

Vascular lesion in a patient of chronic active Epstein-Barr virus infection with hypersensitivity to mosquito bites: vasculitis induced by mosquito bite with the infiltration of nonneoplastic Epstein-Barr virus-positive cells and subsequent development of natural killer/T-cell lymphoma with angiodestruction

This report describes a vasculitis and subsequently developing angiodestructive lymphoma in an 11-year-old Japanese-Filipino girl exhibiting mosquito allergy with the background of chronic active Epstein-Barr virus (EBV) infection. She developed necrotic skin ulcer at the site of mosquito bite, and histopathological examination revealed EBV-positive mononuclear cell infiltration throughout the wall of small-sized muscular artery. These EBV-positive lymphoid cells were oligoclonal in Southern blot analysis for EBV terminal repeats. Effectiveness of steroid therapy also supports the nonneoplastic nature. Approximately 1 year later, she developed progressive large skin ulcer without mosquito bites. Microscopically, the angiocentric or angiodestructive pattern of EBV-positive atypical cells supported the diagnosis of extranodal natural killer/T-cell lymphoma. Southern blot analysis revealed the monoclonal neoplastic nature of EBV-positive cells. In contrast to the primary mosquito bite lesion, natural killer/T-cell lymphoma cells exhibited the higher expression of EBV latent membrane protein 1 mRNA and the apparent protein expression detected by immunohistochemistry.     

EBV-encoded EBNA-5 associates with P14ARF in extranucleolar inclusions and prolongs the survival of P14ARF-expressing cells

Epstein-Barr virus (EBV) carrying lymphoblastoid cells of normal origin express the full program of all 9 virus-encoded, growth transformation associated proteins. They have an intact p53 pathway as a rule. This raises the question of whether any of the viral proteins impair the pathway functionally. Using a yeast 2-hybrid system, we have shown that EBNA-5 but not the other EBNAs interacts with the p14ARF protein, a regulator of the p53 pathway. The interaction was confirmed in vitro using a GST pull-down assay. Moreover, expression of EBNA-5 increased the survival of p14ARF-transfected cells. EBV infection of resting B cells induced the expression of p14ARF mRNA without increased level of the protein. A fraction of the p14ARF localized to the nucleoli but the bulk of the protein accumulated in nuclear but extranucleolar inclusions. Formation of the extranucleolar inclusions led to complete relocalization of EBNA-5 from nucleoplasm to these structures. The inclusions also contained p53 and HDM2, and were surrounded by PML bodies and proteasomes, which suggests that these inclusions could be targets for proteasome dependent protein degradation.     

CD8 T cell recognition of endogenously expressed epstein-barr virus nuclear antigen 1

The Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 contains a glycine-alanine repeat (GAr) domain that appears to protect the antigen from proteasomal breakdown and, as measured in cytotoxicity assays, from major histocompatibility complex (MHC) class I-restricted presentation to CD8+ T cells. This led to the concept of EBNA1 as an immunologically silent protein that although unique in being expressed in all EBV malignancies, could not be exploited as a CD8 target. Here, using CD8+ T cell clones to native EBNA1 epitopes upstream and downstream of the GAr domain and assaying recognition by interferon gamma release, we show that the EBNA1 naturally expressed in EBV-transformed lymphoblastoid cell lines (LCLs) is in fact presented to CD8+ T cells via a proteasome/peptide transporter-dependent pathway. Furthermore, LCL recognition by such CD8+ T cells, although slightly lower than seen with paired lines expressing a GAr-deleted EBNA1 protein, leads to strong and specific inhibition of LCL outgrowth in vitro. Endogenously expressed EBNA1 is therefore accessible to the MHC class I pathway despite GAr-mediated stabilization of the mature protein. We infer that EBNA1-specific CD8+ T cells do play a role in control of EBV infection in vivo and might be exploitable in the control of EBV+ malignancies.     

Epstein-Barr Virus nuclear antigen 1 (EBNA1) confers resistance to apoptosis in EBV-positive B-lymphoma cells through up-regulation of survivin

Resistance to apoptosis is an important component of the overall mechanism which drives the tumorigenic process. EBV is a ubiquitous human gamma-herpesvirus which preferentially establishes latent infection in viral infected B-lymphocytes. EBNA1 is typically expressed in most forms of EBV-positive malignancies and is important for replication of the latent episome in concert with replication of the host cells. Here, we investigate the effects of EBNA1 on survivin up-regulation in EBV-infected human B-lymphoma cells. We present evidence which demonstrates that EBNA1 forms a complex with Sp1 or Sp1-like proteins bound to their cis-element at the survivin promoter. This enhances the activity of the complex and up-regulates survivin. Knockdown of survivin and EBNA1 showed enhanced apoptosis in infected cells and thus supports a role for EBNA1 in suppressing apoptosis in EBV-infected cells. Here, we suggest that EBV encoded EBNA1 can contribute to the oncogenic process by up-regulating the apoptosis suppressor protein, survivin in EBV-associated B-lymphoma cells.     

Analysis of Epstein–Barr virus (EBV) type and variant in spontaneous lymphoblastoid cells and Hu-SCID mouse tumours

Epstein–Barr virus (EBV) type and strain variations were examined using both lymphoblastoid cell lines (LCLs), spontaneously derivedin vitrofrom peripheral blood mononuclear cells (PBMC) of 15 HIV-1-seropositive individuals, and SCID mouse tumours induced by inoculation of PBMC from 11 healthy human donors (Hu-SCID tumours). Polymerase chain reaction (PCR) analysis disclosed that all but one of the 26 EBV+ samples harboured EBV nuclear antigen (EBNA) 2 and 3C type A virus. On the other hand, single strand conformation polymorphism (SSCP) analysis using Epstein–Barr encoded RNA (EBER) specific primers detected an AG876-like (type B) band pattern in 21 of the 26 EBV+ samples. Three Hu-SCID tumours scored as B95.8-like (type A), and two showed neither a type A nor a type B SSCP migration pattern. Sequence analysis of the amplified EBER fragments confirmed the PCR-SSCP findings; moreover, additional mutations were present not only in the two EBV+ samples with anomalous SSCP pattern, but also in two other samples with a standard SSCP profile. Thus, EBER analysis did not correlate with EBNA typing, and appeared to be unsuitable for EBV type assessment. Latent membrane protein (LMP) analysis disclosed, on the whole, seven size variants: as expected, the differences were due to the variable numbers of a 33-bp repeat in the amplified fragment, as assessed by direct sequencing. The broader variability detected by LMP analysis should prove more useful than typing for assessing the presence of single and/or mixed variants resulting from EBV reactivation and/or reinfection.     

EBNA1 expression in a lung transplant recipient with hypocomplementemic urticarial vasculitis syndrome

This article describes a transplant recipient with underlying hypocomplementemic urticarial vasculitis syndrome who expressed persistently Epstein-Barr virus nuclear antigen 1 (EBNA1) in peripheral blood. The patient received a bilateral lung transplant and was subsequently followed with monitoring of EBV expression in peripheral blood. Evaluation of viral expression in peripheral blood, serum, and graft tissue was performed with RT-PCR, Q-PCR, indirect immunofluorescence, anti-peptide assays, and in situ hybridization; samples were collected at various time-points up to 91 days post-transplantation. The patient expressed EBNA1 in 8/10 (80%) of the peripheral blood samples tested during the post-transplantation period, and interestingly, even including the day of transplantation. After analyses of indicative EBV mRNA, EBNA1 expression was found mainly to be Qp-initiated EBNA1, known to be important for EBV maintenance. Anti-EBNA1 epitope mapping showed significantly higher and broader antibody responses to EBNA1 epitopes pre-transplantation when compared to normal controls and a matched lung transplant control. Post-transplantation this response was largely diminished but there were still epitopes significantly higher than controls. Our results show the presence of EBV-positive proliferating cells before onset of intensive immunosuppressive treatment. Although no previous connection between EBV and hypocomplementemic urticarial vasculitis syndrome has been reported, it is tempting to speculate that the continuous EBNA1 expression is not caused by immunosuppression or post-transplant lymphoproliferative disease, but may be a factor involved in the etiology of the autoimmune disease.